J. gen. Virol. (I979), 44, I79-186 Printed in Great Britain
179
Replication o f T w o Influenza Virus Strains and a Recombinant in H E F and LEP Cells By Y. G H E N D O N , E. TUCKOV,~,* V. V O N K A , * A. K L I M O V , V. G I N Z B U R G AND S. M A R K U S H I N Moscow Institute for Viral Preparations, Moscow, U.S.S.R. and * Department of Experimental Virology, Institute of Sera and Vaccines, Prague, Czechoslovakia (Accepted 20 December [978)
SUMMARY The replication of influenza viruses A/NWS-D, A/WS-MK and their rl~ recombinant in human embryo fibroblast (HEF) and human diploid fibroblast (LEP) cell lines was studied. In HEF cells virus NWS-D and recombinant r12 induced synthesis of virus-specific macromolecules and produced infectious virions; virus WS-MK induced synthesis of virus complementary RNA (cRNA), virion RNA (vRNA), proteins, RNP and non-infectious virions, but haemagglutinin cleavage was impaired and the virions formed contained uncleaved haemagglutinin. In LEP cells, infectious virions were formed only by virus NWS-D; viruses WS-MK and r~2 induced synthesis of virus cRNA, vRNA, proteins and RNP; virus rlz had the haemagglutinin cleaved, whereas in virus WS-MK this process was impaired; neither virus WS-MK nor r~2 was capable of forming virions. Analysis of the recombinant rl~ genome showed that it had only inherited a single gene from NWS-D, the one coding for neuraminidase, having inherited all others (P1, P2, P3, HA, NP, M, NS) from WS-MK. The data obtai~ied suggested that the inability of virus WS-MK to form infectious virions in HEF cells is due to the character of its neuraminidase, which is incapable of participating in haemagglutinin cleavage. The deficient reproduction of this virus in the other host-cell system (LEP) is apparently associated with some characteristics of another protein (other proteins) of this virus. INTRODUCTION Previous studies by Tu6kovfi et al. (I971), Tu~kov~i & Vonka 0973) and Anisimov~ et al. (1977) showed that the influenza virus NWS-D replicates well in LEP and HEF cell lines, whereas infectious virion formation by the closely related influenza virus WS-MK was heavily impaired in both cell systems. The crossing of these strains yielded recombinants, some of which (e.g. r12) grew well in HEF cells but did not reproduce in LEP cells. The present study was undertaken to investigate why virus WS-MK is incapable of forming infectious virions in HEF and LEP cells and to study in greater detail the rio recombinant, which behaves like a host-range mutant of virus NWS-D. The data obtained show that the inability of virus WS-MK to form infectious virions in HEF cells is due to the character of its neuraminidase. On the other hand, the deficient reproduction of this strain in LEP cells is associated with peculiarities of another protein (proteins) of this virus.
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Y. GHENDON AND OTHERS METHODS
Viruses. Influenza strains A / N W S - D ( H o N 0 , A / W S - M K ( H o N I ) , and their ra2 recombinants were used. The viruses were grown in I o-day-old chick embryos. Their characteristics and the method of derivation of the recombinant strain have been described (Tu~kovfi et al. 1971; Tu~kovfi & Vonka, 1973). Cell cultures. The human diploid fibroblast line (LEP) and the hamster embryo fibroblast line (HEF) were the same as in previous experiments (Tu~kowi et al. 1971). Cells were cultivated in modified Parker medium containing calf serum growth-promoting protein (Michl, I960. RNA and RNP synthesis. The synthesis of virus-specific cRNA, vRNA and RNP was studied as described elsewhere (Ghendon et al. 1973, I975). Virus-specific polypeptide synthesis. Cells were infected at a multiplicity of 2oo to 3o0 EIDs0/cell and incubated at 36 °C for 5 h. Then a hydrolysate of Chlorella 14C-proteins (I/zCi/ml) was added and the samples were incubated at 36 °C for 60 rain. Analysis of the virus-specific proteins synthesized was done in a 7"5 % cylindrical polyacrylamide gel containing o.t °/o SDS as described previously (Ghendon et al. ~973)In other experiments, ceils were infected at a multiplicity of l oo to 2oo EIDs0/cell and allowed to adsorb for 3o min. Medium 199 without methionine was then added and cultures were incubated at 36 °C for 5 h at which time 35S-methionine (2o/zCi per culture, sp. act. 53o Ci/mmol, Amersham) was added and incubation continued for lo rain. The cell monolayer was collected in a solubilizing solution containing 5 M-urea, 1% (w/w) SDS and o.1% mercaptoethanol, boiled for 3 min and examined by electrophoresis on 25 % polyacrylamide gels using the discontinuous buffer system described by Laemmli 097o). Autoradiography was done as described by Russell & Skehel (1972). Virionsformed in infected cells. Infected cells (Ioo EIDso/cell) were incubated in amino acid-free Eagle's medium at 36 °C for 60 min. Then hydrolysate of Chlorella 14C-proteins (2/zCi/ml) was added and cells were incubated at 36 °C for 5 h. The medium was then removed, warm (36 °C) Parker medium added and incubation continued at 36 °C for 17 h. Virus was sedimented from culture medium by centrifugation, purified (Klimov & Ghendon, I975) and studied electrophoretically in SDS-PAGE as described elsewhere (Ghendon et al. 1973). Effect of trypsin treatment on infectivity of cell-formed virions. Infected cells (3 to 5 EIDs0/ cell) were incubated at 36 °C for 2o h, frozen, thawed and centrifuged. The material obtained was divided into two portions. Trypsin (Calbiochem, Io/zg/ml) was added to one portion and an equal quantity of buffered saline to the other. Samples were incubated at 37 °C for 15 min and virus was titrated in chick embryos. Analysis of recombinant genome. The method of double-stranded R N A analysis by means of electrophoresis in slab polyacrylamide gel was used (Hay et al. I977). A monolayer culture of chick embryo fibroblasts (5 × IO* cells) was incubated with Parker medium containing cycloheximide (IOO/zg/ml) at 36 °C for 3o min. Medium was then decanted and cells were infected with virus (5oo EIDs0/cell) and incubated in the presence of cycloheximide at room temperature for 3o min. Thereafter, medium containing cycloheximide (too/zg/ml) was added, the cells were incubated at 36 °C for 3o min, 3H-uridine (sp. act. 24. 5 Ci/mmol) was added at I o o # C i / m l and incubation was continued for another 3"5 h. Isolation of cRNA was performed as described by Hay et al. (I977). Specimens of c R N A were hybridized by the method of Ito & Joklik (i972) in an excess of unlabelled v R N A (I5/zg per specimen) isolated from concentrated and purified virions. This material was then treated with nuclease S-I (IOOOunits/ml, 37 °C, 3 h), precipitated with ethanol and analysed by
Replication of influenza virus
I8I
Synthes& of complementary RNA (cRNA) in LEP and HEF cells infected by NWS-D, WS-MK or recombinant rlz influenza virus*
T a b l e I.
