Journal o f General Virology (1990), 71, 501-522.

501

Printed in Great Britain

Review article Molecular biology of the Bunyaviridae Richard M. Elliott Institute of Virology, University of Glasgow, Church Street, Glasgow Gll 5JR, U.K.

Introduction More than 300 viruses, mostly arthropod-transmitted, are classified into the family Bunyaviridae, making it one of the largest groupings of animal viruses (Karabatsos, 1985). Until relatively recently these viruses were somewhat the 'Cinderellas' of animal virology, but with the increased recognition of their role in human diseases together with the results generated by the application of molecular techniques, the Bunyaviridae have achieved greater respectability. Rift Valley fever, CrimeanCongo haemorrhagic fever and California encephalitis viruses are serious human pathogens that are classified in the family Bunyaviridae. In the tropics febrile illnesses are often diagnosed under the 'great umbrella' (Downs, 1975) of malaria and treated as such; in fact many cases are probably caused by members of the Bunyaviridae, although true diagnosis is rarely achieved (Shope, 1985). Hantaan and related viruses, the causative agents of haemorrhagic fever with renal syndrome, are now recognized as belonging to the Bunyaviridae (Schmaljohn & Dalrymple, 1983) and cause a severe haemorrhagic disease with significant mortality throughout Asia, especially in China. As will be discussed later the Bunyaviridae have the capacity for sudden dramatic variation comparable to the antigenic shift associated with influenza viruses and thus warrant continual surveillance. At an academic level the Bunyaviridae display novel features in their gene coding arrangements and in their replication to maintain scientific interest. On a lighter note the Bunyaviridae also offer the opportunity to work with viruses having such glorious names as Main Drain, Pongola, Mahogany Hammock, Wongal etc. ; the reader is referred to the International Catalogue of Arboviruses (Karabatsos, 1985) for other gems. The criteria for inclusion in the family Bunyaviridae are the following: virus particles are spherical, 90-100 nm in diameter and enveloped with glycoprotein surface projections; the virions contain three unique segments of negative-sense single-stranded R N A in the form of 0000-9320 © 1990 SGM

circular ribonucleoprotein complexes (nucleocapsids) and a transcriptase enzyme. Viruses have the ability to interact genetically with certain other closely related viruses by genome segment reassortment. The viruses replicate in the cytoplasm of the infected cell and mature by budding into smooth-surface vesicles in or near the Golgi region (Bishop et al., 1980). In this review recent data concerning the molecular characteristics of the Bunyaviridae will be discussed to provide an update on previous accounts of this area (e.g. Obijeski & Murphy, 1977; Bishop & Shope, 1979; Bishop, 1979, 1985a). Important aspects of the biology and pathogenesis of the Bunyaviridae are afforded only cursory treatment, but have been covered elsewhere (e.g. Parsonson & McPhee, 1985; Shope, 1985; Canonico & Pifat, 1987; Beaty & Bishop, 1988; Gonzalez-Scarano et al., 1988; Monath, 1988; Tesh, 1988).

Taxonomy and classification The classification of viruses now in the family Bunyaviridae was originally based on their serological relationships and these data have been supplemented and largely supported by biochemical analyses. Five genera are currently recognized within the family: Bunyavirus, Hantavirus, Nairovirus, Phlebovirus and Uukuvirus (Bishop et al., 1980; Schmaljohn & Dalrymple, 1983). In addition, at least 10 serogroups containing 28 viruses, as well as 26 other viruses, are considered to be members of the family, but have yet to be assigned to a particular genus. In general viruses within a genus share complement-fixation antibodies and are segregated into serogroups on the basis of neutralization and haemagglutination-inhibition antibodies; phleboviruses are exceptions in that the complement-fixation test is specific and the haemagglutination-inhibition is cross-reactive (Calisher et al., 1981 ; Shope, 1985; Calisher & Karabatsos, 1988). Bishop (1985b) has suggested division of the Bunyaviridae into a number of subfamilies (Bunyavirinae, Nairovirinae and Phlebovirinae), but the proposal has not yet

502

R . M . Ell~tt

Table 1. Classification and properties of the Bunyaviridae and properties of tomato spotted wilt virus Bunyaviridae Genus

Bunyavirus

Hantavirus

No. of serogroups No. of viruses Prototype virus

16 156 Bunyamwera

1 9 Hantaan

Examples of viruses associated with human disease

La Crosse California encephalitis Tahyna Jamestown Canyon Oropouche Mosquitoes gnats 6875 4458-4534 850-984

Hantaan Puumula Seoul

L 259K G1 108K-120K G2 29K 41K

L >200K G1 68K-76K G2 52K-58K

N 19K-25K M : NSm, 15K-18K

N 50K 54K

Principal arthropod vectors Size of L RNA M segmentst S (bases) Size of structural proteins Non-structural proteins and RNA segment coding assignment

None (65001-8500) 3616 1696

NDII S: NSs, 10K-13K

Nairovirus 6 32 Crimean-Congo haemorrhagic fever Crimean Congo haemorrhagic fever

Phlebovirus

Uukuvirus

1 (8)*

Tomato spotted wilt virus

1

1

39 Sandfly fever Sicilian Sandfly fever Naples and Sicilian Toscana Rift Valley fever

12 Uukuniemi None

1 Tomato spotted wilt None

Sandflies gnats (6500-8200) 3884-4330 1746-1904

Ticks

Thrips

L >200K G1 72K-84K G2/3 30K-45K:~

L >200K G1 55K-70K G2 50K-60K

L >200K G1 70K-75K G2 65K-70K

N 48K-54K Glycoprotein precursors

N 20K-30K M: 14K, 78K

N 20K-25K

S: NS, 29K 31K

S: NS, 30K

Ticks (11000-14400) (4400-6300) (1760 2050)

(6500-8500) 3231 (1200-1900)

(7940-8200) (5000-5400) (3200-3400) L G1 G2a G2b N

>200K 78K 58K 52K§ 27K

ND

* Phleboviruses comprise a single serogroup (phlebotomus fever) divided into eight antigenic complexes. t Figures in brackets are estimates from analysis of RNA directly, unbracketed figures are from nucleotide sequence data (see Table 2). :~Some nairoviruses may have three structural glycoproteins. § G2b may be a degradation product of G2a. [I ND, Not detected. been adopted. As more data b e c o m e available and as new viruses are discovered the classification m a y change and hence designations described here should be regarded as somewhat fuid. The classification and distinguishing features o f the Bunyaviridae are summarized in Table 1. More detailed discussion and comprehensive lists of viruses are found elsewhere (Bishop & Shope, 1979; Bishop, 1979, 1985a, b, 1986; Karabatsos, 1985; Calisher & Karabatsos, 1988). At this point it should be noted that the term 'bunyavirus' is applied only to m e m b e r s of the Bunyavirus genus and is not used when discussing the family Bunyaviridae in general. It is hoped that others will adhere to this principle to avoid confusion in the literature. As can be seen in Table 1 there are genus-specific patterns in the sizes o f the three genomic R N A segments and in the sizes of the four structural proteins that m a k e up the virion. Bunyaviruses and nairoviruses have distinctive R N A profiles, whereas the R N A segments of hantavirus, phlebovirus and uukuvirus are similar in size. The pattern o f glycoprotein and nucleocapsid

protein sizes is distinct for bunya-, hanta- and nairoviruses, but those of phleboviruses and uukuviruses are very similar. The Y-terminal nucleotide sequences of the three genomic R N A segments are conserved within each genus (Clerx-van Haaster & Bishop, 1980; Obijeski et al., 1980; Parker & Hewlett, 1981 ; Clerx-van Haaster et al., 1982a, b; Schmaljohn & Dalrymple, 1983). As shown in Fig. 1 the conserved sequences of bunyaviruses and hantaviruses are similar and those of phleboviruses and uukuviruses are identical. F r o m the above information it appears that the phleboviruses and uukuviruses are the most closely related of the genera, based on their structural similarities, and recently a weak serological relationship has been demonstrated between members of these two genera (C. H. Calisher, personal c o m m u n i c a tion), although there are distinct differences in their biological properties. The majority o f the Bunyaviridae are transmitted by biting arthropods; bunyaviruses by mosquitoes and gnats, nairoviruses and uukuviruses by ticks, and phleboviruses by sandflies (phlebotomines) and gnats (Bishop & Shope, 1979). Infection apparently has no ill

R e v i e w : Molecular biology o f the Bunyaviridae

Genus

Terminal sequence

Bunyav~z~

(3')

Hantavirus

(Y)

Nairovirus

(3')

u

c

A

U

C

A

C

A

U

G

A

A

U

C

A

U

C

A

U

C

U

G

.

A

G

A

G

A

U

U

C

U

.

.

.

o

,

o

.

503

Collett et al., 1985). Further molecular studies are needed to unravel fully the genome strategy and possible relationships to the Bunyaviridae, but the available data do suggest that tomato spotted wilt virus may be the first phytobunyavirus.

Virion structure

Phh, bovirus

(3')

u

G

U

G

U

U

U

C

. . . .

Uukm'irus

(3')

u

g

u

G

u

u

u

c

u

°

o

° .

Fig. 1. Genus-specificT-terminalconsensusnucleotidesequencesof BunyaviridaeVRNAsegments.

effects on the vector and a life-long persistent infection usually develops. The viruses can be transmitted between adults venereally and transovarially to their offspring; in fact overwintering in eggs is a method of maintenance of the virus in nature (Bishop & Shope, 1979; Beaty & Bishop, 1988). There is no evidence of an arthropod vector for hantaviruses; these viruses cause asymptomatic persistent infections of rodents and rodent-rodent and rodent-human infections are caused by aerosolized rodent excretions. Human disease has been associated with representatives of all genera except the uukuviruses. Disease syndromes include transient mild fevers, encephalitides and severe haemorrhagic fevers, which are detailed in a review by Shope (1985). Whereas most human infections are transmitted by vectors, Crimean-Congo haemorrhagic fever and Rift Valley fever viruses can also be spread by exposure to infected tissue. These agents therefore put medical, nursing and veterinary staff at risk. In fact with Crimean-Congo haemorrhagic fever virus the case fatality rate is higher following nosocomial infection than natural infection (Shope, 1985). Here I should mention tomato spotted wilt virus, an arthropod (thrip)-transmitted virus of plants, which possesses some of the characteristics of the Bunyaviridae, i.e. a similar morphology, tripartite ssRNA genome and cytoplasmic site of particle maturation (Milne & Francki, 1984; de Haan et aI., I989). Preliminary data from de Haan et al. (1989) indicate that the M RNA segment of tomato spotted wilt virus is of negative polarity and that the N-terminal 80 amino acids of the predicted gene product of this segment show 25~ similarity with the NSm protein encoded by the M segment of Rift Valley fever virus (Phlebovirus genus;

Using electron microscopy, negatively stained Bunyaviridae particles appear spherical or pleomorphic, according to thefixation conditions, and are usually surrounded by a fringe of glycoprotein spikes (Pettersson et al., 1971 ; Murphy et al., 1973; Smith & Pifat, 1982; Hung et aL, 1983; Pettersson &von Bonsdorff, 1987). Martin et al. (1985) have described subtle differences in the morphology of different viruses that correlate with their genus; presumably these differences reflect altered interaction of the stain with the surface proteins, or different arrangements of the surface proteins on the particle. More recently Talmon et al. (1987) examined vitrifiedhydrated La Crosse virions using cryoelectron microscopy, which preserves the structure of the particles in their native state (Fig. 2). The virions are uniformly spherical, but of varying diameters (75 to 115 nm). A membrane bilayer (4 nm thick) and spikes (10 nm long) are clearly seen. The virus particle consists of four structural proteins: two internal proteins, the L (transcriptase component) and the N (nucleocapsid) proteins, and two external glycoproteins, termed G1 and G2, which are inserted in the viral membrane. By convention the larger Mr glycoprotein is designated G 1. Some nairoviruses may be exceptions to this pattern, as Foulke et al. (1981) detected three glycoprotein species in Hazara virus. The Bunyaviridae do not encode an internal matrix protein, therefore the virion structure may be stabilized by direct interaction of the internal nucleocapsids with the membrane or with the cytoplasmic domain of the inserted glycoprotein (Pettersson &von Bonsdorff, 1987; Talmon et al., 1987). The tripartite ssRNA genome of the Bunyaviridae forms 1 to 2 ~ by weight of the particle and each genome segment is intimately associated with the N (2100 molecules per particle) and L proteins (25 molecules per particle; Obijeski et al., 1976a) to form a nucleocapsid. A feature of the individual genome segments is the complementarity of the 3' and 5" termini, which presumably accounts for the observation of circular and/or panhandle forms of the RNAs seen by electron microscopy (Bouloy et al., 1973/74; Samso et al., 1976; Hewlett et al., 1977; Pardigon et al., 1982). The terminal 11 bases of the three Bunyamwera virus RNAs are conserved and complementary except for positions 9 and

504

R. M . Elliott

\ 3' 5'

G

/\

C-G-A

/

U-C-A-U-C-A-C-A-U-G-A-G-G-U-G G-A-U U-U-U-G-A-A I I I l I I I i J l I I E I I I i I l I i [ I A-G-U-A-G-U-G-U-G-C-U-C-C-A-C-C-U-A-A-A-A-C-U-U

A-C

\

/

U-A I I A-U

\

/

\

A-A-A

/ 3" 5'

A-G

U-C-A-U-C-A-C-A-U-G-A-U-G-G-C-U-A-U-G-U I r I i I 4 i I I I I I I F I I I I I A-G-U-A-G-U-G-U-G-C-U-A-C-C-G-A-U-A-C-A

\

\

A-C-

U

A-

/\

/

U-G-U-U G-G-A-A r I I I b I r A-C-A-G-C-C-U U

\

/

A-A

A

G-

?I?

3"U-C-A-U-C-A-C-A-U-G-A-G-G-A-U-G-U-A-UU-C-U-U-U-U-AA I I I I I [ I l i t I I I I I I I I 1 1 l 1 1 1 1 1 5"A-G-U-A-G-U-G-U-G-C-U-C-C-U-A-C-A-U-A-A-G-A-A-A-A-U-U

\

U I A

/\

G-UC-

Fig. 3. Complementarysequencesand possible base-paired structures between the 3' and 5' termini of Bunyamwera virus genomic RNA segments.

Fig. 2. Electron micrographs of vitrified-hydratedLa Crosse virions. (a) Small defocus value, which demonstrates membrane bilayer; (b) large defocus value, which demonstrates spikes (see Talmon et al., 1987). Bar marker represents 100 nm. These micrographs were generously supplied by Dr B. V. V. Prasad.

- 9 and the following 20 or so bases show segmentspecific complementarity (Fig. 3). The mismatch at positions 9 and - 9 is found in all bunyavirus RNAs sequenced so far, but not in the complementary sequence of the termini of the phlebovirus M and S segments, the uukuvirus M, or the hantavirus M and S segments.

However, in the sequence of the Hantaan virus M segment reported by Yoo & Kang (1987a) positions 12 and - 12 are mismatched in the terminal 18 complementary nucleotides. Bunyavirus and uukuvirus nucleocapsids are circular (Pettersson & v o n Bonsdorff, 1975; Samso et al., 1975 ; Obijeski et al., 1976b). It is probable that the conserved terminal sequences of the RNAs are important for encapsidation by N protein because, in general, genomic ( - ) R N A and full-length viral complementary ( + ) R N A are encapsidated, whereas viral m R N A , which has 5'-terminal extensions and is shorter at the 3' end, is not (Raju & Kolakofsky, 1986b). However, Raju & Kalakofsky (1987) reported a minor amount of mRNA-like transcripts late in infection that were encapsidated and some full-length ( + ) molecules with 5' extensions that were not encapsidated. The authors concluded that the nucleocapsid recognition sequence was at the 5' end of the R N A and this had to be in the correct context for initiation of encapsidation. More recently Raju & Kolakofsky (1989) have shown that the ends of the fulllength ( - ) and ( + ) strands are base-paired in the viral nucleocapsid. These workers reiterated the importance of unmatched and mismatched bases within doublestranded regions of R N A for recognition by proteins (reviewed by Wickens & Dahlberg, 1987) and also that the ends of the ( - ) and ( + ) RNAs would have significantly different structures, i.e. G - U in genomic R N A is an 'allowed' pair, whereas the C - A in the complementary R N A is not (Fig. 3). I f a protein (e.g. the N protein) could distinguish these structures this may be important in transcription and the selective packaging of ( - ) R N A into virions. Hsu et al. (1987) have shown that the negatively stranded R N A segments of the influenza virus genome are in a circular conformation as well and also suggested that the terminal base-paired structures were cis-acting regulatory elements for the transcription,

R e v i e w : M o l e c u l a r biology o f the Bunyaviridae

505

Table 2. Complete nucleotide sequences o f Bunyaviridae genome segments Genus Bunyavirus

Serogroup

Virus

Bunyamwera Bunyamwera Germiston California

Maguari La Crosse Snowshoe hare

Hantavirus Phlebovirus

Uukuvirus

Simbu Hantaan

Aino Hantaan 76-118

Phlebotomus fever

Rift Valley fever Punta Toro

Uukuniemi

Sandfly fever Sicilian Uukuniemi

Segment

Length (nucleotides)

A+ U (%)

L M S M S S M S S M S S M M S M

6875 4458 961 4534 980 945 4526 984 984 4527 982 850 3616 3616 1696 3884

66.7 61.0 58.2 63-2 57.6 59.9 62-0 58.8 58.8 61-0 56.3 55.9 60.7 60.7 57.1 54.5

Elliott (1989b) Lees et al. (1986) Elliott(1989a) Pardigonet al. (1988) Gerbaud et al. (1987b) Elliott & McGregor (1989) Grady et al. (1987) Akashi& Bishop (1983) Cabradilla et al. (1983) Eshita & Bishop (1984) Bishopet al. (1982) Akashi et al. (1984) Schmaljohnet aL (1987b) Yoo & Kang (1987a) Schmaljohnet aL (1986b) Collettet al. (1985)

X14383 BLCMA D00379 M21951 BLCSA D00380 BLCVMA BLCSRNA BLCNCNP SSHM SSHS AINSRNA HANM HANHANM HANSCNC RVFMRNA

M S S

4330 1904 1746

60.3 60.4 53.2

Ihara et al. (1985b) Ihara et al. (1984) Marriott et al. (1989)

PTPMRNA PTPSRNA

M

3231

52-2

Ronnholm& Pettersson (1987)

UUKGPM

and possibly the packaging, of the influenza virus genome.

