Gene expression using insect cells and viruses David H. L. Bishop Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford, UK Current Opinion in Biotechnology 1990, 1:62--67
Introduction Of the various expression systems in current use, insect cells programmed by recombinant baculoviruses have provided one of the more valuable sources of expressed proteins. Yields of greater than 50 mg per litre of 2 x 10-9 infected cells are not uncommon, although not always realized. Yields vary according to the gene product that is being expressed. The reason why is not always clear, but appears to be attributable to either mRNA translation or post-translational effects, rather than a transcriptional deficiency, provided the complete baculovirus promoter is present in the vector [ 1]. As most of the typical eukaryotic processes of protein post-translational modification also occur in insect cells (an exception is the inability of insect cells to make complex-type glycans and add sialyic acid, etc.[2..]), the system offers the opportunity of mimicking the proteins and structures made in vertebrate cells, as well as those normally made in invertebrate cells. A number of available reviews describe in detail the expression systems based on the baculovirus of autographa californica nuclear polyhedrosis virus (AcNPV) [3,4",5 "]. A useful technical manual is also available (Smith and Summers, Texas Agricultural Experimental Station Bulletin 1555. College Station Texas, 1988). The technology involved in preparing a recombinant baculovirus is facile and involves the coinfection of insect ceils with infectious wild-type baculovirus DNA in the presence of a recombinant (plasmid) transfer vector. Lipofectin-mediated transformation is the procedure of choice. Recombinant viruses are then recovered, for example by virtue of their polyhedrin negative phenotypes. Both positive and negative selection systems are available. The recombinant transfer vector is derived from a plasmid that contains a small part of the 80-150 kb AcNPV genome (e.g. the 8 kbp EcoR1 T fragment containing the AcNPV polyhedrin gene) with a suitable insertion site for introduction of a foreign gene such as a BamHI site. In essence, the preparation of a recombinant (transfer) vector involves either the substitution of a resident viral gene (e.g. the polyhedfi'n) by a foreign gene [P1 .], but under the control of a baculovirus mRNA transcription promoter and other regulatory signals [6], or the addi-
tion of a foreign gene under the control of a suitable positioned, duplicated copy of a baculovirus promoter [P2 **] and either the viral or some acceptable alternative foreign transcription termination signal [7]. Single and multiple gene expression vectors have been made and a variety of plasmid transfer vectors are available for their construction. For high-level expression, either the baculovirus polyhedrin promoter [8,9], or the p l 0 promoter [10] may be used. Other expression vectors are also available, including those based on other AcNPV promoters, e.g. the 'basic' gene promoter [11"] or the baculovirus of Bombyx mori, the silkworm [12]. Commonly, cell lines derived from Spodoptera frugiperda ceils are employed as hosts for AcNPV infections; however, those of Mamestra brassicae provide even higher yields of expressed proteins on a per cell basis.
The infection course of a baculovirus such as AcNPV In order to understand the utility and limitations of the baculovirus expression system, account must be taken of the normal course of infection of the virus. Baculoviruses are naturally occurring viruses whose host range is limited to arthropods. Although different baculoviruses may infect particular arthropod orders (the list includes viruses of decapoda, diptera, hymenoptera, lepidoptera, etc.) none has been shown to affect or replicate in members of alternative orders of arthropod, or in other invertebrates, microorganisms, plants, or vertebrates such as man. Therefore, from the point of view of laboratory work and human safety, baculoviruses are intrinsically safe to use. Depending on the virus and the natural arthropod host, infection may cause the death of the host. Some insectspecific viruses have been used as insecticides for over 100 years, although in many instances their use has been superseded by broad-specificity chemical insecticides. The ability of certain baculoviruses to persist in the environment is a direct consequence of the presence of an occluding protein, the polyhedrin protein. The poly-
Abbreviations AcNPV--Autographa californica nuclear polyhedrosis virus; BTV--blue tongue virus; HIV--human immunodeficiency virus; VSV--vesicular stomatitis virus. 62
C) Current Biology Ltd ISSN 0958-1669
Gene expression using insect cells and viruses Bishop
hedral inclusion body protects the viral genetic information (herein termed the viral nucleocapsid) from degradation as it moves between hosts. Although not all baculoviruses produce polyhedra (e.g. the so-called group C baculoviruses), most do (e.g. groups A and B). The virus polyhedral inclusion bodies allow the nucleocapsids to persist for years in soil (or in other localities) until ingested by a susceptible species. In the alkaline midgut of a caterpillar, the polyhedrin degrades and the released nucleocapsids infect selected midgut cells. Subsequent infection of other cells by non-occluded, lipid- and glycoprotein-enveloped nucleocapsids of the virus (resembling virions of other virus families) can result in the eventual formation of more than 109 polyhedra per infected insect (depending, of course, on the virus, the insect, its stadium, etc). It is this high production of polyhedra that accounts for the ability of the virus to be effectively disseminated and to establish infections in further generations of the host. The polyhedra produced may account for up to 50% of the total protein present in an infected caterpillar by the time of its death (Fig. 1). Most of this protein is the polyhedrin protein, although a second protein, the pl0 protein, is also produced in great abundance in infected cells. The infection of an insect cells by a baculovirus such as AcNPV has been described in four phases, 'immediate
early', 'delayed early', 'late' and 'very late'. Because the Ac NPV DNA is infectious p e r se, immediate early gene expression is understood to represent the transcription of selected viral genes by the insect DNA-directed RNA polymerase. In support of this view, it has been found that these genes are transcribed in the presence of inhibitors of protein synthesis. 'Delayed early' genes include viral genes that are transactivated by the products of the 'immediate early' genes and are believed to include the viralinduced DNA polymerase. The 'delayed early' genes are not synthesized in the presence of protein synthesis inhibitors. 'Late' gene products include the major structural proteins of the virus, in particular the virus-specified glycoprotein, gp64, that is responsible for the formation of the enveloped virions. The 'very late' phase gene products include the pl0 and polyhedrin that are formed in such abundance in infected cells. The function of p l 0 is not clear. Polyhedrin protein is responsible for occluding the viral nucleocapsids and the formation of polyhedra, so it could be that pl0 has a secondary role in this process. However, as far as the formation of virions is concerned, both polyhedrin and pl0 proteins are nonessential. It is the replacement of the genes that encode pl0 and/or polyhedrin by foreign gene(s) that is the basis of most expression vectors. In essence, the promoters [P2 °°] of these genes are retained and a foreign gene placed in lieu of the viral coding sequences.
Fig. 1. Baculovirus-infected insect cell showing morphogenesis of polyhedra 3 days post-infection.
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64 Expressionsystems Single gene expressionmcharacteristics of the products Most of the reported studies involving baculovirus expression vectors have used them to obtain a s i @ e protein product for structure-function analyses. As noted above, high yields of protein are obtained in many cases although not always. Low-level expression ( < 5 mg per 2 x 109 cells) appears to be caused by either translational or post-translational problems, or might represent an inherent property of the particular gene product. There are certain limitations to the baculovirus expression system that is commonly used. The expression of a foreign gene from the polyhedrin or pl0 promoter comes at the end of the infection cycle and at a time when the host cell is in the process of degradation. Not surprisingly, processing of the foreign gene products can be incomplete when 'very late' baculovirus promoters are used, particularly when a foreign gene is expressed to high level. For example, when the glycoproteins of rabies and vesicular stomatitis virus (VSV) were expressed by polyhedrin-based vectors, not all the expressed protein was fully glycosylated [13 ",14"] even though the products exhibited the expected antigenic and biological activities. It is possible that expression from an earlier promoter (e.g. a duplicated copy of an essential 'late gene' promoter such as the basic protein promoter [11..]) would improve the efficiency of post-translational modification. Expression and characterization of a variety of glycoproteins has been reported [13"-27",P3"]; usually at lower levels than those reported for the glycoproteins of rabies and VSV [13% 14.]. In addition to glycoproteins, the biological activities of other expressed proteins have been found to resemble those of their natural counterparts [28"-38.]. Such observations validate the use of baculoviruses as expression vectors and have provided researchers with useful tools for structure-function analyses, particularly when high levels of the desired products are obtained.