RNase resistant radioactivity A
rLEP cells
H E F cells"
F
Virus
•
r
Before hybridization
After hybridization
Before hybridization
After hybridization
4'I 4"4 3"6
42'7 36"2 35 q
5'2 3"7 3"8
46"I 33"4 33"8
NWS-D WS-MK raz
* Cells were infected with purified, aH-uridine-labelled virus and incubated at 36 °C for 5 h. R N A s were isolated, hybridized at 60 °C for 72 h and treated with RNase (a mixture of pancreatic and T-I RNases, final concentrations IO #g/ml and 2o units/ml, respectively; 37 °C, 30 min (for details see Ghendon et al. I975). (a) 28S18S
x
NWS-D]
I A
18
28S 18S
4-
I
WS-MK
I
28S 18S
!
r12
!
t8
15
15
12
12 x
~, 9
9
6
6
3
3
i
WS
$3
S
"~
S 3
25 20-
-
X
X
1
!
5
10
15
20
25
5
10 15 20 Fraction number
25
5
10
15
20
25
Fig. I. Synthesis of v R N A and formation of RNP in H E F and LEP cells infected with NWS-D, WS-MK, or rx~ recombinant virus. (a) Electrophoretic pattern of v R N A in P A G E ; arrows locate 28S and I8S ribosomal RNAs. Cells were infected with virus Uoo EIDsdcell) and incubated at 36 °C for 3 h. Actinomycin D (5 #g/rnl) and, 30 min later, ~H-uridine (15/zCi/ml) were added and thesamptes were againincubated for 3 h. (b) Analysis of virus R N P i n a I5 to 3 o ~ sucrose gradient. Arrows locate corresponding cellular ribosomal subunits. Cells were infected with virus (Ioo EfDsdcell) and incubated at 36 °C for 3 h. Actinomycin D (5 #g/ml) and, 30 min later, 8H-uridine (I5/zCi/ml) were added and incubation was continued for 60 min. @ - - @ , H E F cells; © - - © , LEP cells.
I82
Y. G H E N D O N AND OTHERS [(a) |
HA n& M ~NP~ HA2~
I (b) HA HA,
M ~NP~ HA2~,
]
](c) |
HA HA, M ~NP[ HA2~
] |
~
4~
~
3~
i [
i
i
t
I
I
I
[
I
I
I
I
I
l
I
I
10 20 30 40 50 60 70
I0 20 30 40 5~0 60 70 10 20 30 40 50 60 70 Fraction number Fig. 2. Synthesis of virus polypeptides and cleavage of haemagglutinin in HEF and LEP cells infected with (a) NWS-D, (b) WS-MK or (c) r~ recombinant virus. Cells were infected with virus (200 to 300 EIDs0/cell) and incubated at 36 °C for 5 h. Then a hydrolysate of *4C-proteins of Chlorella (I #Ci/ml) was added and incubation was continued for 60 min. Analysis of the virusspecific polypeptides synthesized was performed in cylindrical 7"5 ~ polyacrylamide gels containing SDS, as described previously (Ghendon et al. 1973). The designations HA, NP, HA,, HAs and M locate the corresponding polypeptides. O - - O , HEF cells; O - - © , LEP cells.
means of electrophoresis in a 7"5% polyacrylamide gel (75V, 24h), as described by Hay et al. (I977)- The gels obtained were prepared for fluorography according to Bonner & Laskey 0974). RESULTS
Synthesis of virus-specific cRNA, vRNA and polypeptides and formation of RNP in LEP and HEF cells As Table I and Fig. I and 2 show, intensive synthesis of cRNA, vRNA, polypeptides and formation of RNP took place in LEP and HEF cells infected with any of the three viruses under study. However, as is apparent from Fig. 2, infection of LEP and HEF cells with NWS-D or recombinant r12 virus involved cleavage of haemagglutinin into the HA1 and HA 2 polypeptides, but this process was impaired in either cell system when infected with virus WS-MK. We could not separate M and NS polypeptides by electrophoresis in cylindrical polyaerylamide gels containing 7"5 % acrylamide (Fig. 2). However, analysis in 25 % polyacrylamide slab gels showed clearly that considerable synthesis of M and NS polypeptides can be observed in both cell systems under the conditions of abortive infection with WS-MK and r12 viruses. Virion formation in LEP and HEF celL~ Fig. 3 shows that in LEP ceils sufficient virions for analysis were formed only after infection by NWS-D virus and that these virions contained readily detectable polypeptides NP and M, as well as HA 1 and HA s haemagglutinin chains. Infection of LEP cells with WS-MK virus or recombinant r12 did not lead to the formation of detectable amounts of virions. It should be pointed out that electron microscopic studies had also failed to detect virion formation in more than 95 % of the LEP cells infected with WS-MK or recombinant r12 viruses (Anisimov~i et al. 1977)- In HEF cells, infection with NWS-D virus again resulted in the formation of virions in which polypeptides NP, M, HA~ and HA 2 were detectable.
Replication o f influenza virus
I83
(a) 4 NWS-DNpI|HAIHA2M I |
WS-MK
r12
3 -
?
