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1.
Immunogold electron microscopy and analysis were used to determine the organization of the major structural proteins of vesicular stomatitis virus (VSV) during virus assembly. We determined that matrix protein (M protein) partitions into plasma membrane microdomains in VSV-infected cells as well as in transfected cells expressing M protein. The sizes of the M-protein-containing microdomains outside the virus budding sites (50 to 100 nm) were smaller than those at sites of virus budding (approximately 560 nm). Glycoprotein (G protein) and M protein microdomains were not colocalized in the plasma membrane outside the virus budding sites, nor was M protein colocalized with microdomains containing the host protein CD4, which efficiently forms pseudotypes with VSV envelopes. These results suggest that separate membrane microdomains containing either viral or host proteins cluster or merge to form virus budding sites. We also determined whether G protein or M protein was colocalized with VSV nucleocapsid protein (N protein) outside the budding sites. Viral nucleocapsids were observed to cluster in regions of the cytoplasm close to the plasma membrane. Membrane-associated N protein was colocalized with G protein in regions of plasma membrane of approximately 600 nm. In contrast to the case for G protein, M protein was not colocalized with these areas of nucleocapsid accumulation. These results suggest a new model of virus assembly in which an interaction of VSV nucleocapsids with G-protein-containing microdomains is a precursor to the formation of viral budding sites. 相似文献
2.
Solubilization and reconstitution of vesicular stomatitis virus envelope using octylglucoside.
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M Paternostre M Viard O Meyer M Ghanam M Ollivon R Blumenthal 《Biophysical journal》1997,72(4):1683-1694
Reconstituted vesicular stomatitis virus envelopes or virosomes are formed by detergent removal from solubilized intact virus. We have monitored the solubilization process of the intact vesicular stomatitis virus by the nonionic surfactant octylglucoside at various initial virus concentrations by employing turbidity measurements. This allowed us to determine the phase boundaries between the membrane and the mixed micelles domains. We have also characterized the lipid and protein content of the solubilized material and of the reconstituted envelope. Both G and M proteins and all of the lipids of the envelope were extracted by octylglucoside and recovered in the reconstituted envelope. Fusion activity of the virosomes tested either on Vero cells or on liposomes showed kinetics and pH dependence similar to those of the intact virus. 相似文献
3.
Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis virus. 总被引:30,自引:14,他引:16
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Mixed infection of a cell by vesicular stomatitis virus (VSV) and retroviruses results in the production of progeny virions bearing the genome of one virus encapsidated by the envelope proteins of the other. The mechanism for the phenomenon of pseudotype formation is not clear, although specific recognition of a viral envelope protein by the nucleocapsid of an unrelated virus is presumably involved. In this study, we used Moloney murine leukemia virus (MoMLV)-based retroviral vectors encoding the gene for neomycin phosphotransferase to investigate the interaction between the VSV G protein and the retroviral nucleocapsid during the formation of MoMLV(VSV) pseudotypes. Our results show that VSV G protein can be incorporated into the virions of retrovirus in the absence of other VSV-encoded proteins or of retroviral envelope protein. Infection of hamster cells by MoMLV(VSV) pseudotypes gave rise to neomycin phosphotransferase-resistant colonies, and addition of anti-VSV serum to the virus preparations completely abolished the infectivity of MoMLV(VSV) pseudotypes. It should be possible to use existing mutants of VSV G protein in the system described here to identify the signals that are important for the formation of MoMLV(VSV) pseudotypes. 相似文献
4.