Characteristics of products that form particles Expression on the complete human immunodeficiency virus (HIV) gag gene results in the synthesis and release of enveloped particles containing unprocessed gag gene products [ 3 9 " , P 4 " ] . The effects of removing parts of the HIV gag coding sequence from the vector, in terms of the formation and location of the particles [33 ",39"] and effects of the products upon the plasma membrane of the infected insect cells, illustrates the power of the expression system for morphogenic analyses. In a study [40..] of the expression of the complete coding region of poliovirus (6.5kb), virus-like particles were synthesized and released. Although only low-level expression was reported, the fact that immunogenic particles could be purified to homogeneity should allow structural analyses to be undertaken as well as manipulation of the genes in-
volved in their synthesis (e.g. the poliovims-coded proteases)
Multiple gene expression - - characteristics of the products A valuable attribute of the baculovims expression system is the ability to express more than one foreign gene at one time. Multiple gene vectors based on duplicated copies of AcNPV 'very late' promoters have been prepared and are an example. For bluetongue vires (BTV), expression of the two major core proteins (VP3, VP7) resulted in high yields of BTV core-like particles [41 ..]. The cores exhibited the same stoichiometries of VP3 and VP7 as those characteristic of BTV cores. Further development along these lines may be expected in the near future especially in relation to vectors that express more foreign genes; in this way it might be possible to induce expression at earlier times in the infection course.
Technical developments that may aid the preparation of expression vectors A continuous cell production system has been described [42 -] to allow insect cells to be produced in a regulated fashion. Encapsidation of vires-infected ceils by alginate can be used to produce cells in high density [43"], although to get recombinant proteins a temperature-sensitive vector is required so that the encapsidated (infected) cells can be induced to express a foreign protein when required. The use of electroporation [44,] allows efficient cell transformation but does not increase the frequency of recombination. Lipofectin is now the procedure of choice for introducing DNA into cells (including insect cells) and causes less cell loss than either the calcium phosphate precipitate or electroporation methods. Yields of protein from caterpillars infected with occluded (wild-type) and non-occluded (recombinant) [45"] vimses may increase the yield of foreign protein from larvae, however, the use of multiple gene expression vectors [7] gives the same result and provides a stable inoculum. Perhaps the most useful recent technical advance has been the vector that has si@e-strand DNA capability [46,,] so that oligonucleotides can be used to make sitedirected mutations in the DNA of the transfer vector.
Conclusion Baculovimses have proved to be valuable tools to express processed proteins. Further developments concerning multiple gene expression vectors will allow research into protein-protein interactions to be undertaken and this will have both fundamental and applied value.
Gene expression using insect cells and viruses
Annotated references and recommended reading • ••
Of interest Of outstanding interest
1.
MATSUURAY, POSSEE RD, OVERTON HA, BISHOP DHL: Bacniovirns e x p r e s s i o n vectors: t h e requirements for high-level expression of proteins, including glycoproteins. J Gen Virol 1987, 68:1233--1250.
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KURODAK, GEYER H, GEYER R, DOERELER W, KLENK H-D: The oligosaccharides of influenza virus haemagglutinin expressed in insect cells by a baculovirus vector. Virology 1990, 174:418-429. Describes the glycan sequences at various sites of the influenza haemagglutinin proteins by comparison with those made in vertebrate cells. 3.
MILLERLK: Baculoviruses as g e n e expression vectors. A n n u Rev Microbiol 1988, 42:177-199.
FRASERMJ: Expression of eucaryotic g e n e s in insect cell cultures. In Vitro Cell Dev Biol 1989, 25:225 235. rovides a review of the baculovirus expression techniques and corn merits on large-scale fermentation including the use of serum-free media. 4.
5. BISHOPDHL, POSSEE RD: Baculovirus expression vectors. Adv • Gene Technol 1990, 1:55-72. A general review of baculovirus expression vectors and their use.
6.
LUCKOWVA, SUMMERS MD: Trends in t h e d e v e l o p m e n t of baculovirus expression vectors. Bio/Technology 1988, 6:47-55.
7.
EMERYVC, BISHOP DHL: The d e v e l o p m e n t of multiple expression v e c t o r s for the high-level synthesis of eukaryotic proteins: e x p r e s s i o n of LCMV-N and AcNPV polyhedrin proteins by a r e c o m b i n a n t bacniovirus. Protein Eng 1987, 1:359-366.
8.
SMITH GE, SUMMERS MD, FRASER MJ: Production of h u m a n [3-interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol 1983, 3:2156-2165
9.
TAKEHARAK, IRELANDD, BISHOP DHL: Co-expression of the hepatitis B surface and core antigens using baculovirus multiple expression vectors. J Gen Virol 1988, 69:2763-2777.
10.
VLAKJM, KLINKENBERGFA, ZAALKJM, USMANYM, KLINGE-ROODE EC, GEERVLIETJBF, ROOSIENJ, VAN LENTJWM: Functional studies of the plO g e n e of A u t o g r a p h a californica nuclear polyhedrosis virus using a r e c o m b i n a n t expressing a p l 0 beta-galactosidase fusion gene. J Gen Virol 1988, 69:765--776.
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HILL-PERKINSMS, POSSEE RD: A bacniovirus expression vector derived from t h e basic protein promoter of A u t o g r a p h a californica nuclear polyhedrosis virus. J Gen Virol 1990, 71:971-976. Describes a new expression system using a duplicated copy of the 'late' gene promoter of the AcNPV basic protein gene. 12.
MAEDA S, KAWAI T, OBINATA M, FUJIWARAH, HOR1UCHI T, NAEKIY, SATO Y, FURUSAWAM: Production of h u m a n a-interferon in silkworm using a baculovirus vector. Nature 1985, 315:592-594.
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protein e x p r e s s e d by baculovirus vectors. Virology 1989, 173:390-399. Demonstrates the synthesis mad immunogenic properties of rabies glycoprotein.