1-
o X
G
(b) NWS-D Np HA1HA2rd 16"-|| | l""
IWS MK NP ; HIA|
m
"r~2
Np~IA~H~21M
1284-
ug-. 10 20 30 40 50 60 70
10 20 30 40 50 60 70 10 20 30 40 50 60 70 Fraction number Fig. 3. Polypeptide composition of influenza virions formed in (a) LEP and (b) HEF cells. Infected cells (Ioo EIDso/cell) were incubated with a hydrolysate of x4C-proteinsof Chlorella(2 #Ci/ml) from I to 5 h after infection. Medium was then exchanged for Parker medium and incubation continued for 17 h. Virus was sedimented from culture fluid by centrifugation, purified and studied by electrophoresis in SDS-PAGE. Similar results were also obtained with recombinant r~2. Virus W S - M K induced in H E F cells the formation of a large number of virions which were shown to contain polypeptides NP and M but only uncleaved haemagglutinin (HA). It should be noted that W S - M K virions grown in chick embryos contain readily detectable H A 1 and HA2 polypeptides (results not shown).
Effect of trypsin treatment on infectivity of virions formed in HEF cells It has been reported (Klenk et al. 1975; Lazarowitz & Choppin, 1975) that treatment with trypsin of non-infectious influenza virions containing uncleaved haemagglutinin results in cleavage of this polypeptide and acquisition of infectivity of the virus. Since in the present experiments impairment of haemagglutinili cleavage (Fig. 2) and formation of virions containing uncleaved haemagglutinin (Fig. 3) was observed in WS-MK-infected H E F cells, the effect of trypsin treatment on the infectivity of these virions was investigated. The data presented in Table 2 shows that in vitro trypsin treatment of W S - M K virions formed in H E F cells resulted in considerable increase of their infectivity. Analogous treatment of strain N W S - D and recombinant rl~ virions was practically without influence on their infectivity.
Recombinant genome analysis Fig. 4 presents the results of an analysis, in a slab-polyacrylamide gel, of doublestranded influenza virus R N A fragments obtained by hybridization of ZH-uridine-labelled virus-specific c R N A isolated from cells infected by the virus strains under study with unlabelled v R N A isolated from purified virions. Prior to the electrophoresis, the double-
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Y. G H E N D O N T a b l e 2.
AND OTHERS
Effect of trypsin treatment on infectivity of influenza virions formed in HEF cells* Virus
Experimental conditions
Infectivity log~0EIDs0/ml
WS-MK
No trypsin Incubation with trypsin No trypsin Incubation with trypsin No trypsin Incubation with trypsin
3o 4"8 5"5 5"8 5"o 5"o
r~2 NWS-D
* Infected cells were incubated at 36 °C for 20 h, frozen and thawed, and incubated at 37 °C for 15 min in the presence or absence of IO #g/ml trypsin; virus was titrated in chick embryos.
W
cWs r
N
1
W
Crl2 r
N
I
W
cNWS r
N
1m 2 m 3 m
ii:i~: 4 m
5m
6n
: :j: :z i!~4 7m
.7
8
5: :
Fig. 4- Analysis of recombinant r12 genome. Cells were infected with virus (5oo EIDs0/ml) and incubated in medium containing cycloheximide and 3H-uridine for 4 h. Then R N A (cRNA) was isolated and hybridized with non-labelled v R N A isolated from purified virions. Samples were treated with S-I nuclease and studied by electrophoresis in a 7"5 ~ slab-polyacrylamide gel. cNWS, crib, cWS are samples of labelled c R N A of the corresponding viruses; N, r, W are samples of unlabelled v R N A of NWS-D, rl~ and WS-MK viruses, respectively, l, 2, 3, 4, 5, 6, 7, 8 give positions of corresponding double-stranded fragments of RNA. Lines have been placed between R N A fragments having similar electrophoretic mobility. (The reproduction shown is a composite of different exposures of the same gel.)
Replication o f influenza virus
185
stranded RNA fragments were treated with S-I nuclease. The data presented in Fig. 4 show that the method of analysis employed was capable of detecting differences between the genes I, 2, 3, 4, 5, 7, 8 and WS-MK virus strains in terms of all the eight genes. These differences are expressed by a change in the electrophoretic mobility of the S-I nucleasetreated duplexes formed in the hybridization of virus NWS-D cRNA with virus WS-MK vRNA, and also of virus WS-MK cRNA with virus NWS-D vRNA, as compared with the electrophoretic mobility of RNA duplexes formed by the cRNAs and vRNAs of homologous strains (compare lines I and 3, and 7 and 9, Fig. 4). The difference of gene 6 between NWS-D and WS-MK strains is so significant that our conditions of S-I nuclease treatment lead to the complete disappearance of the corresponding duplex from the preparations obtained after hybridization of cRNA with vRNA of heterologous strains of viruses (see, for example, lines 3 and 7, Fig. 4). The analysis of recombinant r12 double-stranded RNA fragments indicated that seven fragments of this virus (I, 2, 3, 4, 5, 7, 8) had identical electrophoretic mobility with the respective WS-MK fragments. On the other hand, recombinant rl~ fragment 6 was identical with strain NWS-D fragment 6. Consistent results were obtained in all three series of experiments in which cRNAs isolated from cells infected with NWS-D (lines 7 to 9), rl~ (lines 4 to 6) or WS-MK (lines I to 3) were used as labelled RNA and each of these cRNAs was hybridized with vRNA isolated from virus NWS-D, r12 or WS-MK. These results clearly show that recombinant r12 has inherited a single gene, fragment 6, from virus NWS-D and all the others from virus WS-MK. According to the data of Palese et al. (I977), the order of the RNA fragments of HoNI influenza viruses in terms of decreasing electrophoretic mobility in slab-polyacrylamide gel, is as follows: P3, PI, P2, HA, NP, NA, M, NS. This would imply that recombinant r12 has inherited from NWS-D virus the gene coding for neuraminidase. It should be noted that previous investigations (Anisimov~i et al. I977) indicated that the r~2 neuraminidase in vitro activity resembled that of NWS-D.
