Poliovirus Mutants at Histidine 195 of VP2 Do Not Cleave VP0 into VP2 and VP4

The final stage of poliovirus assembly is characterized by a cleavage of the capsid precursor protein VP0 into VP2 and VP4. This cleavage is thought to be autocatalytic and dependent on RNA encapsidation. Analysis of the poliovirus empty capsid structure has led to a mechanistic model for VP0 cleavage involving a conserved histidine residue that is present in the surrounding environment of the VP0 cleavage site. Histidine 195 of VP2 (2195H) is hypothesized to activate local water molecules, thus initiating a nucleophilic attack at the scissile bond. To test this hypothesis, 2195H mutants were constructed and their phenotypes were characterized. Consistent with the requirement of VP0 cleavage for poliovirus infectivity, all 2195H mutants were nonviable upon introduction of the mutant genomes into HeLa cells. Replacement of 2195H with threonine or arginine resulted in the assembly of a highly unstable 150S virus particle. Further analyses showed that these particles contain genomic RNA and uncleaved VP0, criteria associated with the provirion assembly intermediate. These data support the involvement of 2195H in mediating VP0 cleavage during the final stages of virus assembly.     

Characterization of the mRNA''s for the polyoma virus capsid proteins VP1, VP2, and VP3.

Polyadenylated cytoplasmic RNA from polyoma virus-infected cells can be translated in the wheat germ system to yield all there polyoma virus capsid proteins, VP1, VP2, and VP3. The translation products of RNA selected from total cytoplasmic RNA of infected cells by hybridization to polyoma virus DNA showed a high degree of enrichment for VP1, VP2, and VP3. The identity of the in vitro products with authentic virion proteins was established in two ways. First, tryptic peptide maps of the in vitro products were found to be essentially identical to those of their in vivo counterparts. Second, the mobilities of the in vitro products on two-dimensional gels were the same as those of viral proteins labeled in vivo. VP1, VP2, and vp3 were all labeled with [35S] formylmethionine when they were synthesized in the presence of [35S] formylmethionyl-tRNAfmet. We determined the sizes of the polyadenylated mRNA's for VP1, VP2, and VP3 by fractionation on gels. The sizes of the major mRNA species for the capsid proteins are as follows: VP2, 8.5 X 10(5) daltons; VP3, 7.4 X 10(5) daltons; and VP1, 4.6 X 10(5) daltons. We conclude that all three viral capsid proteins are synthesized independently in vitro, that all three viral capsid proteins are virally coded, and that each of the capsid proteins has a discrete mRNA.     

Partial amino-terminal sequences of the polyoma nonhistone proteins VP1, VP2, and VP3 synthesized in vitro.

The three polyoma virus capsid proteins VP1, VP2, and VP3 were synthesized in vitro in the presence of several radiolabeled amino acids and, after purification on sodium dodecyl sulfate-polyacrylamide gels, were subjected to sequential Edman degradation. The partial amino-terminal amino acid sequences obtained were compared with the sequence of amino acids predicted from the polyoma virus DNA sequencing (Arrand et al., J. Virol. 33:606--618, 1980). Together, these results showed that the 5' ends of the VP1, VP2, and VP3 coding sequences are located 1,217, 289, and 634 nucleotides, respectively, from the junction of HpaII restriction fragments 3 and 5.     

Cell-free synthesis of polyoma virus capsid proteins VP1 and VP2.

Polyadenylated RNA isolated from the cytoplasm of mouse 3T6 cells 28 h after infection with polyoma virus has been isolated and translated in vitro. Polyoma capsid proteins VP1 and VP2 have been identified in the cell-free product by polyacrylamide gel electrophoresis, specific immunoprecipitation, and tryptic peptide fingerprinting. Polyoma mRNA species have been isolated by preparative hybridization to purified viral DNA immobilized on cellulose nitrate filters and shown to code for both VP1 and VP2. These experiments establish conditions for the isolation of late polyoma mRNA and the cell-free synthesis of polyoma capsid proteins and indicate that the active mRNA species are at least partially virus coded.     

Infectious Bursal Disease Virus VP3 Upregulates VP1-Mediated RNA-Dependent RNA Replication

Genome replication is a critical step in virus life cycles. Here, we analyzed the role of the infectious bursal disease virus (IBDV) VP3, a major component of IBDV ribonucleoprotein complexes, on the regulation of VP1, the virus-encoded RNA-dependent RNA polymerase (RdRp). Data show that VP3, as well as a peptide mimicking its C-terminal domain, efficiently stimulates the ability of VP1 to replicate synthetic single-stranded RNA templates containing the 3′ untranslated regions (UTRs) from the IBDV genome segments.     