Nucleotide sequence analysis and protein function This section reviews the results of c D N A cloning and nucleotide sequence determination of various genome segments and relates these data to the functions of the viral proteins. This account is unbalanced as it reflects the different degree of study that some genera have attracted compared to others, e.g. whereas the complete nucleotide sequence of the Bunyamwera virus genome is known, no sequences of nairovirus genome segments have yet been published. A list of available sequences, as of August 1989, is given in Table 2. For some viruses the mapping of proteins to genome segments has been achieved by genetic or biochemical methods (reviewed by Bishop, 1985 a). These results have been confirmed by sequencing studies which have also revealed the coding strategies of the individual genome segments (Fig. 4). Based on the available information some general observations can be made regarding the family Bunyaviridae as a whole, which will be followed by a more detailed discussion of individual cases. 1. The genome is of negative polarity, i.e. the infecting genome must be transcribed into ( + ) sense m R N A s for protein production. A variation of this strategy is the ambisense nature of the phlebovirus S R N A segment (Ihara et al., 1984).

Reference

Database accession no.

2. The genome segments are richer in A + U residues than G + C residues (Table 2). 3. The L R N A segment encodes the L protein. 4. The M R N A segment encodes the viral glycoproteins, as a precursor polyprotein; the glycoproteins themselves are unusually rich in Cys residues ( > 5 %). 5. The S R N A segment encodes the N protein. 6. There is economical use of the genomic R N A to encode protein, e.g. of the 12294 nucleotides in the Bunyamwera virus genome, only 576 (4.7%) are noncoding (Elliott, 1989b; summarized in Table 3). L R N A segment and L protein

It is presumed that the L R N A segment encodes the large ( > 200 000 Mr) protein, termed L, that has been observed in representative viruses of all genera, although this has been formally proved only for members of the Bunyavirus genus (Elliott, 1989b; Endres et al., 1989). The L R N A segment of Bunyamwera virus is 6875 nucleotides in length (Elliott, 1989b). This is rather less than predicted by analysis of genomic R N A directly but is in good agreement with the estimate of 7000 bases made from single-stranded c D N A (see Pringle et al., 1984). In the viral complementary R N A there is a single A U G initiated open reading frame (ORF) that encodes a protein of 2238 amino acids (Mr 259000), thought to be the L protein. In the negative-sense genomic R N A there is a small AUG-initiated O R F potentially encoding a 14.7K protein. This protein has not been found in infected cells and its significance is questionable (Elliott, 1989b).

506

R. M. Elliott

3'

5 r

Bunyavirus --

L segment

~1~ L 3'

5 ~

Bunyavirus • ................ L::,::t................................................. G2~4Sm 3'

G1 5'

Hantavirus ~_ .............................1.................................. G1 I G2 3 ~

M segment 5'

Phlebovirus •

h._ a

NSm

G1/G2

I

G2/G1

3 ~

Uukuvirus III t...........................I.................................... G1 I G2 3 ~

5 ~

Bunyavirus []

v N

:::::::::::::::::::::::::::::::::::::::::::::::::

3'

NSs

5'

Hantavirus i

~:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: =x~a

N 3'

5'

S segment

Nairovirus -u== :+::=:.:+=.:~:-:= :::::::::::::::::::::::= :.:.::=::::==: =::: =-:.=::,:= i

N

3'

5'

= ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

Phlebovirus

other negative strand virus polymerases failed to detect regions of extended homology, with the exception of an area (amino acids 950 to 1220) of weak homology with influenza virus PB1 protein (Elliott, 1989b). The PB1 protein is the polypeptide responsible for R N A polymerization (Braam et al., 1983), thus the homologous region in the Bunyamwera virus L protein may represent the same catalytic domain. The bunyavirus L protein has also been implicated in biological properties of the virus. A La Crosse virus isolate with a non-temperature-sensitive (ts) mutation in the L segment was altered in its virulence for mice (Rozhon et al., 1981) and viruses carrying the same defect were inefficient in establishing productive infection of Aedes triseriatus mosquitoes (Beaty et al., 1981). Janssen et al. (1986) reported that the L (and perhaps the S) R N A gene products also played a role in modulating the virulence of reassortant viruses, depending on the genetic background. The mechanism by which the L protein affects virulence is not understood.

N NSs

Fig. 4. Coding strategies of Bunyaviridaegenomes. Genomic RNAs are representedby thin lines,mRNAs by arrows( • indicates5' end, I~ indicates 3' end)and geneproductsare designatedby solidboxes(not to scale). The L R N A segments of hanta-, phlebo- and uukuviruses are similar in size to the bunyavirus L segment, but the nairovirus L R N A is significantly larger (perhaps twice the size). However, the L proteins encoded by these different viruses are all similar in size and hence the coding potential of the nairovirus L R N A appears to be grossly under-utilized. Perhaps another protein is encoded in the nairovirus L segment, but the lack of molecular data on the nairovirus genome precludes further discussion. The L protein is thought to be a component (at least) of the virion-associated transcriptase or RNA-polymerase. Comparison of the Bunyamwera virus L protein with

M R N A segment and viral glycoproteins Bunyavirus genus. In addition to the virion glycoproteins G1 ( M r 108K to 125K) and G2 ( M r 29K to 41K) the bunyavirus M segments encodes a non-structural protein termed NSm (Mr 11K to 16K) (Gentsch & Bishop, 1979; Fuller & Bishop, 1982; Elliott, 1985). The precursor polypeptide containing these proteins has not been detected in infected cells, suggesting that cleavage may be a cotranslational event (Pennington et al., 1977; Lees et al., 1986). Attempts to translate bunyavirus M segment-specific m R N A in vitro have also been unsuccessful (Abraham & Pattnaik, 1983; Elliott, 1985). The glycoproteins have few oligosaccharide side-chains (Vorndam & Trend, 1979; Cash et al., 1980; Lees et al., 1986; Pardigon et al., 1988). The glycans are mostly of the endoglycosidase H (endo H) complex type (Madoff & Lenard, 1982; Pesonen et al., 1982b), but in addition some endo H-sensitive high-mannose and some small endo H-resistant intermediate type glycans are found. It was suggested that the small endo H-resistant glycans represented glycosylation intermediates that accumulate due to the intracellular maturation of the virus (Pesonen et al., 1982b). The glycoproteins contain covalently attached fatty acid (Madoff & Lenard, 1982). The nucleotide sequences of four bunyavirus M segments have been determined (La Crosse and snowshoe hare viruses of the California serogroup, and Bunyamwera and Germiston viruses of the Bunyamwera serogroup) and range from 4458 to 4534 bases in length; the precursor ORFs contain 1433 to 1441 codons (Table 2; Eshita & Bishop, 1984; Lees et al., 1986; Grady et al., 1987; Pardigon et al., 1988). The gene order of snowshoe hare bunyavirus M segment has been determined by

Review." Molecular biology o f the Bunyaviridae

507

Table 3. Coding capacity of the Bunyamwera virus genome Recognized gene products Number of nucleotides RNA segment Lt M:~ S§ Total

Total

5' NC*

3' NC

Protein L Glycoprotein precursor (G1, G2 and NSm) N NSs

6875 4458

50 56

108 103

961

85

174

12 294

191

385

Number of amino acids

Predicted Mr

2238 1433

258656 162065

233 101 4005

26662 11023 458406

* NC, Non-coding. t Data from Elliott (1989b). :~Data from Lees et al. (1986). § Data from Elliott (1989a).

Fazakerley et al. (1988) using direct amino acid sequence analysis and specific peptide antibodies as 5' G 2 - N S m G1 3' in the viral complementary RNA. The four available amino acid sequences can be readily aligned with a notable conservation of Cys residues (Fig. 5). By extrapolating the data on snowshoe hare virus the following observations can be made" there is a putative signal peptide of 13 to 21 residues at the amino terminus of the polyprotein; the G2 proteins show an overall amino acid homology of about 66 ~ and the positions of two predicted N-linked glycosylation sites (Asn-XxxSer/Thr) are strictly conserved; cleavage at the carboxy terminus of G2 occurs after a conserved Arg residue; the non-structural peptide between G2 and G1 shows about 50~ conservation of residues. Prediction of the amino terminus of G 1 by comparison with snowshoe hare virus is not obvious because of low sequence homology, but cleavage of the precursor does appear to occur after a conserved Ala residue. Overall the G 1 proteins display about 40 ~ similarity, but are more similar in the carboxy half than the amino half of the molecule. There is a single, strictly conserved N-glycosylation site, whereas a second site is conserved between viruses in the same serogroup. The G 1 protein is susceptible to cleavage by trypsin (Kingsford & Hill, 1983; Gonzalez-Scarano, 1985); the major trypsin-sensitive site of G1 was determined directly by Fazakerley et al. (1988) and its position is conserved in all four proteins. The hydropathy profiles of the four polypeptides are very similar (Fig. 6). The G2 protein has a rather long hydrophobic sequence (residues 187 to 245) followed by a charged stop-transfer sequence, but the orientation of G2 relative to the membrane is not known. The G 1 protein has typical carboxy-terminal transmembrane and anchor sequences, which together with the trypsin cleavage data orientate G1 with its amino terminus external to the membrane (Fazakerley et al., 1988). Neither the function nor the intracellular location of NSm are known, but

inspection of the amino acid sequence and hydropathy profile suggests it is a membrane-bound protein. The above analyses demonstrate the evolutionary relatedness of the glycoprotein species of viruses from two serogroups of the Bunyavirus genus and suggest that the antigenic differences that distinguish individual viruses may be located in the (external) amino half of G1. The gene products of the bunyavirus M segment have been implicated in many of the important biological properties of the virus, including virulence, neutralization, haemagglutination and cell fusion. Through the use of reassortant bunyaviruses it was shown that the major determinants of viral virulence in mice and of viral infectivity in mosquitoes cosegregated with the M RNA segment (Beaty et al., 1981, 1982; Shope et al., 1981; Janssen et al., 1986) and analysis of monoclonal antibody (MAb)-selected variant viruses indicated that the G1 glycoprotein maybe the most important gene product determining virulence and infectivity (Gonzalez-Scarano et al., 1985; Sundin eta[., I987). Gonzalez-Scarano et al. (1988) have identified four quantifiable biological markers (neuroinvasiveness and neurovirulence in the mouse, oral and intrathoracic infectivity in the mosquito), which can vary independently, suggesting there may be multiple sites within the M segment that affect virulence. As stated earlier the L and S segments can also have a modulating effect when the M segment is derived from an avirulent parent (Janssen et al., 1986). Analysis of reassortant viruses correlated the M segment with neutralizing antibody (Gentsch et al., 1980) and subsequently neutralizing MAbs have been isolated that are directed against G1 (Gonzalez-Scarano et al., 1982, 1983; Grady et al., 1983a, b; Kingsford & Hill, 1983; Kingsford et al., 1983; Kingsford, 1984; Grady & Kinch, 1985). No MAbs directed against G2 have been reported. Analysis of MAb-resistant mutants isolated by Najjar et al. (1985) suggested that the epitopes

508

R.M.

Elliott

G2NH 2 i



100

Ssh. Gp Lac.Gp Bun.Gp Ger. Gp Consensus

...... Mici•iLfaVT...aASPvyqRC•QDGaivkqnpSkeavtEvc•KDDvSMiKt•arYiKNaTGvFsnnvaiRkW•vsDWhdCrPkkitGGhINv ...... Mirm•vLivvT...aASPVyqRCFQDGaiVkqnpSkeaVtEvC•KDDVSMiKtEarYvKNaTGVFsnnvaiRkW•VsDWhdCrPkkivGGhINv ...... Mri•i•L•aVTqLavsSPVitRC•hgGq•IaerkSqtsIs•fCiKDDVSH•KsEiv•tKNdTGIFghskvfRhWtItDWkaCnPvvtaGGsINV maistsL•iva•Lik•c•vntApPI.skCFQDGi•Iae•kSssgIsEfCiKDDISi•KsEitYsKNdTGIFmhskvfRhWtVaDWkqCnhts.aGGstNV ...... M ..... L--VT .... ASPV--RCFQDG ....... S ..... E-C-KDDVSM-K-E--Y-KN-TG-F ...... R-W-V-DW--C-P I01 IEvgddLsLhTesYvcsaDcTIgVDKEtAQVrLQTDttNHFEIaGTiVksGWFKStTyITLDqTCEH•KVSCGpKSiQFHACFnQHMSCvRFLHRtILPG IEVgddLsLhTesYVcsaDCTIgVDKEtAQVrLQTDttNHFEIaGTTVksGWFKStTyITLDqTCEH•KVSCapKSvQFHACFnQHMScVRFLHRtILPG IEVdknLnLvTrnYVCtgDCTItVDrknAQIiFQTDk•NHFEVtGTTIstGWFKSkasVTLDrTCEHiKvSCGkKt•QFHACFkQHMSCVRFLHRsILPG ~EVdknLnLvaknYmCtrpCvItIDKEnAQl~FQTEqLNqFZVtGTTIstGWFKSkTsVsLDnTC~HiKVtCGkKS~Q~HAC~kQHMSCVRFLHRsVLPG IEV---L-L-T--YVC--DcTI-VDKE-AQV--QTD--NHFE--GTT---GWFKS-T--TLD-TCEH-KVScG-KS-QFHACF-QHMScVRFLHR-ILPG

200

Ssh. Gp Lac.Gp Bun. Gp Ger.Gp Consensus

201 • SiAnSICQNIEiII•VtLTL•IFILLsVLsKTYIcYLLMPVFiPIAYaYGiiYNkSCKKCk•CGLvYHPFTeCGthCvCGaRYDTSDRMkLHRaSGLC•G

300

Ssh. Gp Lac.Gp Bun.Gp Ger.Gp Consensus

SmAiSICQNIE•IIitiLaLcIFIiMiILtKTYICYvLiPvFmPIAFaYGwaYNrSCKKCtcCGLaYHPFTnCGsyCVCGskFETSDR•rMHReSGLCqG ymAssIcQNIE•IIiIiLTLaIFIFMcIitrTYICYLMLP•FaPIAY•YGw•YNrSCKKciccGLaYHPFTncGsyc•CGsRFETsDRMrLHReSGLCqG S-A-SICQNIE II-V-LTL-IFIL--IL-KTYICYLLMPVF-PIAY-YG--YN-SCKKc--CGL-YHPFT-cG--CVCG-R--TSDRM-LHR-$GLC-G

Ssh. Gp Lac.Gp Bun.Gp Ger.Gp Consensus

301 ~ YKSLRaARvMCKSKGpasI•SViTAILILtFVTPInsMvvg.eskevFeLEq••DdMLdmaLrinfyYFVcIMnyavtwGLIIIgL•IgL•FkkyqhrFs YKSLRaARvMcKSKGpasI•SIiTAVLVLtFVTPInsMv•g.esketFeLEE•pDdMLemaLrinsyYFtcILnyavswGLIIagL•VgLiFkkyqhrF• FKSLRvARrLCKSKGss•IiSI••sVLILsFVTPIegtltnyptdqkYtLDEiaDvLqakthedstkYYIi•yts•fgaGLtIIfagVaLgLtii•evLt FKSLRvARsLCKSKGss•ViSI•TAmLILsFITPleaMttnypddkkFtLkEvnDivLgrdMeqe•kssI•ILmsicgiGiI•IfFg•tv•Leiv•e•ia -KSLR-AR--CKSKG---I-SI-TAVLIL-FVTPI--M ......... F-LEE--D-ML---L ..... YFI-IL ...... GLIII-L-V-L

SiAnSICQNIEiII•VtLTL•IFILLsILsKTYICYLLMPIFiPIAYmYGviYNkScKKCk•CGLvYHPFTeCGthCVCGaRYDTsDRMkLHRaSGLC•G

G2COOH,

q--

NSM

40 0

........