Role of the vesicular stomatitis virus matrix protein in maintaining the viral nucleocapsid in the condensed form found in native virions. 总被引:2,自引:16,他引:2
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Vesicular stomatitis virus was extracted with 60 mM octylglucoside in the absence of salts and in the presence of 0.5 M NaCl. The resulting extracted virus particles were examined by electron microscopy, and the proteins present were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extraction in the absence of salts yielded subviral structures which we cell "skeletons" as originally suggested by Cartwright et al. (J. Gen. Virol. 7:19-32, 1970). The skeletons contained the viral N, M, and L proteins, but they lacked the glycoprotein (G) entirely. Morphologically, the skeletons resembled intact vesicular stomatitis virus but they were slightly longer and smaller in diameter. Like native vesicular stomatitis virus, skeletons were found to have lateral striations spaced 5.0 to 6.0 nm apart along the length of the structure. In contrast to extraction in the absence of NaCl, extraction of vesicular stomatitis virus with 60 mM octylglucoside in the presence of 0.5 M NaCl yielded highly extended viral nucleocapsids in which N was the predominant protein; no M or G proteins could be detected. These results support the view that the M protein is involved in maintaining the nucleocapsid in the compact form found in native virions. 相似文献
5.
Insertion of the human immunodeficiency virus CD4 receptor into the envelope of vesicular stomatitis virus particles.
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Enveloped virus particles carrying the human immunodeficiency virus (HIV) CD4 receptor may potentially be employed in a targeted antiviral approach. The mechanisms for efficient insertion and the requirements for the functionality of foreign glycoproteins within viral envelopes, however, have not been elucidated. Conditions for efficient insertion of foreign glycoproteins into the vesicular stomatitis virus (VSV) envelope were first established by inserting the wild-type envelope glycoprotein (G) of VSV expressed by a vaccinia virus recombinant. To determine whether the transmembrane and cytoplasmic portions of the VSV G protein were required for insertion of the HIV receptor, a chimeric CD4/G glycoprotein gene was constructed and a vaccinia virus recombinant which expresses the fused CD4/G gene was isolated. The chimeric CD4/G protein was functional as shown in a syncytium-forming assay in HeLa cells as demonstrated by coexpression with a vaccinia virus recombinant expressing the HIV envelope protein. The CD4/G protein was efficiently inserted into the envelope of VSV, and the virus particles retained their infectivity even after specific immunoprecipitation experiments with monoclonal anti-CD4 antibodies. Expression of the normal CD4 protein also led to insertion of the receptor into the envelope of VSV particles. The efficiency of CD4 insertion was similar to that of CD4/G, with approximately 60 molecules of CD4/G or CD4 per virus particle compared with 1,200 molecules of VSV G protein. Considering that (i) the amount of VSV G protein in the cell extract was fivefold higher than for either CD4 or CD4/G and (ii) VSV G protein is inserted as a trimer (CD4 is a monomer), the insertion of VSV G protein was not significantly preferred over CD4 or CD4/G, if at all. We conclude that the efficiency of CD4 or CD4/G insertion appears dependent on the concentration of the glycoprotein rather than on specific selection of these glycoproteins during viral assembly. 相似文献
6.
Vesicular stomatitis virus-infected cells fuse when the intracellular pool of functional M protein is reduced in the presence of G protein.
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Five highly cytolytic strains of both Indiana and New Jersey serotypes of vesicular stomatitis virus were shown to induce cell fusion in BHK-21 and R(B77) cells. Inhibition of protein synthesis after the eclipse period of viral replication is a prerequisite for vesicular stomatitis virus-induced cell fusion. Pulse-chase experiments showed that inhibition of protein synthesis would lead to a drastic reduction in the intracellular pool of M protein as compared with other proteins. A temperature-sensitive mutant defective in M protein function (G31) was the only mutant of the five complementation groups to spontaneously induce polykaryocytes at the nonpermissive temperature. Previously, G protein has been shown to play a role in vesicular stomatitis virus-induced cell fusion. These results suggest that the combination of the presence of G protein on the virus-infected cell surface and the absence of functional M protein or a reduced level of intracellular M protein promotes cell fusion. On the basis of this study, we propose that vesicular stomatitis virus infection can induce cell fusion when the functional M protein pool declines to a critical level while G protein remains on the cell surface. 相似文献
7.