The VP5 domain of VP4 can mediate attachment of rotaviruses to cells

Some animal rotaviruses require the presence of sialic acid (SA) on the cell surface to infect the cell. We have isolated variants of rhesus rotavirus (RRV) whose infectivity no longer depends on SA. Both the SA-dependent and -independent interactions of these viruses with the cell are mediated by the virus spike protein VP4, which is cleaved by trypsin into two domains, VP5 and VP8. In this work we have compared the binding characteristics of wild-type RRV and its variant nar3 to MA104 cells. In a direct nonradioactive binding assay, both viruses bound to the cells in a saturable and specific manner. When neutralizing monoclonal antibodies directed to both the VP8 and VP5 domains of VP4 were used to block virus binding, antibodies to VP8 blocked the cell attachment of wild-type RRV but not that of the variant nar3. Conversely, an antibody to VP5 inhibited the binding of nar3 but not that of RRV. These results suggest that while RRV binds to the cell through VP8, the variant does so through the VP5 domain of VP4. This observation was further sustained by the fact that recombinant VP8 and VP5 proteins, produced in bacteria as fusion products with glutathione S-transferase, were found to bind to MA104 cells in a specific and saturable manner and, when preincubated with the cell, were capable of inhibiting the binding of wild-type and variant viruses, respectively. In addition, the VP5 and VP8 recombinant proteins inhibited the infectivity of nar3 and RRV, respectively, confirming the results obtained in the binding assays. Interestingly, when the infectivity assay was performed on neuraminidase-treated cells, the VP5 fusion protein was also found to inhibit the infectivity of RRV, suggesting that RRV could bind to the cell through two sequential steps mediated by the interaction of VP8 and VP5 with SA-containing and SA-independent cell surface receptors, respectively.     

Characterization of bovine rotavirus VP6 and VP7 as glycoproteins using monoclonal antibodies

Bovine rotavirus proteins were analysed by a panel of monoclonal antibodies. Glycosylated epitopes were identified on both inner and outer capsid proteins (VP6 and VP7 respectively). VP7 possessed a periodate insensitive epitope which was, however, sensitive to endoglycosidase H, mixed glycosidases and to protease treatment. This epitope was not detected on viruses grown in the presence of 2-deoxy-D-glucose or tunicamycin. An epitope was detected on VP6 which was sensitive to periodate oxidation. The blotted protein reacted with a glycan assay kit; yet the epitope was not affected by endoglycosidase H and was found on viruses grown in the presence of 2-deoxy-D-glucose or tunicamycin. These results suggest that VP7 and VP6 epitopes are carbohydrate dependent. The VP7 epitope contains an N-linked carbohydrate moiety in contrast to the VP6 epitope which appears to contain O-linked glycosyl units.     

Nuclear localizations of the herpes simplex virus type 1 tegument proteins VP13/14, vhs, and VP16 precede VP22-dependent microtubule reorganization and VP22 nuclear import

Herpes simplex virus type 1 (HSV-1) induces microtubule reorganization beginning at approximately 9 h postinfection (hpi), and this correlates with the nuclear localization of the tegument protein VP22. Thus, the active retention of this major virion component by cytoskeletal structures may function to regulate its subcellular localization (A. Kotsakis, L. E. Pomeranz, A. Blouin, and J. A. Blaho, J. Virol. 75:8697-8711, 2001). The goal of this study was to determine whether the subcellular localization patterns of other HSV-1 tegument proteins are similar to that observed with VP22. To address this, we performed a series of indirect immunofluorescence analyses using synchronously infected cells. We observed that tegument proteins VP13/14, vhs, and VP16 localized to the nucleus as early as 5 hpi and were concentrated in nuclei by 9 hpi, which differed from that seen with VP22. Microtubule reorganization was delayed during infection with HSV-1(RF177), a recombinant virus that does not produce full-length VP22. These infected cells did not begin to lose microtubule-organizing centers until 13 hpi. Repair of the unique long 49 (UL49) locus in HSV-1(RF177) yielded HSV-1(RF177R). Microtubule reorganization in HSV-1(RF177R)-infected cells occurred with the same kinetics as HSV-1(F). Acetylated tubulin remained unchanged during infection with either HSV-1(F) or HSV-1(RF177). Thus, while alpha-tubulin reorganized during infection, acetylated tubulin was stable, and the absence of full-length VP22 did not affect this stability. Our findings indicate that the nuclear localizations of tegument proteins VP13/14, VP16, and vhs do not appear to require HSV-1-induced microtubule reorganization. We conclude that full-length VP22 is needed for optimal microtubule reorganization during infection. This implies that VP22 mainly functions to reorganize microtubules later, rather than earlier, in infection. That acetylated tubulin does not undergo restructuring during VP22-dependent, virus-induced microtubule reorganization suggests that it plays a role in stabilizing the infected cells. Our results emphasize that VP22 likely plays a key role in cellular cytopathology during HSV-1 infection.     

