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1.
Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of BTV. 总被引:19,自引:15,他引:4
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The L3 and M7 genes of bluetongue virus (BTV), which encode the two major core proteins of the virus (VP3 and VP7, respectively), were inserted into a baculovirus dual-expression transfer vector and a recombinant baculovirus expressing both foreign genes isolated following in vivo recombination with wild-type Autographa californica nuclear polyhedrosis virus DNA. Spodoptera frugiperda insect cells infected with the recombinant synthesized large amounts of BTV corelike particles. These particles have been shown to be similar to authentic BTV cores in terms of size, appearance, stoichiometric arrangement of VP3 to VP7 (ratio, 2:15), and the predominance of VP7 on the surface of the particles. In infected insect cells, the corelike particles were observed in paracrystalline arrays. The formation of these structures indicates that neither the BTV double-stranded viral RNA species nor the associated minor core proteins are necessary for assembly of cores in insect cells. Furthermore, the three BTV nonstructural proteins NS1, NS2, and NS3, are not required to assist or direct the formation of empty corelike particles from VP3 and VP7. 相似文献
2.
《Gene》1997,190(1):119-129
The baculovirus expression vector is a eukaryotic DNA viral vector for the cloning and expression of foreign genes in cultured lepidopteran insect cells and insects. It has become an important tool for the large-scale production of recombinant proteins for a variety of applications including the structure-function analysis of genes and their gene products. We have developed a number of baculovirus multigene expression vectors and utilized these to understand the assembly process of multicomponent capsid structures of large viruses such as bluetongue virus (BTV), a member of the Orbivirus genus within the family Reoviridae. BTV is some 810 Å in diameter and comprised of two protein shells containing four major proteins, VP2, VP5, VP7 and VP3, surrounding a genome of ten double-stranded RNA segments and three minor proteins (VP2, VP4 and VP6). BTV is the etiological agent of a sheep disease that is sometimes fatal in certain parts of the world (e.g., Africa, Asia, and the Americas). Using baculovirus multigene vectors, we have co-expressed various combinations of BTV genes in insect cells and produced structures that mimic the various stages of BTV assembly. For example, co-expressed VP3 and VP7 form BTV core-like particles, while co-expressed VP2, VP5, VP7 and VP3 form BTV virus-like particles. Using deletion, point and domain switching analyses of each protein, we have been able to identify certain sequences in the VP7 and VP3 proteins that are essential for the assembly of core-like particles. These expression and biochemical studies have been complemented by collaboration studies using cryoelectron microscopy and image processing analyses to provide the three-dimensional structure of the expressed particles. In addition and with other associates, we have used X-ray crystallography of VP7 to deduce its atomic structure. Extensive studies on the immune responses elicited by these self-assembled particles, and chimeric derivatives involving various foreign antigens, have been carried out. Finally, using as little as 10 μg of the self-assembled virus-like particles, we have shown that they can confer long-lasting protection in sheep against BTV. 相似文献
3.
Assembly of double-shelled, viruslike particles of bluetongue virus by the simultaneous expression of four structural proteins. 总被引:9,自引:7,他引:2
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Bluetongue is a disease of ruminants. The etiologic agent is bluetongue virus (BTV), a gnat-transmitted member of the Orbivirus genus of the Reoviridae. The virus has a genome of 10 double-stranded RNA species L1 to L3, M4 to M6, S7 to S10). The L2 and M5 genes of BTV which encode the outer capsid proteins VP2 and VP5, respectively, were inserted into a recombinant baculovirus downstream of duplicated copies of the baculovirus polyhedrin promoter. Insect cells coinfected with this virus plus a recombinant baculovirus expressing the two major core proteins VP3 and VP7 of BTV (T.J. French and P. Roy, J. Virol. 64:1530-1536, 1990) synthesized noninfectious, double-shelled, viruslike particles. When purified, these particles were found to have the same size and appearance as authentic BTV virions and exhibited high levels of hemagglutination activity. Antibodies raised to the expressed particles contained high titers of neutralizing activity against the homologous BTV serotype. The assembly of these bluetongue viruslike particles after the simultaneous expression of four separate proteins is indicative of the potential of this technology for the production of a new generation of viral vaccines and for the study of complex, multiprotein structures. 相似文献
4.