Noninfectious vesicular stomatitis virus particles deficient in the viral nucleocapsid. 总被引:6,自引:2,他引:4
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Several temperature-sensitive mutants of vesicular stomatitis virus in complementation group III produce, at nonpermissive temperature, noninfectious particles which contain the viral M (matrix) and G (glycoprotein) proteins but less than 10% of the normal proportion of N protein or RNA. Since group III mutants are thought to be defective in the structural gene for the virus M protein, these findings demonstrate that an interaction between M and the nucleocapsid is of importance in virus budding. Taken together with earlier results, they suggest that M is the key protein in bud formation. 相似文献
8.
Semliki forest virus and Sindbis virus (Alphaviruses belonging to the togavirus group) grown in BHK-21 cells possessed very low levels of virion-associated protein kinase activity. For comparison, vesicular stomatitis virus, also grown in BHK-21 cells, contained a virion-bound protein kinase which had a specific activity 80 times greater than that of the Alphaviruses. The Alphavirus protein kinase was unmasked by the nonionic detergent Nonidet P-40 but was not activated by cyclic nucleotides. Phosvitin was the best exogenous phosphate acceptor for assaying the viral enzyme in vitro. Phosphoprotein phosphatase activity was also detected in the Alphaviruses. Both in vivo and in vitro, all of the viral structural polypeptides were phosphorylated, and the phosphorylated amino acids were found to be serine and threonine. The viral nucleocapsid protein was about four times more efficient as a phosphate acceptor than were the envelope proteins. From 33 to 50% of the total protein kinase was bound to the viral nucleocapsid, and the specific activity of this enzyme was 4 to 10 times greater than that associated with the viral envelope. 相似文献
9.
Enhanced Gene Transfer with Fusogenic Liposomes Containing Vesicular Stomatitis Virus G Glycoprotein 总被引:1,自引:0,他引:1
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Exposure of Lipofectin-DNA complexes to the partially purified G glycoprotein of the vesicular stomatitis virus envelope (VSV-G) results in loss of serum-mediated inhibition and in enhanced efficiency of gene transfer. Sucrose density gradient sedimentation analysis indicated that the VSV-G associates physically with the DNA-lipid complex to produce a VSV-G liposome. The ability to incorporate surrogate viral or cellular envelope components such as VSV-G into liposomes may allow more-efficient and possibly targeted gene delivery by lipofection, both in vitro and in vivo. 相似文献
10.
Maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus. 总被引:42,自引:19,他引:23
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Maturation of viral proteins in cells infected with mutants of vesicular stomatitis virus was studied by surface iodination and cell fractionation. The movement of G, M, and N proteins to the virion bud appeared to be interdependent. Mutations thought to be in G protein prevented its migration to the cell surface, allowed neither M nor N protein to become membrane bound, and blocked formation of viral particles. Mutant G protein appeared not to leave the endoplasmic reticulum at the nonpermissive temperature, but this defect was partially reversible. In cells infected with mutants that caused N protein to be degraded rapidly or prevented its assembly into nucleocapsids, M protein did not bind to membranes and G protein matured to the cell surface, but never entered structures with the density of virions. Mutations causing M protein to be degraded prevented virion formation, and G protein behaved as in cells infected by mutants in N protein. These results are consistent with a model of virion formation involving coalescence of soluble nucleocapsid and soluble M protein with G protein already in the plasma membrane. 相似文献
11.
During measles virus (MV) replication, approximately half of the internal M and N proteins, together with envelope H and F glycoproteins, are selectively enriched in microdomains rich in cholesterol and sphingolipids called membrane rafts. Rafts isolated from MV-infected cells after cold Triton X-100 solubilization and flotation in a sucrose gradient contain all MV components and are infectious. Furthermore, the H and F glycoproteins from released virus are also partly in membrane rafts (S. N. Manié et al., J. Virol. 74:305-311, 2000). When expressed alone, the M but not N protein shows a low partitioning (around 10%) into rafts; this distribution is unchanged when all of the internal proteins, M, N, P, and L, are coexpressed. After infection with MGV, a chimeric MV where both H and F proteins have been replaced by vesicular stomatitis virus G protein, both the M and N proteins were found enriched in membrane rafts, whereas the G protein was not. These data suggest that assembly of internal MV proteins into rafts requires the presence of the MV genome. The F but not H glycoprotein has the intrinsic ability to be localized in rafts. When coexpressed with F, the H glycoprotein is dragged into the rafts. This is not observed following coexpression of either the M or N protein. We propose a model for MV assembly into membrane rafts where the virus envelope and the ribonucleoparticle colocalize and associate. 相似文献
12.
