Factors involved in the generation and replication of rhabdovirus defective T particles.

Previous indications that cloned B virions might be genetically predisposed to generate a particular defective T particle are shown to be inaccurate. T particle generation was found to be a much more random process than was previously believed. We show that the previously observed generation of particular sizes of T particles by B virion pools is due to the random generation of T particles during preparation of first-passage pools of cloned B virions, and these breed true during the additional passages needed to produce visible quantities of T particles. It is also shown that different host cell lines selectively amplify different T particles, suggesting a strong role of host cell factors in T particle replication. Surprisingly, our line of HeLa cells did not generate or replicate detectable T particles of vesicular stomatitis virus (VSV) Indiana after either serial undiluted passage or direct addition of T particles, even though the added T particles strongly interfered with B virion replication. In contrast to VSV, rabies virus generates large amounts of T particles during the first passage of cloned B virions, and every rabies-infected baby hamster kidney-21 cell culture evolves into a persistent carrier state. We find that T particle RNA is biologically inactive although T particle nucleocapsid ribonucleoprotein replicates and interferes in cells coinfected with B virions. Efforts to study the mechanism of T particle generation by in vitro attempts to generate T particles or modify their size (using sheared ribonucleoprotein or chemical or UV mutagenesis) were unsuccessful. The kinetics of UV and nitrous acid inactivation of T particles indicate a smaller target size relative to B virions, even after correcting for lengths of RNA molecules. The intercalating dye proflavine does not photosensitize VSV B virions or T particles when present during replication, indicating that there is little or no RNA base pairing in the helical nucleocapsids of either.     

Specific incorporation of host cell surface proteins into budding vesicular stomatitis virus particles

The specific incorporation of cell surface proteins into budding Vesicular Stomatitis Virus (VSV) particles was shown by two approaches. In the first, monolayer cultures of Vero or L cells were labeled by lactoperoxidase-catalyzed iodination and the cells were then infected with VSV. Approximately 2% of the cell surface ¹²⁵1 radioactivity was incorporated into particles which co-purify with normal, infectious virions by both velocity and equilibrium gradient centrifugation and which are precipitated by antiserum specific for the VSV glycoprotein. Control experiments establish that these ¹²⁵I-labeled particles are not cell debris or cellular material which aggregate with or adhere to VSV virions. VSV virions contain only a subset of the 10–15 normal ¹²⁵1-labeled cell surface polypeptides resolved by SDS gel electrophoresis; VSV grown in L cells and Vero cells incorporate different host polypeptides. In a second approach, Vero cells were labeled with ³⁵S-methione, then infected with VSV. Two predominant host polypeptides (molecular weights 110,000 and 20,000) were incorporated into VSV virions. These proteins, like VSV G protein, are exposed to the surface of the virion. They co-migrate with the major incorporated ¹²⁵1 host polypeptides. These host proteins are present in approximately 10 and 80 copies, respectively, per virion. Specific incorporation of host polypeptides into VSV virions does not require the presence of viral glycoprotein. This was shown by use of a ts VSV mutant defective in maturation of VSV G protein to the cell surface. Budding from infected cells are noninfectious particles which contain all the viral proteins except for G; these particles contain the same proportion and spectrum of ¹²⁵1-labeled host surface polypeptides as do wild-type virions. These results extend previous conclusions implicating the submembrane viral matrix protein, or the viral nucleocapsid, as being of primary importance in selecting cell surface proteins for incorporation into budding VSV virions.     

Proteins of Vesicular Stomatitis Virus and of Phenotypically Mixed Vesicular Stomatitis Virus-Simian Virus 5 Virions

