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2.
Previous genetic and biochemical studies performed with several members of the Alphaherpesvirus subfamily have shown that the UL31 and UL34 proteins are essential components of the molecular machinery that mediates the primary egress of newly assembled capsids across the nuclear membrane. Further, there is substantial evidence that BFLF2 and BFRF1, the respective positional homologs of UL31 and UL34 in the Epstein-Barr virus (EBV) genome, are also their functional homologs, i.e., that the UL31/UL34 pathway is common to distant herpesviruses. However, the low degree of protein sequence identity between UL31 and BFLF2 would argue against such a hypothesis. To further clarify this issue, we have constructed a recombinant EBV strain devoid of BFLF2 (DeltaBFLF2) and show that BFLF2 is crucial for efficient virus production but not for lytic DNA replication or B-cell transformation. This defective phenotype could be efficiently restored by trans complementation with a BFLF2 expression plasmid. Detailed analysis of replicating cells by electron microscopy revealed that, as expected, DeltaBFLF2 viruses not only failed to egress from the nucleus but also showed defective DNA packaging. Nonfunctional primary egress did not, however, impair the production and extracellular release of enveloped but empty viral particles that comprised L particles containing tegument-like structures and a few virus-like particles carrying empty capsids. The DeltaBFLF2 and DeltaUL31 phenotypes therefore only partly overlap, from which we infer that BFLF2 and UL31 have substantially diverged during evolution to fulfil related but distinct functions. 相似文献
3.
The molecular mechanisms that underlie maturation and egress of Epstein-Barr virus (EBV) virions are only partially characterized. We have recently shown that the BFRF1 gene, the EBV positional homolog of herpes simplex virus type 1 and pseudorabies virus UL34, is expressed early during EBV lytic replication and that it is found predominantly on the nuclear membrane (A. Farina, R. Santarelli, R. Gonnella, R. Bei, R. Muraro, G. Cardinali, S. Uccini, G. Ragona, L. Frati, A. Faggioni, and A. Angeloni, J. Virol. 74:3235-3244, 2000). These data suggest that the BFRF1 protein might be involved in viral primary envelopment. To precisely determine the function of this protein, we have constructed an EBV mutant devoid of the BFRF1 gene (BFRF1-KO). 293 cells carrying BFRF1-KO showed no differences in comparison with wild-type EBV in terms of DNA lytic replication or expression of late viral proteins upon induction of the lytic cycle. However, binding assays and infection experiments using cell lines or human cord blood lymphocytes showed a clear reduction in the viral mutant titers. Complementation experiments with BFRF1-KO and a BFRF1 expression vector restored viral titers to levels similar to those for the wild-type control, showing that the modifications that we introduced were limited to the BFRF1 gene. Electron microscopic observations showed that the reduction in viral titers was due to sequestration of EBV nucleocapsids in the nuclei of lytically induced cells. This suggests that BFRF1 is involved in transport of the maturing virion across the nuclear membrane. This hypothesis was further supported by the observation that BFRF1 is present in maturing intracellular virions but not in their extracellular counterparts. This implies that BFRF1 is a key protein for EBV maturation. 相似文献
4.
Viral egress and autophagy are two mechanisms that seem to be strictly connected in Herpesviruses’s biology. Several data suggest that the autophagic machinery facilitates the egress of viral capsids and thus the production of new infectious particles. In the Herpesvirus family, viral nuclear egress is controlled and organized by a well conserved group of proteins named Nuclear Egress Complex (NEC). In the case of EBV, NEC is composed by BFRF1 and BFLF2 proteins, although the alterations of the nuclear host cell architecture are mainly driven by BFRF1, a multifunctional viral protein anchored to the inner nuclear membrane of the host cell. BFRF1 shares a peculiar distribution with several nuclear components and with them it strictly interacts. In this study, we investigated the possible role of BFRF1 in manipulating autophagy, pathway that possibly originates from nucleus, regulating the interplay between autophagy and viral egress. 相似文献
6.