Darpel KE Langner KF Nimtz M Anthony SJ Brownlie J Takamatsu HH Mellor PS Mertens PP 《PloS one》2011,6(3):e17545
Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.). The larger of the BTV outer-capsid proteins, 'VP2', can be cleaved by proteases (including trypsin or chymotrypsin), forming infectious subviral particles (ISVP) which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis). We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector), cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent/non-vector species). Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ~10 fold, while infectivity for BHK cells was reduced by 2-6 fold. Treatment of an 'eastern' strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a 'western' strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species), can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to increased infectivity specifically for Culicoides cells and, in turn, efficiency of transmission to the insect vector. 相似文献
5.
Identification of domains in bluetongue virus VP3 molecules essential for the assembly of virus cores. 总被引:2,自引:1,他引:1
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Bluetongue virus (BTV) cores consist of the viral genome and five proteins, including two major components (VP3 and VP7) and three minor components (VP1, VP4, and VP6). VP3 proteins form an inner scaffold for the deposition on the core of the surface layer of VP7. VP3 also encapsidates and interacts with the three minor proteins. The BTV VP3 protein consists of 901 amino acids and has a sequence that is a highly conserved among BTV serotypes and other orbiviruses (e.g., epizootic hemorrhagic disease virus and African horse sickness virus). To locate sites of interaction between VP3 and the other structural proteins, we have analyzed the effects of a number of VP3 deletion mutants representing conserved regions of the protein, using as an assay the formation of core-like particles (CLPs) expressed by recombinant baculoviruses. Five of the VP3 deletion mutants interacted with the coexpressed VP7 and made CLPs. These CLPs also incorporated the three minor proteins. One mutant, lacking VP3 amino acid residues 499 to 508, failed to make CLPs. Further mutational analyses have demonstrated that a methionine at residue 500 of VP3 and an arginine at residue 502 were both required for CLP formation. 相似文献
6.
Identification of bluetongue virus VP6 protein as a nucleic acid-binding protein and the localization of VP6 in virus-infected vertebrate cells. 总被引:2,自引:8,他引:2
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Recently the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has been effectively adapted as a highly efficient vector in insect cells for the expression of various genes. A cDNA sequence of RNA segment 9 of bluetongue virus serotype 10 (BTV-10, an orbivirus member of the Reoviridae family) encoding a minor core protein (VP6) has been inserted into the BamHI site of the pAcYM1 transfer vector derived from AcNPV. Spodoptera frugiperda cells were cotransfected with the derived vector in the presence of authentic AcNPV DNA to produce recombinant viruses. These synthesized significant amounts of a protein (representing ca. 50% of the stained cellular protein) similar in size and antigenicity to the authentic BTV VP6. The expressed protein was identified as a nucleic acid-binding protein by using an RNA overlay-protein blot assay. A polyclonal anti-VP6 serum prepared by using the expressed VP6 protein has been used in an immunogold procedure to locate VP6 in BTV-infected mammalian cells. Gold was found to be associated with the matrix of virus inclusion bodies (VIB), with viruslike particles in the VIB, as well as with mature virion particles that were in close proximity to the VIB or were released from cells and adsorbed to cell surfaces. The recombinant virus antigen has also been used to identify antibodies to different BTV serotypes in infected sheep sera, indicating the potential of the expressed protein as a group-reactive antigen for the diagnosis of BTV infections. 相似文献
7.
8.