Proteolysis of Monomeric Recombinant Rotavirus VP4 Yields an Oligomeric VP5* Core

Rotavirus particles are activated for cell entry by trypsin cleavage of the outer capsid spike protein, VP4, into a hemagglutinin, VP8*, and a membrane penetration protein, VP5*. We have purified rhesus rotavirus VP4, expressed in baculovirus-infected insect cells. Purified VP4 is a soluble, elongated monomer, as determined by analytical ultracentrifugation. Trypsin cleaves purified VP4 at a number of sites that are protected on the virion and yields a heterogeneous group of protease-resistant cores of VP5*. The most abundant tryptic VP5* core is trimmed past the N terminus associated with activation for virus entry into cells. Sequential digestion of purified VP4 with chymotrypsin and trypsin generates homogeneous VP8* and VP5* cores (VP8CT and VP5CT, respectively), which have the authentic trypsin cleavages in the activation region. VP8CT is a soluble monomer composed primarily of beta-sheets. VP5CT forms sodium dodecyl sulfate-resistant dimers. These results suggest that trypsinization of rotavirus particles triggers a rearrangement in the VP5* region of VP4 to yield the dimeric spikes observed in icosahedral image reconstructions from electron cryomicroscopy of trypsinized rotavirus virions. The solubility of VP5CT and of trypsinized rotavirus particles suggests that the trypsin-triggered conformational change primes VP4 for a subsequent rearrangement that accomplishes membrane penetration. The domains of VP4 defined by protease analysis contain all mapped neutralizing epitopes, sialic acid binding residues, the heptad repeat region, and the membrane permeabilization region. This biochemical analysis of VP4 provides sequence-specific structural information that complements electron cryomicroscopy data and defines targets and strategies for atomic-resolution structural studies.     

鸡贫血病毒VP1和VP2蛋白在家蚕中的联合表达

将鸡贫血病毒vp1和vp2基因分别克隆入转移载体pBacPAK8中,获得重组转移质粒pBac-vp1和pBac-vp2。以上两质粒分别与Cvn Ⅰ酶切线性化的亲本病毒Bm\|BacPAK6 DNA共转染家蚕细胞,通过蓝白斑筛选,纯化得到重组病毒Bm-vp1和Bm-vp2。PCR分析表明vp1和vp2基因已整合进杆状病毒基因组中。将Bm-vp1和Bm-vp2共感染5龄家蚕,通过表达产物免疫SPF鸡产生的抗血清与CAV感染的MDCCMSB1细胞的间接荧光抗体分析,证明表达产物能诱导鸡产生相应的抗体,而且能够保护子代鸡免受CAV的攻击。该研究表明,表达VP1和VP2蛋白的重组家蚕杆状病毒(Recombinant BmNPV)是很有前途的CAV亚单位疫苗的生产系统。     

鸡贫血病毒VP1和VP1蛋白在家蚕中的联合表达

将鸡贫血病毒vp1和vp2基因分别克隆入转移载体pBacPAK8中,获得重组转移质粒pBac-vp1和pBac-vp2。以上两质粒分别与CunI酶切线性化的亲本病毒Bm-Bacpak6DNA共转染家蚕细胞,通过蓝白斑筛选,纯化得到重组病毒Bm-vp1和Bm-vp2。PCR分析表明vp1和vp2基因已整合进杆状病毒基因组中。将Bm-vp1和Bm-vp2共感染5龄家蚕,通过表达产物免疫SPF鸡产生的抗血清与CAV感染的MDCC－MSB1细胞的间接荧光抗体分析,证明表达产物能诱导鸡产生的抗体,而且能够保护子代鸡免受CAV的攻击。该研究表明,表达VP1和VP2蛋白的重组家蚕杆状病毒（Recombinant BmNP)是很有前途的CAV亚单位疫苗的生产系统。     