DNA viruses adopt various strategies to modulate the cellular environment for efficient genome replication and virion production. Previously, we demonstrated that the BGLF4 kinase of Epstein-Barr virus (EBV) induces premature chromosome condensation through the activation of condensin and topoisomerase IIα (C. P. Lee, J. Y. Chen, J. T. Wang, K. Kimura, A. Takemoto, C. C. Lu, and M. R. Chen, J. Virol. 81:5166-5180, 2007). In this study, we show that BGLF4 interacts with lamin A/C and phosphorylates lamin A protein in vitro. Using a green fluorescent protein (GFP)-lamin A system, we found that Ser-22, Ser-390, and Ser-392 of lamin A are important for the BGLF4-induced disassembly of the nuclear lamina and the EBV reactivation-mediated redistribution of nuclear lamin. Virion production and protein levels of two EBV primary envelope proteins, BFRF1 and BFLF2, were reduced significantly by the expression of GFP-lamin A(5A), which has five Ser residues replaced by Ala at amino acids 22, 390, 392, 652, and 657 of lamin A. Our data indicate that BGLF4 kinase phosphorylates lamin A/C to promote the reorganization of the nuclear lamina, which then may facilitate the interaction of BFRF1 and BFLF2s and subsequent virion maturation. UL kinases of alpha- and betaherpesviruses also induce the disassembly of the nuclear lamina through similar sites on lamin A/C, suggesting a conserved mechanism for the nuclear egress of herpesviruses. 相似文献
7.
We have previously reported that Potato virus X-expressed coat protein of Cucumber mosaic virus (CMV) formed virus-like particles (VLPs), which served as carriers for display of different neutralizing epitopes of Newcastle disease virus (NDV). In this work, we further modified the purification protocol of recombinant VLPs carrying short neutralizing epitopes of the NDV proteins and demonstrated that self-contained capsid protein subunits of CMV transiently expressed from heterologous virus packaged into individual virions morphologically resembling and/or indistinguishable from wild type CMV particles. Homogeneity of the final preparation represents an advance over our previous study, where VLPs were found to be of variable size. Chickens immunized with purified VLPs developed antigen-specific response. 相似文献
8.
The Epstein-Barr virus (EBV) lytic program includes lytic viral DNA replication and the production of a viral particle into which the replicated viral DNA is packaged. The terminal repeats (TRs) located at the end of the linear viral DNA have been identified as the packaging signals. A TR-negative (TR(-)) mutant therefore provides an appropriate tool to analyze the relationships between EBV DNA packaging and virus production. Here, we show that supernatants from lytically induced 293 cells carrying TR mutant EBV genomes (293/TR(-)) contain large amounts of viral particles devoid of viral DNA which are nevertheless able to bind to EBV target cells. This shows that viral DNA packaging is not a prerequisite for virion formation and egress. Rather surprisingly, supernatants from lytically induced 293/TR(-) cells also contained rare infectious viruses carrying the viral mutant DNA. This observation indicates that the TRs are important but not absolutely essential for virus encapsidation. 相似文献
9.
We report the identification and characterization of p33, the product of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 69 (ORF69), a positional homolog of the conserved herpesvirus protein UL31. p33 is expressed upon induction of viral lytic cycle with early kinetics. Immunofluorescence analysis revealed that in infected cell lines, the protein is localized in the nucleus, both in dotted spots and along the nuclear membrane. Nuclear fractionation experiments showed that p33 partitions with the nuclear matrix, and both immunoblotting of purified virions and immunoelectron microscopy indicated that the novel protein is not a component of the mature virus. Following ectopic expression in KSHV-negative cells, the protein was never associated with the nuclear membrane, suggesting that p33 needs to interact with additional viral proteins to reach the nuclear rim. In fact, after cotransfection with the ORF67 gene, the KSHV positional homolog of UL34, the p33 intranuclear signal changed and the two proteins colocalized on the nuclear membrane. A similar result was obtained when ORF69 was cotransfected with BFRF1, the Epstein-Barr virus (EBV) positional homolog of UL34 and ORF67. Finally, upon cotransfection, ORF69 significantly increased nuclear membrane reduplications induced by BFRF1. The above results indicate that KSHV p33 shares many similarities with its EBV homolog BFLF2 and suggest that functional cross-complementation is possible between members of the gammaherpesvirus subfamily. 相似文献
10.