The complete VP2 gene of bovine rotavirus strain RF has been inserted into the baculovirus transfer vector pVL941 under the control of the polyhedrin promoter. Cotransfection of Spodoptera frugiperda 9 cells with wild-type baculovirus DNA and transfer vector DNA led to the formation of recombinant baculoviruses which contain bovine rotavirus gene 2. Infection of S. frugiperda cells with this recombinant virus resulted in the production of a protein similar in size and antigenic properties to the authentic rotavirus VP2. The protein binds double-stranded RNA and DNA in an overlay protein blot assay. Expressed VP2 assembles in the cytoplasm of infected cells in corelike particles 45 nm in diameter. These corelike particles were purified by sucrose gradient centrifugation and found to be devoid of nucleic acid. Coexpression of VP2 and VP6 from heterologous rotavirus strains (bovine and simian) resulted in the formation of single-shelled particles. These results definitively show the existence of an innermost protein shell in rotavirus which is formed independently of other rotavirus proteins. These results have implications for schemes of rotavirus morphogenesis. 相似文献
9.
Van Dijk AA 《Biotechnology advances》1993,11(1):1-12
Bluetongue virus is the aetiological agent of bluetongue, a disease of domestic and wild ruminants. Twenty-four serotypes are recognized. Novel subunit vaccines, that complement existing modified live polyvalent vaccines, are being developed. Serotype-specific viral neutralizing antibodies that are able to protect sheep against virulent homologous virus challenge can be induced by immunizing with the BTV outer capsid protein VP2 purified from virions or with VP2 expressed by baculovirus recombinants. Presentation of VP2 on virus-like particles, which assemble upon co-expression of the four major structural viral proteins (VP2, VP5, VP3 and VP7), improves the protective effect of VP2. Sheep immunized with core-like particles, comprised of VP3 and VP7, developed only limited clinical signs after virulent virus challenge, demonstrating that not only the outer capsid proteins, but also the core proteins are involved in protection against bluetongue. 相似文献
10.
The bluetongue virus (BTV) core protein VP3 plays a crucial role in the virion assembly and replication process. Although the structure of the protein is well characterized, much less is known about the intracellular processing and localization of the protein in the infected host cell. In BTV-infected cells, newly synthesized viral core particles accumulate in specific locations within the host cell in structures known as virus inclusion bodies (VIBs), which are composed predominantly of the nonstructural protein NS2. However, core protein location in the absence of VIBs remains unclear. In this study, we examined VP3 location and degradation both in the absence of any other viral protein and in the presence of NS2 or the VP3 natural associate protein, VP7. To enable real-time tracking and processing of VP3 within the host cell, a fully functional enhanced green fluorescent protein (EGFP)-VP3 chimera was synthesized, and distribution of the fusion protein was monitored in different cell types using specific markers and inhibitors. In the absence of other BTV proteins, EGFP-VP3 exhibited distinct cytoplasmic focus formation. Further evidence suggested that EGFP-VP3 was targeted to the proteasome of the host cells but was dispersed throughout the cytoplasm when MG132, a specific proteasome inhibitor, was added. However, the distribution of the chimeric EGFP-VP3 protein was altered dramatically when the protein was expressed in the presence of the BTV core protein VP7, a normal partner of VP3 during BTV assembly. Interaction of EGFP-VP3 and VP7 and subsequent assembly of core-like particles was further examined by visualizing fluorescent particles and was confirmed by biochemical analysis and by electron microscopy. These data indicated the correct assembly of EGFP-VP3 subcores, suggesting that core formation could be monitored in real time. When EGFP-VP3 was expressed in BTV-infected BSR cells, the protein was not associated with proteasomes but instead was distributed within the BTV inclusion bodies, where it colocalized with NS2. These findings expand our knowledge about VP3 localization and its fate within the host cell and illustrate the assembly capability of a VP3 molecule with a large amino-terminal extension. This also opens up the possibility of application as a delivery system. 相似文献
11.