Vesicular stomatitis virus was disrupted by a combination of freezing and thawing, osmotic shock, and sonic treatment. Subviral components were separated by isopycnic centrifugation. The low-density, lipid-rich fractions were pooled and shown to contain primarily viral glycoprotein. Further purification of this material resulted in the isolation of a preparation of vesicles which contained only the G protein and the same phospholipids as in the intact virions and exhibited spikelike structures similar to those on intact vesicular stomatitis virions. We conclude that we have isolated fragments of native vesicular stomatitis virus envelopes. 相似文献
13.
Cytoplasmic Compartmentalization of the Protein and Ribonucleic Acid Species of Vesicular Stomatitis Virus 总被引:23,自引:23,他引:0
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Robert R. Wagner Michael P. Kiley Ruth M. Snyder Carl A. Schnaitman 《Journal of virology》1972,9(4):672-683
The cytoplasmic sites of synthesis in L cells of the protein and ribonucleic acid species of vesicular stomatitis virus were studied by polyacrylamide gel electrophoresis after fractionation of membrane and other cytoplasmic components by the Caliguiri-Tamm technique. The viral spike protein (glycoprotein G) was found primarily associated with a smooth membrane fraction which is rich in plasma membrane; the G protein was also present in fractions containing rough endoplasmic reticulum. The nonglycosylated envelope protein S (also called M) was found in the smooth membrane fractions but was more abundant in endoplasmic reticulum-enriched fractions. Longer labeling resulted in detection of nucleoprotein N, as well as other minor nucleocapsid proteins L and NS1, in the cellular membrane fractions. The N protein appeared to be made in membrane-free cytoplasm along with progeny ribonucleic acid and later became associated with membrane containing G and S viral proteins. 相似文献
14.
Organization of the vesicular stomatitis virus glycoprotein into membrane microdomains occurs independently of intracellular viral components 总被引:2,自引:0,他引:2
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The glycoprotein (G protein) of vesicular stomatitis virus (VSV) is primarily organized in plasma membranes of infected cells into membrane microdomains with diameters of 100 to 150 nm, with smaller amounts organized into microdomains of larger sizes. This organization has been observed in areas of the infected-cell plasma membrane that are outside of virus budding sites as well as in the envelopes of budding virions. These observations raise the question of whether the intracellular virion components play a role in organizing the G protein into membrane microdomains. Immunogold-labeling electron microscopy was used to analyze the distribution of the G protein in arbitrarily chosen areas of plasma membranes of transfected cells that expressed the G protein in the absence of other viral components. Similar to the results with virus-infected cells, the G protein was organized predominantly into membrane microdomains with diameters of approximately 100 to 150 nm. These results indicate that internal virion components are not required to concentrate the G protein into membrane microdomains with a density similar to that of virus envelopes. To determine if interactions between the G protein cytoplasmic domain and internal virion components were required to create a virus budding site, cells infected with recombinant VSVs encoding truncation mutations of the G protein cytoplasmic domain were analyzed by immunogold-labeling electron microscopy. Deletion of the cytoplasmic domain of the G protein did not alter its partitioning into the 100- to 150-nm microdomains, nor did it affect the incorporation of the G protein into virus envelopes. These data support a model for virus assembly in which the G protein has the inherent property of partitioning into membrane microdomains that then serve as the sites of assembly of internal virion components. 相似文献
15.
Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes. 总被引:21,自引:1,他引:20
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H Vennema G J Godeke J W Rossen W F Voorhout M C Horzinek D J Opstelten P J Rottier 《The EMBO journal》1996,15(8):2020-2028
Budding of enveloped viruses has been shown to be driven by interactions between a nucleocapsid and a proteolipid membrane. By contrast, we here describe the assembly of viral envelopes independent of a nucleocapsid. Membrane particles containing coronaviral envelope proteins were assembled in and released from animal cells co-expressing these proteins' genes from transfected plasmids. Of the three viral membrane proteins only two were required for particle formation, the membrane glycoprotein (M) and the small envelope protein (E). The spike (S) protein was dispensable but was incorporated when present. Importantly, the nucleocapsid protein (N) was neither required not taken into the particles when present. The E protein, recently recognized to be a structural protein, was shown to be an integral membrane protein. The envelope vesicles were found by immunogold labelling and electron microscopy to form a homogeneous population of spherical particles indistinguishable from authentic coronavirions in size (approximately 100 nm in diameter) and shape. They were less dense than virions and sedimented slightly slower than virions in sucrose velocity gradients. The nucleocapsid-independent formation of apparently bona fide viral envelopes represents a novel mode of virus assembly. 相似文献
16.
Assembly of the coronavirus envelope: homotypic interactions between the M proteins 总被引:1,自引:0,他引:1
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The viral membrane proteins M and E are the minimal requirements for the budding of coronavirus particles. Since the E protein occurs in particles only in trace amounts, the lateral interactions between the M proteins apparently generate the major driving force for envelope formation. By using coimmunoprecipitation and envelope incorporation assays, we provide extensive evidence for the existence of such M-M interactions. In addition, we determined which domains of the M protein are involved in this homotypic association, using a mutagenetic approach. Mutant M proteins which were not able to assemble into viruslike particles (VLPs) by themselves (C. A. M. de Haan, L. Kuo, P. S. Masters, H. Vennema, and P. J. M. Rottier, J. Virol. 72:6838-6850, 1998) were tested for the ability to associate with other M proteins and to be rescued into VLPs formed by assembly-competent M proteins. We found that M proteins lacking parts of the transmembrane cluster, of the amphipathic domain, or of the hydrophilic carboxy-terminal tail, or M proteins that had their luminal domain replaced by heterologous ectodomains, were still able to associate with assembly-competent M proteins, resulting in their coincorporation into VLPs. Only a mutant M protein in which all three transmembrane domains had been replaced lost this ability. The results indicate that M protein molecules interact with each other through multiple contact sites, particularly at the transmembrane level. Finally, we tested the stringency with which membrane proteins are selected for incorporation into the coronavirus envelope by probing the coassembly of some foreign proteins. The observed efficient exclusion from budding of the vesicular stomatitis virus G protein and the equine arteritis virus M protein indicates that envelope assembly is indeed a highly selective sorting process. The low but detectable incorporation of CD8 molecules, however, demonstrated that this process is not perfect. 相似文献
17.
An enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late Golgi compartments. 总被引:42,自引:5,他引:37
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The expression of viral envelope proteins on the plasma membrane domains of the epithelial cell line, MDCK, is polar. Influenza virus infection of these cells leads to expression of the viral haemagglutinin and neuraminidase glycoproteins on the apical domain of the plasma membrane while vesicular stomatitis virus (VSV) infection yields basolateral expression of the sialic acid-bearing G protein. We have exploited the ability of the influenza neuraminidase to desialate the G protein of VSV to test for contact between these proteins during their intracellular transport to separate plasma membrane domains. We were able to select for VSV-G protein expression in doubly-infected cells because VSV protein production was accelerated in cells pre-infected with influenza virus. During double infection the envelope proteins of both viruses displayed the same polar localization as during single infection but the VSG-G protein was undersialated due to the action of the influenza neuraminidase. Incubation of singly-infected cells at 20 degrees C blocked the transport of VSV-G protein to the cell surface and resulted in increased sialation of the protein over that seen at 37 degrees C. This suggests that G protein is held in contact with the sialyl transferase at this temperature. 20 degrees C incubations of doubly-infected cells also produced the undersialated G protein characteristic of interaction with the neuraminidase. We conclude that most of the newly synthesised basolaterally-directed G protein is in physical contact with the majority of the neuraminidase through the terminal steps of Golgi processing. 相似文献
18.