~9

Ns. 500 n•yamYCeECdMYHDrsgLKrnGDFTNKCrqcTCGqyEDatGLmtHrktynC•vrYKakWvmnfLIaYmLLtLIkdsaIvVqAagtdFTtC•etEnInwn niyamYCeECNMYHDKsgLKrhGDFTNKCrqCTCGqyEDatGLItHrktynC•vqYKakWmmnfLIiYiFLiLIkdsaIvgqAtgtdFTtC•etEsInwn .

Ssh. Gp Lac.Gp Bun. Gp Ger.Gp Consensus

.... GG-INV

.

.

.

kinviFCnECNMYHsKksiKyvGDFTNKCgf•TCG••EDpeGvVvHkakksCtysYqinWvrgiMIfvaFLfvIqntiImVaAee .... d C w k n E e l k e d krstiFCkECNLiHDKksMtyrGDFTNKCgfCpCGe•EDpeGLviHttrksCtyyiKirn•k•iM•iFsiviLmqnatm•VvAgencWT .... n t e I k a d ...... C-ECNMYHDK--LK--GDFTNKC--CTCG--ED--GLV-H ..... C---YK--W ..... I-Y-FL-LI .... I-V-A .... FT-C---E-I---

Ssh.Gp Lac. Gp Bun.Gp Ger. Gp Consensus

501 CtGPF•n•gnCqkqqkKepYanIAtqLkg•qa•svLDmpmIasipEdIagALRyIEeketFHvq•taEyamLsrycDYYaqFsdNSGYSQTtWRvy•rsh CtGPF•n•gnCqkqqkKepYtnIAtqLk••kaIsvLDipiItsipDdIagALRyIEekedFHvq•ttEyamLsKyCDYYtqFsdNSGYSQTtWRvy•rsh CvGPLiapkdCtdkdhK.tY•seAs•LatakkItqvDaenvei•gktmesAiRvIErqktYHrmh••EavfLnKhCDYYkmFehNSGYSQvkWRmmiktq CvGPLigpsaCtnkgsK.tYktVAqeLvtaskItqLDadkyv•lgDtIesALda•tsqkhYsamh••EtmfLmKhCDYYkvYehNSGYSQTkWR•iaian C-GP ...... C .... K--Y--IA--L ..... I--LD---I .... D-I--ALR-IE ..... H ..... E---L-K-CDYY--F--NSGYSQT-WR

Ssh.Gp Lac.Gp Bun. Gp Ger. Gp Consensus

601 ~ 700 dFDaCiLyPNqhFCrCvkrgdkCSssngDFAneMknyYSgKqnkFdkDLnLaLmaLhhAFrGTssAYiatm•skKsndd•IaytnKIKeKFPgNaLLkaI dFEaCiLYPNqhFCkCvkngekCSssnwDFAngMknyYSgKqakFdkDLnLaLtaLhhAFrGTssAYiaam•skKsndd•IaytnKIKaKFPgNaLLkaI hFDiCaLqaNspFCaqciadnsCaqgswEFdthMnstYSsKvdnFkhDFsLfLriFeaAFpGTayvh••tnikeKkpyqaVsmieKIKkKFPnNkLLigy sFDictntPtpnFCkC•s.dssCSttt•nFAtsMnatYtsKvefFnhDFtLfLdiFeaAFpGsatAFlfkkikeKn•yqafemmgKIanKYPnNkLLvvI -FD-C-L-PN--FC-C ...... CS .... ,,,--,---,,-,---,--,--,-, ..... AF-GT--AY ....... K ..... I .... KIK-KFP-N-LL--I

7o~ Ssh. Gp Lac. Gp Bun. Gp Ger. Gp Consensus

Ssh. Gp Lac.Gp Bun.Gp Ger. Gp Consensus

~trypsin

600

......

8o0

vDYiaYMksLSemSsFkydefWdD•LYksaptKapsLsRgsepsynfkL..vVssrsiKsCKnvKsVvCLSPRsGvsydsiIaCGDpngpsVYrKPsdgv iDYiaYMkGLpemanFkydefWdE•LYkpnpaKasnLaRgkessynfkL..aIssksiKtCKnvKdVacLSPRsGaiyssiIaCGEpngpsVYrKPsggV iDFgkYLiGLShaStYelqqrqlDkLYqpt ..... eLtRsggq..qtsLansVvgqatKeCKkyKdVsCLSPRfGIpledlIsCcDqpnynIYkKPkk.V ikYgqYMvGLShaStYqlkqeW ........ vaKslsLtRaqrtglkmsManaepgpatKeCsdaKtIaCLtPkfqVevnnlmsCGaspnfkIYvKtge.l -DY--YM-GLS--S ....... W-D-LY ..... K---L-R ......... L---V ..... K-CK--K-V-CLSPR-GV ..... I-CGD ...... Y-KP---V 801 900 FqsnadqStYCLaDsHCLeDFevvsqEe•DaiKKsKCWeaEyPdvk•sk•tdgvKSCRMKDsGnCNVaanrWpIIqCENdKFYYSE•qkdyDktqDIG.H FqsstdrSiYCL•DsHCLeEFeaisqEe•DavKKsKCWeiEyPdvrp•qesdgaKSCRMKDsGncNVatnrWpVmqCENdKFYYSE•qkdyDktqDIG.H YkahdkeetWcinDqHCLvDFvpaeaDtvEk•KpmKCW•vD.PgknddvysiaiKtCRvvDkGvCtVnsqkWnIIkCDs••LYYSDhipgeDtgnDIG.H YkahdrnSvWCLnDmHCLtpYtpanaEiittmKKmdCWq.DnPkqptde•aipk•SCqMKDrG•CNsgadkWkIIkCDNhKLFYtDa•errDpasiVGsn ....... S--CL-D-HCL-DF ..... E--D--KK-KCW .... P ........... KSCRMKD-G-CNV .... W-II-C-N-K-YYS ...... D---DIG-H

on G 1 were clustered within a single immunodominant antigenic site; nucleotide sequence data on the variants are required to support this hypothesis. Many of the neutralizing MAbs also inhibit haemagglutination, implicating G 1 in the attachment to cellular receptors (Gonzalez-Scarano et al., 1982; Grady et al., 1983a; Kingsford et al., 1983). Gonzalez-Scarano et al. (1984) demonstrated that La Crosse bunyavirus was able to fuse BHK cells at moderately acidic pH and subsequently it was shown that this activity was accompanied by a conformational change in G1, resulting in an altered reactivity of GI with MAbs and different cleavage pattern by proteolytic enzymes (Gon-

zalez-ScaranO, 1985). Further evidence implying a role for G1 in the fusion event was provided by a variant (V22) of La Crosse virus, selected with a G1 MAb, which had reduced neuroinvasiveness in mice and had altered fusion activity, i.e. reduced efficiency of fusion and requirement for a lower pH (Gonzalez-Scarano et al., 1985). However, it appears that G 1 itself is not the actual fusing protein; Pobjecky et al. (1989) reported that G1containing liposomes were unable to induce fusion, whereas virus particles that had most of their G1 removed by protease treatment, but still contained G2, were able to fuse liposomes. The authors suggested that the large hydrophobic domain in G2 (residues 187 to 245;

Review: Molecular biology of the Bunyaviridae

Ssh. Gp Lac.Gp Bun.Gp Ger.Gp Consensus

Ssh. Gp Lac.Gp Bun. Gp Ger. Gp Consensus

509

901 1000 FcLSpGCsTvRFPINPkhIsNCnWqvsrssiakIdvhniedIDqYrKAITqKLqtsLsLFkYAk.tkNLPHIKPIYKYITieGTETAEGIEsAYIeseIP YCLSpGCtTiRYPINPkhIsNcnWqvsrssiakIdvhnvedIEqYkKAITqKLqtsLsLFkYAk.tkNLPHIrPIYKYITmkeTETAEGIEsAYIesevP YCvsaGCkTdRYPINPdvVtdCvWeftsrksqyIgkismqs•EdYeKA•TdrLthtLetYsFAp•.eNLPHIKPVYKYITaqGvEnsDGI•gAFItasIP hCFSekcqieRYPINPts•tNCeW•yravrpeyIkk•s•qtIEeYkKAIadKLthtLqLYhFApl•eNLPHIKPtYKYITaqGTyTADGIEgAsIttsIP Z C L S - G C - T - R Y P I N P - - I - N C - W . . . . . . . . . I . . . . . . . I E - Y - K A I T - K L - - - L - L - - - A .... N L P H I K P I Y K Y I T - - G T E T A - G I E - A Y I - - - I p i001 ii00 ALaGTSI•FKItSKEGkhLLDvIgYVKSAscssIYtk•YtTGPTsgINTKHDELCTGpcPakInHqtgWLTFakERTSsWGcEEFGCLAIsdGCVFGSCQ ALaGTsVG•KInSKEGkhLLDVIaYvKSAsysSvYaklYsTGPTsgINTKHDELCTG•cPanInHqvgWLTFarERTSsWGc•EFGcLAVsdGCVFGSCQ AagGTSIGYnVrSKDGfpLLD•IvFVKSAvikStYnhiYdTGPTisINTKHDEhCTGqCPsnIeHeanWLTFsqERTSrWGCEEFGCLAVntGCVFGSCQ

ALsGTSVGFKInaKD•tdLLDIVvYIKasvvkSIYnhi•dTGPTinINsKHDELCTGqCPkkIpad•nWLTFsqERTSrWGCEE•GCLAIntGcvYGSCQ AL-GTS-GFKI-SK-G--LLDvI-YVKSA---SIY---Y-TGPT--INTKHDELcTG-CP--I-H---WLTF--ERTS-WGCEEFGcLA---GCVFGSCQ

Consensus

ii01 • 1200 DIIRdE•tvYRKetDEVtdve•C•TFsdkTYCTn•NpItPiITD•FEvQFKTvETySLPrIVAIqNHeikiGQVNDLGvYskgCGNVQKvNgtVyGnGvP DIIkeE•svYRKetEEVtnve•C•TFsdkTYcTn•NpVtPiITD•FEvQFKTVETySLPrIVAVqNHeikiGQINDLGvYskgCGNVQKvNgtVyGnGvP DVIRpEtkvYRKavDEvvi•tvCiTYpghTFCTeiNaIePkITEeiE•QFKTVDTktLPyIvAVnNHk•ysGQINDLGtFgqmcGNVQKtNssI•GtGt• DvIRtEtkVYRKan•Etvm•tvciTYpghT•cTdvNahePkITDeLE•QFKTIDikSLPn•VAVtNHk•ytGQINDLGtFgqmCGNVQKtNtshtGaGtP D - I R - E - - V Y R K - - - E V ..... C-T .... T - C T - - N - I - P - I T D - F E - Q F K T V - T - S L P - I V A V - N H .... G Q I N D L G ..... C G N V Q K - N - - V - G - G - P

Ssh. Gp Lac.Gp Bun. Gp Ger. Gp Consensus

1201 1300 KFDY•cH•ASRKEvIvRkcFdNdYqaCKFLqspasyrLEEDsgTVTVidyKki•GtikMKaILGDVkYKtFaDnvDMtaEgsctGCInCFEnihceLt•h rFDY•CH•ASRKEVIvRkcFdNdYqaCKFLqspasyrL-EDsgTVTIidyKki•GtikMKaILGDVkYKtFaDsvDitaEgscaGcInCFqnihCeLtih KFDYtCHgAsRKDIIVRrcYnNnFdsCKLLkeetq•iFnDDhdTITVyntnh•iGe•aiK•ILGDIqYK•FtEt•DLqiDakcvGCpdcF•sysCnFqiv KFDYtCysASRKDIIIRrCYnNnYdscrLLnqesd••FDDnheT•vVynnKr•nGe•aLK••LGD•qYK•YtEnmELe•EakcvGCVgcFEsyqCnLqit K F D Y - C H - A S R K - - I V R - C - - N - Y - - C K - L . . . . . . . . E - D - - T V T V - - - K - - - G - - - M K - I L G D - - Y K - F .... , , - - , - - , - , , , - , , , - - - , - , - - -

Ssh. Gp Lac.Gp Bun.Gp Ger. Gp

ConsensHs

1301 1400 ttIEascpivstCtvFHDRI•VtpnehkYALKVvCTekPGnt•TirIcNtkvEasLa•Vdakpi•E•apvDQTaYIr•KD•RcKTWMcRVRDEGLqVILE ttIEasCpikssctvFHDRI•VtpnehkYALKIvcTekPGnt•TikvCNtriEasMa•VdakpiIE•a•vDQTaYIrEKDERCKTWMcRVRDEGLqVILE snIDtiCs•egpCdtFHnRIsIkamqqnYAvK•sCqkdPrpsgTfkICNreytvvFhtVakddkIEinvgDQTsFIkEKDDRCKTWLCRVRDEGisVIF• ss•Deta•y•vpvshFHDRIqIkttkkdYAMKIsCTrdPGdkasfrVcgksyDfnFhtv•kndkIEvnvgDeTsYIkEKDnRCgrWLCRvRDEGLsVIFE - - I - - - C ..... C - - F H D R I . . . . . . . . , , , , , - , , - - , , - - - , - - - , , - - - , - - , - - , ..... IE .... D Q T - Y I - E K D E R C K T W - C R V R D E G L - V I - E

Ssh. Gp Lac. Gp Bun. Gp Ger. Gp Consensus

1401 1456 PFKNLFGSYigIFYtfIIsIIaLLiIIYIvLPiCfKLRDtLrkhEdaYkrEMKiR* PFKNLFGSYigIFYtfIIsIIaLLvIIYVLLPiCfKLRDtLrkhDdaYkrEMKiR* PiKaFFGSYfsIFFyiIVvVVvgFIIIYIFMPmfmKLkEvLkanEklYIqEiKqk* PLnNFFGnYInmFLyilggIIILFIalYILMPmCarLRDeLkrnErlhqmEMKkR* PFKN-FGSY--IFZ--II-II-L--IIYIL-P-C-KLRD-L---E--Y--EMK-R-

Ssh. Gp ,ac.Gp Bun.Gp ,er. Gp

Fig. 5. Alignmentof the glycoproteinprecursorsof snowshoehare(Ssh.gp), La Crosse(Lac.gp), Bunyamwera(Bun.gp)and Germiston (Ger. gp) viruses. The amino and carboxy termini of G2 and the amino terminus of G1, as determined for snowshoe hare virus (Fazakerleyet al., 1988), are indicated. The exact boundariesof NSm are not known. N-linked glycosylationsites conserved in all four proteinsare indicatedby • and in two proteins(serogroupconserved)by ~ or ~ . Thereis a potentialglycosylationsite at position 89 in the Germiston virus sequence, which is not conserved.

Fig. 6) was responsible for fusion activity and hence the role of G1 concerned interaction with the cell surface receptor rather than directly with the lipid bilayer. The nature of the bunyavirus receptor(s) either in vertebrate or invertebrate cells is not known. Grady & Kinch (1985) described a specific epitope on G1 that was associated with infection of cultured mosquito cells and James & Millican (1986) isolated mutants of Bunyamwera virus by passage through mosquito cells that were better adapted to replicate in the invertebrate cells than the initiating virus and which reacted differently to G1specific MAbs raised against the parental virus. These data, together with those of Kingsford & Hill (1983) and Kingsford et al. (1983), point to G1 being involved in attachment. However, Ludwig et al. (1989) reported that protease removal of G1 increased the affinity of La Crosse virus for mosquito cells (although the rate of viral replication was unchanged). These authors proposed a model whereby the major determinants for attachment to mosquito cells are on G2 and are usually masked by G1 ; removal of G1 by proteolysis (as could occur in the insect

midgut) is required for efficient attachment and infection of mosquito midgut cells. Hantavirus genus. The M segment of the prototype Hantaan virus strain 76-118 is 3616 nucleotides long and encodes an ORF of 1135 amino acids (Schmaljohn et al., 1987a; Yoo & Kang, 1987a). The gene order has been established as 5' G 1 - G 2 3'. There is an 18-residue signal peptide preceding G1 and a small peptide of Mr 6K between GI and G2, which has a mainly hydrophobic character and may act as a signal sequence for G2 (Schmaljohn et al., 1987a). Asparagine-linked sugars of predominantly the high-mannose type are associated with the glycoproteins; the Mr of unglycosylated G1 is about 65K and unglycosylated G2 about 54K, whereas the glycosytated species have M r of 68K to 72K for G 1 and 56K to 57K for G2 (L. H. Elliott et al., 1984; Schmaljohn et al., 1986a). The Hantaan envelope proteins show haemagglutinating activity (Tsai et al., 1984; Okuno et al., 1986) and induce cell fusion at low pH (Arikawa et al., 1985), but it is not known whether the

510

R. M. Elliott 14 0

_,

G2

DI

NSm

I,~

GI 500

,

.