DISCUSSION
Previous experiments (Tu~kovfi et al. 197 I) indicated that the NWS-D strain of influenza virus reproduces readily in HEF and LEP cells whereas the ability of virus WS-MK to produce infectious virions in these cells is seriously impaired. The data obtained in the present work furnish evidence that the mechanisms responsible for the impairment of infectious virion production by WS-MK virus in HEF and LEP cells are not identical. Thus, the virus is capable of carrying out transcription, replication and translation in HEF cells but the cleavage of the haemagglutinin (HA) polypeptide into HA1 and HA2 is impaired. Only uncleaved haemagglutinin was also found in non-infectious virions forming in WS-MKinfected HEF cells. Treatment of these virions with trypsin, which cleaves the haemagglutinin (Klenk et al. I975; Lazarowitz & Choppin, I975), resulted in a marked increase in their infectivity. On the other hand, recombinant rl~, whose genome consists entirely of WS-MK genes except for the neuraminidase gene, which has been inherited from NWS-D, proved capable of forming infectious virions and its haemagglutinin cleavage was not impaired in HEF cells. The data obtained imply that the marked decrease in the ability of virus WS-MK to form infectious virions in HEF cells is linked with impairment of haemagglutinin cleavage, this defect being due to the properties of virus WS-MK neuraminidase. It should be pointed out that similar data on influenza virus-neuraminidase possibly being involved in haemagglutinin cleavage in MDBK cells have recently been reported by Schulman & Palese 0977)The mechanism responsible for the inability of strain WS-MK to produce infectious virions in LEP cells is different. In WS-MK-infected LEP cells, as in HEF cells, the synthesis of cRNA, vRNA, polypeptides and the formation of RNP do take place, but virions, even
I86
Y. G H E N D O N
AND OTHERS
non-infectious virions, are not formed in detectable amounts. This kind of reproduction defect in strain WS-MK in LEP cells is presumably not due to its neuraminidase. This can be concluded from the behaviour of rl~ virus which has inherited its neuraminidase gene from strain NWS-D and all its other genes from virus WS-MK. It is likewise incapable of virion formation in LEP cells, in spite of haemagglutinin cleavage in rl,-infected cells not being impaired (in contrast to WS-MK-infected cells). Apparently the inability of virus WS-MK to form virions in LEP cells is due to another (or several) of its protein, whose function is impaired in LEP cells but which is capable of fulfilling its role in virion assembly in HEF cells. In the recently published paper by Almond (i 977) it was shown that the inability of fowl plague virus (Rostock strain) to form plaques in BHK cells is associated with the peculiarity of P3 protein participating in RNA synthesis (Palese et al. I977). According to our data the WS-MK virus in LEP cells induces the synthesis of cRNA and vRNA (Fig. I). The inability of WS-MK virus to make up infected virions in LEP cells does not appear to depend on the features of the proteins participating in the synthesis of virus-specific RNA. The data obtained allow the conclusion to be drawn that the inability of influenza virus to form infectious virions in certain cell lines may be due to impairment of function of different virus proteins and that these defects, although associated with genetically encoded characters of virus proteins, are largely co-dependent on the host cell for their phenotypic expression. The present results furnish evidence of an intimate dependence of influenza virus reproduction on the host cell not only at the early but also at late reproduction stages, when virions are being assembled. REFERENCES ALMOND,J. W. (1977). A single gene determines the host range of influenza virus. Nature, London z7o, 617- 6 t 8. ANtSIMOV~,,E., XU~KOV.~,,E., VOtqr:A,V. & ZAVADOVA,H. (I977). Ultrastructural changes induced by influenza virus in permissive and nonpermissive cells. Virology 77, 330--336. BO~NER, Wo M. & LAS~EY,g. a. (I974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. European Journal of Biochemistry 46, 83-88. GHENDON, Y., MARKUSHIN, S., MARCHENKO, A., SITNIKOV, B. & GINSBURG, V. (1973). Biochemical characteristics of fowl plague virus ts mutants. Virology 55, 3o5-319. GrtENDON, V., MAR~USmN,S., aLAGOVESH~r~SKAVA,O. & GENKtNA,D. (t975)- Study of fowl plague virus RNA synthesis in temperature-sensitive mutants. Virology 66, 454-463. HAY, A. J., LOMN~CZl,n., BELLAMY,A. R. & SKEHEL,J. J. 0977). Transcription of the influenza virus genome. Virology 83, 337-355. l'rO, Y. & JOKLIK, W. K. 0972). Temperature-sensitive mutants of reovirus. I. Patterns of gene expression by mutants of groups C, D and E. Virology 50, 189-20 I. KLENK,H.-D., ROTT, R., ORLICH, M. & eL6DORN, J. (I975). Activation of influenza A virus by trypsin treatment. Virology 68, 426-439. gLIMOV, A. & GHENDON, V. 0975). The effect of the isolation procedure on the transcriptase activity of fowl plague virus RNP. ,4cta Virologica x9, 9I. LAEMML1,U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 2z7, 680-685. LAZAROWITZ,S. & CaOPPlN, P. (I975)- Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the haemagglutinin polypeptide. Virology 68, 440-454. MICHL, J. (196T). Metabolism of cell in tissue culture in vitro. I. The influence of serum protein fractions on the growth of normal and neoplastic cells. Experimental Cell Research 23, 324-334. PALESE, P., RITCHEY, M. B. & SCHULMAN,J. L (X977). Pl and P3 proteins of influenza virus are required for complementary R N A synthesis. Journal of Virology 2x, 1187-I 195. RUSSELL,W. G. & SKEF1EL,J. J. (I972). The polypeptides of adenovirus infected cells. JournalofGeneral Virology xs, 45-57. SCHULMAN, J. L. & PALESE, P. 0977)- Virulence factors of influenza A viruses: WSN virus neuraminidase required for plaque production in MDBK cells. Journal of Virology 24, I7o-I77. TU(:KOVA, E. & VONKA, V. (I973). Genetic interaction between two influenza A / W S virus mutants. HI. Capacity of WS/NWS recombinants to reproduce in various cell systems. ,4cta Virologica x7, 5oi-5o4. TUg:KOV.~,~., VONKA,V. & ST~,.EK, M. (197I)- Genetic interaction between two influenza A / W S virus mutants. I. Characterization of the mutants. Acta Virologica xS, 337-344.