To investigate the role of protein-protein and protein-nucleic acid interactions in virus assembly, we compared the stabilities of native bacteriophage MS2, virus-like particles (VLPs) containing nonviral RNAs, and an assembly-defective coat protein mutant (dlFG) and its single-chain variant (sc-dlFG). Physical (high pressure) and chemical (urea and guanidine hydrochloride) agents were used to promote virus disassembly and protein denaturation, and the changes in virus and protein structure were monitored by measuring tryptophan intrinsic fluorescence, bis-ANS probe fluorescence, and light scattering. We found that VLPs dissociate into capsid proteins that remain folded and more stable than the proteins dissociated from authentic particles. The proposed model is that the capsid disassembles but the protein remains bound to the heterologous RNA encased by VLPs. The dlFG dimerizes correctly, but fails to assemble into capsids, because it lacks the 15-amino acid FG loop involved in inter-dimer interactions at the viral fivefold and quasi-sixfold axes. This protein was very unstable and, when compared with the dissociation/denaturation of the VLPs and the wild-type virus, it was much more susceptible to chemical and physical perturbation. Genetic fusion of the two subunits of the dimer in the single-chain dimer sc-dlFG stabilized the protein, as did the presence of 34-bp poly(GC) DNA. These studies reveal mechanisms by which interactions in the capsid lattice can be sufficiently stable and specific to ensure assembly, and they shed light on the processes that lead to the formation of infectious viral particles. 相似文献
11.
Isometric virus-like particles (VLPs) with diameter of approximately 34 nm containing ss-RNA were purified from abnormal mycelium of Shiitake mushroom, Lentinus edodes (Berk.) Sing. SDS-polyacrylamide gel elec-trophoresis demonstrated that the virions contain a single capsid polypeptide with molecular weight of about 22 000 daltons. The nucleic acid extract from purified VLPs preparations showed only one band with a size of approximately 7.3 kilobases. The susceptibility to RNase 1 and S1 digestions, resistance to DNase and thermal denaturation behaviour of the viral genome indicated that it is a single-stranded RNA. To our knowledge, isometric single-stranded RNA VLPs isolated from Shiitake mushroom mycelia have not been reported before. 相似文献
12.
We have used viruslike particles (VLPs) of human papillomaviruses to study the structure and assembly of the viral capsid. We demonstrate that mutation of either of two highly conserved cysteines of the major capsid protein L1 to serine completely prevents the assembly of VLPs but not of capsomers, whereas mutation of all other cysteines leaves VLP assembly unaffected. These two cysteines form intercapsomeric disulfides yielding an L1 trimer. Trimerization comprises about half of the L1 molecules in VLPs but all L1 molecules in complete virions. We suggest that trimerization of L1 is indispensable for the stabilization of intercapsomeric contacts in papillomavirus capsids. 相似文献
13.
Recent studies have established that several nonenveloped viruses utilize virus-encoded lytic peptides for host membrane disruption. We investigated this mechanism with the “gamma” peptide of the insect virus Flock House virus (FHV). We demonstrate that the C terminus of gamma is essential for membrane disruption in vitro and the rescue of immature virus infectivity in vivo, and the amphipathic N terminus of gamma alone is not sufficient. We also show that deletion of the C-terminal domain disrupts icosahedral ordering of the amphipathic helices of gamma in the virus. Our results have broad implications for understanding membrane lysis during nonenveloped virus entry.The presence of membrane lytic peptides in many nonenveloped viruses is well established ( 3, 16), but how these peptides are deployed from the virus capsid during host cell entry and disrupt membranes remains unclear. These peptides are typically generated by a postassembly proteolytic processing event ( 1, 11) and are exposed from a previously buried position during conformational alterations in the capsid triggered by host cell conditions ( 2, 18). Flock House virus (FHV), an insect nodavirus, contains a 4-kDa peptide called “gamma” (γ), which shares many of the characteristics of other nonenveloped virus lytic peptides ( 3). The FHV capsid is made from 180 copies of a single-coat protein (α) enclosing a single-stranded bipartite RNA genome ( 9). Gamma is generated by the autocatalytic cleavage of α during virus maturation (α → β + γ) ( 15), remains localized in the capsid interior ( 9) with occasional