.

.

.

"-I 1000 I

.

.

tr

(a) O

!~

_3-~

3-

--3 J

_3.-i

i

r-

_3-~

s;0

ld00

Fig. 6. Hydropathyprofilesof(a) snowshoehare, (b) La Crosse,(c) Bunyamweraand (d) Germistonvirusglycoproteinprecursors. The boundariesof G2, NSm and G 1are indicated.H designatesthe hydrophobicdomainin G2 suggestedto be involvedin fusion(seetext).

fusogenic property resides on G 1 or G2. Both G 1 and G2 contain sites that may be neutralized by MAbs and whereas all neutralizing MAbs also inhibit haemagglutination, some MAbs to G2 have been identified that haemagglutinate, but do not neutralize (Yamanishi et al., 1984; Dantas et al., 1986; Arikawa et al., 1989). Schmaljohn et al. (1988 a) compared two virus isolates from patients with Korean haemorrhagic fever to the prototype strain; the three strains were clearly antigenically similar and nucleotide sequence analysis of the M R N A segments predicted 1-3 to 2-3% amino acid changes among the three viruses, with most occurring as conservative changes in G 1. Using radioimmunoprecipitation as an assay Sheshberadaran et al. (1988) showed that G 1 was the least cross-reactive protein and G2 was only weakly cross-reactive, when comparing seven hantaviruses. The Hantaan virus M segment has been expressed via a vaccinia virus vector and authentic G 1 and G2 proteins were synthesized, which localized in the Golgi region of recombinant infected cells. Mice inoculated with the recombinant vaccinia virus raised neutralizing antibodies to Hantaan virus even though no surface expression was detected (Pensiero et al., 1988). Nairovirus genus. The coding of nairovirus M segment has not been determined and estimates of the size of the M R N A of different nairoviruses range from 4400 to 6300 bases, the latter value being significantly larger than the corresponding segment of the other genera (Clerx & Bishop, 1981; Clerx et al., 1981; Watret &

Elliott, 1985 b). The glycoproteins of nairoviruses present a more complicated picture than in the other genera. The pattern of the virion glycoproteins is distinctive [G t, Mr 72K to 84K and G2, Mr 30K to 45K (Clerx & Bishop, 1981 ; Clerx et al., 1981 ; Cash, 1985; Watret & Elliott, 1985b)], but for Hazara virus three glycoproteins of Mr 84K, 45K and 30K have been detected in virion preparations (Foulke et al., 1981). Analyses of nairovirus-infected cells have identified a number of nonstructural glycoprotein precursors which, by pulse-chase experiments, are related to the structural glycoproteins (Clerx & Bishop, 1981; Cash, 1985; Watret & Elliott, 1985b). The protein designated G2 is apparently only produced during the final stages of virus maturation (Clerx & Bishop, 1981; Watret & Elliott, 1985b). No details of the cleavage events or interrelationships between the identified proteins are known. Phlebovirus genus. The M R N A segments of two phleboviruses have been sequenced; Punta Toro virus M segment is 4330 nucleotides in length and encodes a single ORF of 1313 amino acids (Ihara et al., 1985b) and Rift Valley fever virus M segment contains 3885 nucleotides and encodes an ORF of 1197 residues (Collett et al., 1985; with corrections in Battles & Dalrymple, 1988 and Takehara et al., 1989). The fulllength precursor proteins have not been detected in phlebovirus-infected cells. The gene order of Punta Toro virus is 5' N S m - G 1 - G 2 3', whereas that of Rift Valley fever virus is 5' NSm-G2-G1 3'. The G1 protein of Punta Toro virus shows 35% similarity with the G2

Review: Molecular biology o f the Bunyaviridae

protein of Rift Valley fever virus, and the Punta Toro virus G2 protein shows 4 9 ~ similarity with the Rift Valley fever virus G 1 (Ihara et al., 1985 b). The homology of the G 1 protein of one virus with the G2 protein of the other reflects the nomenclature system for the glycoproteins, whereby the slowest migrating protein on SDSpolyacrylamide gels is designated G1, and the fact that both the phlebovirus glycoproteins are of similar Mr (Robeson et al., 1979). The N-terminal amino acid of both G1 and G2 follows an Ala residue in the precursor protein, suggesting a common cleavage event by an alanine-specific protease (Ihara et al., 1985b). Interestingly an alanine-specific protease is implicated in the cleavage of the bunyavirus glycoprotein precursor to generate G1 (see above). The predicted NSm proteins, which should perhaps be referred to as preglycoprotein regions, of the two viruses are significantly different in size, Mr 30K for Punta Toro virus and Mr 17K for Rift Valley fever virus; the 30K protein has not been observed in Punta Toro virus-infected cells (Ihara et al., 1985b). The expression of the Rift Valley fever virus M segment has been studied in detail: in addition to G 1 (Mr 65K) and G2 (Mr 56K), a glycosylated Mr 78K and a nonglycosylated Mr 14K protein are produced and all four proteins can be detected in Rift Valley fever virusinfected cells (Kakach et al., 1988; Wasmonen et al., 1988). Upstream of the amino terminus of G2 there are five in-frame A U G codons (Fig. 7). Synthesis of the four M segment-specified proteins involves independent initiation of translation at each of the first and second A U G codons, yielding primary translation products which are cotranslationally cleaved. The 78K protein initiates at the first A U G and probably represents an unprocessed polyprotein of NSm and G2 sequences; the 14K protein initiates at the second A U G and contains only pre-G2 sequences (Kakach et al., 1988; Suzich & Collett, 1988). It is noteworthy that the common glycosylation site at position 285 is not utilized in the 14K protein, but is used in the 78K protein (Kakach et al., 1989). The 37 amino acids between the first and second AUG codons appear to be important in the utilization of this glycosylation site and also prevent proteolytic cleavage at the amino terminus of G2. The authors speculate that these 37 amino acids affect the conformation or intracellular transport of the resulting protein (Collett et al., 1989; Kakach et al., 1989). What functions the 78K and 14K proteins play in the virus replication cycle await elucidation. The nucleotide sequences of the M segments of two other Rift Valley fever virus strains have been reported (Takehara et al., 1989): ZH-548 and ZH-548M12, a mutagenized derivative that is an attenuated candidate vaccine strain (Caplen et al., 1985). Eight nucleotide and

i

511

i

pre-G i (NSm) i

G2

i

G1 3'

78K 14K G2

G1

Fig. 7. Expression strategy of the Rift Valley fever virus M R N A segment. At the top is shown the R N A with the coding region indicated. A U G codons are shown as ~ and N-linked glycosylation sites as V . The four protein products detected in infected cells are indicated below. This figure is modified from Collett et al. (1989).

three amino acid differences were found between ZH548 and the ZH-501 strain sequenced by Collett et al. (1985). A further 12 nucleotide and seven amino acid differences were present in ZH-548M12, although their role in attenuation of this strain is unknown. Interestingly the ZH-548M12 strain M R N A had a short ORF (Met-Val-His-Stop) immediately preceding the initiation codon of the major ORF. The presence or absence of this new ORF affected the expression of G1 and G2 in recombinant baculovirus vectors (Takehara et al., 1989). Neutralizing epitopes are present on the G2 protein of Rift Valley fever virus (Keegan & Collett, 1986) and Pifat et al. (1988) isolated MAbs directed against G1 and G2 of Punta Toro virus that both neutralized and inhibited haemagglutination. Using either vaccinia virus- or baculovirus-expressed Rift Valley fever virus glycoproteins it has been demonstrated that inoculation with a combination of G1 and G2, or with G2 alone, protects animals against disease when challenged with live virus (Collett et al., 1987; Dalrymple et al., 1989; Schmaljohn et al., 1989). Uukuvirus genus. The uukuvirus glycoproteins are similar in size, G1 Mr 75K and G2 Mr 62K to 65K (Pettersson et al., 1971 ; Watret & Elliott, 1985a; Watret et al., 1985). Translation in vitro of M segment-specific mRNA extracted from infected cells yielded an Mr 110K protein, which was cleaved roughly in the middle in the presence of microsomal membranes to give G1 and G2 (Ulmanen et al., 1981). The 110K protein has not been detected in infected cells. Using in vivo pulse-labelling experiments Kuismanen (1984) determined the gene order of the M segment to be 5' G I-G2 3'. Analysis of the carbohydrate moieties of the individual glycoproteins showed that G1 contains mainly complex and small endo H-resistant intermediate glycans and G2 contains mainly high-mannose, as well as some complex, glycans (Pesonen et al., 1982a). The Uukuniemi virus M segment is 3231 bases long

512

R.M.

Ell~tt

and encodes a single ORF of 1008 residues (R6nnholm & Pettersson, 1987). The gene order was confirmed as 5' G1-G2 3' and no evidence for a non-structural protein was found. Both G1 and G2 are preceded by stretches of predominantly hydrophobic amino acids, which probably act as signal sequences. Comparison of the amino acid sequences of the Uukuniemi and phlebovirus M gene products revealed a low but significant homology, particularly towards the carboxy end of the precursor (i.e. G2 of Uukuniemi and Punta Toro andG1 of Rift Valley fever viruses), with a marked conservation of Cys residues. No homology was detected with the NSm region of the phleboviruses. Using the same parameters for comparison no similarity was detected between the uukuvirus and bunyavirus glycoproteins (R6nnholm & Pettersson, 1987). Together with the identical terminal nucleotide sequences (Fig. 1) the above data suggest a distant evolutionary relationship between genomes of uukuviruses and phleboviruses. S R N A segment and nucleocapsid protein Bunyavirus genus. The bunyavirus S segment specifies

the N protein (Mr 19K to 26K) and a non-structural protein termed NSs (Mr 10K to 12K), which are encoded in overlapping reading frames (Fig. 4) (Gentsch & Bishop, 1978; Cash et at., 1979; Bishop et al., 1982; Fuller & Bishop, 1982; Akashi & Bishop, 1983; Cabradilla et al., 1983; Fuller et al., 1983; Akashi et al., 1984; Bouloy et al., 1984; Elliott, 1985; Gerbaud et al., 1987b; Elliott & McGregor, 1989). Patterson et al. (1983) claimed that multiple leader and messenger RNAs were transcribed from the La Crosse virus S R N A segment, thereby generating different mRNAs for synthesis of N and NSs. This suggestion is erroneous and later work showed that the plethora of S RNA-specific transcripts were in fact degradation products (Raju & Kolakofsky, 1986b). The N and NSs proteins are probably made from the same mRNA species, the result of alternative initiation of translation (Bishop et al., 1983b; Elliott & McGregor, 1989). The S segments of Germiston and Maguari bunyaviruses have an additional ORF downstream of, and in the same frame as, NSs (Gerbaud et al., 1987b; Elliott & McGregor, 1989). Although the product of the third ORF can be translated in cell-free systems from RNA transcribed in vitro from cloned cDNA, the protein has not been detected in vivo (Elliott & McGregor, 1989). The nucleotide sequences of six bunyavirus S segments have been determined and range from 850 to 984 bases in length (Table 2). The N proteins of these six viruses, which represent three serogroups in the Bunyavirus genus, show 4 0 ~ sequence similarity overall, whereas the N proteins of viruses in the same serogroup show 80~ similarity (Elliott, 1989a). Certain regions of the N protein are strictly conserved between all six sequences;

these domains are presumably of functional importance, perhaps binding to the viral RNA, although homology to designated RNA-binding proteins (Chan et al., 1989; Query et al., 1989) is not obvious. The conserved regions may also elicit the complement-fixing antibodies that cross-react throughout the Bunyavirus genus (Shope, 1985). The S segment cDNAs of snowshoe hare and Maguari viruses have been cloned into a baculovirus vector and efficient expression of the N, but not NSs, proteins was obtained (Urakawa et al., 1988; Elliott & McGregor, 1989). This system should provide large amounts of N protein for functional and structural studies. No function has yet been ascribed to NSs. Hantavirus genus. The Hantaan virus S RNA segment is 1696 nucleotides in length and encodes a single ORF in the complementary RNA of 429 amino acids (Schmaljohn et al., 1986b). The predicted Mr of the product of this ORF, 48-1K, is in good agreement with that of Hantaan N protein (Schmaljohn & Dalrymple, 1983; L. H. Elliott et al., 1984). No evidence for a nonstructural protein encoded by the S R N A was found. The N protein ORF contains two A U G codons, separated by two codons, at the amino terminus. Expression of the Hantaan virus S segment in a recombinant baculovirus showed that the first AUG initiated translation of N, and no N protein was made if the first A U G was removed (Schmaljohn et al., 1988b). The hantavirus N protein is the major cross-reactive antigen between different antigenic groups of viruses detected by immunoprecipitation (Sheshberadaran et al., 1988), which presumably reflects areas of sequence conservation on the S segment detected by cross-hybridization studies (Schmaljohn et al., 1987b). Nairovirus genus. The nairovirus N protein has a similar size to that of hantaviruses (Mr 48K to 52K) and is the major viral protein detected in infected cells (David-West, 1974; Clerx & Bishop, 1981; Clerx et al., 1981 ; Foulke et al., 1981 ; Cash, 1985; Watret & Elliott, 1985b; Watret et al., 1985). The N protein has a net positive charge (Cash, 1985). The coding strategy of the nairovirus S segment is similar to that of hantaviruses, i.e. a single ORF in the viral complementary R N A corresponding to the N protein (V. K. Ward & P. A. Nuttall, personal communication). Phlebovirus genus. Two phlebovirus S segment sequences have been determined, Punta Toro virus (1904 nucleotides) and sandfiy fever Sicilian virus (1746 nucleotides) (Ihara et al., 1984; Marriott et al., 1989). Both sequences contain ORFs corresponding to N and NSs proteins, but in a novel ambisense coding strategy; the N protein is encoded in a complementary ( + ) R N A corresponding to the 3' half of the genomic S RNA,

Review: Molecular biology o f the Bunyaviridae

whereas the NSs coding sequence is encoded in the 5' half of the genomic RNA (Fig. 4). The proteins are translated from separate subgenomic mRNAs (Ihara et al., 1984, 1985a). The Mr 30K to 34K NSs protein has been seen in cells infected with Rift Valley fever, Karimabad and sandfly fever Sicilian virus-infected cells (Smith & Pifat, 1982; Struthers & Swanepoel, 1982; Watret et al., 1985), but not in cells infected with Punta Toro virus. The NSs protein of Rift Valley fever virus was observed in the nuclei of infected cells (Struthers & Swanepoel, 1982) and appeared to correspond to the virus-induced antigen demonstrated in eosinophilic intranuclear inclusions described by Swanepoel & Blackburn (1977). In contrast the large amounts of the 31K NSs protein synthesized in Karimabad virusinfected cells remained entirely cytoplasmic (Smith & Pifat, 1982). Struthers et al. (1984) later reported that the Rift Valley fever virus NSs protein was phosphorylated, which so far seems to be a unique feature of any Bunyaviridae protein. The Punta Toro NSs protein has been identified using antisera raised against a recombinant baculovirus expressing an appropriate cDNA construct (Overton et al., 1987); NSs was also found to be specifically associated with nucleocapsids. Comparison of the Punta Toro and sandfly fever Sicilian virus S segment products showed 47 ~ similarity of the N proteins and 21 ~ similarity of the NSs proteins (Marriott et al., 1989). MAbs directed against the Punta Toro virus N protein did not neutralize viral infectivity and showed very limited cross-reactivity with other phleboviruses (Pifat et al., 1988). This is in line with previous data that showed that complement-fixing antibodies, which are mediated by N protein, were not cross-reactive among phleboviruses (Tesh et al., 1975, 1982). Uukuvirus genus. The uukuvirus S R N A segment is estimated to be about 1900 nucleotides long, similar to the phlebovirus S segment, but no nucleotide sequences are yet available (Pettersson et al., 1977; Ulmanen et al,, 1981 ; Watret & Elliott, 1985a). Translation in vitro of 12S subgenomic mRNA, specific for the S segment of Uukuniemi virus, yielded the Mr 25K N protein and an Mr 30K non-structural protein (Ulmanen et al., 1981). The coding strategy employed by the uukuvirus S segment is not known, but in view of the similarities discussed above between phleboviruses and uukuviruses, one might expect an ambisense strategy.