(Received 4 September I978)
179
Replication o f T w o Influenza Virus Strains and a Recombinant in H E F and LEP Cells By Y. G H E N D O N , E. TUCKOV,~,* V. V O N K A , * A. K L I M O V , V. G I N Z B U R G AND S. M A R K U S H I N Moscow Institute for Viral Preparations, Moscow, U.S.S.R. and * Department of Experimental Virology, Institute of Sera and Vaccines, Prague, Czechoslovakia (Accepted 20 December [978)
SUMMARY The replication of influenza viruses A/NWS-D, A/WS-MK and their rl~ recombinant in human embryo fibroblast (HEF) and human diploid fibroblast (LEP) cell lines was studied. In HEF cells virus NWS-D and recombinant r12 induced synthesis of virus-specific macromolecules and produced infectious virions; virus WS-MK induced synthesis of virus complementary RNA (cRNA), virion RNA (vRNA), proteins, RNP and non-infectious virions, but haemagglutinin cleavage was impaired and the virions formed contained uncleaved haemagglutinin. In LEP cells, infectious virions were formed only by virus NWS-D; viruses WS-MK and r~2 induced synthesis of virus cRNA, vRNA, proteins and RNP; virus rlz had the haemagglutinin cleaved, whereas in virus WS-MK this process was impaired; neither virus WS-MK nor r~2 was capable of forming virions. Analysis of the recombinant rl~ genome showed that it had only inherited a single gene from NWS-D, the one coding for neuraminidase, having inherited all others (P1, P2, P3, HA, NP, M, NS) from WS-MK. The data obtai~ied suggested that the inability of virus WS-MK to form infectious virions in HEF cells is due to the character of its neuraminidase, which is incapable of participating in haemagglutinin cleavage. The deficient reproduction of this virus in the other host-cell system (LEP) is apparently associated with some characteristics of another protein (other proteins) of this virus. INTRODUCTION Previous studies by Tu6kovfi et al. (I971), Tu~kov~i & Vonka 0973) and Anisimov~ et al. (1977) showed that the influenza virus NWS-D replicates well in LEP and HEF cell lines, whereas infectious virion formation by the closely related influenza virus WS-MK was heavily impaired in both cell systems. The crossing of these strains yielded recombinants, some of which (e.g. r12) grew well in HEF cells but did not reproduce in LEP cells. The present study was undertaken to investigate why virus WS-MK is incapable of forming infectious virions in HEF and LEP cells and to study in greater detail the rio recombinant, which behaves like a host-range mutant of virus NWS-D. The data obtained show that the inability of virus WS-MK to form infectious virions in HEF cells is due to the character of its neuraminidase. On the other hand, the deficient reproduction of this strain in LEP cells is associated with peculiarities of another protein (proteins) of this virus.
oo22-1317/79/oooo-3457 $oz.oo © I979 SGM
I80
Y. GHENDON AND OTHERS METHODS
Viruses. Influenza strains A / N W S - D ( H o N 0 , A / W S - M K ( H o N I ) , and their ra2 recombinants were used. The viruses were grown in I o-day-old chick embryos. Their characteristics and the method of derivation of the recombinant strain have been described (Tu~kovfi et al. 1971; Tu~kovfi & Vonka, 1973). Cell cultures. The human diploid fibroblast line (LEP) and the hamster embryo fibroblast line (HEF) were the same as in previous experiments (Tu~kowi et al. 1971). Cells were cultivated in modified Parker medium containing calf serum growth-promoting protein (Michl, I960. RNA and RNP synthesis. The synthesis of virus-specific cRNA, vRNA and RNP was studied as described elsewhere (Ghendon et al. 1973, I975). Virus-specific polypeptide synthesis. Cells were infected at a multiplicity of 2oo to 3o0 EIDs0/cell and incubated at 36 °C for 5 h. Then a hydrolysate of Chlorella 14C-proteins (I/zCi/ml) was added and the samples were incubated at 36 °C for 60 rain. Analysis of the virus-specific proteins synthesized was done in a 7"5 % cylindrical polyacrylamide gel containing o.t °/o SDS as described previously (Ghendon et al. ~973)In other experiments, ceils were infected at a multiplicity of l oo to 2oo EIDs0/cell and allowed to adsorb for 3o min. Medium 199 without methionine was then added and cultures were incubated at 36 °C for 5 h at which time 35S-methionine (2o/zCi per culture, sp. act. 53o Ci/mmol, Amersham) was added and incubation continued for lo rain. The cell monolayer was collected in a solubilizing solution containing 5 M-urea, 1% (w/w) SDS and o.1% mercaptoethanol, boiled for 3 min and examined by electrophoresis on 25 % polyacrylamide gels using the discontinuous buffer system described by Laemmli 097o). Autoradiography was done as described by Russell & Skehel (1972). Virionsformed in infected cells. Infected cells (Ioo EIDso/cell) were incubated in amino acid-free Eagle's medium at 36 °C for 60 min. Then hydrolysate of Chlorella 14C-proteins (2/zCi/ml) was added and cells were incubated at 36 °C for 5 h. The medium was then removed, warm (36 °C) Parker medium added and incubation continued at 36 °C for 17 h. Virus was sedimented from culture medium by centrifugation, purified (Klimov & Ghendon, I975) and studied electrophoretically in SDS-PAGE as described elsewhere (Ghendon et al. 1973). Effect of trypsin treatment on infectivity of cell-formed virions. Infected cells (3 to 5 EIDs0/ cell) were incubated at 36 °C for 2o h, frozen, thawed and centrifuged. The material obtained was divided into two portions. Trypsin (Calbiochem, Io/zg/ml) was added to one portion and an equal quantity of buffered saline to the other. Samples were incubated at 37 °C for 15 min and virus was titrated in chick embryos. Analysis of recombinant genome. The method of double-stranded R N A analysis by means of electrophoresis in slab polyacrylamide gel was used (Hay et al. I977). A monolayer culture of chick embryo fibroblasts (5 × IO* cells) was incubated with Parker medium containing cycloheximide (IOO/zg/ml) at 36 °C for 3o min. Medium was then decanted and cells were infected with virus (5oo EIDs0/cell) and incubated in the presence of cycloheximide at room temperature for 3o min. Thereafter, medium containing cycloheximide (too/zg/ml) was added, the cells were incubated at 36 °C for 3o min, 3H-uridine (sp. act. 24. 5 Ci/mmol) was added at I o o # C i / m l and incubation was continued for another 3"5 h. Isolation of cRNA was performed as described by Hay et al. (I977). Specimens of c R N A were hybridized by the method of Ito & Joklik (i972) in an excess of unlabelled v R N A (I5/zg per specimen) isolated from concentrated and purified virions. This material was then treated with nuclease S-I (IOOOunits/ml, 37 °C, 3 h), precipitated with ethanol and analysed by
Replication of influenza virus
I8I
Synthes& of complementary RNA (cRNA) in LEP and HEF cells infected by NWS-D, WS-MK or recombinant rlz influenza virus*
T a b l e I.