The identity of the glycoprotein of vesicular stomatitis virus (VSV) as the spike protein has been confirmed by the removal of the spikes with a protease from Streptomyces griseus, leaving bullet-shaped particles bounded by a smooth membrane. This treatment removes the glycoprotein but does not affect the other virion proteins, apparently because they are protected from the enzyme by the lipids in the viral membrane. The proteins of phenotypically mixed, bullet-shaped virions produced by cells mixedly infected with VSV and the parainfluenza virus simian virus 5 (SV5) have been analyzed by polyacrylamide gel electrophoresis. These virions contain all the VSV proteins plus the two SV5 spike proteins, both of which are glycoproteins. The finding of the SV5 spike glycoproteins on virions with the typical morphology of VSV indicates that there is not a stringent requirement that only the VSV glycoprotein can be used to form the bullet-shaped virion. On the other hand, the SV5 nucleocapsid protein and the major non-spike protein of the SV5 envelope were not detected in the phenotypically mixed virions, and this suggests that a specific interaction between the VSV nucleocapsid and regions of the cell membrane which contain the nonglycosylated VSV envelope protein is necessary for assembly of the bullet-shaped virion.     

Proteomic Characterization of Bovine Herpesvirus 4 Extracellular Virions

Gammaherpesviruses are important pathogens in human and animal populations. During early events of infection, these viruses manipulate preexisting host cell signaling pathways to allow successful infection. The different proteins that compose viral particles are therefore likely to have critical functions not only in viral structures and in entry into target cell but also in evasion of the host''s antiviral response. In this study, we analyzed the protein composition of bovine herpesvirus 4 (BoHV-4), a close relative of the human Kaposi''s sarcoma-associated herpesvirus. Using mass spectrometry-based approaches, we identified 37 viral proteins associated with extracellular virions, among which 24 were resistant to proteinase K treatment of intact virions. Analysis of proteins associated with purified capsid-tegument preparations allowed us to define protein localization. In parallel, in order to identify some previously undefined open reading frames, we mapped peptides detected in whole virion lysates onto the six frames of the BoHV-4 genome to generate a proteogenomic map of BoHV-4 virions. Furthermore, we detected important glycosylation of three envelope proteins: gB, gH, and gp180. Finally, we identified 38 host proteins associated with BoHV-4 virions; 15 of these proteins were resistant to proteinase K treatment of intact virions. Many of these have important functions in different cellular pathways involved in virus infection. This study extends our knowledge of gammaherpesvirus virions composition and provides new insights for understanding the life cycle of these viruses.     

Display of heterologous proteins on gp64null baculovirus virions and enhanced budding mediated by a vesicular stomatitis virus G-stem construct

The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) GP64 envelope glycoprotein is essential for virus entry and plays an important role in virion budding. An AcMNPV construct that contains a deletion of the gp64 gene is unable to propagate infection from cell to cell, and this defect results from both a severe reduction in the production of budded virions and the absence of GP64 on virions. In the current study, we examined GP64 proteins containing N- and C-terminal truncations of the ectodomain and identified a minimal construct capable of targeting the truncated GP64 to budded virions. The minimal budding and targeting construct of GP64 contained 38 amino acids from the mature N terminus of the GP64 ectodomain and 52 amino acids from the C terminus of GP64. Because the vesicular stomatitis virus (VSV) G protein was previously found to rescue infectivity of a gp64null AcMNPV, we also examined a small C-terminal construct of the VSV G protein. We found that a construct containing 91 amino acids from the C terminus of VSV G (termed G-stem) was capable of rescuing AcMNPV gp64null virion budding to wild-type (wt) or nearly wt levels. We also examined the display of chimeric proteins on the gp64null AcMNPV virion. By generating viruses that expressed chimeric influenza virus hemagglutinin (HA) proteins containing the GP64 targeting domain and coinfecting those viruses with a virus expressing the G-stem construct, we demonstrated enhanced display of the HA protein on gp64null AcMNPV budded virions. The combined use of gp64null virions, VSV G-stem-enhanced budding, and GP64 domains for targeting heterologous proteins to virions should be valuable for biotechnological applications ranging from targeted transduction of mammalian cells to vaccine production.     

Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome

Human cytomegalovirus (HCMV), a member of the herpesvirus family, is a large complex enveloped virus composed of both viral and cellular gene products. While the sequence of the HCMV genome has been known for over a decade, the full set of viral and cellular proteins that compose the HCMV virion are unknown. To approach this problem we have utilized gel-free two-dimensional capillary liquid chromatography-tandem mass spectrometry (MS/MS) and Fourier transform ion cyclotron resonance MS to identify and determine the relative abundances of viral and cellular proteins in purified HCMV AD169 virions and dense bodies. Analysis of the proteins from purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than previously known. In this study, we identified 71 HCMV-encoded proteins that included 12 proteins encoded by known viral open reading frames (ORFs) previously not associated with virions and 12 proteins from novel viral ORFs. Analysis of the relative abundance of HCMV proteins indicated that the predominant virion protein was the pp65 tegument protein and that gM rather than gB was the most abundant glycoprotein. We have also identified over 70 host cellular proteins in HCMV virions, which include cellular structural proteins, enzymes, and chaperones. In addition, analysis of HCMV dense bodies indicated that these viral particles are composed of 29 viral proteins with a reduced quantity of cellular proteins in comparison to HCMV virions. This study provides the first comprehensive quantitative analysis of the viral and cellular proteins that compose infectious particles of a large complex virus.     

Proteomic Analysis of Mamestra Brassicae Nucleopolyhedrovirus Progeny Virions from Two Different Hosts

Mamestra brassicae nucleopolyhedrovirus (MabrNPV) has a wide host range replication in more than one insect species. In this study, a sequenced MabrNPV strain, MabrNPV-CTa, was used to perform proteomic analysis of both BVs and ODVs derived from two infected hosts: Helicoverpa armigera and Spodoptera exigua. A total of 82 and 39 viral proteins were identified in ODVs and BVs, respectively. And totally, 23 and 76 host proteins were identified as virion-associated with ODVs and BVs, respectively. The host proteins incorporated into the virus particles were mainly involved in cytoskeleton, signaling, vesicle trafficking, chaperone and metabolic systems. Some host proteins, such as actin, cyclophilin A and heat shock protein 70 would be important for viral replication. Several host proteins involved in immune response were also identified in BV, and a C-type lectin protein was firstly found to be associated with BV and its family members have been demonstrated to be involved in entry process of other viruses. This study facilitated the annotation of baculovirus genome, and would help us to understand baculovirus virion structure. Furthermore, the identification of host proteins associated with virions produced in vivo would facilitate investigations on the involvement of intriguing host proteins in virus replication.     

Characterization of vesicular stomatitis virus recombinants that express and incorporate high levels of hepatitis C virus glycoproteins

We generated recombinant vesicular stomatitis viruses (VSV) expressing genes encoding hybrid proteins consisting of the extracellular domains of hepatitis C virus (HCV) glycoproteins fused at different positions to the transmembrane and cytoplasmic domains of the VSV G glycoprotein (E1G and E2G). We show that these chimeric proteins are transported to the cell surface and incorporated into VSV virions efficiently. We also generated VSV recombinants in which the gene encoding the VSV G protein was deleted and replaced by one or both of the E1G and E2G genes, together with a green fluorescent protein gene. These DeltaG viruses incorporated E1G and E2G proteins at levels approximately equivalent to the normal level of VSV G itself, or about 1,200 molecules of each protein per virion. Given the potency of VSV recombinants as vaccines in other studies, this high-level expression and incorporation of HCV proteins into virions could be very important for development of an HCV vaccine. Despite the presence of E1G and E2G proteins at high levels in the virions, these virions did not infect cell lines that have been reported to support at least a low level of HCV infection and replication.     

Mutations in the PPPY motif of vesicular stomatitis virus matrix protein reduce virus budding by inhibiting a late step in virion release

The N terminus of the matrix (M) protein of vesicular stomatitis virus (VSV) and of other rhabdoviruses contains a highly conserved PPPY sequence (or PY motif) similar to the late (L) domains in the Gag proteins of some retroviruses. These L domains in retroviral Gag proteins are required for efficient release of virus particles. In this report, we show that mutations in the PPPY sequence of the VSV M protein reduce virus yield by blocking a late stage in virus budding. We also observed a delay in the ability of mutant viruses to cause inhibition of host gene expression compared to wild-type (WT) VSV. The effect of PY mutations on virus budding appears to be due to a block at a stage just prior to virion release, since electron microscopic examination of PPPA mutant-infected cells showed a large number of assembled virions at the plasma membrane trapped in the process of budding. Deletion of the glycoprotein (G) in addition to these mutations further reduced the virus yield to less than 1% of WT levels, and very few particles were assembled at the cell surface. This observation suggested that G protein aids in the initial stage of budding, presumably during the formation of the bud site. Overall, our results confirm that the PPPY sequence of the VSV M protein possesses L domain activity analogous to that of the retroviral Gag proteins.     