externalization or “breathing” ( 6), and is exposed under low-pH conditions in the endosomes during entry (Odegard et al., submitted for publication). Covalently independent gamma is necessary for virus infection, since maturation-defective FHV (D75N/N363T FHV), which does not undergo the autocleavage of α, is not infectious ( 15, 17). The N-terminal ∼21 residues of gamma (corresponding to residues 364 to 384 of α) constitute an amphipathic helix which can disrupt membranes in vitro when synthetically produced ( 4, 5) and is recognized as the host membrane-interacting region of FHV during entry. The hydrophobic, ∼23-residue-long C terminus of gamma, especially certain phenylalanine residues (at positions 402, 405, and 407), is responsible for specifically packaging viral RNA into capsids during assembly ( 14).It was recently demonstrated that a supply of full-length gamma from noninfectious virus-like particles (VLPs) of FHV ( 13) during entry can restore infectivity to maturation-defective FHV ( 17), suggesting that gamma can function in trans to mediate access into host cells. This trans-complementation assay ( 17) was utilized to provide a quantitative readout of the effect of gamma mutations specifically on virus entry and to thus assess the region(s) of gamma required during virus entry. To determine the minimal sequence of gamma required for trans-complementation, FHV VLPs were produced that included the first 384, 390, or 395 amino acids of capsid protein α (designated Δγ384, Δγ390, and Δγ395, respectively). These VLPs contained only the amphipathic region of gamma, or that and additional parts of the C-terminal region (Fig. ), and underwent normal assembly and maturation cleavage (Fig. ). The amount of progeny virus produced by trans-complementing maturation-defective D75N/N363T FHV with these mutated VLPs was negligible (∼5%) compared to that produced by the wild-type (WT) VLPs (considered 100%) (Fig. ). Thus, the amphipathic region of gamma was not sufficient by itself to restore infectivity to maturation-defective FHV, and the C terminus of gamma, beyond residue 395, was essential for rescue. Three phenylalanines located beyond residue 395 in gamma were separately mutated to alanines, and mature VLPs were generated (Fig. ) and tested in the trans-complementation assay. The relative efficiencies of rescue demonstrated by the F402A, F405A, and F407A VLPs were 15%, 29%, and 43%, respectively (Fig. ), compared to that by the WT VLPs (100%). This rescue efficiency was similar to the relative infectivity previously determined for FHV containing the same point mutations ( 14). This suggests that the phenylalanines at the gamma C terminus are not only involved in the specific packaging of genomic RNA during FHV assembly ( 14) but are also required during virus entry. Open in a separate windowC-terminal region of gamma required for trans-complementation during the entry of maturation-defective FHV. (A) Schematic of truncations or single mutations in the gamma region of FHV capsid protein α in VLPs. The sequence of gamma in each of the mutated VLPs is shown, with the N-terminal amphipathic region boxed. The single F→A mutations in F402A, F405A, and F407A are indicated in boldface. (B) A total of 5 μl of WT, Δγ384, phenylalanine mutant, or D75N VLPs, at a concentration of 5 mg/ml, was subjected to SDS-PAGE on a 4 to 20% Tris-glycine gel (Invitrogen) and stained with Coomassie brilliant blue. The position of gamma is indicated. The cleavage-defective D75N VLPs do not have gamma. (C) Drosophila DL-1 cells (1 × 10 8) were coinfected with 1.5 × 10 3 particles/cell of D75N/N363T FHV and 9 × 10 3 particles/cell of WT, Δγ384, Δγ390, Δγ395, F402A, F405A, or F407A VLPs. [ 35S]methionine-cysteine-labeled progeny virus was quantified, with the amount of progeny produced during coinfection with D75N/N363T FHV and WT VLPs normalized at 100%. The standard deviation was calculated from three replicates.Since the primary function of gamma during FHV entry is expected to be host membrane disruption, the ability of the mutated VLPs to disrupt DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)-treated liposomes and release enclosed fluorescent dye was determined in comparison to the WT VLPs. We found that the in vivo rescue behavior of VLPs containing truncated gamma correlated with their in vitro membrane disruption activities. WT VLPs, at a concentration of 0.1 mg/ml (6.37 × 10 11 particles), released dye from liposomes at pH 7.0 (Fig. ) and at a much higher rate at pH 6.0 (Fig. ), which mimics the acidic endosomal environment. In contrast, the Δγ384, Δγ390, and Δγ395 VLPs were severely impaired in disrupting liposomes at