RNA synthesis The classical scheme for the replication of a negativestrand RNA virus is that the infecting genome is first transcribed into mRNA by the virion-associated R N A

513

polymerase or transcriptase. This primary transcription does not require ongoing protein synthesis. After translation of the primary m R N A transcripts, replication of the genome and amplified or secondary transcription of m R N A occurs. Transcriptase activity has been detected in detergent-disrupted preparations of Lumbo (Bouloy & Hannoun, 1976), La Crosse (Patterson et al., 1984), Germiston (Gerbaud et al., 1987a), Uukuniemi (Ranki & Pettersson, 1975) and Hantaan (Schmaljohn & Dalrymple, 1983) viruses, which represent three of the five Bunyaviridae genera. The transcriptase activity reported was weak compared to other viral transcriptases (e.g. that of vesicular stomatitis virus), which has hampered comprehensive analysis of the enzymic events. However, Patterson et al. (1984) using purified La Crosse virions demonstrated that polymerase activity was stimulated by oligonucleotides such as ApG, cap analogues (e.g. mTGpppAm) and natural mRNAs (e.g. alfalfa mosaic virus R N A 4) and evidence was obtained that these acted as primers for m R N A synthesis. Furthermore they detected an endonuclease activity which cleaved methylated capped mRNAs in vitro. These results correlated with analyses of viral mRNAs extracted from infected cells, which indicated the presence of heterogeneous non-viral sequences at their 5' ends (Bishop et al., 1983b; Patterson & Kolakofsky, 1984; Eshita et al., 1985; Collett, 1986). Thus bunyavirus transcription resembles that of influenza virus in using a 'cap-snatch' mechanism to prime m R N A synthesis (reviewed by Krug et al., 1987). In contrast to influenza virus, bunyavirus transcription is not affected by actinomycin D or ~-amanitin (Vezza et al., 1979) and takes place in the cytoplasm of infected cells (Rossier et al., 1986; Kolakofsky et al., 1987). Bunyavirus, phlebovirus and uukuvirus mRNAs are probably not 3' polyadenylated, as judged by their inability to bind to oligo(dT)-cellulose (Ulmanen et al., 1981 ; Abraham & Pattnaik, 1983; Pattnaik & Abraham, 1983; Bouloy et al., 1984; Elliott, 1985; Emery & Bishop, 1987), but Cash et al. (1979) did claim to select a proportion of snowshoe hare bunyavirus m R N A molecules by this method. There does not appear to be a universal signal for bunyavirus transcription termination, although only a few termination sites have actually been mapped. The mRNAs are about 60 to 100 nucleotides shorter than full-length transcripts. The S mRNAs of snowshoe hare and La Crosse bunyaviruses terminate near the sequence G U U U U U in the negativesense RNA (Patterson & Kolakofsky, 1984; Eshita et al., 1985), which is similar to the termination-polyadenylation signal containing poly(U) stretches used by other negative-strand R N A viruses (Gupta & Kingsbury, 1982). However, this sequence is not ubiquitous among the RNA segments for which information is available (Table 2). Collett (1986) and Raju & Kolakofsky (1986b)

514

R.M.

Ell~tt

have suggested that purine-rich sequences, which seem to occur around the termination sites, might be involved, but further mapping studies are required to delineate these in detail. Attempts to analyse bunyavirus primary transcription have given conflicting results. Very low levels of primary transcripts were detected in Bunyamwera or snowshoe hare bunyavirus-infected cells in the presence of protein synthesis inhibitors (Kascsak & Lyons, 1977; Vezza et al., 1979). Using cDNA hybridization probes Eshita et al. (1985) detected only S segment m R N A in snowshoe hare bunyavirus-infected cells treated with puromycin. In contrast, primary transcripts were not detected in the absence of protein synthesis in cells infected with Akabane (Pattnaik & Abraham, 1983), Bunyamwera (Abraham & Pattnaik, 1983), Germiston (Gerbaud et al., 1987a) or La Crosse (Raju & Kolakofsky, 1986a) bunyaviruses. Whether these contrary results reflect the intrinsically low levels of primary transcription and/or differences in the sensitivities of the assays used is unclear and I do not believe that this issue has been satisfactorily resolved. The latter results have no precedent among other negative-strand viruses and are paradoxical considering the presence of an active transcriptase in virions. This prompted a reexamination by Bellocq et al. (1987) of the products of the in vitro transcription reaction; they found that only incomplete transcripts were made under standard conditions. When transcription was carried out in the presence of rabbit reticulocyte lysate complete S m R N A was detected. Addition of protein synthesis inhibitors to the supplemented reaction prevented full-length mRNA production and only incomplete transcripts were detected. The incomplete transcripts were similar to those observed in infected cells treated with protein synthesis inhibitors (Raju & Kolakofsky, 1986b). A model to explain these findings has been proposed, which suggests that interactions between the nascent m R N A chain and its template cause the polymerase to pause and terminate, generating the incomplete transcripts. In the presence of concomitant protein synthesis ribosomes moving along the nascent mRNA behind the polymerase prevent the mRNA from hybridizing to its template and the polymerase reads through to the major termination site (Bellocq & Kolakofsky, 1987; Kolakofsky et al., 1987). This hypothesis requires further experimental confirmation, but it should be noted that Gerbaud et al. (1987a) were able to detect complete S mRNA in their in vitro transcription reactions, using Germiston bunyavirus, without the need to supplement with reticulocyte lysate. The ambisense nature of the phlebovirus S RNA provides an additional level of control over the production of its encoded proteins. In the absence of puromycin or cycloheximide four types of S segment-specific R N A

were identified in Punta Toro phlebovirus-infected cells; genomic RNA, fullqength viral complementary ( + ) RNA, N mRNA and NSs mRNA. Both the N and NSs mRNAs had 5'-terminal extensions of 12 to 17 nucleotides, similar to the situation with bunyavirus mRNAs. Subgenomic S segment mRNAs have also been identified in sandfly fever Sicilian phlebovirus-infected cells (Marriott et al., 1989). In the presence of protein synthesis inhibitors only newly synthesized N mRNA was detected, which accumulated and could be translated in vitro to yield N protein (Ihara et al., 1985a). Hence NSs m R N A synthesis requires the onset of RNA replication, i.e. the production of the full length viral complementary RNA. Therefore, in the case of the ambisense S genome segment, the full-length viral complementary RNA acts as a template for both the subgenomic NSs mRNA and for new genome synthesis. Furthermore, the synthesis of NSs mRNA is regulated independently to that of N mRNA. This implies that the NSs protein is not involved in the early stages of viral complementary RNA synthesis (Bishop, 1986). The 3' termini of the N and NSs mRNAs have been mapped to a common region of the Punta Toro phlebovirus S R N A between bases 977 and 1017 (Emery &'Bishop, 1987). Examination of the sequence in this intergenic region suggests that a stable A-U-rich hairpin can be formed (nucleotides 886 to 1092) with its peak at position 996, which correlates with the region involved in termination of mRNA transcription. However, the S RNA of sandfly fever Sicilian phlebovirus has a predominantly C-rich intergenic region, which cannot be folded into a large base-paired structure (Marriott et al., 1989). Therefore phlebovirus transcription termination signals may be more subtle then pronounced secondary structure in the template.

Virus maturation Maturation of the Bunyaviridae characteristically occurs at intracellular smooth membranes, principally in the Golgi complex (Lyons & Heyduk, 1973; Murphy et al., 1973; Kuismanen et al., 1982, 1984; Smith & Pifat, 1982) and is inhibited by monensin, a monovalent ionophore (Cash, 1982; Kuismanen et al., 1985; Schmaljohn et al., 1986a; G, E. Watret & R. M. Elliott, unpublished observations). Exceptionally, Rift Valley fever phlebovirus has been observed to mature at the cell surface of infected rat hepatocytes (Anderson & Smith, 1987). The glycoproteins accumulate in the Golgi complex, causing a progressive vacuolization (Kuismanen et al., 1984), an effect mediated by the glycoproteins alone (Gahmberg et al., 1986a). However, the morphologically altered Golgi

Review: Molecular biology o f the Bunyaviridae

complex remains functionally active, as evidenced by its ability to glycosylate and transport authentic plasma membrane glycoproteins (Gahmberg et al., 1986b). Virions bud into Golgi vesicles, which are transported to the cell surface where the particles are released by exocytosis. Some G1 protein has been detected on the cell surface, but its transport from the endoplasmic reticulum is about two- to threefold slower than that of vesicular stomatitis virus G protein (Madoff & Lenard, 1982). Expression of hantavirus and phlebovirus glycoproteins in recombinant vaccinia vectors indicates that Golgi targeting is also a property of the glycoproteins themselves (Matsuoka et al., 1988; Pensiero et al., 1988). The signal responsible for Golgi localization has not yet been determined.

Genetics of Bunyaviridae Genome reassortment

A problem faced by all segmented genome viruses is the production of virus particles that package the correct genetic complement. With the Bunyaviridae the RNAs extracted from purified virus preparations are rarely equimolar and the S segment usually predominates (Bouloy et al., 1973/74; Pettersson & K/i/iri~iinen, 1973; Obijeski et al., 1976b; Gentsch et al., 1977a; Hewlett et al., 1977; Pettersson et al., 1977; Yoo & Kang, 1987b). It is unclear how much of the deviation from an equimolar ratio is due to degradation of the larger RNAs during sample preparation, but it does appear that virions containing different numbers of genome segments are produced (Talmon et al., 1987). Analysis of RNAprotein interactions should throw light on the packaging system and this will be a major challenge in the future. A potential advantage of a segmented genome is the possibility for RNA segment reassortment to occur, which could confer new and desirable genetic traits on the progeny reassortants. In the Bunyaviridae reassortment has been demonstrated in the laboratory between certain related viruses, but not between viruses in different genera, nor between viruses in different serogroups in the same genus. Even within a serogroup some viruses appear to be incompatible with each other (Gentsch & Bishop, 1976; Gentsch et al., 1977b, 1979; Ozden & Hannoun, 1978, 1980; Iroegbu & Pringle, 1981 ; Rozhon et al., 1981; Pringle & Iroegbu, 1982; R. M. Elliott et al., 1984; Pringle et al., 1984; Janssen et al., 1986). There are further restrictions on reassortment, as judged by the difficulty in obtaining reassortants with a certain genotype, e.g. with Batai, Bunyamwera and Maguari bunyaviruses the L and S segments appeared to cosegregate in heterologous crosses, but this linkage

515

could be broken when heterologous reassortants were used as parental viruses (R. M. Elliott et al., 1984; Pringle et al., 1984). Genome segment reassortment has also been demonstrated experimentally in dually infected mosquitoes (Beaty et al., 1981, 1982, 1985; Shope et aL, 1981) and evidence that reassortment occurs in nature has been obtained (Klimas et al., 1981 ; Ushijima et al., 1981). Thus the Bunyaviridae have the capability for rapid evolution by genome segment reassortment, but the relative importance of reassortment in the generation of new virus types in nature is open to question. Temperature-sensitive mutants More than 200 ts mutants of 10 bunyaviruses and of Uukuniemi virus have been isolated following chemical mutagenesis (Gentsch et al., 1977b, 1979; Bishop, 1979; Ozden & Hannoun, 1978, 1980; Iroegbu & Pringle, 1981 ; Pringle & Iroegbu, 1982; Gahmberg, 1984). A surprising feature of these mutants was that the vast majority fell into two reassortment groups, instead of three as expected for a tripartite genome. In fact there is only a single mutant, Maguari virus ts23, that defines the third group (Pringle & Iroegbu, 1982). Mutants of the California and group C bunyaviruses were classified into L or M segment reassortment groups and no mutants with lesions in the S segment were obtained (Gentsch et al., 1977b, 1979; Bishop et al., 1983a). It has been suggested that the paucity of S segment mutants reflects the overlapping reading frame strategy employed by this segment. However, Murphy & Pringle (1987) assigned the ts lesion of Maguari virus ts23 to the L segment. Thus for Maguari virus it seems that L gene mutants are rare, contrary to studies with other negative-strand viruses. Further experimental work is required to resolve this discrepancy. It is worth noting that all the Uukuniemi virus ts mutants, which were also classed into only two reassortment groups, synthesized RNA at the nonpermissive temperature, implying the possession of a functional polymerase (Gahmberg, 1984). Unequivocal mapping of the mutations has not yet been achieved. Genome variation

Many RNA virus genomes have been shown to evolve rapidly, both in nature and in the laboratory, by point mutations, most probably because of the apparently high error rate of RNA polymerases (Domingo & Holland, 1988). Analysis by oligonucleotide fingerprinting of a number of isolates of La Crosse bunyavirus from various ecological niches showed that every isolate had distinguishable L, M and S genome segments, suggesting genetic drift as a major means of bunyavirus evolution (El Said et al., 1979; Klimas et al., 1981). On the other hand the stability of viral genomes has also been demonstrated. The genomes of two isolates of La Crosse

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R. M . Elliott

virus obtained from the brains of fatally infected individuals 18 years apart had very similar oligonucleotide fingerprints (Bishop & Shope, 1979) and no changes in the genome of Toscana phlebovirus were detected over the course of 12 successive transovarial transmissions in its natural sandfly host (Bilsel et al., 1988). The effects on genome stability and the selection of viral variants during passage between the invertebrate and vertebrate hosts would be a rewarding area of study.

Persistent infection A common biological property of arboviruses is their ability to replicate in cells of disparate phylogeny, although the outcome of these infections is often markedly different. In permissive vertebrate cells the infection is usually cytopathic and leads to cell death, whereas in invertebrate cells the infection can be asymptomatic, self-limiting and a persistent infection is readily established. Mosquito cell cultures persistently infected with Bunyamwera, La Crosse and Marituba bunyaviruses and Toscana phlebovirus have been established. The cells showed no differences in their metabolic activities, but continued to shed infectious virus (Newton et al., 1981 ; Nicoletti & Verani, 1985; Carvalho et al., 1986; Elliott & Wilkie, 1986; Rossier et al., 1988; M. Scallan & R. M. Elliott, unpublished observations). The persistently infected cells were refractory to superinfection by homologous or closely related viruses and the virus shed from the carrier cultures contained ts and plaque-morphology mutants (Newton et al., 1981 ; Elliott & Wilkie, 1986). No inhibition of host protein synthesis was observed in infected mosquito cells, in sharp contrast to the situation in infected mammalian cells. It has been suggested that the shut-off of host macromolecular synthesis in the latter cells is because of a general mRNA instability induced by the virus, perhaps mediated by the endonuclease activity of the viral transcriptase (Raju & Kolakofsky, 1988). Rossier et al. (1988) detected less L mRNA in mosquito compared to mammalian cells (although the levels of L mRNA in both cell types were very low), but it seems unlikely that this difference alone can account for the lack of host shut-off in mosquito cells. A feature of the persistently infected cells was the excess amount of S segment-specific RNA they contained (Elliott & Wilkie, 1986; Rossier et al., 1988). However, whereas the amount of S mRNA remained high, the amount of N protein synthesis declined, indicative perhaps of some block at the level of translation (Rossier et al., 1988; M. Scallan & R. M. Elliott, unpublished observations). This suggests that the self-limiting nature of the infection of mosquito cells is related to control of N

protein translation, but as yet we do not know how this control operates. Some features of the persistently infected mosquito cells, and also of persistently infected mammalian cells (Dugbe nairovirus in pig kidney cells; David-West & Porterfield, 1974, and Toscana phlebovirus in Vero cells; Verani et al., 1984), suggest the involvement of defective interfering (DI) RNAs, but these have not been demonstrated directly. Elliott & Wilkie (1986) described interfering particles from the carrier cultures that appeared to contain only S segment RNA rather than RNA molecules containing internal deletions, as is characteristic of 'typical' DI particles. Similar S RNAcontaining interfering particles were isolated by Kascsak & Lyons (1978) following repeated high multiplicity passage of Bunyamwera virus in BHK cells. In both cases interference of wild-type virus replication was more apparent in the homologous cell system. More typical DI RNAs, derived from the L RNA segment, have been observed in Germiston bunyavirus-infected cells (Cunningham & Szilagyi, 1987).