15. •
SISSOMJ, ELLIS L: Secretion of the extracellniar domain of t h e h u m a n insulin receptor from insect cells by u s e of a baculovirus vector. Biochem J 1989, 261:119-126. A truncated derivative of the extracellular domain of dimers of glycosylated h u m a n insulin receptor was effectively secreted from recombinant baculovirus-infected cells. The product was inefficiently processed and only slowly secreted. 16. •
WHITEFLEET-SMITHJ, ROSEN E, MCL1NDENJ, PLOPLISVA, FRASER MJ, TOMHNSONJE, MCLEANJW, CASTELIMqOFJ: Expression of h u m a n plasminogen eDNA in a baculovirus vector-infected insect cell system. Arch Biochem Biophys 1989, 271:390-399. Relatively low-level secretion of h u m a n plasminogen with biological activity is reported. Uroldnase activated change from the plasminogen to plasmin, indicating that biological activity was retained. 17. •
SCHMALJOHNCS, PARKER MD, ENN1S WH, DALRYMPLE JM, COLLETrMS, SUZICHJA~ SCHMALJOHNAL: Baculovirus expression of the M g e n o m e s e g m e n t of Rift Valley fever virus and examination of antigenic and i m m u n o g e n i c properties of t h e e x p r e s s e d proteins. Virology 1989, 170:184-192. Proteolytic processing and low-level expression of the Rift Valley fever vires glycoprotein precursor is reported, together with evidence for the protective properties of the product. 18. •
GEORGE ST, ARBABIANMA, RUOHO AE, KIELYJ, MALBON CC: High-efficiency expression o f mammalian [3-adrenergic receptors in baculovirus-infected insect cells. Biochem Biophys Res C o m m u n 1990, 163:1265 1269. Expression of glycosylated and non-glycosylated versions of the receptor are reported. 19. •
WEBB NR, MADOULET C, TOSl P-F, BROUSSARDSDR, SNEED L, NICOLAUC, SUMMERS MD: Cell-surface expression and purification of h u m a n CD4 p r o d u c e d in baculovirus-infected insect cells. Proc Natl Acad Sci USA 1989, 86:7731-7735. Cell associated glycosylated and non-glycosylated derivatives of CD4 were expressed at low levels. 20. •
MORIKAWAY, OVERTON HA, MOORE JP, WILKINSONAJ, BRADY RL, LEWIS SJ, JONES IM Expression of HIV-1 g p l 2 0 and hum a n soluble CD4 by r e c o m b i n a n t baculoviruses and their interaction in vitro. Aids Res H u m a n Retroviruses 1990, 6:765-773. The glycosylated derivatives of baculovims-expressed soluble gpl20 and soluble CD4 interact in vitro to form complexes that resemble their natural interactions. Again, low-level expression is reported. 21. •
MOOREJP, MCKEATINGJA, JONES IM, STEPHENS PE, CLEMENTS G, THOMPSON S, WEISS RA: Characterization of recombinant g p l 2 0 and gp160 from HIV-I: binding to monoclonal antibodies and soluble CD4. AIDS 1990, 4:30~315. Expression vectors that synthesize low levels of the HIV1 gpl60 and a secreted version of gp120 are described. In addition, the antigenic and biological properties of the products are discussed. 22. •
MORIKAWA Y, OHKI K, IKUTAK, JONES IM: O n the specificity of g p l 2 0 - C D 4 binding reaction. Antiviral Chem Chemotherapy 1990, 1:73. Analyses of the H1V-1 gpl20 and CD4 baculovirus-expressed products are reported.
BA~LEYMJ, McLKEOD DA, KANG C-Y, BISHOP DHL: Glycosylation is not required for t h e fusion activity of the G protein of vesicular stomatitis virus in insect cells. Virology 1989, 169:323-331. Reports the high-level expression and phenotype of VSV glycoprotein expressed by recombinant AcNPV, including various mutant G proteins that lack one or both glycan sites.
STEINERH, POHL G, HELLERSM, EIMAMMERA, HANSSON L: Hum a n tissue-type plasminogen activator synthesized by using a baculovirus in insect cells c o m p a r e d with h u m a n plasm i n o g e n activator p r o d u c e d in m o u s e cells. Gene 1988, 73:449-457. Glycosylated tissue plasminogen activator produced by recombinant baculoviruses was secreted in the culture medium and exhibited biological activity. The level of expression was low and two forms of the glycosylated product were detected.
14. •
24. ,
13. •
PREHAUDC, TAKEHARAK, FLAMANDA, BISHOP DHL: I m m u n o genic and protective properties of rabies virus glyco-
23. •
QUELLEFW, CASLAKELF, BURKERTRE, WOJCHOWSKIDM: Highlevel expression and purification of a r e c o m b i n a n t h u m a n
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Expressionsystems erythropoietin p r o d u c e d using a baculovirus vector. Blood 1989, 74:652-657. An 890-fold purification of h u m a n erythropoietin expressed and efficiently secreted from a recombinant baculovirus is reported. Character ization of the product indicated a uniform oligosacchatide composition (30%) and contrasts with the heterogeneously glycosylated mammalian products (40-50%). 25. •
KRISHNAS, BLACKLAWSBA, OVERTON HA,. BISHOP DHL, NASH A& Expression of glycoprotein D of h e r p e s simplex virus type 1 in a r e c o m b i n a n t baculovirus: protective r e s p o n s e s and T cell recognition of the recombinant-infected cell extracts. J Gen Virol 1989, 70:1805-1814. Low-level expression and protection of mice by the herpes D glycoprotein is reported. 26. .
WATHENMW, BRIDEAU RJ, THOMSEN DR, MURPHY BR: Characterization of a novel h u m a n respiratory syncytial virus chimeric FG glycoprotein e x p r e s s e d u s e d a baculovirus vector. J Gen Virol 1989, 70:2625-2636. Secretion of a novel chimaeric construct of the h u m a n respiratory syn~ cytial vires F and G proteins is reported. The products were heterogeneous in size and may have contained O-linked glycans, although this was not determined. Cleavage of the F portion of the chimaera was indicated. OKER-BLOMC, PETTERSSONRE, SUMMERSMD: Baculovirus polyhedrin promoter-directed expression of rubella virus envelope glycoproteins, E1 and E2, in Spodoptera frugiperda cells. Virology 1989, 172:82-91. Low-level expression and proteolytic cleavage of the rubella glycoprorein precursor to the E1 and E2 proteins is reported. 27. •
28.
ASSEFFAA, SMITH SJ, NAGATA K, G1LLETrE J, GELBOIN HV, GONZALEZFJ: Novel e x o g e n o u s h e m e - d e p e n d e n t expression of mammalian c y t o c h r o m e P450 using baculovirus. Arch Biochem Biophys 1989, 274:481. Cytochrome P450 IIA1 synthesized by insect cells was activated by the addition of haem to the culture medium and then exhibited testosterone hydroxylase activity. •
29.
PENDERGASTAM, CLARKR, KAWASAKIES, McCORMICKFP, WITFE ON: Baculovirus expression of functional P210 BCR-ABL o n c o g e n e product. Oncogene 1989, 4:759-766. The tyrosine kinase product of the chronic myelogenous leukaemiaassociated P210 BCR-ABL oncogene was shown to phosphorylate angiotensin with a Km similar to that of the enzyme isolated from h u m a n sources. The kinase is capable of autophosphorylation. •
30. •
BARBERGN, CLEGG JCS, LLOYD G: Expression of the Lassa virus nucleocapsid protein in insect cells infected w i t h a recombinant baculovirus: application to diagnostic assays for Lassa virus infection. J Gen Virol 1990, 71:19-28. The usefulness of the expressed Lassa protein for diagnosis is d e m o n strated. 31. •
URAKAWAT, RITTER DG, ROY P: Expression of largest RNA s e g m e n t and synthesis o f VP1 protein of b l u e t o n g u e virus in insect cells by r e c o m b i n a n t baculovirus: association of VP1 protein with RNA polymerase activity. Nucl Acids Res 1989, 17:7395-7402. Evidence that the BTV VP1 protein is a c o m p o n e n t of the viral RNAdirected RNA polymerase is presented. 32. •
MAKINOY, TADANOM, ANZAIT, MA S-P, YASUDAS, FUKUNAGAT: Detection of d e n g u e 4 virus core protein in the nucleus. II Antibody against d e n g u e 4 core protein p r o d u c e d by a r e c o m b i n a n t baculovirus reacts w i t h t h e antigen in t h e nucleus. J Gen VirO1 1989, 70:1417-1425. Antibody raised to the expressed antigen was used to investigate the course of dengue virus infection of cells. 33. •
OVERTONHA, FUJII Y, PRICE IR, JONES IM: T h e protease and gag g e n e products of t h e h u m a n immunodeficiency virus: authentic cleavage and post-translational modification in an insect cell expression system. Virology 1989, 170:107-116.