both pH conditions (Fig. ), indicating that diminished endosomal membrane lysis by truncated gamma peptides could be responsible for the inability of mutated VLPs to rescue the infectivity of maturation-defective particles. The maturation-defective D75N VLP, which is unable to rescue infection ( 17), was also inefficient in liposome disruption at a neutral or low pH (Fig. ), indicating that in vitro membrane disruption by VLPs is a reliable indicator of their in vivo entry behavior. Open in a separate windowDisruption of liposomes and fluorescent dye release by VLPs in vitro. In each case, total fluorescence is normalized to dye release achieved by the addition of 0.1% Triton X-100 to liposomes under the same conditions. In the case of panels A, B, and D, closely similar results were obtained in three different experiments, whereas the standard deviations in panel C were calculated from three replicates. Fluorescence measurements were carried out at excitation/emission maxima of 492/514 nm for 6-carboxyfluorescein and 535/585 nm for SulfoB. (A) Kinetic study of 6-carboxyfluorescein release from DOPC-treated liposomes upon the addition of 6.37 × 10 11 particles (lipid/particle molar ratio, 481:1) of WT, gamma-truncated, or maturation-defective D75N VLPs to liposomes in 50 mM HEPES (pH 7.0). (B) SulfoB release from DOPC-treated liposomes in 50 mM Bis-Tris (pH 6.0) upon the addition of 6.37 × 10 11 particles of VLPs. (C) SulfoB release from DOPC-treated liposomes by 6.37 × 10 11 particles of WT, F402A, F405A, or F407A VLPs after 1 h at pH 7.0 (black) or by 2 × 10 11 particles (lipid/particle molar ratio, 1,387:1) of each of the VLPs after 15 min at pH 6.0 (gray). (D) SulfoB fluorescence upon the addition of heat-released gamma peptide with the WT sequence or with phenylalanine mutations at F402, F405, and F407 to dye-filled liposomes.VLPs containing single phenylalanine mutations in gamma were able to disrupt liposomes but were overall less effective than the WT. At a concentration of 0.1 mg/ml at pH 7.0, F402A and F405A VLPs displayed ∼60% liposome disruption (1 h postincubation) compared to that of the WT (Fig. ). At pH 6.0, these VLPs caused significantly less liposome disruption than the WT at an early time point (15 min postincubation) at a concentration of 0.035 mg/ml (2 × 10 11 particles) (Fig. ), although after 45 min of incubation, the extent of the liposome disruption approached the WT levels (data not shown). The F407A mutant VLPs, approximately half as competent as the WT VLPs in rescuing infection (Fig. ), were nonetheless as efficient as the WT VLPs in membrane disruption at low pH, although they were ∼80% as competent at a neutral pH (Fig. ). When gamma was isolated from the WT and phenylalanine mutant VLPs, by heating equal amounts (80 μg) at 65°C in Bis-Tris (pH 6.0) and centrifuging the heated material on a 30% sucrose cushion, the top fraction, which contained WT or mutated gamma peptides, caused the immediate release of fluorescent dye from liposomes (Fig. ). Given the dissimilar behavior of particle-associated phenylalanine mutant gamma and free mutant gamma, we hypothesized that the mutations in the C terminus could affect the organization of gamma within the particle.To test this hypothesis, cryoelectron microscopy (CryoEM) and image reconstruction were carried out to locate and compare the gamma peptides of WT FHV VLPs and Δγ384 VLPs, which had the most drastic truncation in gamma and could provide robust structural evidence. Data were collected on frozen hydrated samples of the VLPs (concentrated to 11 mg/ml in 50 mM HEPES [pH 7.0]) at the National Resource for Automated Molecular Microscopy on an FEI Tecnai F20 electron microscope operating at 120 kV. Images were processed using the EMAN suite ( 10), with a map calculated from the protein atomic coordinates of FHV (PDB entry no. 2Z2Q) as the starting model for image reconstructions. Real space and rigid body refinement was carried out using Chimera ( 12) and CNS ( 7), respectively. The estimated resolution for each image reconstruction was 8.8 Å, and the final R/ Rfree were 28.4%/28.3% for WT and 29.5%/29.3% for Δγ384 VLPs. While the WT and Δγ384 reconstructions were nearly indistinguishable at the surface, striking changes were detected in the gamma region (Fig. ). To quantify these changes, the electron densities for the pseudoatomic models of FHV, lacking the interior RNA, the gamma helices, and the capsid protein N termini, and calculated at the resolution of the CryoEM image reconstructions, were subtracted from the WT and Δγ384 VLP image reconstructions. The volume of the density associated with the gamma helices and the capsid