RNase resistant radioactivity A
rLEP cells
H E F cells"
F
Virus
•
r
Before hybridization
After hybridization
Before hybridization
After hybridization
4'I 4"4 3"6
42'7 36"2 35 q
5'2 3"7 3"8
46"I 33"4 33"8
NWS-D WS-MK raz
* Cells were infected with purified, aH-uridine-labelled virus and incubated at 36 °C for 5 h. R N A s were isolated, hybridized at 60 °C for 72 h and treated with RNase (a mixture of pancreatic and T-I RNases, final concentrations IO #g/ml and 2o units/ml, respectively; 37 °C, 30 min (for details see Ghendon et al. I975). (a) 28S18S
x
NWS-D]
I A
18
28S 18S
4-
I
WS-MK
I
28S 18S
!
r12
!
t8
15
15
12
12 x
~, 9
9
6
6
3
3
i
WS
$3
S
"~
S 3
25 20-
-
X
X
1
!
5
10
15
20
25
5
10 15 20 Fraction number
25
5
10
15
20
25
Fig. I. Synthesis of v R N A and formation of RNP in H E F and LEP cells infected with NWS-D, WS-MK, or rx~ recombinant virus. (a) Electrophoretic pattern of v R N A in P A G E ; arrows locate 28S and I8S ribosomal RNAs. Cells were infected with virus Uoo EIDsdcell) and incubated at 36 °C for 3 h. Actinomycin D (5 #g/rnl) and, 30 min later, ~H-uridine (15/zCi/ml) were added and thesamptes were againincubated for 3 h. (b) Analysis of virus R N P i n a I5 to 3 o ~ sucrose gradient. Arrows locate corresponding cellular ribosomal subunits. Cells were infected with virus (Ioo EfDsdcell) and incubated at 36 °C for 3 h. Actinomycin D (5 #g/ml) and, 30 min later, 8H-uridine (I5/zCi/ml) were added and incubation was continued for 60 min. @ - - @ , H E F cells; © - - © , LEP cells.
I82
Y. G H E N D O N AND OTHERS [(a) |
HA n& M ~NP~ HA2~
I (b) HA HA,
M ~NP~ HA2~,
]
](c) |
HA HA, M ~NP[ HA2~
] |
~
4~
~
3~
i [
i
i
t
I
I
I
[
I
I
I
I
I
l
I
I
10 20 30 40 50 60 70
I0 20 30 40 5~0 60 70 10 20 30 40 50 60 70 Fraction number Fig. 2. Synthesis of virus polypeptides and cleavage of haemagglutinin in HEF and LEP cells infected with (a) NWS-D, (b) WS-MK or (c) r~ recombinant virus. Cells were infected with virus (200 to 300 EIDs0/cell) and incubated at 36 °C for 5 h. Then a hydrolysate of *4C-proteins of Chlorella (I #Ci/ml) was added and incubation was continued for 60 min. Analysis of the virusspecific polypeptides synthesized was performed in cylindrical 7"5 ~ polyacrylamide gels containing SDS, as described previously (Ghendon et al. 1973). The designations HA, NP, HA,, HAs and M locate the corresponding polypeptides. O - - O , HEF cells; O - - © , LEP cells.
means of electrophoresis in a 7"5% polyacrylamide gel (75V, 24h), as described by Hay et al. (I977)- The gels obtained were prepared for fluorography according to Bonner & Laskey 0974). RESULTS
Synthesis of virus-specific cRNA, vRNA and polypeptides and formation of RNP in LEP and HEF cells As Table I and Fig. I and 2 show, intensive synthesis of cRNA, vRNA, polypeptides and formation of RNP took place in LEP and HEF cells infected with any of the three viruses under study. However, as is apparent from Fig. 2, infection of LEP and HEF cells with NWS-D or recombinant r12 virus involved cleavage of haemagglutinin into the HA1 and HA 2 polypeptides, but this process was impaired in either cell system when infected with virus WS-MK. We could not separate M and NS polypeptides by electrophoresis in cylindrical polyaerylamide gels containing 7"5 % acrylamide (Fig. 2). However, analysis in 25 % polyacrylamide slab gels showed clearly that considerable synthesis of M and NS polypeptides can be observed in both cell systems under the conditions of abortive infection with WS-MK and r12 viruses. Virion formation in LEP and HEF celL~ Fig. 3 shows that in LEP ceils sufficient virions for analysis were formed only after infection by NWS-D virus and that these virions contained readily detectable polypeptides NP and M, as well as HA 1 and HA s haemagglutinin chains. Infection of LEP cells with WS-MK virus or recombinant r12 did not lead to the formation of detectable amounts of virions. It should be pointed out that electron microscopic studies had also failed to detect virion formation in more than 95 % of the LEP cells infected with WS-MK or recombinant r12 viruses (Anisimov~i et al. 1977)- In HEF cells, infection with NWS-D virus again resulted in the formation of virions in which polypeptides NP, M, HA~ and HA 2 were detectable.
Replication o f influenza virus
I83
(a) 4 NWS-DNpI|HAIHA2M I |
WS-MK
r12
3 -
?