Active HW protease expressed by recombinant baculovimses either as a polyprotein or as an independently expressed product has been demonstrated. 34. .
FRENCHTJ, INUMARUS, ROY P: Expression of two related nonstructural proteins of BTV-10 in insect cells by a recombinant baculovirus: production of polyclonal ascitic fluid and characterization o f the g e n e p r o d u c t in BTV infected BHK cells. J virol 1989, 63:3270-3278. Two forms of the NS3 protein of BTV synthesized by baculovirus vectors are described. ROY P, ADACHIA, URAKAWAT, BOOTH TF, THOMAS CP: Identification of b l u e t o n g u e virus VP6 protein as a nucleic acid binding protein and the localization of VP6 in virus infected vertebrate cells. J Virol 1990, 64:1-8. Functional analyses were undertaken of the nucleic acid binding VP6 protein of BTV expressed to high level by a baculovims vector. 35. •
36. •
MARSHALL JA, ROY P: High level expression of the two o u t e r capsid proteins o f bluetongue virus serotype 10: their relationship w i t h t h e neutralization of virus infection. Vir Res 1990, 15:189-195. The VP2 and VP5 outer capsid proteins of BTV were analysed with respect to their immunogenic properties and ability to raise neutralizing sera. 37. The
RoY P, VAN DIJK AA, ERASMUSBA: Recombinant virus vaccine for b l u e t o n g u e disease in sheep. J virol 1990, 64:1198-2003. ability of baculovirus-expressed VP2 (with or without other baculovirus-expressed BTV proteins) to dicit neutralizing antibodies and protection of sheep against virulent BTV challenge were investigated. Combinations of VP2 plus VP5 in the presence of adjuvant were the most effective. 38. .
DODSON MS, CRUTE JJ, BRUCKNER RC, LEHMAN IR: Overexpression and assembly of t h e herpes simplex virus type 1 helicase-primase in insect cells. J Biol Chem 1989, 264:20835-20838. Expression and characterization of the HSV-1 helicase-primase proteins is reported. 39.
GHEYSEND, JACOBS E, DE FRESTA F, THIRIART C, FRANCOTFE DE WILDE M: Assembly and release of HIV1 p r e c u r s o r Pr55gag virus-like particles from r e c o m b i n a n t baculovirus-infected insect cells. Cell 1989, 59:103112. Enveloped particles containing the unprocessed H1V gag gene product are released from infected cells. This property is dependent on the presence of a myristoylated N-terminal glycine of gag since absence of the N-terminal amino acids led to accumulation of particles of the cytoplasm and nucleus. Inclusion of the pol gene led to processing of the gag antigen and decreased particle secretion. Deletion of the carboxyterminal p16 sequences from gag abolished particle formation but resulted in membrane evagination. • •
M, THINES D ,
40.
URAKAWAT, GERGUSON M, MINOR PD, COOPER J, SULLIVANM, ALMONDJW, BISHOP DHL: Synthesis of immunogenic, b u t non-infectious, poliovirus particles in insect cells by a baculovirus expression vector. J Gen Virol 1989, 70:1453-1463. Low-level synthesis of intact, immunogenic but non-infectious virus-like particles of poliovims containing VPO, VP1 and VP3 is reported by expression of the 6.5 kb coding region of poliovirus cDNA. The data indicate that both viral-coded proteases are functional and support the view that inclusion of poliovirus RNA is required for the cleavage of VPO to VP2 and VP4. •,
41. , •
FRENCHTJ, ROY P: Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing t h e two major structural core proteins of BTV. J Virol 1990, 64:1530-1536. Multigene expression vectors were employed to obtain high yields of the two core proteins of BTV, VP3 and VP7. The expressed proteins self-assembled into core-like proteins which were purified by gradient centrifugation procedures.
42. •
DEGOOIJERCD, VANHERFLJ, VANDENENDEJ, VLAKJM, TRAMPERJ: A model for baculovirus production with c o n t i n u o u s insect cell cultures. Appl Microbiol Biotechnol 1989, 30:497-501,
Gene expression using insect cells and viruses A system for continuous cell production is described. KING G& DAUGUUS AJ, GOOSEN MFA, FAULKENER P, BAYLY • D: Alginate concentration: a key factor in g r o w t h of temperature-sensitive baculovirus infected insect cells in microcapsules. Biotechnol Bioeng 1989, 34:1085-1091. Encapsidation of insect cells infected with temperature sensitive baculovims allows the expression of a foreign gene at high cell density using temperature induction.
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Annotated patents
43.
44. •
MANNSG, KING LA: Efficient transfection of insect cells with baculovirus DNA using electroporation. J Gen Virol 1989, 70:35010505. This procedure has been superseded by the use of lipofectin. 45. •
PRICEPM, REICHELDERFERCF, JOHANSSON BE, KILBOURNE ED, Acs G: C o m p l e m e n t a t i o n of r e c o m b i n a n t baculoviruses by coinfection w i t h wild-type virus facilitates production of insect larvae of antigenic proteins of hepatitis B virus and influenza virus. Proc Nail Acad Sci USA 1989, 86:1453-1456_ Occluded viruses are more infective for caterpillars than non occluded viruses and can raise production levels. This procedure is superseded by the use of multiple expression vectors [7] that express polyhedrin and a foreign gene product. 46. LIVINGSTONEC, JONES I: Baculovirus expression vectors with •• single stranded capability. Nucl Acid Res 1989, 17:2366. This vector allows site-directed mutagenesis to be undertaken on singlestranded DNA (based on the pAcYM1 transfer vector) and gives a recombinant DNA that may be directly transferred into the recombinant vector.
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Of interest Of outstanding interest
P1. •
INSTITUTE NAT RECH SCIENCE: ,Modified baculovirus as cxpression vector for foreign g e n e s inserted directly w i t h o u t transfer vector, contains restriction fragment u n d e r control of p r o m o t e r of baculovirus-associated protein gene. 31/5/88 88FR~007207. 6/12/89 EP 345152A. Proposes improved method for obtaining recombinants. P2. ••
NATURALENVIRON RES: N e w plasmid replicon for inserting several g e n e s into vector containing two polypeptide expression structures, and derived viral vectors for infecting insect cells. 12/8/87 87GB019108. 16/8/89 EP-327626 A. Multigene expression vector technology for baculovimses. P3. •
NIPPON ZEON KK: Production of envelope protein of Japanese encephalitis virus using recombinant baculovirus infected into insect cells. 12/5/88 88JP-115316. 15/11/89 GB2218421 A. Expressioh of Japanese encephalitis vires glycoprotein as a candidate vaccine. P4. ••
SMITHKLINEBIOLOGIC: N e w r e c o m b i n a n t DNA encoding HIV gag p r e c u r s o r protein w i t h o u t flanking sequences, exp r e s s e d in insect, yeast and mammalian cells, producing particles useful in vaccines and diagnosis. 3/6/88 88US202271. 6/12/89 EP-345242 A. HIV-1 antigens made from various expression vectors can be used as antigens for H1V diagnosis and as potential vaccines.