Cloning and sequencing of envelope proteins (VP19, VP28) and nucleocapsid proteins (VP15, VP35) of a white spot syndrome virus isolate from Korean shrimp

Since our first report in 1998, white spot syndrome virus (WSSV) has become wide-spread on the southern and western coasts of Korea. Almost all shrimp in ponds die within 3 to 4 d after the first dead shrimp are observed with gross lesions ranging from abnormal red body discoloration to white spots in the cuticle. From one isolate, we cloned and sequenced WSSV genomic DNA coding for VP19 and VP28 envelope proteins and VP15 and VP35 nucleocapsid proteins. Putative protein sequences were submitted to GenBank and assigned accession numbers AY316119 (VP19), AY324881 (VP28), AY374120 (VP15) and AY325896 (VP35). At the nucleotide level, VP19, VP28 and VP15 sequences were, respectively, 99, 100 and 100% identical to those of China, Indonesia, Japan and the United States and the VP35 sequence was 100% identical to that of a Taiwanese isolate. The deduced amino-acid sequences were 99 to 100% identical to those from other countries. In VP19, C and T in the foreign isolates were replaced by T and A in the Korean isolate at Positions 57 and 218 nt, respectively, downstream of A (+) of the VP19 start codon. The change at Position 218 nt resulted in valine in the foreign isolates being replaced by aspartate in the Korean isolate.     

Specific interactions between rotavirus outer capsid proteins VP4 and VP7 determine expression of a cross-reactive, neutralizing VP4-specific epitope.

We previously reported that the expression of rotavirus phenotypes by reassortants was affected by recipient genetic background and proposed specific interactions between the outer capsid proteins VP4 and VP7 as the basis for the phenotypic effects (D. Chen, J. W. Burns, M. K. Estes, and R. F. Ramig, Proc. Natl. Acad. Sci. USA 86:3743-3747, 1989). A neutralizing, cross-reactive VP4-specific monoclonal antibody (MAb), 2G4, was used to probe the protein-protein interactions. The VP4 specificity of 2G4 was confirmed by immunoblot analysis. MAb 2G4 reacted with both standard (SA11-C13) and variant rotavirus SA11 (SA11-4F) but did not react with bovine rotavirus B223 as determined by plaque reduction neutralization (PRN) and enzyme-linked immunosorbent assay (ELISA). When a panel of SA11-4F/B223 and SA11-Cl3/B223 reassortants in purified or crude lysate form that had been grown in the presence or absence of trypsin was analyzed with MAb 2G4 by PRN and ELISA, the results with some reassortants were unexpected. That is, MAb 2G4 reacted with VP4 of SA11 parental origin (4F or C13) when it was assembled into capsids with the homologous SA11 VP7 but failed to react with VP4 of SA11 assembled into capsids with heterologous B223 VP7. Conversely, MAb 2G4 failed to react with VP4 of B223 parental origin when it was assembled into capsids with homologous B223 VP7 but did react with B223 VP4 assembled into capsids with the heterologous SA11 VP7. Similar reactivity was observed when 2G4 was used to immunoprecipitate purified double-shelled virions. When soluble unassembled viral proteins were analyzed by ELISA, the 2G4 reactive pattern was as predicted from the parental origin of VP4. That is, 2G4 reacted with the soluble VP4 of reassortants having VP4 from SA11-Cl3 or SA11-4F and failed to react with VP4 of B223 origin, regardless of the origin of VP7. PRN and ELISA results obtained with nonglycosylated viruses revealed that the unexpected reactivity of 2G4 with virus particles was not the result of differential glycosylation of VP7 and epitope masking. These results indicate that the 2G4 epitope existed in the soluble form of VP4 encoded by SA11-Cl3 or SA11-4F but not in soluble B223 VP4. On the other hand, in assembled virions, the presentation of the 2G4 epitope on VP4 was unexpected in some reassortants and was affected by the specific interactions between VP4 and VP7 of heterologous parental origin.     