protein N termini was calculated above a threshold of 0.4σ. While the densities of the capsid protein N termini (residues 59 to 72), calculated as controls, appeared similar for the A, B, and C subunits in the icosahedral asymmetric unit (iASU) between the two reconstructions, the densities of the gamma helices (residues 364 to 381) in the A and B subunits were increased approximately fourfold and twofold, respectively, in the WT reconstruction, whereas the density of C-subunit gamma was fourfold stronger in the Δγ384 reconstruction (Fig. ). The magnitude of the difference in density represents a clear structural dissimilarity between the WT and Δγ384 VLPs, with the gamma amphipathic helices becoming less icosahedrally ordered overall in the Δγ384 reconstruction. Interestingly, the pentameric helical bundles formed by gamma N termini at the fivefold axis of symmetry of the FHV capsid (Fig. ), and thought to be primarily involved in membrane interaction ( 8), were visible in the WT VLP reconstruction, but the corresponding density weakened significantly in the Δγ384 VLP (Fig. ), indicating significant disorder. Open in a separate windowCryoEM image reconstructions of WT and Δγ384 VLPs. (A) Model of WT FHV showing the iASU consisting of the A, B, and C subunits, as well as the A subunits at the fivefold axis of symmetry. An expanded view of the fivefold axis shows the amphipathic region of the gamma peptides (red) forming a pentameric helical bundle. (B) Panel I, inner surface of WT VLP and Δγ384 VLP image reconstructions contoured at 0.4σ. The coloring is as follows: density for the capsid protein N termini (residues 59 to 72) in the iASU is in yellow, density for the gamma amphipathic helices (residues 364 to 381) is in cyan, and a potential pocket factor is in purple. Modeled into the density for the gamma amphipathic helices are the corresponding residues from the FHV crystal structure (PDB entry 2Z2Q) in red. Panel II, inner view, looking down on the fivefold axis of symmetry of WT and Δγ384 VLP image reconstructions. The density for the gamma amphipathic helices from the A subunits is in cyan, with the corresponding residues from the FHV crystal structure modeled in red. A pocket factor is in purple. (C) Volume ratios corresponding to the gamma amphipathic helices and the capsid protein N termini for subunits A, B, and C in the iASU. The reported ratio for each volume pair is the WT VLP density to the corresponding Δγ384 VLP density.Our data show that the N-terminal amphipathic helix of gamma, previously thought to be the key to infection, is not sufficient for membrane permeabilization during FHV entry and is coincidentally less icosahedrally ordered in the capsid interior in the absence of the C terminus. Although the role of the gamma C terminus in entry is not clear, one interesting possibility is that the phenylalanine residues in this region interact with other capsid elements or packaged RNA to maintain the N-terminal helices in a structural conformation essential for biological activity. We demonstrate that other regions of nonenveloped virus lytic peptides, in addition to the membrane-interacting domains, might be essential in order to gain maximum leverage during entry. 相似文献
14.
Although envelope glycoprotein M (gM) is highly conserved among herpesviruses, the varicella-zoster virus (VZV) gM homolog has never been investigated. Here we characterized the VZV gM homolog and analyzed its function in VZV-infected cells. The VZV gM homolog was expressed on virions as a glycoprotein modified with a complex N-linked oligosaccharide and localized mainly to the Golgi apparatus and the trans-Golgi network in infected cells. To analyze its function, a gM deletion mutant was generated using the bacterial artificial chromosome system in Escherichia coli, and the virus was reconstituted in MRC-5 cells. VZV is highly cell associated, and infection proceeds mostly by cell-to-cell spread. Compared with wild-type VZV, the gM deletion mutant showed a 90% reduction in plaque size and 50% of the cell-to-cell spread in MRC-5 cells. The analysis of infected cells by electron microscopy revealed numerous aberrant vacuoles containing electron-dense materials in cells infected with the deletion mutant virus but not in those infected with wild-type virus. However, enveloped immature particles termed L particles were found at the same level on the surfaces of cells infected with either type of virus, indicating that envelopment without a capsid might not be impaired. These results showed that VZV gM is important for efficient cell-to-cell virus spread in cell culture, although it is not essential for virus growth. 相似文献
15.