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Introduction Of the various expression systems in current use, insect cells programmed by recombinant baculoviruses have provided one of the more valuable sources of expressed proteins. Yields of greater than 50 mg per litre of 2 x 10-9 infected cells are not uncommon, although not always realized. Yields vary according to the gene product that is being expressed. The reason why is not always clear, but appears to be attributable to either mRNA translation or post-translational effects, rather than a transcriptional deficiency, provided the complete baculovirus promoter is present in the vector [ 1]. As most of the typical eukaryotic processes of protein post-translational modification also occur in insect cells (an exception is the inability of insect cells to make complex-type glycans and add sialyic acid, etc.[2..]), the system offers the opportunity of mimicking the proteins and structures made in vertebrate cells, as well as those normally made in invertebrate cells. A number of available reviews describe in detail the expression systems based on the baculovirus of autographa californica nuclear polyhedrosis virus (AcNPV) [3,4",5 "]. A useful technical manual is also available (Smith and Summers, Texas Agricultural Experimental Station Bulletin 1555. College Station Texas, 1988). The technology involved in preparing a recombinant baculovirus is facile and involves the coinfection of insect ceils with infectious wild-type baculovirus DNA in the presence of a recombinant (plasmid) transfer vector. Lipofectin-mediated transformation is the procedure of choice. Recombinant viruses are then recovered, for example by virtue of their polyhedrin negative phenotypes. Both positive and negative selection systems are available. The recombinant transfer vector is derived from a plasmid that contains a small part of the 80-150 kb AcNPV genome (e.g. the 8 kbp EcoR1 T fragment containing the AcNPV polyhedrin gene) with a suitable insertion site for introduction of a foreign gene such as a BamHI site. In essence, the preparation of a recombinant (transfer) vector involves either the substitution of a resident viral gene (e.g. the polyhedfi'n) by a foreign gene [P1 .], but under the control of a baculovirus mRNA transcription promoter and other regulatory signals [6], or the addi-
tion of a foreign gene under the control of a suitable positioned, duplicated copy of a baculovirus promoter [P2 **] and either the viral or some acceptable alternative foreign transcription termination signal [7]. Single and multiple gene expression vectors have been made and a variety of plasmid transfer vectors are available for their construction. For high-level expression, either the baculovirus polyhedrin promoter [8,9], or the p l 0 promoter [10] may be used. Other expression vectors are also available, including those based on other AcNPV promoters, e.g. the 'basic' gene promoter [11"] or the baculovirus of Bombyx mori, the silkworm [12]. Commonly, cell lines derived from Spodoptera frugiperda ceils are employed as hosts for AcNPV infections; however, those of Mamestra brassicae provide even higher yields of expressed proteins on a per cell basis.
The infection course of a baculovirus such as AcNPV In order to understand the utility and limitations of the baculovirus expression system, account must be taken of the normal course of infection of the virus. Baculoviruses are naturally occurring viruses whose host range is limited to arthropods. Although different baculoviruses may infect particular arthropod orders (the list includes viruses of decapoda, diptera, hymenoptera, lepidoptera, etc.) none has been shown to affect or replicate in members of alternative orders of arthropod, or in other invertebrates, microorganisms, plants, or vertebrates such as man. Therefore, from the point of view of laboratory work and human safety, baculoviruses are intrinsically safe to use. Depending on the virus and the natural arthropod host, infection may cause the death of the host. Some insectspecific viruses have been used as insecticides for over 100 years, although in many instances their use has been superseded by broad-specificity chemical insecticides. The ability of certain baculoviruses to persist in the environment is a direct consequence of the presence of an occluding protein, the polyhedrin protein. The poly-
Abbreviations AcNPV--Autographa californica nuclear polyhedrosis virus; BTV--blue tongue virus; HIV--human immunodeficiency virus; VSV--vesicular stomatitis virus. 62
C) Current Biology Ltd ISSN 0958-1669
Gene expression using insect cells and viruses Bishop
hedral inclusion body protects the viral genetic information (herein termed the viral nucleocapsid) from degradation as it moves between hosts. Although not all baculoviruses produce polyhedra (e.g. the so-called group C baculoviruses), most do (e.g. groups A and B). The virus polyhedral inclusion bodies allow the nucleocapsids to persist for years in soil (or in other localities) until ingested by a susceptible species. In the alkaline midgut of a caterpillar, the polyhedrin degrades and the released nucleocapsids infect selected midgut cells. Subsequent infection of other cells by non-occluded, lipid- and glycoprotein-enveloped nucleocapsids of the virus (resembling virions of other virus families) can result in the eventual formation of more than 109 polyhedra per infected insect (depending, of course, on the virus, the insect, its stadium, etc). It is this high production of polyhedra that accounts for the ability of the virus to be effectively disseminated and to establish infections in further generations of the host. The polyhedra produced may account for up to 50% of the total protein present in an infected caterpillar by the time of its death (Fig. 1). Most of this protein is the polyhedrin protein, although a second protein, the pl0 protein, is also produced in great abundance in infected cells. The infection of an insect cells by a baculovirus such as AcNPV has been described in four phases, 'immediate
early', 'delayed early', 'late' and 'very late'. Because the Ac NPV DNA is infectious p e r se, immediate early gene expression is understood to represent the transcription of selected viral genes by the insect DNA-directed RNA polymerase. In support of this view, it has been found that these genes are transcribed in the presence of inhibitors of protein synthesis. 'Delayed early' genes include viral genes that are transactivated by the products of the 'immediate early' genes and are believed to include the viralinduced DNA polymerase. The 'delayed early' genes are not synthesized in the presence of protein synthesis inhibitors. 'Late' gene products include the major structural proteins of the virus, in particular the virus-specified glycoprotein, gp64, that is responsible for the formation of the enveloped virions. The 'very late' phase gene products include the pl0 and polyhedrin that are formed in such abundance in infected cells. The function of p l 0 is not clear. Polyhedrin protein is responsible for occluding the viral nucleocapsids and the formation of polyhedra, so it could be that pl0 has a secondary role in this process. However, as far as the formation of virions is concerned, both polyhedrin and pl0 proteins are nonessential. It is the replacement of the genes that encode pl0 and/or polyhedrin by foreign gene(s) that is the basis of most expression vectors. In essence, the promoters [P2 °°] of these genes are retained and a foreign gene placed in lieu of the viral coding sequences.
Fig. 1. Baculovirus-infected insect cell showing morphogenesis of polyhedra 3 days post-infection.
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64 Expressionsystems Single gene expressionmcharacteristics of the products Most of the reported studies involving baculovirus expression vectors have used them to obtain a s i @ e protein product for structure-function analyses. As noted above, high yields of protein are obtained in many cases although not always. Low-level expression ( < 5 mg per 2 x 109 cells) appears to be caused by either translational or post-translational problems, or might represent an inherent property of the particular gene product. There are certain limitations to the baculovirus expression system that is commonly used. The expression of a foreign gene from the polyhedrin or pl0 promoter comes at the end of the infection cycle and at a time when the host cell is in the process of degradation. Not surprisingly, processing of the foreign gene products can be incomplete when 'very late' baculovirus promoters are used, particularly when a foreign gene is expressed to high level. For example, when the glycoproteins of rabies and vesicular stomatitis virus (VSV) were expressed by polyhedrin-based vectors, not all the expressed protein was fully glycosylated [13 ",14"] even though the products exhibited the expected antigenic and biological activities. It is possible that expression from an earlier promoter (e.g. a duplicated copy of an essential 'late gene' promoter such as the basic protein promoter [11..]) would improve the efficiency of post-translational modification. Expression and characterization of a variety of glycoproteins has been reported [13"-27",P3"]; usually at lower levels than those reported for the glycoproteins of rabies and VSV [13% 14.]. In addition to glycoproteins, the biological activities of other expressed proteins have been found to resemble those of their natural counterparts [28"-38.]. Such observations validate the use of baculoviruses as expression vectors and have provided researchers with useful tools for structure-function analyses, particularly when high levels of the desired products are obtained.