Outlook Our knowledge of the molecular biology of the Bunyaviridae has expanded rapidly over the last few years, revealing the different coding strategies used by individual genome segments and providing some clues about the evolutionary relationships between the viruses, but these data are far from complete. As might be expected from such a large taxonomic grouping of viruses considerable diversity exists within the family. At the very least we need to know the entire genome sequences of a representative of each of the five genera (and putative members of the Bunyaviridae, such as tomato spotted wilt virus) to assess the extent of this diversity in terms of coding and replicative strategies. This information is required before re-evaluation of the classification within the family can be undertaken. More extensive sequencing projects are needed to provide data for comparative purposes to further our understanding of the phylogenetic relationships within the family. Of particular interest would be analyses of the polymerase proteins, since the bunyavirus polymerase appears unrelated to other negative-strand virus polymerases, whereas the homologous proteins of rhabdoviruses and paramyxoviruses show a clear evolutionary relationship (Tordo et al., 1988). Data on the Bunyaviridae polymerase may supply evidence to suggest whether there was a single progenitor, or whether there were multiple ancestral viruses. Nucleotide sequence data provide the foundation to elucidate gene function and this must be a major avenue

Review." Molecular biology of the Bunyaviridae for future research. In order to design effective strategies to control the important pathogens in the family we need an understanding of the functions of the viral proteins in replication, the role played by these proteins in biological aspects such as virulence or interaction with vectors and the recognition of these proteins by the immune system. A serious obstacle to some of this work is our inability to produce viruses containing specific genetic alterations the development of such a system presents a great challenge. I thank Nigel Stow for critically reading the manuscript, members of my laboratory for useful discussions and colleagues elsewhere for communicating unpublished results. I also thank C. Diamond, M. McArthur and E. Robertson for typing the manuscript. I am a Medical Research Council Senior Fellow and work in my laboratory is supported by project grants from the MRC. Note added in proof Since acceptance of this review the following relevant papers have appeared. Nucleotide sequence analysis and protein Junction section: Simons et al. (1990; Journal of Virology 64, 247-255) have shown by nucleotide sequence determination that the uukuvirus S RNA segment has an ambisense coding strategy, and that the uukuvirus and phlebovirus N proteins share >30% identical amino acids. Complete nucleotide sequences have been determined for two of the genome segments of nephropathia epidemica hantavirus, the M segment (Giebel et al., 1989; Virology 172, 498-505) and the S segment (Stohwasser et al., 1990; Virology 174, 79-86). RNA synthesis section: Raju et al. (1989; Journal of Virology 63, 5159-5165) have suggested that cell-type dependent host factors are involved in La Crosse bunyavirus mRNA synthesis, and that these factors may not be present in all BHK cell lines which would account for the discrepancies noted regarding primary transcription. Persistent infection section: Hacker et al. (1989; Journal of Virology 63, 5166-5174) have demonstrated that in mosquito cells La Crosse bunyavirus S mRNA becomes encapsidated by N protein, thereby preventing its translation.

References ABRAHAM, G. & PATTNAIK, A. K. (1983). Early R N A synthesis in Bunyamwera virus-infected cells. Journal of General Virology 64, 1277-1290. AKASHI, H. & BISHOP, D. H. L. (1983). Comparison of the sequences and coding of La Crosse and snowshoe hare bunyavirus S RNA species. Journal of Virology 45, 1155-1158. AKASHI, H., GAY, M., IHARA,T. & BISHOP, D. H. L. (1984). Localized conserved regions of the S RNA gene products of bunyaviruses are revealed by sequence analyses of the Simbu serogroup Aino virus. Virus Research 1, 51-63. ANDERSON, G. W. & SMITH, J. V. (1987). Immunoelectron microscopy of Rift Valley fever viral morphogenesis in primary rat hepatocytes. Virology 161, 91-100. ARIKAWA, J., TAKASHIMA,I. & HASHIMOTO, N. (1985). Cell fusion by haemorrhagic fever with renal syndrome (HFRS) viruses and its application for titration of virus infectivity and neutralizing antibody. Archives of Virology 86, 303-313. ARIKAWA, J., SCHMALJOHN,A. L., DALRYMPLE, J. i . & SCHMALJOHN, C. S. (1989). Characterization of Hantaan virus envelope glycoprotein antigenic determinants defined by monoclonal antibodies. Journal of General Virology 70, 615-624. BATTLES, J. & DALRYMPLE, J. M. (1988). Genetic variation among geographic isolates of Rift Valley fever virus. American Journal of Tropical Medicine and Hygiene 39, 623-637. BEATY, B. J. & BISHOP, n . H. L. (1988). Bunyavirus-vector interaction. Virus Research 10, 289-302.

517

BEATY, B. J., ROZHON, E. J., GENSEMER,P. & BISHOP, D. H. L. (1981). Formation of reassortant bunyaviruses in dually infected mosquitoes. Virology 111, 662-665. BEATY, B. J., MILLER, B. R., SHOPE, R. E., ROZHON, E. J. & BISHOP, D. H. L. (1982). Molecular basis of bunyavirus per os infection of mosquitoes: rote of the middle-sized RNA segment. Proceedings of the National Academy of Sciences, U.S.A. 79, 1295-1297. BEATY, B. J., SUNDIN, D. R., CHANDLER, L. J. & BISHOP, D. H. L. (1985). Evolution of bunyaviruses by genome reassortment in dually infected mosquitoes (Aedes triseriatus). Science 230, 548-550. BELLOCQ, C. & KOLAKOFSKY,D. (1987). Translational requirement for La Crosse virus S-mRNA synthesis: a possible mechanism. Journal of Virology 61, 3960-3967. BELLOCQ, C., RAJU, R,, PATTERSON, J. L. & KOLAKOFSKY, D. (1987). Translational requirement of La Crosse virus S-mRNA synthesis: in vitro studies. Journal of Virology 61, 87-95. BILSEL, P. A., TESH, R. B. & NICHOL, S. T. (1988). RNA genome stability of Toscana virus during serial transovarial transmission in the Handily Phlebotomus perniciosus. Virus Research 11, 87-94. BISHOP, D. H. L. (1979). Genetic potential of bunyaviruses. Current Topics in Microbiology and Immunology 86, 1-33. BISHOP, D. H. L. (1985a). Replication of arenaviruses and bunyaviruses. In Virology, pp. 1083-1110. Edited by B. N. Fields. New York: Raven Press. BISHOP, D. H. L. (1985b). The genetic basis for describing viruses as species. Intervirology 24, 79-93. BISHOP, D. H. L. (1986). Ambisense RNA genomes of arenaviruses and phleboviruses. Advances in Virus Research 31, 1-51. BISHOP, D. H. L. & SHOPE, R. E. (1979). Bunyaviridae. Comprehensive Virology 14, 1-36. BISHOP, D. H. L., CALISHER, C., CASALS, J., CHUMAKOV, M. P., GAIDAMOVICH,S. Y., HANNOUN, C., Lvov, D. K., MARSHALL,I. D., OKER-BLOM, N. M., PETTERSSON, R. F., PORTERFIELD, J. S., RUSSELL, P. K., SHOPE, R. E. & WESTAWAY, E. G. (1980). Bunyaviridae. Intervirotogy 14, 125-143. BISHOP, D. H. L., GOULD, K. G., AKASHI, H. & CLERX-VANHAASTER, C. M. (1982). The complete sequence and coding content of snowshoe hare bunyavirus small (S) viral RNA species. Nucleic Acids Research 10, 3703 3713. BISHOP, D. H. L., FULLER, F., AKASHI, H., BEATY,B. J. & SHOPE, R. E. (1983a). The use of reassortment bunyaviruses to deduce their coding and pathogenic potentials. In Mechanisms of Viral Pathogenesis: From Gene to Pathogen, pp. 49-60. Edited by A. K o h n & P. Fuchs. Boston: Martinus Nijhoff. BISHOP, D. H. L., GAY, i . E. & MATSUOKO, Y. (1983b). Nonviral heterogeneous sequences are present at the 5' ends of one species of snowshoe hare bunyavirus S complementary RNA. Nucleic Acids Research 11, 6409-6418. BOULOY, i . & HANNOUN, C. (1976). Studies on Lumbo virus replication. I. RNA dependent RNA polymerase associated with virions. Virology 69, 258-268. BOULOY, M., KRAMS-OZDEN, S., HORODNICEANU, F. & HANNOUN, C. (1973/74). Three segment RNA genome of Lumbo virus (bunyavirus). Intervirology 2, 173-180. BOULOY, M., VIALAT, P., GIRARD, M. & PARDIGON, N. (1984). A transcript from the S segment of the Germiston bunyavirus is uncapped and codes for the nucleoprotein and a nonstructural protein. Journal of Virology 49, 717-723. BRAAM, J., ULMANEN, 1. & KRUG, R. M. (1983). Molecular model of a eukaryotic transcription complex: functions and movements of influenza P proteins during capped RNA primed transcription. Cell 34, 609-618. CABRADILLA, C. D., HOLLOWAY, B. P. & OmJESKI, J. F. (1983). Molecular cloning and sequencing of the La Crosse virus S RNA. Virology 128, 463-468. CALISHER, C. I-I. & KARABATSOS, N. (1988). Arbovirus serogroups: definition and geographic distribution. In The Arboviruses: Epidemiology and Ecology, pp. 19-57. Edited by T. P. Monath. Boca Raton: CRC Press. CALISHER, C. H., MONATH, T. P., KARABATSOS,N. & TRENT, D. W. (1981). Arbovirus subtyping: applications to epidemiologic studies,

518

R.M.

Ellwtt

availability of reagents and testing services. American Journal of

Epidemiology 114, 619-630. CANONICO, P. G. & PIEAT, D. Y. (1987). Highly pathogenic viruses of Africa and China. In HIV and Other Highly Pathogenic Viruses, pp. 63-80. Edited by R. A. Smith. San Diego: Academic Press. CAPLEN, H., PETERS, C. J. & BISHOP, D. H. L. (1985). Mutagen-directed attenuation of Rift Valley fever virus as a method for vaccine development. Journal of General Virology 66, 2271-2277. CARVALHO, M. G. C., FRUGULHETTI, I. C. & REBELLO, M. A. (1986). Marituba (Bunyaviridae) virus replication in cultured Aedes albopictus cells and in L-A9 cells. Archives of Virology 90, 325 335. CASH, P. (1982). Inhibition of La Crosse virus replication by monensin, a monovalent ionophore. Journal of General Virology 59, 193-196. CASH, P. (1985). Polypeptide synthesis of Dugbe virus, a member of the Nairovirus genus of the Bunyaviridae. Journal of General Virology 66, 141-148. CASH, P., VEZZA, A. C., GENTSCH, J. R. • BISHOP, D. H. L. (1979). Genome complexities of the three mRNA species of snowshoe b_are bunyavirus and in vitro translation of S mRNA to viral N polypeptide. Journal of Virology 31,685-694. CASH, P., HENDERSHOT, L. & BISHOP, D. H. L. (1980). The effects of glycosylation inhibitors on the maturation and intracellular polypeptide synthesis induced by snowshoe hare bunyavirus. Virology 103, 235-240. CHAN, E. K. L., SULLIVAN,K. F. & TAN, E. M. (1989). Ribonucleoprotein SS-B/La belongs to a protein family with consensus for RNAbinding. Nucleic Acids Research 17, 2233 2244. CLERX, J. P. M. & BISHOP, D. H. L. (1981). Qalyub virus, a member of the newly proposed Nairovirus genus (Bunyaviridae). Virology 108, 361-372. CLERX, . l . P . M . , CASALS, J. & BISHOP, D. H. L. (1981). Structural characteristics of nairoviruses (genus Nairovirus, Bunyaviridae). Journal of General Virology 55, 165-178. CLERX-VANHAASTER, C. M. & BISHOP, D. H. L. (1980). Analysis of the Y-terminal sequences of snowshoe hare and La Crosse bunyaviruses. Virology 105, 564-574. CLERX-VAN HAASTER, C. M., AKASHI, H., AUPERIN, D. D. & BISHOP, D. H. L. (1982a). Nucleotide sequence analyses and predicted coding of bunyavirus genome RNA species. Journal of Virology 41, 119 128. CLERX-VAN HAASTER, C. M., CLERX, J. P. M., USHIJIMA,H., AKASHI, H., FULLER, F. & BISHOP, n . H. L. (1982b). The 3' terminal RNA sequences of bunyaviruses and nairoviruses (Bunyaviridae): evidence of end sequence generic differences within the virus family. Journal of General Virology 61, 289-292, COLLETT,M. S. (1986). Messenger RNA of the M segment RNA of Rift Valley fever virus. Virology 151, 151-156. COLLETT, M. S., PURCHIO, A. F., KEEGAN, K., FRAZIER, S., HAYS, W., ANDERSON, D. K., PARKER, M. D., SCHMALJOHN,C., SCHMIDT,J. & DALRYMPLE, J. M. (1985). Complete nucleotide sequence of the M RNA segment of Rift Valley fever virus. Virology 114, 228-245. COLLETT,M. S., KEEGAN, K., HU, S.-L., SRIDHAR, P., PURCHIO, A. F., ENNIS, W. H. & DALRYMPLE, J. M. (1987). Protective subunit immunogens to Rift Valley fever virus from bacteria and recombinant vaccinia virus. In The Biology of Negative Strand Viruses, pp. 321329. Edited by B. W. J. Mahy & D. Kolakofsky. Amsterdam: Elsevier. COLLETT, M. S., KAKACH,L. T., SUZICH, J. A. & WASMOEN,T. L. (1989). Gene products and expression strategy of the M segment of the phlebovirus Rift Valley fever virus. In Genetics and Pathogenesis of Negative Strand Viruses, pp. 49-57. Edited by D. Kolakofsky & B. W. J. Mahy. Amsterdam: Elsevier Biomedical. CUNNINGHAM, C. & SZILAGYI,J. F. (1987). Viral RNAs synthesized in cells infected with Germiston bunyavirus. Virology 157, 431-439. DALRYMPLE, J. M., HASTY, S. E., KAKACH, L. T. & COLLETT, M. S. (1989). Mapping protective determinants of Rift Valley fever virus using recombinant vaccinia viruses. In Vaccines "89, pp. 371-375. Edited by R. A. Lerner;H. Ginsberg, R. Chanock & F. Brown. New York: Cold Spring Harbor Laboratory. DANTAS, J. R., OKUNO, Y., ASADA,H., TAMURA, M , TAKAHASHI,i . , TANISHITA,O., TAKAHISHI,Y., KURATA,T. & YAMANISHI,K. (1986). Characterization of glycoproteins of viruses causing hemorrhagic

fever with renal syndrome (HFRS) using monoclonal antibodies. Virology 151, 379-384. DAVID-WEST, T. S. (1974). Polyacrylamide gel electrophoresis of Dugbe virus infected cells. Microbios 11, 21-23. DAVID-WEST, T. S. & PORTEREIELD, J. S. (1974). Dugbe virus: a tickborne arbovirus from Nigeria. Journal of General Virology 23, 297 307. DE HAAN, P., WAGEMAKERS,L., GOLDBACH, R. & PETERS, D. (1989). Tomato-spotted wilt virus, a new member of the Bunyaviridae? In Genetics and Pathogenicity of Negative Strand Viruses, pp. 287-291. Edited by D. Kolakofsky & B. W. J. Mahy. Amsterdam: Elsevier. DOMINGO, E. & HOLLAND, J. J. (1988). High error rates, population equilibrium and evolution of RNA replicating systems. In RNA Genetics, vol. III, pp. 3-36. Edited by E. Domingo, J. J. Holland & P. Alquist. Boca Raton: CRC Press. DOWNS, W. G. (1975). Malaria, the great umbrella. Bulletin of the New York Academy of Medicine 51, 984-990. EL SAID, L. H., VORNDAM, V., GENTSCH, J. R., CLEWLEY, J. P., CALISHER, C. O., KLIMAS, R. A., THOMPSON, W. H., GRAYSON, M., TRENT, D. W. & BISHOP,D. H. L. (1979). A comparison of La Crosse virus isoIates obtained from different ecological niches and an analysis of the structural components of California encephalitis serogroup viruses and other bunyaviruses. American Journal of Tropical Medicine and Hygiene 28, 364-386. ELLIOTT, L. H., KILEY, M. P. & MCCORMICK, J. B. (1984). Hantaan virus : identification of virion proteins. Journal of General Virology 65, 1285-1293. ELLIOa-r, R. M. (1985). Identification of non-structural proteins encoded by viruses of the Bunyamwera serogroup (family Bunyaviridae). Virology 143, 119 126. ELLIOTT, R. M. (1989a). Nucleotide sequence analysis of the small (S) RNA segment of Bunyamwera virus, the prototype of the family Bunyaviridae. Journal of General Virology 70, 1281- 1285. ELLIOTX, R. M. (1989b). Nucleotide sequence analysis of the large (L) genomic RNA segment of Bunyamwera virus, the prototype of the family Bunyaviridae. Virology 173, 426-436. ELLIOTr, R. M. & MCGREGOR, A. (1989). Nucleotide sequence and expression of the small (S) RNA segment of Maguari bunyavirus. Virology 171, 516-524. ELLIOT'r, R. M. & WILKIE, M. L. (1986). Persistent infection of Aedes albopictus C6/36 cells by Bunyamwera virus. Virology 150, 21 32. ELLIOTT, R. M., LEES, J. F., WATRET, G. E., CLARK, W. & PRINGLE, C. R. (1984). Genetic diversity of bunyaviruses and mechanisms of genetic variation. In Mechanisms of Viral Pathogenesis : From Gene to Pathogen, pp. 61 76. Edited by A. Kohn & P. Fuchs. Boston: Martinus Nijhoff. EMERY, V. C. & BISHOP, D. H. L. (1987). Characterization of Punta TOEDS mRNA species and identification of an inverted complementary sequence in the intergenic region of Punta Toro phlebovirus ambisense S RNA that is involved in mRNA transcription termination. Virology 156, 1 11. ENDRES, M. J., JACOBY, D. R., JANSSEN, R. S., GONZALEZ-SCARANO,F. & NATHANSON, N. (1989). The large viral RNA segment of California serogroup bunyaviruses encodes the large viral protein. Journal of General Virology 70, 223-228. ESHITA, Y. & BISHOP, D. H. L. (1984). The complete sequence of the M RNA of snowshoe hare bunyavirus reveals the presence of internal hydrophobic domains in the viral glycoprotein. Virology 137, 227240. ESHITA, Y., ERICSON, B., ROMANOWSKI,V. & BISHOP, D. H. L. (1985). Analyses of the M RNA transcription processes of snowshoe hare bunyavirus S and M RNA species. Journal of Virology 55, 681-689. FAZAKERLEY, J. K., GONZALEZ-SCARANO,F., STRICKLER, J., DIETZSCHOLD, B., KARUSH, F. & NATHANSON, N. (1988). Organization of the middle RNA segment of snowshoe hare bunyavirus. Virology 167, 422-432. FOULKE, R. S., ROSAXO, R. R. & FRENCH, G. R. (1981). Structural polypeptides of Hazara virus. Journal of General Virology 53, 169172. FULLER, F. & BISHOP, D. H. L. (1982). Identification of virus-coded non-structural polypeptides in bunyavirus-infected cells. Journal of Virology 41, 643-648.