Novel activities of cyclophilin A and cyclosporin A during HIV-1 infection of primary lymphocytes and macrophages

Studies conducted in cell lines indicate that cyclophilin A (CypA) is a component of HIV type 1 (HIV-1) virions, and that when CypA incorporation into virions is inhibited by treatment of infected cells with the immunosuppressive agent cyclosporin A (CsA), HIV-1 infection also is inhibited. Because HIV-1 particles assemble along a different pathway and incorporate different host proteins in macrophages than in other cell types, we investigated CypA and CsA activities in HIV-1-infected primary human macrophages, compared with primary human lymphocytes. We tested virus protein production, virion composition and infectivity, and progress through the virus life cycle under perturbation by drug treatment or mutagenesis in infected cells from multiple donors. Our findings from both primary cell types are different from that previously reported in transformed cells and show that the amount of CypA incorporated into virions is variable and that CsA inhibits HIV-1 infection at both early and late phases of virus replication, the stage affected is determined by the sequence of HIV-1 Gag. Because the cell type infected determines the identity of host proteins active in HIV-1 replication and can influence the activity of some viral inhibitors, infection of transformed cells may not recapitulate infection of the native targets of HIV-1.     

Proteomic Profiling of Purified Rabies Virus Particles

While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus, these characteristics have been largely neglected in studies on rabies virus(RABV). Here, we purified the RABV virions with good purity and integrity, and analyzed their proteome by nano LC–MS/MS, followed by the confirmation with immunoblot and immuno-electronic microscopy. In addition to the 5 viral proteins, 49 cellular proteins were reproducibly identified to be incorporated into matured RABV virions. Function annotation suggested that 24 of them were likely involved in virus replication. Furthermore, cryo-EM was employed to observe the purified RABV virions, generating high-resolution pictures of the bullet-shaped virion structure of RABV. This study has provided new insights into the host proteins composition in RABV virion and shed the light for further investigation on molecular mechanisms of RABV infection, as well as the discovery of new anti-RABV therapeutics.     

Inhibition of pseudorabies virus replication by vesicular stomicles virus I. Activity of infectious and inactivated B particles.

Infectious B particles of vesicular stomatitis virus (VSV) are capable of inhibiting the replication of pseudorabies virus (PSR) in a variety of cell lines. Even under conditions of an abortive infection in a continuous line of rabbit cornea cells (RC-6O), B particles interfere with the replication of PSR with high efficiency. Particle per cell dose-response analysis of B particle populations revealed that the number of VSV particles capable of inhibiting PSR replication exceeds the number of PFU by a factor of 32 to 64. When B particles are treated with UV irradiation, a drastic increase in the multiplicity of infection is required to inhibit PSR replication. Whereas one infective B particles per cell is sufficient to prevent replication of PSR, 800 to 1,000 VSV particles rendered noninfective by UV irradiation are required to compensate for the loss of VSV synthetic activity that results from irradiation. Temperature-sensitive mutants representing five complementation groups of VSV were tested at low multiplicities of infection for their effect on PSR replication at the nonpermissive temperature. Generally, the ability of the different complementation groups to amplify virion products at the nonpermissive temperature is associated with their ability to inhibit PSR replication. These results imply that at low multiplicities of infection, amplification of infecting VSV components is necessary for inhibition of PSR replication., but at high multiplicities of infection with VSV, a virion component can prevent PSR replication in the absence of de novo VSV RNA or protein synthesis.     