Characteristics of products that form particles Expression on the complete human immunodeficiency virus (HIV) gag gene results in the synthesis and release of enveloped particles containing unprocessed gag gene products [ 3 9 " , P 4 " ] . The effects of removing parts of the HIV gag coding sequence from the vector, in terms of the formation and location of the particles [33 ",39"] and effects of the products upon the plasma membrane of the infected insect cells, illustrates the power of the expression system for morphogenic analyses. In a study [40..] of the expression of the complete coding region of poliovirus (6.5kb), virus-like particles were synthesized and released. Although only low-level expression was reported, the fact that immunogenic particles could be purified to homogeneity should allow structural analyses to be undertaken as well as manipulation of the genes in-
volved in their synthesis (e.g. the poliovims-coded proteases)
Multiple gene expression - - characteristics of the products A valuable attribute of the baculovims expression system is the ability to express more than one foreign gene at one time. Multiple gene vectors based on duplicated copies of AcNPV 'very late' promoters have been prepared and are an example. For bluetongue vires (BTV), expression of the two major core proteins (VP3, VP7) resulted in high yields of BTV core-like particles [41 ..]. The cores exhibited the same stoichiometries of VP3 and VP7 as those characteristic of BTV cores. Further development along these lines may be expected in the near future especially in relation to vectors that express more foreign genes; in this way it might be possible to induce expression at earlier times in the infection course.
Technical developments that may aid the preparation of expression vectors A continuous cell production system has been described [42 -] to allow insect cells to be produced in a regulated fashion. Encapsidation of vires-infected ceils by alginate can be used to produce cells in high density [43"], although to get recombinant proteins a temperature-sensitive vector is required so that the encapsidated (infected) cells can be induced to express a foreign protein when required. The use of electroporation [44,] allows efficient cell transformation but does not increase the frequency of recombination. Lipofectin is now the procedure of choice for introducing DNA into cells (including insect cells) and causes less cell loss than either the calcium phosphate precipitate or electroporation methods. Yields of protein from caterpillars infected with occluded (wild-type) and non-occluded (recombinant) [45"] vimses may increase the yield of foreign protein from larvae, however, the use of multiple gene expression vectors [7] gives the same result and provides a stable inoculum. Perhaps the most useful recent technical advance has been the vector that has si@e-strand DNA capability [46,,] so that oligonucleotides can be used to make sitedirected mutations in the DNA of the transfer vector.
Conclusion Baculovimses have proved to be valuable tools to express processed proteins. Further developments concerning multiple gene expression vectors will allow research into protein-protein interactions to be undertaken and this will have both fundamental and applied value.
Gene expression using insect cells and viruses
Annotated references and recommended reading • ••
Of interest Of outstanding interest
1.
MATSUURAY, POSSEE RD, OVERTON HA, BISHOP DHL: Bacniovirns e x p r e s s i o n vectors: t h e requirements for high-level expression of proteins, including glycoproteins. J Gen Virol 1987, 68:1233--1250.
2. ••
KURODAK, GEYER H, GEYER R, DOERELER W, KLENK H-D: The oligosaccharides of influenza virus haemagglutinin expressed in insect cells by a baculovirus vector. Virology 1990, 174:418-429. Describes the glycan sequences at various sites of the influenza haemagglutinin proteins by comparison with those made in vertebrate cells. 3.
MILLERLK: Baculoviruses as g e n e expression vectors. A n n u Rev Microbiol 1988, 42:177-199.
FRASERMJ: Expression of eucaryotic g e n e s in insect cell cultures. In Vitro Cell Dev Biol 1989, 25:225 235. rovides a review of the baculovirus expression techniques and corn merits on large-scale fermentation including the use of serum-free media. 4.
5. BISHOPDHL, POSSEE RD: Baculovirus expression vectors. Adv • Gene Technol 1990, 1:55-72. A general review of baculovirus expression vectors and their use.
6.
LUCKOWVA, SUMMERS MD: Trends in t h e d e v e l o p m e n t of baculovirus expression vectors. Bio/Technology 1988, 6:47-55.
7.
EMERYVC, BISHOP DHL: The d e v e l o p m e n t of multiple expression v e c t o r s for the high-level synthesis of eukaryotic proteins: e x p r e s s i o n of LCMV-N and AcNPV polyhedrin proteins by a r e c o m b i n a n t bacniovirus. Protein Eng 1987, 1:359-366.
8.
SMITH GE, SUMMERS MD, FRASER MJ: Production of h u m a n [3-interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol 1983, 3:2156-2165
9.
TAKEHARAK, IRELANDD, BISHOP DHL: Co-expression of the hepatitis B surface and core antigens using baculovirus multiple expression vectors. J Gen Virol 1988, 69:2763-2777.
10.
VLAKJM, KLINKENBERGFA, ZAALKJM, USMANYM, KLINGE-ROODE EC, GEERVLIETJBF, ROOSIENJ, VAN LENTJWM: Functional studies of the plO g e n e of A u t o g r a p h a californica nuclear polyhedrosis virus using a r e c o m b i n a n t expressing a p l 0 beta-galactosidase fusion gene. J Gen Virol 1988, 69:765--776.
11. ••
HILL-PERKINSMS, POSSEE RD: A bacniovirus expression vector derived from t h e basic protein promoter of A u t o g r a p h a californica nuclear polyhedrosis virus. J Gen Virol 1990, 71:971-976. Describes a new expression system using a duplicated copy of the 'late' gene promoter of the AcNPV basic protein gene. 12.
MAEDA S, KAWAI T, OBINATA M, FUJIWARAH, HOR1UCHI T, NAEKIY, SATO Y, FURUSAWAM: Production of h u m a n a-interferon in silkworm using a baculovirus vector. Nature 1985, 315:592-594.
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protein e x p r e s s e d by baculovirus vectors. Virology 1989, 173:390-399. Demonstrates the synthesis mad immunogenic properties of rabies glycoprotein.