The oligomerization domain of VP3, the scaffolding protein of infectious bursal disease virus,plays a critical role in capsid assembly 总被引:1,自引:0,他引:1
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Maraver A Oña A Abaitua F González D Clemente R Ruiz-Díaz JA Castón JR Pazos F Rodriguez JF 《Journal of virology》2003,77(11):6438-6449
Infectious bursal disease virus (IBDV) capsids are formed by a single protein layer containing three polypeptides, pVP2, VP2, and VP3. Here, we show that the VP3 protein synthesized in insect cells, either after expression of the complete polyprotein or from a VP3 gene construct, is proteolytically degraded, leading to the accumulation of product lacking the 13 C-terminal residues. This finding led to identification of the VP3 oligomerization domain within a 24-amino-acid stretch near the C-terminal end of the polypeptide, partially overlapping the VP1 binding domain. Inactivation of the VP3 oligomerization domain, by either proteolysis or deletion of the polyprotein gene, abolishes viruslike particle formation. Formation of VP3-VP1 complexes in cells infected with a dual recombinant baculovirus simultaneously expressing the polyprotein and VP1 prevented VP3 proteolysis and led to efficient virus-like particle formation in insect cells. 相似文献
12.
Crystal structure of the top domain of African horse sickness virus VP7: comparisons with bluetongue virus VP7. 总被引:2,自引:1,他引:1
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The baculovirus-expressed core protein VP7 of African horse sickness virus serotype 4 (AHSV-4) has been purified to homogeneity and crystallized in the presence of 2.8 M urea. The X-ray structure has been solved to a 2.3-Angstroms (1 Angstrom = 0.1 nm) resolution with an Rfactor of 19.8%. The structure of AHSV VP7 reveals that during crystallization, the two-domain protein is cleaved and only the top domain remains. A similar problem was encountered previously with bluetongue virus (BTV) VP7 (whose structure has been reported), showing that the connections between the top and the bottom domains are rather weak for these two distinct orbiviruses. The top domains of both BTV and AHSV VP7 are trimeric and structurally very similar. The electron density maps show that they both possess an extra electron density feature along their molecular threefold axes, which is most likely due to an unidentified ion. The characteristics of the molecular surface of BTV and AHSV VP7 suggest why AHSV VP7 is much less soluble than BTV VP7 and indicate the possibility of attachment to the cell via attachment of an Arg-Gly-Asp (RGD) motif in the top domain of VP7 to a cellular integrin for both of these orbiviruses. 相似文献
13.
DNA encapsidation by viruslike particles assembled in insect cells from the major capsid protein VP1 of B-lymphotropic papovavirus. 总被引:5,自引:2,他引:3
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Capsids of polyomaviruses--small, nonenveloped DNA viruses--consist of the major structural protein VP1 and the minor structural proteins VP2 and VP3. The contributions of the individual capsid proteins to functions of the viral particle, such as DNA encapsidation, cell receptor attachment, entry, and uncoating, are still not clear. Here we show that viruslike particles assembled in nuclei of insect cells from VP1 of the monkey B-lymphotropic papovavirus (LPV) are sufficient to unspecifically encapsidate DNA. LPV VP1 expressed in large amounts in insect cells by a baculovirus vector assembled spontaneously in the nuclei to form viruslike particles. After metrizamide equilibrium density gradient purification and nuclease digestion, a fraction of these particles was shown to contain VP1-associated linear, double-stranded DNA with a predominant size of 4.5 kb. The fraction of DNA-containing VP1 particles increased with time and dose of baculovirus vector infection. The DNA-containing particles, further purified by sucrose gradient centrifugation, appeared as "full" particles in negative-staining electron microscopy. As shown by DNA hybridization, the encapsidated DNA consisted of insect cell and baculoviral sequences with no apparent strong homology to LPV sequences. Three non-LPV VP1-derived host proteins with apparent molecular masses of approximately 14, 15, and 16 kDa copurified with the DNA-containing particles and may represent insect cell histones encapsidated together with the DNA. A similar species of host DNA was also found in purified LPV wild-type virions. These data suggest that LPV VP1 alone can be sufficient to encapsidate linear DNA in a sequence-independent manner. 相似文献
14.
The hepatitis A virus polyprotein expressed by a recombinant vaccinia virus undergoes proteolytic processing and assembly into viruslike particles.