African green monkey kidney cells infected by simian virus 40 were analyzed for the presence of the major capsid protein (capsid protein I) by immunological and radiolabeling techniques. Antisera with different specificities were prepared by immunization with intact or denatured viral particles. Antisera prepared against intact virus reacted by complement fixation with viral particles and with an 8S subunit containing the capsid protein I. Antisera prepared against denatured viral particles reacted with unassembled capsid protein(s) as well as with viral particles. These antisera were used to detect 8S viral subunits or unassembled viral capsid protein in soluble extracts of infected cells after centrifugation at 100,000 x g to remove viral particles. The soluble antigen pool was found to be small during infection with wild-type virus or a temperature-sensitive mutant deficient in the synthesis of viral particles. Pulse-chase experiments, performed at a high multiplicity of infection, also indicated a small pool of nonparticle capsid protein I. Radioactive lysine was incorporated into capsid protein I of virus particles during a 2-hr pulse. A subsequent chase with excess unlabeled lysine resulted in only a slight increase in the radio-activity found in capsid protein I of viral particles. Furthermore, in the same experiments, capsid protein I was incorporated preferentially into empty shells during the pulse with a shift in radioactivity to intact virions during the chase period, indicating a possible precursor relationship between the two types of virus particles. 相似文献
16.
Foot-and-mouth disease (FMD) is an economically important, global disease of cloven-hoofed animals. The conventional vaccine could bring down the incidence of disease in many parts of the world but has many limitations and in India, the disease is enzootic. More promisingly, the alternate vaccine candidates, virus-like particles (VLPs) are as immunogenic as a native virus but are more labile to heat than the live virus capsids. To produce stable VLPs, a single amino acid residue was mutated at 93 and 98 positions at VP2 inter-pentamer region of the P1-2A gene of FMD virus serotype O (IND/R2/75). The mutated capsid protein was expressed in insect cells and characterized for temperature and varying pH stability. Out of S93Y, S93F, S93C, S93H, and Y98F mutant, VLPs, S93Y, S93F, and Y98F showed improved stability at 37 °C for 75 days compared to wild capsid, which was evaluated by sandwich ELISA. Further, the stability analysis of purified VLPs either by differential scanning fluorescence (DSF) stability assay at different temperatures and pH conditions or by dissociation kinetics showed that the Y98F mutant VLPs were more stable than S93Y, S93F, S93C, and S93H mutant and wild-type VLPs. Immunization of guinea pigs with Y98F VLPs induced neutralizing antibodies and 60% of the animals were protected from the FMDV “O” 100 GPID50 challenge virus. 相似文献
17.
Human norovirus is a leading cause of acute gastroenteritis worldwide in a plethora of residential and commercial settings, including restaurants, schools, and hospitals. Methods for easily detecting the virus and for treating and preventing infection are critical to stopping norovirus outbreaks, and inactivation via nanoparticles (NPs) is a more universal and attractive alternative to other physical and chemical approaches. Using norovirus GI.1 (Norwalk) virus-like particles (VLPs) as a model viral system, this study characterized the antiviral activity of Au/CuS core/shell nanoparticles (NPs) against GI.1 VLPs for the rapid inactivation of HuNoV. Inactivation of VLPs (GI.1) by Au/CuS NPs evaluated using an absorbance-based ELISA indicated that treatment with 0.083 μM NPs for 10 min inactivated ~50% VLPs in a 0.37 μg/ml VLP solution and 0.83 μM NPs for 10 min completely inactivated the VLPs. Increasing nanoparticle concentration and/or VLP-NP contact time significantly increased the virucidal efficacy of Au/CuS NPs. Changes to the VLP particle morphology, size, and capsid protein were characterized using dynamic light scattering, transmission electron microscopy, and Western blot analysis. The strategy reported here provides the first reported proof-of-concept Au/CuS NPs-based virucide for rapidly inactivating human norovirus. 相似文献
18.