15. •
SISSOMJ, ELLIS L: Secretion of the extracellniar domain of t h e h u m a n insulin receptor from insect cells by u s e of a baculovirus vector. Biochem J 1989, 261:119-126. A truncated derivative of the extracellular domain of dimers of glycosylated h u m a n insulin receptor was effectively secreted from recombinant baculovirus-infected cells. The product was inefficiently processed and only slowly secreted. 16. •
WHITEFLEET-SMITHJ, ROSEN E, MCL1NDENJ, PLOPLISVA, FRASER MJ, TOMHNSONJE, MCLEANJW, CASTELIMqOFJ: Expression of h u m a n plasminogen eDNA in a baculovirus vector-infected insect cell system. Arch Biochem Biophys 1989, 271:390-399. Relatively low-level secretion of h u m a n plasminogen with biological activity is reported. Uroldnase activated change from the plasminogen to plasmin, indicating that biological activity was retained. 17. •
SCHMALJOHNCS, PARKER MD, ENN1S WH, DALRYMPLE JM, COLLETrMS, SUZICHJA~ SCHMALJOHNAL: Baculovirus expression of the M g e n o m e s e g m e n t of Rift Valley fever virus and examination of antigenic and i m m u n o g e n i c properties of t h e e x p r e s s e d proteins. Virology 1989, 170:184-192. Proteolytic processing and low-level expression of the Rift Valley fever vires glycoprotein precursor is reported, together with evidence for the protective properties of the product. 18. •
GEORGE ST, ARBABIANMA, RUOHO AE, KIELYJ, MALBON CC: High-efficiency expression o f mammalian [3-adrenergic receptors in baculovirus-infected insect cells. Biochem Biophys Res C o m m u n 1990, 163:1265 1269. Expression of glycosylated and non-glycosylated versions of the receptor are reported. 19. •
WEBB NR, MADOULET C, TOSl P-F, BROUSSARDSDR, SNEED L, NICOLAUC, SUMMERS MD: Cell-surface expression and purification of h u m a n CD4 p r o d u c e d in baculovirus-infected insect cells. Proc Natl Acad Sci USA 1989, 86:7731-7735. Cell associated glycosylated and non-glycosylated derivatives of CD4 were expressed at low levels. 20. •
MORIKAWAY, OVERTON HA, MOORE JP, WILKINSONAJ, BRADY RL, LEWIS SJ, JONES IM Expression of HIV-1 g p l 2 0 and hum a n soluble CD4 by r e c o m b i n a n t baculoviruses and their interaction in vitro. Aids Res H u m a n Retroviruses 1990, 6:765-773. The glycosylated derivatives of baculovims-expressed soluble gpl20 and soluble CD4 interact in vitro to form complexes that resemble their natural interactions. Again, low-level expression is reported. 21. •
MOOREJP, MCKEATINGJA, JONES IM, STEPHENS PE, CLEMENTS G, THOMPSON S, WEISS RA: Characterization of recombinant g p l 2 0 and gp160 from HIV-I: binding to monoclonal antibodies and soluble CD4. AIDS 1990, 4:30~315. Expression vectors that synthesize low levels of the HIV1 gpl60 and a secreted version of gp120 are described. In addition, the antigenic and biological properties of the products are discussed. 22. •
MORIKAWA Y, OHKI K, IKUTAK, JONES IM: O n the specificity of g p l 2 0 - C D 4 binding reaction. Antiviral Chem Chemotherapy 1990, 1:73. Analyses of the H1V-1 gpl20 and CD4 baculovirus-expressed products are reported.
BA~LEYMJ, McLKEOD DA, KANG C-Y, BISHOP DHL: Glycosylation is not required for t h e fusion activity of the G protein of vesicular stomatitis virus in insect cells. Virology 1989, 169:323-331. Reports the high-level expression and phenotype of VSV glycoprotein expressed by recombinant AcNPV, including various mutant G proteins that lack one or both glycan sites.
STEINERH, POHL G, HELLERSM, EIMAMMERA, HANSSON L: Hum a n tissue-type plasminogen activator synthesized by using a baculovirus in insect cells c o m p a r e d with h u m a n plasm i n o g e n activator p r o d u c e d in m o u s e cells. Gene 1988, 73:449-457. Glycosylated tissue plasminogen activator produced by recombinant baculoviruses was secreted in the culture medium and exhibited biological activity. The level of expression was low and two forms of the glycosylated product were detected.
14. •
24. ,
13. •
PREHAUDC, TAKEHARAK, FLAMANDA, BISHOP DHL: I m m u n o genic and protective properties of rabies virus glyco-
23. •
QUELLEFW, CASLAKELF, BURKERTRE, WOJCHOWSKIDM: Highlevel expression and purification of a r e c o m b i n a n t h u m a n
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Expressionsystems erythropoietin p r o d u c e d using a baculovirus vector. Blood 1989, 74:652-657. An 890-fold purification of h u m a n erythropoietin expressed and efficiently secreted from a recombinant baculovirus is reported. Character ization of the product indicated a uniform oligosacchatide composition (30%) and contrasts with the heterogeneously glycosylated mammalian products (40-50%). 25. •
KRISHNAS, BLACKLAWSBA, OVERTON HA,. BISHOP DHL, NASH A& Expression of glycoprotein D of h e r p e s simplex virus type 1 in a r e c o m b i n a n t baculovirus: protective r e s p o n s e s and T cell recognition of the recombinant-infected cell extracts. J Gen Virol 1989, 70:1805-1814. Low-level expression and protection of mice by the herpes D glycoprotein is reported. 26. .
WATHENMW, BRIDEAU RJ, THOMSEN DR, MURPHY BR: Characterization of a novel h u m a n respiratory syncytial virus chimeric FG glycoprotein e x p r e s s e d u s e d a baculovirus vector. J Gen Virol 1989, 70:2625-2636. Secretion of a novel chimaeric construct of the h u m a n respiratory syn~ cytial vires F and G proteins is reported. The products were heterogeneous in size and may have contained O-linked glycans, although this was not determined. Cleavage of the F portion of the chimaera was indicated. OKER-BLOMC, PETTERSSONRE, SUMMERSMD: Baculovirus polyhedrin promoter-directed expression of rubella virus envelope glycoproteins, E1 and E2, in Spodoptera frugiperda cells. Virology 1989, 172:82-91. Low-level expression and proteolytic cleavage of the rubella glycoprorein precursor to the E1 and E2 proteins is reported. 27. •
28.
ASSEFFAA, SMITH SJ, NAGATA K, G1LLETrE J, GELBOIN HV, GONZALEZFJ: Novel e x o g e n o u s h e m e - d e p e n d e n t expression of mammalian c y t o c h r o m e P450 using baculovirus. Arch Biochem Biophys 1989, 274:481. Cytochrome P450 IIA1 synthesized by insect cells was activated by the addition of haem to the culture medium and then exhibited testosterone hydroxylase activity. •
29.
PENDERGASTAM, CLARKR, KAWASAKIES, McCORMICKFP, WITFE ON: Baculovirus expression of functional P210 BCR-ABL o n c o g e n e product. Oncogene 1989, 4:759-766. The tyrosine kinase product of the chronic myelogenous leukaemiaassociated P210 BCR-ABL oncogene was shown to phosphorylate angiotensin with a Km similar to that of the enzyme isolated from h u m a n sources. The kinase is capable of autophosphorylation. •
30. •
BARBERGN, CLEGG JCS, LLOYD G: Expression of the Lassa virus nucleocapsid protein in insect cells infected w i t h a recombinant baculovirus: application to diagnostic assays for Lassa virus infection. J Gen Virol 1990, 71:19-28. The usefulness of the expressed Lassa protein for diagnosis is d e m o n strated. 31. •
URAKAWAT, RITTER DG, ROY P: Expression of largest RNA s e g m e n t and synthesis o f VP1 protein of b l u e t o n g u e virus in insect cells by r e c o m b i n a n t baculovirus: association of VP1 protein with RNA polymerase activity. Nucl Acids Res 1989, 17:7395-7402. Evidence that the BTV VP1 protein is a c o m p o n e n t of the viral RNAdirected RNA polymerase is presented. 32. •
MAKINOY, TADANOM, ANZAIT, MA S-P, YASUDAS, FUKUNAGAT: Detection of d e n g u e 4 virus core protein in the nucleus. II Antibody against d e n g u e 4 core protein p r o d u c e d by a r e c o m b i n a n t baculovirus reacts w i t h t h e antigen in t h e nucleus. J Gen VirO1 1989, 70:1417-1425. Antibody raised to the expressed antigen was used to investigate the course of dengue virus infection of cells. 33. •
OVERTONHA, FUJII Y, PRICE IR, JONES IM: T h e protease and gag g e n e products of t h e h u m a n immunodeficiency virus: authentic cleavage and post-translational modification in an insect cell expression system. Virology 1989, 170:107-116.