Utilization of homotypic and heterotypic proteins of vesicular stomatitis virus by defective interfering particle genomes for RNA replication and virion assembly: implications for the mechanism of homologous viral interference

Defective interfering (DI) particles of Indiana serotype of vesicular stomatitis virus (VSV(Ind)) are capable of interfering with the replication of both homotypic VSV(Ind) and heterotypic New Jersey serotype (VSV(NJ)) standard virus. In contrast, DI particles from VSV(NJ) do not interfere with the replication of VSV(Ind) standard virus but do interfere with VSV(NJ) replication. The differences in the interfering activities of VSV(Ind) DI particles and VSV(NJ) DI particles against heterotypic standard virus were investigated. We examined the utilization of homotypic and heterotypic VSV proteins by DI particle genomic RNAs for replication and maturation into infectious DI particles. Here we show that the RNA-nucleocapsid protein (N) complex of one serotype does not utilize the polymerase complex (P and L) of the other serotype for RNA synthesis, while DI particle genomic RNAs of both serotypes can utilize the N, P, and L proteins of either serotype without serotypic restriction but with differing efficiencies as long as all three proteins are derived from the same serotype. The genomic RNAs of VSV(Ind) DI particles assembled and matured into DI particles by using either homotypic or heterotypic viral proteins. In contrast, VSV(NJ) DI particles could assemble only with homotypic VSV(NJ) viral proteins, although the genomic RNAs of VSV(NJ) DI particles could be replicated by using heterotypic VSV(Ind) N, P, and L proteins. Thus, we concluded that both efficient RNA replication and assembly of DI particles are required for the heterotypic interference by VSV DI particles.     

Heterogeneity of vesicular stomatitis virus particles: implications for virion assembly

Vesicular stomatitis virus (VSV) particles formed at early times after infection contain only one-third the amount of viral glycoportein (G protein), relative to the major internal structural proteins M and N, as is found in particles released later. These "early" particles also have a lower density in equilibrium sucrose gradients than do those formed later; however, the sedimentation velocity and specific infectivity of these two classes of particles are the same. VSV-infected cells also release virus-like particles which sediment considerably faster than authentic virions and contain a higher-than-normal proportion of the VSV G protein relative to internal VSV proteins. These particles have a reduced specific infectivity but a normal density in sucrose gradients. All classes of VSV virions contain a constant proportion of M and N polypeptides. The ratio of G protein to M or N protein, in contrast, can vary over a sixfold range; this implies that an interaction between a precise number of surface G proteins with either of the underlying M and N proteins is not a prerequisite for budding of infectious viral particles from the cell surface.     

Low infectivity of vesicular stomatitis virus (VSV) particles released from interferon-treated cells is related to glycoprotein deficiency

We have investigated the mechanism for the low infectivity of vesicular stomatitis virus (VSV) released from interferon (IFN) -treated cells. With 10-30 units/ml of IFN there was an approximately 5-30 fold reduction in the production of virus particles, as measured by VSV proteins; however, the infectivity of the VSV released from IFN-treated mouse LB, JLS-V9R, or human GM2504 was drastically reduced (2 to 4 logs). The low infectivity of VSV was directly related to a deficiency in virion glycoprotein (G). IFN treatment did not change the specific infectivity of the VSV particles released by HeLa cells; their G protein was also not reduced. A further effect of IFN to reduce the amount of virion M protein appeared to be secondary and was probably not related to the reduced infectivity of VSV.     

Functional analysis of the simian immunodeficiency virus Vpx protein: identification of packaging determinants and a novel nuclear targeting domain