Human cytomegalovirus (HCMV) employs numerous strategies to combat, subvert, or co-opt host immunity. One evolutionary strategy for this involves capture of a host gene and then its successive duplication and divergence, forming a family of genes, many of which have immunomodulatory activities. The HCMV US12 family consists of 10 tandemly arranged sequence-related genes in the unique short (US) region of the HCMV genome (US12 to US21). Each gene encodes a protein possessing seven predicted transmembrane domains, patches of sequence similarity with cellular G-protein-coupled receptors, and the Bax inhibitor 1 family of antiapoptotic proteins. We show that one member, US17, plays an important role during virion maturation. Microarray analysis of cells infected with a recombinant HCMV isolate with a US17 deletion (the ΔUS17 mutant virus) revealed blunted host innate and interferon responses at early times after infection (12 h postinfection [hpi]), a pattern opposite that previously seen in the absence of the immunomodulatory tegument protein pp65 (pUL83). Although the ΔUS17 mutant virus produced numbers of infectious particles in fibroblasts equal to the numbers produced by the parental virus, it produced >3-fold more genome-containing noninfectious viral particles and delivered increased amounts of pp65 to newly infected cells. These results suggest that US17 has evolved to control virion composition, to elicit an appropriately balanced host immune response. At later time points (96 hpi), ΔUS17 mutant-infected cells displayed aberrant expression of several host endoplasmic reticulum stress response genes and chaperones, some of which are important for the final stages of virion assembly and egress. Our results suggest that US17 modulates host pathways to enable production of virions that elicit an appropriately balanced host immune response. 相似文献
19.
Vif is a human immunodeficiency virus type 1 (HIV-1) protein that is essential for the production of infectious virus. Most of Vif synthesized during HIV infection localizes within cells, and the extent of Vif packaging into virions and its function there remain controversial. Here we show that a small but detectable amount of Vif remains associated with purified virions even after their treatment with the protease subtilisin. However, treatment of these virions with 1% Triton X-100 revealed that most of the virion-associated Vif segregated with detergent-resistant virus particles consisting of unprocessed Gag, indicating that detergent-soluble, mature virions contain very little Vif. To investigate the control of Vif packaging in immature virus particles, we tested its association with Gag-containing virus-like particles (VLPs) in a Vif and Gag coexpression system in human cells. Only a small proportion of Vif molecules synthesized in this system became packaged into VLPs, and the VLP-associated Vif was protected from exogenous protease and detergent treatment, indicating that it is stably incorporated into immature virion-like cores. About 10-fold more Vpr than Vif was packaged into VLPs but most of the VLP-associated Vpr was removed by treatment with detergent. Mutagenesis of the C-terminal sequences in Gag previously shown to be responsible for interaction with Vif did not reduce the extent of Vif packaging into Gag VLPs. Surprisingly, short deletions in the capsid domain (CA) of Gag (amino acid residues 284 to 304 and 350 to 362) increased Vif packaging over 10-fold. The 350 to 363 deletion introduced into CA in HIV provirus also increased Vif incorporation into purified virions. Our results show that Vif can be packaged at low levels into aberrant virus particles or immature virions and that Vif is not present significantly in mature virions. Overall, these results indicate that the Vif content in virions is tightly regulated and also argue against a function of virion-associated Vif. 相似文献
20.
Virion glycoproteins such as glycoprotein D (gD) are believed to be the dominant antigens of herpes simplex virus 2 (HSV-2). We have observed that mice immunized with a live HSV-2 ICP0- mutant virus, HSV-2 0ΔNLS, are 10 to 100 times better protected against genital herpes than mice immunized with a HSV-2 gD subunit vaccine ( PLoS ONE 6:e17748). In light of these results, we sought to determine which viral proteins were the dominant antibody-generators (antigens) of the live HSV-2 0ΔNLS vaccine. Western blot analyses indicated the live HSV-2 0ΔNLS vaccine elicited an IgG antibody response against 9 or more viral proteins. Many antibodies were directed against infected-cell proteins of >100 kDa in size, and only 10 ± 5% of antibodies were directed against gD. Immunoprecipitation (IP) of total HSV-2 antigen with 0ΔNLS antiserum pulled down 19 viral proteins. Mass spectrometry suggested 44% of immunoprecipitated viral peptides were derived from two HSV-2 infected cells proteins, RR-1 and ICP8, whereas only 14% of immunoprecipitated peptides were derived from HSV-2’s thirteen glycoproteins. Collectively, the results suggest the immune response to the live HSV-2 0ΔNLS vaccine includes antibodies specific for infected cell proteins, capsid proteins, tegument proteins, and glycoproteins. This increased breadth of antibody-generating proteins may contribute to the live HSV-2 vaccine’s capacity to elicit superior protection against genital herpes relative to a gD subunit vaccine. 相似文献
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