猪细小病毒结构蛋白VP1和VP2的基因免疫研究

利用真核表达载体pCIneo和pcDNA3.1( )分别构建了含有猪细小病毒VP1基因的pCIneo VP1和含有VP2基因的pCIneo VP2与pcDNA VP2三种真核表达质粒。将上述三种真核表达质粒分别转染ＩＢＲＳ－２细胞,利用间接ELISA检测表达情况,结果表明上述三种质粒均能在ＩＢＲＳ－２细胞表达,表达产物位于细胞中。在此基础上,利用这三种质粒分别以肌内注射的方式,间隔2周2次免疫小鼠,结果发现所有表达质粒均能诱导产生明显的细胞免疫和体液免疫,其中pCIneo VP1质粒诱导的体液免疫最强,与猪细小病毒灭活疫苗免疫组相当,pCIneo VP2诱导的细胞免疫应答强于PPV灭活组,pCIneo VP1和pCIneo VP2联合免疫并没有加强作用。     

Rotavirus proteins VP7, NS28, and VP4 form oligomeric structures.

Sucrose gradient sedimentation analysis of rotavirus SA11-infected Ma104 cells revealed the presence of oligomers of VP7, the structural glycoprotein, and NS28, the nonstructural glycoprotein. Cross-linking the proteins, either before or after sucrose gradient centrifugation, stabilizes oligomers, which can be analyzed by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after immunoprecipitation. The major NS28 oligomer was tetrameric, though dimers and higher-order structures were observed as well. VP7 formed predominantly dimers, and again tetramers and higher oligomeric forms were present. Each oligomer of VP7 and NS28 sedimented at the same characteristic rate through the sucrose gradient either in the presence or absence of cross-linking. Monomers could not be cross-linked to form oligomers, demonstrating that cross-linked oligomers were not artifactually derived from monomers. Reversing the cross-linking of immunoprecipitated VP7 on reducing SDS-PAGE resulted in the appearance of only the monomeric form of VP7. Dissociation of the NS28 oligomers resulted in stable dimers as well an monomers. In the faster-sedimenting fractions, a 16S to 20S complex, which contained the rotavirus outer shell proteins VP7 and VP4 cross-linked to NS28, was observed. These complexes were shown not to be associated with any known subviral particle. The association of VP4, NS28, and VP7 may represent sites on the endoplasmic reticulum membrane that participate in the budding of the single-shelled particles into the lumen of the endoplasmic reticulum, where maturation to double-shelled particles occurs.     

Expression of the polyomavirus minor capsid proteins VP2 and VP3 in Escherichia coli: in vitro interactions with recombinant VP1 capsomeres.

The polyomavirus VP2 and VP3 capsid proteins were expressed in Escherichia coli. The majority of the expressed proteins were in an insoluble fraction, and they were extracted and initially purified in 8 M urea before renaturation. Soluble VP2 and VP3 were mixed with purified recombinant VP1 capsomeres, and their interactions were assayed by immunoprecipitation and ion-exchange chromatography. Coimmunoprecipitation could be demonstrated with antibodies to either VP1 or VP2/VP3. Mixing recombinant VP1 with VP2 and VP3 modified the recognition of VP1 by domain-specific antipeptide antibodies and altered the chromatographic behavior of the individual proteins. Similar results were observed when a truncated VP1 protein, delta NCOVP1, with 62 amino acids deleted from the carboxy terminus was mixed with VP2/VP3. After the mixing, equilibrium dissociation constants for their binding to either VP1 or delta NCOVP1 were determined to be 0.37 +/- 0.23 microM for VP2 and 0.18 +/- 0.21 microM for VP3. These studies demonstrate that the recombinant VP2 and VP3 proteins interact with VP1 to affect the biochemical properties of VP1 capsomeres and to change the epitope accessibility of VP1 pentamers. These changes may reflect conformational alterations in VP1 capsomeres which are necessary for viral genome encapsidation.     

Interaction of polyomavirus internal protein VP2 with the major capsid protein VP1 and implications for participation of VP2 in viral entry.

A complex of the polyomavirus internal protein VP2/VP3 with the pentameric major capsid protein VP1 has been prepared by co-expression in Escherichia coli. A C-terminal segment of VP2/VP3 is required for tight association, and a crystal structure of this segment, complexed with a VP1 pentamer, has been determined at 2.2 A resolution. The structure shows specific contacts between a single copy of the internal protein and a pentamer of VP1. These interactions were not detected in the previously described structure of the virion, but the location of VP2 in the recombinant complex is consistent with features in the virion electron-density map. The C-terminus of VP2/VP3 inserts in an unusual, hairpin-like manner into the axial cavity of the VP1 pentamer, where it is anchored strongly by hydrophobic interactions. The remainder of the internal protein appears to have significant flexibility. This structure restricts possible models for exposure of the internal proteins during viral entry.