Active HW protease expressed by recombinant baculovimses either as a polyprotein or as an independently expressed product has been demonstrated. 34. .
FRENCHTJ, INUMARUS, ROY P: Expression of two related nonstructural proteins of BTV-10 in insect cells by a recombinant baculovirus: production of polyclonal ascitic fluid and characterization o f the g e n e p r o d u c t in BTV infected BHK cells. J virol 1989, 63:3270-3278. Two forms of the NS3 protein of BTV synthesized by baculovirus vectors are described. ROY P, ADACHIA, URAKAWAT, BOOTH TF, THOMAS CP: Identification of b l u e t o n g u e virus VP6 protein as a nucleic acid binding protein and the localization of VP6 in virus infected vertebrate cells. J Virol 1990, 64:1-8. Functional analyses were undertaken of the nucleic acid binding VP6 protein of BTV expressed to high level by a baculovims vector. 35. •
36. •
MARSHALL JA, ROY P: High level expression of the two o u t e r capsid proteins o f bluetongue virus serotype 10: their relationship w i t h t h e neutralization of virus infection. Vir Res 1990, 15:189-195. The VP2 and VP5 outer capsid proteins of BTV were analysed with respect to their immunogenic properties and ability to raise neutralizing sera. 37. The
RoY P, VAN DIJK AA, ERASMUSBA: Recombinant virus vaccine for b l u e t o n g u e disease in sheep. J virol 1990, 64:1198-2003. ability of baculovirus-expressed VP2 (with or without other baculovirus-expressed BTV proteins) to dicit neutralizing antibodies and protection of sheep against virulent BTV challenge were investigated. Combinations of VP2 plus VP5 in the presence of adjuvant were the most effective. 38. .
DODSON MS, CRUTE JJ, BRUCKNER RC, LEHMAN IR: Overexpression and assembly of t h e herpes simplex virus type 1 helicase-primase in insect cells. J Biol Chem 1989, 264:20835-20838. Expression and characterization of the HSV-1 helicase-primase proteins is reported. 39.
GHEYSEND, JACOBS E, DE FRESTA F, THIRIART C, FRANCOTFE DE WILDE M: Assembly and release of HIV1 p r e c u r s o r Pr55gag virus-like particles from r e c o m b i n a n t baculovirus-infected insect cells. Cell 1989, 59:103112. Enveloped particles containing the unprocessed H1V gag gene product are released from infected cells. This property is dependent on the presence of a myristoylated N-terminal glycine of gag since absence of the N-terminal amino acids led to accumulation of particles of the cytoplasm and nucleus. Inclusion of the pol gene led to processing of the gag antigen and decreased particle secretion. Deletion of the carboxyterminal p16 sequences from gag abolished particle formation but resulted in membrane evagination. • •
M, THINES D ,
40.
URAKAWAT, GERGUSON M, MINOR PD, COOPER J, SULLIVANM, ALMONDJW, BISHOP DHL: Synthesis of immunogenic, b u t non-infectious, poliovirus particles in insect cells by a baculovirus expression vector. J Gen Virol 1989, 70:1453-1463. Low-level synthesis of intact, immunogenic but non-infectious virus-like particles of poliovims containing VPO, VP1 and VP3 is reported by expression of the 6.5 kb coding region of poliovirus cDNA. The data indicate that both viral-coded proteases are functional and support the view that inclusion of poliovirus RNA is required for the cleavage of VPO to VP2 and VP4. •,
41. , •
FRENCHTJ, ROY P: Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing t h e two major structural core proteins of BTV. J Virol 1990, 64:1530-1536. Multigene expression vectors were employed to obtain high yields of the two core proteins of BTV, VP3 and VP7. The expressed proteins self-assembled into core-like proteins which were purified by gradient centrifugation procedures.
42. •
DEGOOIJERCD, VANHERFLJ, VANDENENDEJ, VLAKJM, TRAMPERJ: A model for baculovirus production with c o n t i n u o u s insect cell cultures. Appl Microbiol Biotechnol 1989, 30:497-501,
Gene expression using insect cells and viruses A system for continuous cell production is described. KING G& DAUGUUS AJ, GOOSEN MFA, FAULKENER P, BAYLY • D: Alginate concentration: a key factor in g r o w t h of temperature-sensitive baculovirus infected insect cells in microcapsules. Biotechnol Bioeng 1989, 34:1085-1091. Encapsidation of insect cells infected with temperature sensitive baculovims allows the expression of a foreign gene at high cell density using temperature induction.
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Annotated patents
43.
44. •
MANNSG, KING LA: Efficient transfection of insect cells with baculovirus DNA using electroporation. J Gen Virol 1989, 70:35010505. This procedure has been superseded by the use of lipofectin. 45. •
PRICEPM, REICHELDERFERCF, JOHANSSON BE, KILBOURNE ED, Acs G: C o m p l e m e n t a t i o n of r e c o m b i n a n t baculoviruses by coinfection w i t h wild-type virus facilitates production of insect larvae of antigenic proteins of hepatitis B virus and influenza virus. Proc Nail Acad Sci USA 1989, 86:1453-1456_ Occluded viruses are more infective for caterpillars than non occluded viruses and can raise production levels. This procedure is superseded by the use of multiple expression vectors [7] that express polyhedrin and a foreign gene product. 46. LIVINGSTONEC, JONES I: Baculovirus expression vectors with •• single stranded capability. Nucl Acid Res 1989, 17:2366. This vector allows site-directed mutagenesis to be undertaken on singlestranded DNA (based on the pAcYM1 transfer vector) and gives a recombinant DNA that may be directly transferred into the recombinant vector.
• ••
Of interest Of outstanding interest
P1. •
INSTITUTE NAT RECH SCIENCE: ,Modified baculovirus as cxpression vector for foreign g e n e s inserted directly w i t h o u t transfer vector, contains restriction fragment u n d e r control of p r o m o t e r of baculovirus-associated protein gene. 31/5/88 88FR~007207. 6/12/89 EP 345152A. Proposes improved method for obtaining recombinants. P2. ••
NATURALENVIRON RES: N e w plasmid replicon for inserting several g e n e s into vector containing two polypeptide expression structures, and derived viral vectors for infecting insect cells. 12/8/87 87GB019108. 16/8/89 EP-327626 A. Multigene expression vector technology for baculovimses. P3. •
NIPPON ZEON KK: Production of envelope protein of Japanese encephalitis virus using recombinant baculovirus infected into insect cells. 12/5/88 88JP-115316. 15/11/89 GB2218421 A. Expressioh of Japanese encephalitis vires glycoprotein as a candidate vaccine. P4. ••
SMITHKLINEBIOLOGIC: N e w r e c o m b i n a n t DNA encoding HIV gag p r e c u r s o r protein w i t h o u t flanking sequences, exp r e s s e d in insect, yeast and mammalian cells, producing particles useful in vaccines and diagnosis. 3/6/88 88US202271. 6/12/89 EP-345242 A. HIV-1 antigens made from various expression vectors can be used as antigens for H1V diagnosis and as potential vaccines.
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