Review." Molecular biology o f the Bunyaviridae

FULLER, F., BHOWN, A. S. & BISHOP, D. H. L. (1983). Bunyavirus nucleoprotein, N, and a non-structural protein, NSs, are coded by overlapping reading frames in the S RNA. Journal of General Virology 64, 1705-1714. GAHMBERG, N. (1984). Characterization of two recombinatio~ complementation groups of Uukuniemi virus temperature-sensitive mutants. Journal of General Virology 65, 1079-1090. GAHMBERG, N., KUISMANEN, E., KERANEN, S. & PETTERSSON, R. F. (1986a). Uukeniemi virus glycoproteins accumulate in and cause morphological changes of the Golgi complex in the absence of virus maturation. Journal of Virology 57, 89%906. GAHMBERG, N., PETTERSSON, R. F. & K.~,.RI.~INEN, L. (1986b). Efficient transport of Semliki forest virus glycoproteins through a Golgi complex morphologically altered by Uukeniemi virus glycoproteins. EMBO Journal 5, 3111-3118. GENTSCH, J. R. & BISHOP, D. H. L. (1976). Recombination and complementation between temperature-sensitive mutants of the bunyavirus snowshoe hare virus. Journal of Virology 20, 351-354. GENTSCH, J. R. & BISHOP, n . H. L.(1978). Small viral RNA segment of bunyaviruses codes for viral nucleocapsid protein. Journal of Virology 28, 417-419. GENTSCH, J. R. & BISHOP, D. H. L.(1979). M viral RNA segment of bunyaviruses codes for two glycoproteins, G1 and G2. Journal of Virology 30, 767-770. GENTSCH, J. R., BISHOP, D. H. L. & OBIJESKI, J. F. (1977a). The virus particle nucleic acids and proteins of four bunyaviruses. Journal of General Virology 34, 257 268. GENTSCH, J. R., WYNNE, L. R., CLEWLEY,J. P., SHOPE, R. E. & BISHOP, D. H. L. (1977b). Formation of recombinants between snowshoe hare and La Crosse bunyaviruses. Journal of Virology 24, 893402. GEN'rSCH, J. R., ROBESON, G. & BISHOP, D. H. L. (1979). Recombination between snowshoe hare and La Crosse Bunyaviruses. Journal of Virology 31, 707-717. GENTSCH, J. R , ROZHON, E. J., KLIMAS, R. A., EL SAID, L. H., SHOPE, R. E. & BISHOP, D. H. L. (1980). Evidence from recombinant bunyavirus studies that the M RNA gene products elicit neutralizing antibodies. Virology" 102, 190-204. GERBAUD,S., PARDIGON, N., VIALAT,P. & BOULOY, i . (1987a). The S segment of the Germiston bunyavirus genome: coding strategy and transcription. In The Biology of Negative Strand Viruses, pp. 191-198. Edited by B. W. J. Mahy & D. Kolakofsky. Amsterdam: Elsevier. GERBAUD,S., VIALAT,P., PARDIGON, N., WYCHOWSKI, C., GIRARD, M. & BOULOY, M. (1987b). The S segment of Germiston virus RNA genome can code for three proteins. Virus Research 8, 1-13. GONZALEZ-SCARANO, F. (1985). La Crosse virus GI glycoprotein undergoes a conformational change at the pH of fusion. Virology140, 209-216. GONZALEZ-SCARANO,F., SHOPE, R. E., CALISHER,C. E. & NATHANSON, N. (1982). Characterization of monoclonal antibodies against the G1 and N proteins of La Crosse and Tahyna, two California serogroup Bunyaviruses. Virology 120, 42-53. GONZALEZ-SCARANO, F., SHOPE, R. E., CALISHER, C. H. & NATHANSON, N. (1983). Monoclonal antibodies against the G1 and nucleocapsid proteins of La Crosse and Tahyna viruses. Progress in Clinical and Biological Research 123, 145-156. GONZALEZ-SCARANO, F., POBJECKY, N. & NATHANSON, N. (1984). La Crosse bunyavirus can mediate pH-dependent fusion from without. Virology 132, 222-225. GONZALEZ-SCARANO,F., JANSSEN, R. S., NAJJAR, J. A., POBJECKY, N. & NATHANSON, N. (1985). An avirulent G1 glycoprotein variant of La Crosse bunyavirus with defective fusion function. Journal of Virology 54, 757-763. GONZALEZ-SCARANO,F., BEATY,B., SUNDIN, D., JANSSEN, R., ENDRES, M. J. & NATHANSON, N. (1988). Genetic determinants of the virulence and infectivity of La Crosse virus. MicrobialPathogenesis4, 1-7. GRADY, L. J. & KINCa, W. (1985). Two monoclonal antibodies against La Crosse virus show host-dependent neutralizing activity. Journalof General Virology 66, 2773-2776. GRADY, L. J., SANDERS, M. L. & CAMPBELL, W. P. (1983a). Evidence for three separate antigenic sites on the G1 protein of La Crosse virus. Virology 126, 395-397.

519

GRADY, L. J., SRIHONGSE, S., GRAYSON, M. A. & DEIBEL, R. (1983b). Monoclonal antibodies against La Crosse virus. Journal of General Virology 64, 1699-1704. GRADY, L. J., SANDERS, i . L. & CAMPBELL, W. P. (1987). The sequence of the M RNA of an isolate of La Crosse virus. Journal of General Virology 68, 3057 3971. GUPTA, K. C. & KINGSBURY,n . W. (1982). Conserved polyadenylation signals in two negative-strand RNA virus families. Virology 120, 518 523. HEWLETT, M. J., PETTERSSON,R. F. & BALTIMORE,D. (1977). Circular forms of Uukuniemi virion RNA: an electron microscopic study. Journal of Virology 21, 1085-1093. Hsu, M.-T., PARVXN, J. D., GUPTA, S., KRYSTAL, M. & PALESE, P. (1987). Genomic RNAs of influenza viruses are held in a circular conformation in virions and in infected ceils by a terminal panhandle. Proceedings of the National Academy of Sciences, U.S.A. 84, 8140-8144. HUNG, T., XIA, S. M., ZHAO,T. X., ZHOU, J. Y., SONG, G., LIAO, G. X., YE, W. W., CHU, Y. L. & HANG, C. S. (1983). Morphological evidence for identifying the viruses of hemorrhagic fever with renal syndrome as candidate members of the Bunyaviridae family. Archives of Virology 78, 137-144. IHARA,T., AKASHI,H. & BISHOP,D. H. L. (1984). Novel coding strategy (ambisense genomic RNA) revealed by sequence analyses of Punta ToED phlebovirus S RNA. Virology 136, 293-306. IHARA, T., MATSUURA,Y. & BISHOP, n . H. L. (1985a). Analyses of the mRNA transcription processes of Punto Toro phlebovirus (Bunyaviridae). Virology 147, 317-325. IHARA, T., SMITH, J., DALRYMPLE, J. M. & BISHOP, D. H. L. (19853). Complete sequences of the glycoproteins and M RNA of Punta Toro phlebovirus compared to those of Rift Valley fever virus. Virology 144, 246-259. IROEGBU, C. U. & PRINGLE, C. R. (1981). Genetic interactions among viruses of the Bunyamwera complex. Journal of Virology 37, 383 394. JAMES,W. S. & MILLICAN,D. (1986). Host-adaptive antigenic variation in bunyaviruses. Journal of General Virology 67, 2803 2806. JANSSEN, R. S., NATHANSON, N., ENDRES, M. J. & GONZALEZSCARANO, F. (1986). Virulence of La Crosse virus is under polygenic control. Journal of Virology 59, 1 7. KAKACH, L. T., WASMOEN,T. L. & COLLETT, M. S. (1988). Rift Valley fever virus M segment: use of recombinant vaccinia viruses to study phlebovirus gene expression. Journal of Virology 62, 826~833. KAKACH, L. T.~ SUZICH, J. A. & COLLETT, M. S. (1989). Rift Valley fever virus M segment: phlebovirus expression strategy and protein glycosylation. Virology 170, 505 510. KARABATSOS, N. (1985). International Catalogue of Arboviruses 1985 Including Certain Other Viruses of Vertebrates. San Antonio: The American Society of Tropical Medicine and Hygiene. KASCSAK, R. J. & LYONS, M. J. (1977). Bunyamwera virus. I. The molecular complexity of the virion RNA. Virology 82, 37-47. KASCSAK, R. J. & LYONS, M. J. (1978). Bunyamwera virus. II. The generation and nature of defective interfering particles. Virology89, 539-546. KEEGAN, K. & COLLETT, M. S. (1986). Use of bacterial expression cloning to define the amino acid sequences of antigenic determinants on the G2 glycoprotein of Rift Valley fever virus. Journal of Virology 58, 263-270. KINGSFORD, L. (1984). Enhanced neutralization of La Crosse virus by the binding of specific pairs of monoclonal antibodies to the G1 glycoprotein. Virology 136, 265-273. KINGSFORD, L. & HILL, D. W. (1983). The effect of proteolytic cleavage of La Crosse virus G1 glycoprotein on antibody neutralization. Journal of General Virology 64, 2147-2156. KINGSFORD, L., ISHIZAWA, L. D. & HILL, D. W. (1983). Biological activities of monoclonal antibodies reactive with antigenic sites mapped on the Gl glycoprotein of La Crosse virus. Virology 129, 443-455. KLIMAS, R. A., THOMPSON, W. A., CALISHER, C. H., CLARK, G. G., GRIMSTAD, P. R. & BISHOP, D. H. L. (1981). Genotypic varieties of La Crosse virus isolated from different geographic regions of the continental United States and evidence for a naturally occurring

520

R. M. Elliott

intertypic recombinant La Crosse virus. American Journal of

Epidemiology 114, 112-131. KOLAKOFSKY, D., BELLOCQ, C. & RAJU, R. (1987). The translational requirement of La Crosse virus S-mRNA synthesis. Cold Spring Harbor Symposia on Quantitative Biology 52, 373-379. KRUG, R. M., ST. ANGELO, C., BRONI, B. & SHAPIRO, G. (1987). Transcription and replication of influenza virion RNA in the nucleus of infected cells. Cold Spring Harbor Symposia on Quantitative Biology 52, 353-358. KUISMANEN, E. (1984). Posttranslational processing of Uukuniemi virus glycoproteins G1 and G2. Journal of Virology 51, 806812. KUISMANEN, E., HEDMAN,K., SARaSTE, J. & PETTERSSON, R. F. (1982). Uukuniemi virus maturation: accumulation of virus particles and viral antigens in the Golgi complex. Molecular and Cellular Biology 2, 1444-1458. KUISMANEN, E., BANG, B., HURME, M. & PETTERSSON, R. F. (1984). Uukuniemi virus maturation: immunofluorescence microscopy with monoclonal glycoprotein-specific antibodies. Journal of Virology 51, 137-146. KUISMANEN, E., SARASTE, J. & PETTERSSON, R. F. (1985). Effect of monensin on the assembly of Uukuniemi virus in the Golgi complex. Journal of Virology 55, 813 822. LEES, J. F., PRINGLE, C. R. & ELLIOTT, R. M. (1986). Nucleotide sequence of the Bunyamwera virus M RNA segment: conservation of structural features in the Bunyavirus glycoprotein gene product. Virology 148, 1-14. LUDWIG, G. V., CHRISTENSEN, B. M., YUILL, T. M. & SCHULTZ, K. T. (1989). Enzyme processing of La Crosse virus glycoprotein G I : a bunyavirus-vector infection model. Virology 171, 108-113. LYONS, M. J. & HEYDUK, J. (1973). Aspects of the developmental morphology of California encephalitis virus in cultured vertebrate and arthropod cells and in mouse brain. Virology 54, 37-52. MADOFF, D. H. & LENARD, J. (1982). A membrane glycoprotein that accumulates intracellularly: cellular processing of the large glycoprotein of La Crosse virus. Cell 28, 821-829. MARRIOTT, A. C., WARD, V. K. & NUTTALL, P. A. (1989). The S RNA segment of sandfly fever Sicilian virus: evidence for an ambisense genome. Virology 169, 341-345. MARTIN, M. L., LINDSEY-REGNERY, n., SASSO, O. R., McCORMICK, J. B. & PALMER,E. (1985). Distinction between Bunyaviridae genera by surface structure and comparison with Hantaan virus using negative stain electron microscopy. Archives of Virology 86, 17-28. MATSUOKA,Y., IHARA, T., BISHOP, D. H. L. & COMPANS,R. W. (1988). Intracellular accumulation of Punta Toro virus glyoproteins expressed from cloned cDNA. Virology 167, 251-260. MILNE, R. G. & FRANCKI, R. I. B. (1984). Should tomato spotted wilt virus be considered as a possible member of the family Bunyaviridae? Intervirology 22, 72-76. MONATH, T. P. (editor) (1988). The Arboviruses: Epidemiology and Ecology, vol. I to V. Boca Raton: CRC Press. MURPHY, F. A., HARRISON, A. K. & WHITFIELD, S. G. (1973). Bunyaviridae: morphologic and morphogenetic similarities of Bunyamwera serologic supergroup viruses and several other arthropod-borne viruses. Intervirology 1, 297-316. MURPHY, J. & PRINGLE, C. R. (1987). Bunyavirus mutants: reassortment group assignment and G1 protein variants. In The Biology of Negative Strand Viruses, pp. 357-362. Edited by B. W. J. Mahy & D. Kolakofsky. Amsterdam: Elsevier. NAJJAR, J. A., GENTSCH, J. R., NATHANSON, N. & GONZALEZSCARANO, F. (1985). Epitopes of the G1 glycoprotein of La Crosse virus from overlapping clusters within a single antigenic site. Virology 144, 426-432. NEWTON, S. E., SHORT, N. J. & DALGARNO, L. (1981). Bunyamwera virus replication in cultured Aedes albopictus (mosquito) cells: establishment of a persistent viral infection. Journal of Virology 38, 1015-1024. NICOLETTI, L. & VERANI, P. (1985). Growth ofPhlebovirus Toscana in a mosquito (Aedespseudoscutellaris)cell line (AP-6 l): establishment of a persistent infection. Archives of Virology 85, 35~45. OBIJESKI, J. F. & MURPHY, F. A. (1977). Bunyaviridae: recent biochemical developments. Journal of General Virology 37, 1-14.