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Hepatitis A virus (HAV) contains a single-stranded, plus-sense RNA genome with a single long open reading frame encoding a polyprotein of approximately 250 kDa. Viral structural proteins are generated by posttranslational proteolytic processing of this polyprotein. We constructed recombinant vaccinia viruses which expressed the HAV polyprotein (rV-ORF) and the P1 structural region (rV-P1). rV-ORF-infected cell lysates demonstrated that the polyprotein was cleaved into immunoreactive 29- and 33-kDa proteins which comigrated with HAV capsid proteins VP0 and VP1. The rV-P1 construct produced a 90-kDa protein which showed no evidence of posttranslational processing. Solid-phase radioimmunoassays with human polyclonal anti-HAV sera and with murine or human neutralizing monoclonal anti-HAV antibodies recognized the rV-ORF-infected cell lysates. Sucrose density gradients of rV-ORF-infected cell lysates contained peaks of HAV antigen with sedimentation coefficients of approximately 70S and 15S, similar to those of HAV empty capsids and pentamers. Immune electron microscopy also demonstrated the presence of viruslike particles in rV-ORF-infected cell lysates. Thus, the HAV polyprotein expressed by a recombinant vaccinia virus demonstrated posttranslational processing into mature capsid proteins which assembled into antigenic viruslike particles. 相似文献
15.
Virus-like particles (VLPs) are empty particles consisting of virus capsid proteins that closely resemble native virus but are devoid of the native viral nucleic acids and therefore have attracted significant attention as noninfectious vaccines. A recombinant baculovirus, vIBD-7, which encodes the structural proteins (VP2, VP3, and VP4) of infectious bursal disease virus (IBDV), produces native IBD VLPs in infected Spodoptera frugiperda insect cells. Another baculovirus, vEDLH-22, encodes VP2 that is fused with a histidine affinity-tag (VP2H) at the C-terminus. By co-infection with these two baculoviruses, hybrid VLPs with histidine tags were formed and purified by immobilized metal affinity chromatography (Hu et al., 1999). Also, we demonstrated that varying the MOI ratio of these infecting viruses altered the extent of VP2H incorporated into the particles. A dynamic mathematical model that described baculovirus infection and VLP synthesis (Hu and Bentley, 2000) was adapted here for co-infection and validated by immunofluorescence labeling. It was shown to predict the VLP composition as a dynamic function of MOI. A constraint in the VP2H content incorporated into the particles was predicted and shown by experiments. Also, the MOI ratio of both infecting viruses was shown to be the major factor influencing the composition of the hybrid particles and an important factor in determining the overall yield. ELISA results confirmed that VP2H was exhibited to a varied extent on the outer surface of the particles. This model provides insight on the use of virus co-infection in virus-mediated recombinant protein expression systems and aids in the optimization of chimeric VLP synthesis. 相似文献
16.
The virion of Leishmania RNA virus is predicted to be composed of a 742-amino-acid major capsid protein and a small percentage of capsid-polymerase fusion molecules. Recently, the capsid protein alone was expressed and shown to spontaneously assemble into viruslike particles. Since the major structural protein of the virion shell self-assembles into viruslike particles when expressed in the baculovirus expression system, assembly of the virion can be studied by mutational analysis and expression of a single open reading frame. In this study, several deletions and one addition of the capsid protein of Leishmania RNA virus LRV1-4 were generated. These mutants show different degrees of assembly. Assembly domains are being identified such that the capsid protein may be used as a macromolecular packaging and delivery system for Leishmania species. 相似文献
17.
P Roy 《FEMS microbiology immunology》1990,2(4):223-234
The productivity and flexibility of insect baculovirus expression vectors and the ability of the baculovirus genome to incorporate (and express) large amounts of foreign DNA allows this system to be used for both single and multiple gene expression. Using the system, bluetongue virus (BTV) genes have been expressed to develop diagnostic reagents and vaccines as well as to understand the basic structures of the virions. BTV which causes disease in ruminants in many parts of the world, consists of 10 double-stranded RNA segments enclosed by double capsids that are composed by 7 structural proteins. Since each protein is encoded by a single RNA species, DNA clones of all 10 RNA species were synthesized and individually expressed in baculovirus vectors at high levels. This has yielded proteins that have been shown to be excellent diagnostic and vaccine reagents. In addition, multiple expression vectors have been used to synthesize morphological structures (viral and subviral) representing BTV. 相似文献
18.