1-
o X
G
(b) NWS-D Np HA1HA2rd 16"-|| | l""
IWS MK NP ; HIA|
m
"r~2
Np~IA~H~21M
1284-
ug-. 10 20 30 40 50 60 70
10 20 30 40 50 60 70 10 20 30 40 50 60 70 Fraction number Fig. 3. Polypeptide composition of influenza virions formed in (a) LEP and (b) HEF cells. Infected cells (Ioo EIDso/cell) were incubated with a hydrolysate of x4C-proteinsof Chlorella(2 #Ci/ml) from I to 5 h after infection. Medium was then exchanged for Parker medium and incubation continued for 17 h. Virus was sedimented from culture fluid by centrifugation, purified and studied by electrophoresis in SDS-PAGE. Similar results were also obtained with recombinant r~2. Virus W S - M K induced in H E F cells the formation of a large number of virions which were shown to contain polypeptides NP and M but only uncleaved haemagglutinin (HA). It should be noted that W S - M K virions grown in chick embryos contain readily detectable H A 1 and HA2 polypeptides (results not shown).
Effect of trypsin treatment on infectivity of virions formed in HEF cells It has been reported (Klenk et al. 1975; Lazarowitz & Choppin, 1975) that treatment with trypsin of non-infectious influenza virions containing uncleaved haemagglutinin results in cleavage of this polypeptide and acquisition of infectivity of the virus. Since in the present experiments impairment of haemagglutinili cleavage (Fig. 2) and formation of virions containing uncleaved haemagglutinin (Fig. 3) was observed in WS-MK-infected H E F cells, the effect of trypsin treatment on the infectivity of these virions was investigated. The data presented in Table 2 shows that in vitro trypsin treatment of W S - M K virions formed in H E F cells resulted in considerable increase of their infectivity. Analogous treatment of strain N W S - D and recombinant rl~ virions was practically without influence on their infectivity.
Recombinant genome analysis Fig. 4 presents the results of an analysis, in a slab-polyacrylamide gel, of doublestranded influenza virus R N A fragments obtained by hybridization of ZH-uridine-labelled virus-specific c R N A isolated from cells infected by the virus strains under study with unlabelled v R N A isolated from purified virions. Prior to the electrophoresis, the double-
I8 4
Y. G H E N D O N T a b l e 2.
AND OTHERS
Effect of trypsin treatment on infectivity of influenza virions formed in HEF cells* Virus
Experimental conditions
Infectivity log~0EIDs0/ml
WS-MK
No trypsin Incubation with trypsin No trypsin Incubation with trypsin No trypsin Incubation with trypsin
3o 4"8 5"5 5"8 5"o 5"o
r~2 NWS-D
* Infected cells were incubated at 36 °C for 20 h, frozen and thawed, and incubated at 37 °C for 15 min in the presence or absence of IO #g/ml trypsin; virus was titrated in chick embryos.
W
cWs r
N
1
W
Crl2 r
N
I
W
cNWS r
N
1m 2 m 3 m
ii:i~: 4 m
5m
6n
: :j: :z i!~4 7m
.7
8
5: :
Fig. 4- Analysis of recombinant r12 genome. Cells were infected with virus (5oo EIDs0/ml) and incubated in medium containing cycloheximide and 3H-uridine for 4 h. Then R N A (cRNA) was isolated and hybridized with non-labelled v R N A isolated from purified virions. Samples were treated with S-I nuclease and studied by electrophoresis in a 7"5 ~ slab-polyacrylamide gel. cNWS, crib, cWS are samples of labelled c R N A of the corresponding viruses; N, r, W are samples of unlabelled v R N A of NWS-D, rl~ and WS-MK viruses, respectively, l, 2, 3, 4, 5, 6, 7, 8 give positions of corresponding double-stranded fragments of RNA. Lines have been placed between R N A fragments having similar electrophoretic mobility. (The reproduction shown is a composite of different exposures of the same gel.)
Replication o f influenza virus
185
stranded RNA fragments were treated with S-I nuclease. The data presented in Fig. 4 show that the method of analysis employed was capable of detecting differences between the genes I, 2, 3, 4, 5, 7, 8 and WS-MK virus strains in terms of all the eight genes. These differences are expressed by a change in the electrophoretic mobility of the S-I nucleasetreated duplexes formed in the hybridization of virus NWS-D cRNA with virus WS-MK vRNA, and also of virus WS-MK cRNA with virus NWS-D vRNA, as compared with the electrophoretic mobility of RNA duplexes formed by the cRNAs and vRNAs of homologous strains (compare lines I and 3, and 7 and 9, Fig. 4). The difference of gene 6 between NWS-D and WS-MK strains is so significant that our conditions of S-I nuclease treatment lead to the complete disappearance of the corresponding duplex from the preparations obtained after hybridization of cRNA with vRNA of heterologous strains of viruses (see, for example, lines 3 and 7, Fig. 4). The analysis of recombinant r12 double-stranded RNA fragments indicated that seven fragments of this virus (I, 2, 3, 4, 5, 7, 8) had identical electrophoretic mobility with the respective WS-MK fragments. On the other hand, recombinant rl~ fragment 6 was identical with strain NWS-D fragment 6. Consistent results were obtained in all three series of experiments in which cRNAs isolated from cells infected with NWS-D (lines 7 to 9), rl~ (lines 4 to 6) or WS-MK (lines I to 3) were used as labelled RNA and each of these cRNAs was hybridized with vRNA isolated from virus NWS-D, r12 or WS-MK. These results clearly show that recombinant r12 has inherited a single gene, fragment 6, from virus NWS-D and all the others from virus WS-MK. According to the data of Palese et al. (I977), the order of the RNA fragments of HoNI influenza viruses in terms of decreasing electrophoretic mobility in slab-polyacrylamide gel, is as follows: P3, PI, P2, HA, NP, NA, M, NS. This would imply that recombinant r12 has inherited from NWS-D virus the gene coding for neuraminidase. It should be noted that previous investigations (Anisimov~i et al. I977) indicated that the r~2 neuraminidase in vitro activity resembled that of NWS-D.