The vpx gene products of human immunodeficiency virus type 2 (HIV-2) and of the closely related simian immunodeficiency viruses from sooty mangabeys (SIVsm) and macaques (SIVmac) comprise a 112-amino-acid virion-associated protein that is critical for efficient virus replication in nondividing cells such as macrophages. When expressed in the absence of other viral proteins, Vpx localizes to the nuclear membrane as well as to the nucleus; however, in the context of virus replication Vpx is packaged into virions via interaction with the p6 domain of the Gag precursor polyprotein (p55(gag)). To identify the domains essential for virion incorporation and nuclear localization, site-directed mutations were introduced into the vpx gene of SIVsmPBj1.9 and functionally analyzed. Our results show that (i) mutation of two highly conserved L74 and I75 residues impaired both virion incorporation and nuclear localization of Vpx; (ii) substitution of conserved H82, G86, C87, P103, and P106 residues impaired Vpx nuclear localization but not virion incorporation; (iii) mutations of conserved Y66, Y69, and Y71 residues impaired virion incorporation but not the translocation of Vpx to the nucleus; and (iv) a mutation at E30 (predicted to disrupt an N-terminal alpha-helix) had no effect on either virion incorporation or nuclear localization of Vpx. Importantly, mutations in Vpx which impaired nuclear localization also reduced virus replication in macaque macrophages, suggesting an important role of the carboxyl terminus of Vpx in nuclear translocation of the viral preintegration complex. Analyzing this domain in greater detail, we identified a 26-amino-acid (aa 60 to 85) fragment that was sufficient to mediate the transport of a heterologous protein (green fluorescent protein [GFP]) to the nucleus. Taken together, these results indicate that virion incorporation and nuclear localization are encoded by two partially overlapping domains in the C-terminus of Vpx (aa 60 to 112). The identification of a novel 26-amino-acid nuclear targeting domain provides a new tool to investigate the nuclear import of the HIV-2/SIV preintegration complex.     

Effect of membrane protein on lipid bilayer structure: a spin-label electron spin resonance study of vesicular stomatitis virus.

Spin-label electron spin resonance (ESR) methods have been used to study the structure of the envelope of vesicular stomatitis virus (VSV). The data indicate that the lipid is organized in a bilayer structure. Proteolytic digestion of the glycoproteins which are the spike-like projections on the outer surface of the virus particle increases the fluidity of the lipid bilayer. Since the lipid composition of the virion reflects the composition of the host plasma membrane and the protein composition is determined by the viral genome, VSV was grown in both MDBK and BHK21-F cells to determine the effect of a change in lipid composition on the structure of the lipid bilayer of VSV. The lipid bilayer of the virion was found to be more rigid when derived from MDBK cells than from BHK21-F cells. Studies comparing spin-labeled intact cells and cell membrane fractions suggest that upon labeling the whole cell the spin label probes the plasma membrane. Comparison of spin-labeled VSV particles and their host cells indicates that the lipid bilayer of the plasma membrane is considerably more fluid than that of the virion. These results are discussed in terms of the effect of membrane-associated protein on the structure of the lipid bilayer.     

Assembly of infectious Herpes simplex virus type 1 virions in the absence of full-length VP22

VP22, the 301-amino-acid phosphoprotein product of the herpes simplex virus type 1 (HSV-1) U(L)49 gene, is incorporated into the tegument during virus assembly. We previously showed that highly modified forms of VP22 are restricted to infected cell nuclei (L. E. Pomeranz and J. A. Blaho, J. Virol. 73:6769-6781, 1999). VP22 packaged into infectious virions appears undermodified, and nuclear- and virion-associated forms are easily differentiated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (J. A. Blaho, C. Mitchell, and B. Roizman, J. Biol. Chem. 269:17401-17410, 1994). As VP22 packaging-associated undermodification is unique among HSV-1 tegument proteins, we sought to determine the role of VP22 during viral replication. We now show the following. (i) VP22 modification occurs in the absence of other viral factors in cell lines which stably express its gene. (ii) RF177, a recombinant HSV-1 strain generated for this study, synthesizes only the amino-terminal 212 amino acids of VP22 (Delta212). (iii) Delta212 localizes to the nucleus and incorporates into virions during RF177 infection of Vero cells. Thus, the carboxy-terminal region is not required for nuclear localization of VP22. (iv) RF177 synthesizes the tegument proteins VP13/14, VP16, and VHS (virus host shutoff) and incorporates them into infectious virions as efficiently as wild-type virus. However, (v) the loss of VP22 in RF177 virus particles is compensated for by a redistribution of minor virion components. (vi) Mature RF177 virions are identical to wild-type particles based on electron microscopic analyses. (vii) Single-step growth kinetics of RF177 in Vero cells are essentially identical to those of wild-type virus. (viii) RF177 plaque size is reduced by nearly 40% compared to wild-type virus. Based on these results, we conclude that VP22 is not required for tegument formation, virion assembly/maturation, or productive HSV-1 replication, while the presence of full-length VP22 in the tegument is needed for efficient virus spread in Vero cell monolayers.