Jiang P Liu Y Yin X Yuan F Nie Y Luo M Aihua Z Liyin D Ding M Deng H 《Biochemical and biophysical research communications》2006,339(2):526-532
Recombinant viral vectors are useful tools for AIDS vaccine development. However, expression of HIV-1 envelope genes using viral vectors has not been successful in the induction of potent neutralizing antibodies in vivo. We took advantage of the strong immunogenicity of vesicular stomatitis virus (VSV)-based vector and expressed HIV-1 HXB2 gp120 gene in the recombinant VSV. Our results showed that HIV-1 gp120 protein expressed by the recombinant VSV retained the native conformation of the protein to some degree and was recognized by two well-characterized broad anti-HIV-1 neutralizing monoclonal antibodies b12, 2G12. We further showed that only one time intranasal immunization with the recombinant VSV led to production of anti-HIV-1 anti-sera in mice. In addition, we found that the anti-sera had the ability to neutralize not only HXB2 envelope-pseudotyped HIV-1 viruses but also HIV-1 pseudotyped viruses with JRFL envelopes. These results suggest that HIV-1 gp120 expressed by the recombinant VSV, in combination with the route of intranasal administration, is an effective strategy to evaluate the immunogenicity of HIV-1 envelope protein and its variants in mice. 相似文献
19.
Dynamic nature of the quaternary structure of the vesicular stomatitis virus envelope glycoprotein 总被引:6,自引:0,他引:6
The envelope glycoprotein (G protein) of vesicular stomatitis virus probably exists in the viral envelope as a trimer of identical subunits. Depending on the conditions of solubilization, G protein may dissociate into monomers. G protein solubilized with the detergent octyl glucoside was shown to exist as oligomeric forms by sedimentation velocity analysis and chemical cross-linking. G protein was modified with either fluorescein isothiocyanate or rhodamine isothiocyanate. Resonance energy transfer between fluorescein and rhodamine labels was observed upon mixing the two labeled G proteins in octyl glucoside. This result provided further evidence that G protein in octyl glucoside is oligomeric and indicated that the subunits are capable of exchange to form mixed oligomers. Resonance energy transfer was independent of G protein concentration in the range examined (10-80 nM) and was not observed when labeled G proteins were mixed with fluorescein or rhodamine that was not conjugated to protein. Resonance energy transfer decreased upon incorporation of G protein into Triton X-100, consistent with sedimentation velocity data that G protein in Triton X-100 is primarily monomeric. Kinetic analysis showed that the subunit exchange reaction had a half-time of about 3 min at 27 degrees C that was independent of G protein concentration. These data indicate that the exchange occurs through dissociation of G protein trimers into monomers and dimers followed by reassociation into timers. Thus, in octyl glucoside, G protein must exist as an equilibrium between monomers and oligomers. This implies that monomers are capable of self-assembly into trimers. 相似文献
20.
Early steps in the assembly of vesicular stomatitis virus nucleocapsids in infected cells. 总被引:2,自引:1,他引:1
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The assembly of nucleocapsids is an essential step in the replicative cycle of vesicular stomatitis virus (VSV). In this study, we have examined the early events of vesicular stomatitis virus nucleocapsid assembly in BHK-21 cells. Nuclease-resistant intracellular nucleocapsids were isolated at various stages of assembly and analyzed for RNA and protein contents. The smallest ribonucleoprotein complex formed during nucleocapsid assembly contains the 5'-terminal 65 nucleotides of nascent viral RNA complexed with the viral proteins N and NS. Elongation of the assembling nucleocapsids proceeds unidirectionally towards the 3' terminus by the sequential addition of viral proteins which incrementally protect short stretches of the growing RNA chain. Pulse-chase studies show that the assembling nucleocapsids can be chased into full-length nucleocapsids which are incorporated into mature virions. Our results also suggest an involvement of the cytoskeletal framework during nucleocapsid assembly. 相似文献