DISCUSSION
Previous experiments (Tu~kovfi et al. 197 I) indicated that the NWS-D strain of influenza virus reproduces readily in HEF and LEP cells whereas the ability of virus WS-MK to produce infectious virions in these cells is seriously impaired. The data obtained in the present work furnish evidence that the mechanisms responsible for the impairment of infectious virion production by WS-MK virus in HEF and LEP cells are not identical. Thus, the virus is capable of carrying out transcription, replication and translation in HEF cells but the cleavage of the haemagglutinin (HA) polypeptide into HA1 and HA2 is impaired. Only uncleaved haemagglutinin was also found in non-infectious virions forming in WS-MKinfected HEF cells. Treatment of these virions with trypsin, which cleaves the haemagglutinin (Klenk et al. I975; Lazarowitz & Choppin, I975), resulted in a marked increase in their infectivity. On the other hand, recombinant rl~, whose genome consists entirely of WS-MK genes except for the neuraminidase gene, which has been inherited from NWS-D, proved capable of forming infectious virions and its haemagglutinin cleavage was not impaired in HEF cells. The data obtained imply that the marked decrease in the ability of virus WS-MK to form infectious virions in HEF cells is linked with impairment of haemagglutinin cleavage, this defect being due to the properties of virus WS-MK neuraminidase. It should be pointed out that similar data on influenza virus-neuraminidase possibly being involved in haemagglutinin cleavage in MDBK cells have recently been reported by Schulman & Palese 0977)The mechanism responsible for the inability of strain WS-MK to produce infectious virions in LEP cells is different. In WS-MK-infected LEP cells, as in HEF cells, the synthesis of cRNA, vRNA, polypeptides and the formation of RNP do take place, but virions, even
I86
Y. G H E N D O N
AND OTHERS
non-infectious virions, are not formed in detectable amounts. This kind of reproduction defect in strain WS-MK in LEP cells is presumably not due to its neuraminidase. This can be concluded from the behaviour of rl~ virus which has inherited its neuraminidase gene from strain NWS-D and all its other genes from virus WS-MK. It is likewise incapable of virion formation in LEP cells, in spite of haemagglutinin cleavage in rl,-infected cells not being impaired (in contrast to WS-MK-infected cells). Apparently the inability of virus WS-MK to form virions in LEP cells is due to another (or several) of its protein, whose function is impaired in LEP cells but which is capable of fulfilling its role in virion assembly in HEF cells. In the recently published paper by Almond (i 977) it was shown that the inability of fowl plague virus (Rostock strain) to form plaques in BHK cells is associated with the peculiarity of P3 protein participating in RNA synthesis (Palese et al. I977). According to our data the WS-MK virus in LEP cells induces the synthesis of cRNA and vRNA (Fig. I). The inability of WS-MK virus to make up infected virions in LEP cells does not appear to depend on the features of the proteins participating in the synthesis of virus-specific RNA. The data obtained allow the conclusion to be drawn that the inability of influenza virus to form infectious virions in certain cell lines may be due to impairment of function of different virus proteins and that these defects, although associated with genetically encoded characters of virus proteins, are largely co-dependent on the host cell for their phenotypic expression. The present results furnish evidence of an intimate dependence of influenza virus reproduction on the host cell not only at the early but also at late reproduction stages, when virions are being assembled. REFERENCES ALMOND,J. W. (1977). A single gene determines the host range of influenza virus. Nature, London z7o, 617- 6 t 8. ANtSIMOV~,,E., XU~KOV.~,,E., VOtqr:A,V. & ZAVADOVA,H. (I977). Ultrastructural changes induced by influenza virus in permissive and nonpermissive cells. Virology 77, 330--336. BO~NER, Wo M. & LAS~EY,g. a. (I974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. European Journal of Biochemistry 46, 83-88. GHENDON, Y., MARKUSHIN, S., MARCHENKO, A., SITNIKOV, B. & GINSBURG, V. (1973). Biochemical characteristics of fowl plague virus ts mutants. Virology 55, 3o5-319. GrtENDON, V., MAR~USmN,S., aLAGOVESH~r~SKAVA,O. & GENKtNA,D. (t975)- Study of fowl plague virus RNA synthesis in temperature-sensitive mutants. Virology 66, 454-463. HAY, A. J., LOMN~CZl,n., BELLAMY,A. R. & SKEHEL,J. J. 0977). Transcription of the influenza virus genome. Virology 83, 337-355. l'rO, Y. & JOKLIK, W. K. 0972). Temperature-sensitive mutants of reovirus. I. Patterns of gene expression by mutants of groups C, D and E. Virology 50, 189-20 I. KLENK,H.-D., ROTT, R., ORLICH, M. & eL6DORN, J. (I975). Activation of influenza A virus by trypsin treatment. Virology 68, 426-439. gLIMOV, A. & GHENDON, V. 0975). The effect of the isolation procedure on the transcriptase activity of fowl plague virus RNP. ,4cta Virologica x9, 9I. LAEMML1,U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 2z7, 680-685. LAZAROWITZ,S. & CaOPPlN, P. (I975)- Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the haemagglutinin polypeptide. Virology 68, 440-454. MICHL, J. (196T). Metabolism of cell in tissue culture in vitro. I. The influence of serum protein fractions on the growth of normal and neoplastic cells. Experimental Cell Research 23, 324-334. PALESE, P., RITCHEY, M. B. & SCHULMAN,J. L (X977). Pl and P3 proteins of influenza virus are required for complementary R N A synthesis. Journal of Virology 2x, 1187-I 195. RUSSELL,W. G. & SKEF1EL,J. J. (I972). The polypeptides of adenovirus infected cells. JournalofGeneral Virology xs, 45-57. SCHULMAN, J. L. & PALESE, P. 0977)- Virulence factors of influenza A viruses: WSN virus neuraminidase required for plaque production in MDBK cells. Journal of Virology 24, I7o-I77. TU(:KOVA, E. & VONKA, V. (I973). Genetic interaction between two influenza A / W S virus mutants. HI. Capacity of WS/NWS recombinants to reproduce in various cell systems. ,4cta Virologica x7, 5oi-5o4. TUg:KOV.~,~., VONKA,V. & ST~,.EK, M. (197I)- Genetic interaction between two influenza A / W S virus mutants. I. Characterization of the mutants. Acta Virologica xS, 337-344.
(Received 4 September I978)