Use of baculovirus expression vectors: development of diagnostic reagents, vaccines and morphological counterparts of bluetongue virus 总被引:1,自引:0,他引:1
Polly Roy 《FEMS microbiology letters》1990,64(4):223-234
Abstract The productivity and flexibility of insect baculovirus expression vectors and the ability of the baculovirus genome to incorporate (and express) large amounts of foreign DNA allows this system to be used for both single and multiple gene expression. Using the system, bluetongue virus (BTV) genes have been expressed to develop diagnostic reagents and vaccines as well as to understand the basic structures of virions. BTV which causes disease in ruminants in many parts of the world, consists of 10 double-stranded RNA segments enclosed by double capsids that are composed by 7 structural proteins. Since each protein is encoded by a single RNA species, DNA clones of all 10 RNA species were synthesized and individually expressed in baculovirus vectors at high levels. This has yielded proteins that have been shown to be excellent diagnostic and vaccine reagents. In addition, multiple expression vectors have been used to synthesize morphological structures (viral and subviral) representing BTV. 相似文献
19.
A filovirus-unique region of Ebola virus nucleoprotein confers aberrant migration and mediates its incorporation into virions
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Shi W Huang Y Sutton-Smith M Tissot B Panico M Morris HR Dell A Haslam SM Boyington J Graham BS Yang ZY Nabel GJ 《Journal of virology》2008,82(13):6190-6199
The Ebola virus nucleoprotein (NP) is an essential component of the nucleocapsid, required for filovirus particle formation and replication. Together with virion protein 35 (VP35) and VP24, this gene product gives rise to the filamentous nucleocapsid within transfected cells. Ebola virus NP migrates aberrantly, with an apparent molecular mass of 115 kDa, although it is predicted to encode an approximately 85-kDa protein. In this report, we show that two domains of this protein determine this aberrant migration and that this region mediates its incorporation into virions. These regions, amino acids 439 to 492 and amino acids 589 to 739, alter the mobility of Ebola virus NP by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by 5 and 15 kDa, respectively, and confer similar effects on a heterologous protein, LacZ, in a position-independent fashion. Furthermore, when coexpressed with VP40, VP35, and VP24, this region mediated incorporation of NP into released viruslike particles. When fused to chimeric paramyxovirus NPs derived from measles or respiratory syncytial virus, this domain directed these proteins into the viruslike particle. The COOH-terminal NP domain comprises a conserved highly acidic region of NP with predicted disorder, distinguishing Ebola virus NPs from paramyxovirus NPs. The acidic character of this domain is likely responsible for its aberrant biochemical properties. These findings demonstrate that this region is essential for the assembly of the filamentous nucleocapsids that give rise to filoviruses. 相似文献
20.
The three capsid proteins VP1, VP2, and VP3 of the adeno-associated virus type 2 (AAV-2) are encoded by overlapping sequences of the same open reading frame. Separate expression of these proteins by recombinant baculoviruses in insect cells was achieved by mutation of the internal translation initiation codons. Coexpression of VP1 and VP2, VP2 and VP3, and all three capsid proteins and the expression of VP2 alone in Sf9 cells resulted in the production of viruslike particles resembling empty capsids generated during infection of HeLa cells with AAV-2 and adenovirus. These results suggest a requirement for VP2 in the formation of empty capsids. Individual expression of the AAV capsid proteins in HeLa cells showed that VP1 and VP2 accumulate in the cell nucleus and VP3 is distributed between nucleus and cytoplasm. Coexpression of VP3 with the other structural proteins also led to nuclear localization of VP3, indicating that the formation of a complex with VP1 or VP2 is required for accumulation of VP3 in the nucleus. 相似文献