OBHESKI, J. F., BISHOP, D. H. L., MURPHY, F. A. & PALMER, E. L. (1976a). Structural proteins of La Crosse virus. Journal of Virology 19, 985-997. OBIJESKI, J. F., BISHOP, D. H. L., PALMER, E. L. & MURPHY, F. A. (1976b). Segmented genome and nucleocapsid of La Crosse virus. Journal of Virology 20, 664~675. OBIJESKI, J. F , MCCAULEY, J. & SKEHEL, J. J. (1980). Nucleotide sequences at the termini of La Crosse virus RNAs. Nucleic Acids Research 8, 2431-2438. OKUNO, Y., YAMANISHI,K , TAKAHASHI,Y., TANISHITA,O., NAGAI,T., DANTAS, J. R., JR, OKAMOTO, Y., TADANO, M. & TAKAHASHI, M. (1986). Haemagglutination-inhibition test for haemorrhagic fever with renal syndrome using viral antigen prepared from infected tissue culture fluid. Journal of General Virology 67, 149-156. OVERTON, H. A., IHARA,T. & BISHOP, D. H. L. (1987). Identification of the N and NSs proteins encoded by the ambisense RNA of Punta Toro phlebovirus using monospecific antisera raised to baculovirus expressed N and NSs proteins. Virology 157, 338-350. OZDEN, S. & HANNOUN, C. (1978). Isolation and preliminary characterization of temperature-sensitive mutants of Lumbo virus. Virology 81, 21(~212. OZDEN, S. & HANNOUN, C. (1980). Biochemical and genetic characteristics of Germiston virus. Virology 103, 232-234. PARDIOON, N., VIALAT, P., GIRARD, M. & BOULOY, M. (1982). Panhandles and hairpin structures at the termini of Germiston virus RNAs (Bunyavirus). Virology 122, 191-197. PARDIGON, N., VIALAT, P., GERBAUD,S., GIRARD, M. & BOULOY, M. (1988). Nucleotide sequence of the M segment of Germiston virus: comparison of the M gene product of several bunyaviruses. Virus Research 11, 73-85. PARKER, M. D. & HEWLETT, M. J. (1981). The 3'-terminal sequences of Uukuniemi and Inkoo virus RNA genome segments. In The Replication of Negative Strand Viruses, pp. 349-354. Edited by D. H. L. Bishop & R. W. Compans. New York & Amsterdam: Elsevier. PARSONSON, I. M. & MCPHEE, D. A. (1985). Bunyavirus pathogenesis. Advances in Virus Research 30, 279-316. PATTERSON, J. L. & KOLAKOFSKY, D. (1984). Characterization of La Crosse virus small-genome transcripts. Journal of Virology 49, 680685. PATTERSON,J. L., CABRADILLA,C., HOLLOWAY, B. P., OBIJESKI, J. F. & KOLAKOFSKY, D. (1983). Multiple leader RNAs and messenger RNAs are transcribed from the La Crosse virus small genome Segment. Cell 33, 791-799. PATTERSON,J. L., HOLLOWAY,B. & KOLAKOFSKV,D. (1984). La Crosse virions contain a primer-stimulated RNA polymerase and a methylated cap-dependent endonuclease. Journal of Virology 52, 215-222. PATTNA1K, A. K. & ABRAHAM, G. (1983). Identification of four complementary RNA species in Akabane virus-infected cells. Journal of Virology 47, 452-462. PENNINGTON, T. H., PRINGLE, C. R. & MCCRAE, M. A. (1977). Bunyamwera virus-induced polypeptide synthesis. Journal of Virology 24, 397-400. PENSIERO, M. N., JENNINGS, G. B., SCHMALJOHN, C. S. & HAY, J. (1988). Expression of the Hantaan virus M genome segment by using a vaccinia virus recombinant. Journal of Virology 62, 696-702. PESONEN, M., KUISMANEN,E. & PETTERSSON, R. F. (1982a). Monosaccharide sequence of protein-bound glycans of Uukuniemi virus. Journal of Virology 41, 390~400. PESONEN, M., R6NNHOLM, R., KUISMANEN, E. & PETTERSSON, R. F. (1982b). Characterization of the oligosaccharides of Inkoo virus envelope glycoproteins. Journal of General Virology 63, 425-434. PETTERSSON, R. & K~fW,IgINEN, L. (1973). The ribonucleic acids of Uukuniemi virus, a non-cubical tick-borne arbovirus. Virology 56, 608-619. PETTERSSON, R. F. & YON BONSDORFF, C. H. (1975). Ribonucleoproteins of Uukuniemi virus are circular. Journal of Virology 15, 386 392. PETTERSSON, R. F. & YON BONSDORFF, C. H. (1987). Bunyaviridae. In Animal Virus Structure, pp. 147-157. Edited by M. V. Nermut & A. C. Steven. Amsterdam: Elsevier.

Review." Molecular biology of the Bunyaviridae PETTERSSON, R., K.~[RIAINEN, L., VON BONSDORFF, C. H. & OKERBLOM, N. (1971). Structural components of Uukuniemi virus, a noncubical tick-borne arbovirus. Virology 46, 721 729. PETTERSSON, R. F., HEWLETT, M. J., BALTIMORE,D. ~ COFFIN, J. M. (1977). The gcnome of Uukuniemi virus consists of three unique RNA segments. Cell 11, 51 63. PIFAT, D. Y., OSTERLING, M. C. & SMITH, J. F. (1988). Antigenic analysis of Punta Toro virus and identification of protective determinants with monoclonal antibodies. Virology 167, 442-450. POBJECKY, N., NATHANSON,N, & GONZALEZ-SCARANO,F. (1989). Use of resonance energy transfer assay to investigate the fusion function of La Crosse virus. In Genetics and Pathogenicity of Negative Strand Viruses, pp. 24-32. Edited by D. Kolakofsky & B. W. J. Mahy. Amsterdam: Elsevier. PRINGLE, C. R. & IROEGBU,C. U. (1982). A mutant identifying a third recombination group in a bunyavirus. Journal of Virology 42, 873879. PRINGLE, C. R., LEES, J. F., CLARK, W. & ELLIOTT, R. M. (1934). Genome sub-unit reassortment among bunyaviruses analysed by dot hybridization using molecularly cloned complementary DNA probes. Virology 135, 244-256. QUERY, C. C., BENTLEY, R. C. & KEENE, J. D. (1989). A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein. Cell 57, 89-101. RAJU, R. & KOLAKOESKY, D. (1986a). Inhibitors of protein synthesis inhibit both La Crosse virus S-mRNA and S genome syntheses in vivo. Virus Research 5, 1-9. RAJU, R. & KOLAKOFSKY, D. (1986b). Translational requirement of La Crosse virus S-mRNA synthesis: in vivo studies. Journal of Virology 61, 96-103. RAJU, R. & KOLAKOFSKY, D. (1987). Unusual transcripts in La Crosse virus-infected ceils and the site for nucleocapsid assembly. Journalof Virology 61, 667-672. RAJU, R. & KOLAKOESKY, D. (1988). La Cross virus infection of mammalian cells induces mRNA instability. Journalof Virology 62, 27-32. RAJU, R. & KOLAKOESKY, D. (1989). The ends of La Crosse virus genome and antigenome RNAs within nucleocapsids are base paired. Journal of Virology 63, 122 128. RANKI, M. & PETTERSSON, R. (1975). Uukuniemi virus contains an RNA polymerase. Journal of Virology 16, 1420-1425. ROBESON~G., EL SAID, L. H., BRANDT, W., DALRYMPLE, J. & BISHOP, D. H. L. (1979). Biochemical studies on the phlebotomus fever group viruses (Bunyaviridae family). Journal of Virology 30, 339350. Rt)NNHOLM, R. & PETTERSSON, R. F. (1987). Complete nucleotide sequence of the M RNA segment of Uukuniemi virus encoding the membrane glycoproteins G1 and G2. Virology 160, 191-202. ROSS1ER, C. J., PATTERSON,J. L. & KOLAKOFSKY, D. (1986). La Crosse virus small genome mRNA is made in the cytoplasm. Journal of Virology 58, 647-650. ROSSIER, C., RAIU, R. & KOLAKOFSKY, D. (1988). La Crosse virus gene expression in mammalian and mosquito cells. Virology 165, 539548. ROZHON, E. J., GENSEMER,P., SHOPE, R. E. & BISHOP, D. H. L. (1981). Attenuation of virulence of a bunyavirus involving an L RNA defect and isolation of LAC/SSH/LAC and LAC/SSH/SSH reassortants. Virology 111, 125-138. SAMSO, A., BOULOY, M. & HANNOUN, C. (1975). Pr6sence de ribonucl6oproteins circulaires dans le virus Lumbo (Bunyavirus). Compte rendue de l'Academie des sciences D280, 779 782. SAMSO, A., BOULOY, M. & HANNOUN, C. (1976). Mise en +vidence de molecules d'acide ribonucleique circulaire dans le virus Lumbo (Bunyavirus). Compte rendue de l'Academie des sciences D282, 16531655. SCHMALJOHN, C. S. & DALRYMPLE, J. M. (1983). Analysis of Hantaan virus RNA : evidence for a new genus of Bunyaviridae. Virology 131, 482-491. SCHMALJOHN, C. S., HASTY, S. E., RASMUSSEN,L. & DALRYMPLE, J. M. (1986a). Hantaan virus replication: effects of monensin, tunicamycin and endoglycosidases on the structural glycoproteins. Journal of General Virology 67, 707-717.

521

SCHMALJOHN, C. S., JENNINGS, G. B., HAY, J. • DALRYMPLE, J. M. (1986b). Coding strategy of the S genome segment of Hantaan virus. Virology 155, 633 643. SCHMALJOHN, C. S., LEE, H. W. & DALRYMPLE, J. M. (1987a). Detection of hantaviruses with RNA probes generated from recombinant DNA. Archives of Virology 95, 291-301. SCHMALJOHN, C. S., SCHMALJOHN,A. L. & DALRYMPLE, J. M. (1987b). Hantaan virus M RNA: coding strategy, nucleotide sequence, and gene order. Virology 157, 31-39. SCHMALJOHN, C. S., ARIKAWA, J., HASTY, S. E., RASMUSSEN, L., LEE, H. W., LEE, P. W. t~ DALRYMPLE, J. M. (1988a). Conservation of antigenic properties and sequences encoding the envelope proteins of prototype Hantaan virus and two virus isolates from Korean haemorrhagic fever patients. Journal of General Virology 69, 1949 1955. SCHMALJOHN, C. S., SUGIYAMA, K., SCHMALJOHN, A. L. & BISHOP, D. H. L. (1988b). Baculovirus expression of the small genome segment of Hantaan virus and potential use of the expressed nucleocapsid protein as a diagnostic antigen. Journal of General Virology 69, 777-786. SCHMALJOHN,C. S., PARKER, M. O., ENNIS, W. H., DALRYMPLE, J. M., COLLETT, M. S., SUZICH, J. A. t~ SCHMALJOHN, A. L. (1989). Baculovirus expression of the M genome segment of Rift Valley lever virus and examination of antigenic and immunogenic properties of expressed proteins. Virology 170, 184-192. SHESHBERADARAN, H., NIKLASSON, B. & TKACHENKO, E. A. 0988). Antigenic relationship between hantaviruses analysed by immunoprecipitation. Journal of General Virology 69, 2645-2651. SHOPE, R. E. (1985). Bunyaviruses. In Virology, pp. 1055-1082. Edited by B. N. Fields. New York: Raven Press. SHOPE, R. E., ROZHON, E. J. & BISHOP, D. H. L.(1981). Role of the middle-sized bunyavirus RNA segment in mouse virulence. Virology 114, 273 276. SMITH, J. F. & PIFAT, D. Y. (1982). Morphogenesis of sandfly fever virus (Bunyaviridae family). Virology 121, 61-81. STRUTHERS, J. K. & SWANEPOEL, R. (1982). Identification of a major non-structural protein in the nuclei of Rift Valley fever virus-infected cells. Journal of General Virology 60, 381-384. STRUTHERS, J. K., SWANEPOEL,R. & SHEPHERD, S. P. (1984). Protein synthesis in Rift Valley fever virus-infected cells. Virology 134, 118124. SUNDIN, D. R., BEATY, B. J., NATHANSON, N. & GONZALEZ-SCARANO, F. (1987). A G1 glycoprotein epitope of La Crosse virus: a determinant of infection of Aedes triseriatus. Science 235, 591 593. SUZICH, J. A. fig.COLLETT, M. S. (1988). Rift Valley Fever virus M segment: cell-free transcription and translation of virus-complementary RNA. Virology 164, 478-486. SWANEPOEL,R. & BLACKBURN,N. K. (1977). Demonstration of nuclear immunofluorescence in Rift Valley fever virus-infected cells. Journal of General Virology 34, 557-561. TAKEHARA, K., M1N, M.-K., BATTLES, J. K., SUGIYAMA,K., EMERY, V. C., DALRYMPLE,J. M. & BISHOP,D. H. L. (i989). Identification of mutations in the M RNA of a candidate vaccine strain of Rift Valley fever virus. Virology 169, 452-457. TALMON, Y., PRASAD, B. V. V., CLERX, J. P. M., WANG, G.-J., CHIU, W. & HEWLET'r, M. J. (1987). Electron microscopy of vitrifiedhydrated La Crosse virus. Journal of Virology 61, 2319 2321. TBSH, R. B. (1988). The genus Phlebovirus and its vectors. Annual Review of Entomology 33, 169 181. TESH, R. B., PERALTA, P. H., SHOPE, R. E., CHANIOTIS,B. N. & JOHNSON, K. M. (1975). Antigenic relationships among phlebotomus fever group arboviruses and their implications for the epidemiology of sandfly fever. American Journal of Tropical Medicine and Hygiene 2,4, 135-144. TESH, R. B., PETERS, C. J. & MEEGAN, J. M. (1982). Studies on the antigenic relationships among phleboviruses. American Journal of Tropical Medicine and Hygiene 31, 149-155. TORDO, N., POCH, O., ERMINE, A., KEITH, G. & ROUGEON, F. (1988). Completion of the rabies virus genome sequence determination: highly conserved domains among the L (polymerase) proteins of unsegmented negative-strand RNA viruses. Virology 165, 565-576.

522

R. M . Elliott

TSAI, T. F., TANG, Y. W., Hu, S. L., YE, K. L., CHEN, G. L. & XU, Z. Y. (1984). Hemagglutination-inhibiting antibody in hemorrhagic fever with renal syndrome. Journal of Infectious Diseases 150, 895-898. ULMANEN, I., SEPP.~,.LX, P. & PETTERSSON, R. F. (1981). In vitro translation of Uukuniemi virus-specific RNAs: identification of a non-structural protein and a precursor to the membrane glycoproteins. Journal of Virology 37, 72-79. URAKAWA, T., SMALL,D. A. & BISHOP, D. H. L. (1988). Expression of snowshoe hare bunyavirus S RNA coding proteins by recombinant baculoviruses. Virus Research 11, 303-317. USHIJIMA, H., CLERX-VANHAASTER, C. M. & BISHOP, D. H. L. (1981). Analyses of Patois group bunyaviruses: evidence for naturally occurring recombinant bunyaviruses and existence of immune precipitable and nonprecipitable nonvirion proteins induced in bunyavirus-infected ceils. Virology 110, 318-332. VERANI, P., NICOLETTI, L. & MARCHI, A. (1984). Establishment and maintenance of persistent infection by the Phlebovirus Toscana in Vero cells. Journal of General Virology 65, 367 375. VEZZA, A. C., REPIK, P. M., CASH, P. & BISHOP, D. H. L. (1979). In vivo transcription and protein synthesis capabilities of bunyaviruses: wild-type snowshoe hare Virus and its temperature-sensitive group I, group II and group I/II mutants. Journal of Virology 31, 426-436. VORNDAM, A. V. ~ TRENT, D. W. (1979). Oligosaccharides of the California encephalitis viruses. Virology 95, 1-7. WASMONEN, T., KAKAC8, L. T. & COLLETT, M. S. (1988). Rift Valley

fever virus M segment: cellular localization of M segment-encoded proteins. Virology 166, 275-280. WATRET, G. E. & ELLIOTT, R. M. (1985a). The proteins and RNAs of St. Abb's Head virus, a Scottish uukuvirus. Journal of General Virology 66, 1001-1010. WATRET, G. E. & ELHOTr, R. M. (1985b). The proteins and RNAs specified by Clo Mor virus, a Scottish nairovirus. Journalof General Virology 66, 2513-2516. WATRET, G. E., PRINGLE, C. R. & ELLIOTT, R. M. (1985). Synthesis of bunyavirus-specific proteins in a continuous cell line (XTC-2) derived from Xenopuslaevis. Journalof General Virology66, 473-482. WICKENS, M. P. 86 DAHLBERG, J. E. (1987). RNA-protein interactions. Cell 51, 339-342. YAMANISHI, K., DANTAS, J. R., TAKAHASHI, M., YAMANOUCHI, T., DOMAE, K., TAKAnASm, Y. & TANISHITA, O. (1984). Antigenic differences between two viruses, isolated in Japan and Korea, that cause haemorrhagic fever with renal syndrome. Journal of Virology 52, 231-237. Yoo, D. & KANG, C. Y. (1987a). Nucleotide sequence of the M segment of the genomic RNA of Hantaan virus 76- l 18. NucleicAcids Research 15, 6299-6300. Yoo, D. & KANG, C. Y. (1987b). Genomic comparison among members of Hantavirus group. In The Biology of Negative Strand Viruses, pp. 424-431. Edited by B. W. J. Mahy & D. Kolakofsky. Amsterdam : Elsevier.

Molecular biology of the Bunyaviridae.

Journal o f General Virology (1990), 71, 501-522. 501 Printed in Great Britain Review article Molecular biology of the Bunyaviridae Richard M. Elli...
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