Proteins in intracellular simian virus 40 nucleoportein complexes: comparison with simian virus 40 core proteins.

Intracellular nucleoprotein complexes containing SV40 supercoiled DNA were purified from cell lysates by chromatography on hydroxyapatite columns followed by velocity sedimentation through sucrose gradients. The major protein components from purified complexes were identified as histone-like proteins. When analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels, complex proteins comigrated with viral core polypeptides VP4, VP5, VP6, and VP7. (3H) tryptophan was not detected in polypeptides from intracellular complexes or in the histone components from purified SV40 virus. However, a large amount of (3H) tryptophan was found in the viral polypeptide VP3 relative to that incorporated into the capsid polypeptides VP1 and VP2. Intracellular complexes contain 30 to 40% more protein than viral cores prepared by alkali dissociation of intact virus, but when complexes were exposed to the same alkaline conditions, protein also was removed from complexes and they subsequently co-sedimented with and had the same buoyant density as viral cores. The composition and physical similarities of nucleoprotein complex and viral cores indicate that complexes may have a role in the assembly of virions.     

Purification and characterization of adult diarrhea rotavirus: identification of viral structural proteins.

Adult diarrhea rotavirus (ADRV) is a newly identified strain of noncultivable human group B rotavirus that has been epidemic in the People's Republic of China since 1982. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western (immuno-) blot analysis to examine the viral proteins present in the outer and inner capsids of ADRV and compared these with the proteins of a group A rotavirus, SA11. EDTA treatment of double-shelled virions removed the outer capsid and resulted in the loss of three polypeptides of 64, 61, and 41, kilodaltons (kDa). Endo-beta-N-acetylglucosaminidase H digestion of double-shelled virions identified the 41-kDa polypeptide as a glycoprotein. CaCl2 treatment of single-shelled particles removed the inner capsid and resulted in the loss of one polypeptide with a molecular mass of 47 kDa. The remaining core particle had two major structural proteins of 136 and 113 kDa. All of the proteins visualized on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were antigenic by Western blot analysis when probed with convalescent-phase human and animal antisera. A 47-kDa polypeptide was most abundant and was strongly immunoreactive with human sera, animal sera raised against ADRV and against other group B animal rotaviruses (infectious diarrhea of infant rat virus, bovine and porcine group B rotavirus, and bovine enteric syncytial virus) and a monoclonal antibody prepared against infectious diarrhea of infant rat virus. This 47-kDa inner capsid polypeptide contains a common group B antigen and is similar to the VP6 of the group A rotaviruses. Human convalescent-phase sera also responded to a 41-kDa polypeptide of the outer capsid that seems similar to the VP7 of group A rotavirus. Other polypeptides have been given tentative designations on the basis of similarities to the control preparation of SA11, including a 136-kDa polypeptide designated VP1, a 113-kDa polypeptide designated VP2, 64- and 61-kDa polypeptides designated VP5 and VP5a, and several proteins in the 110- to 72-kDa range that may be VP3, VP4, or related proteins. The lack of cross-reactivity on Western blots between antisera to group A versus group B rotaviruses confirmed that these viruses are antigenically quite distinct.     

Proteolytic enhancement of rotavirus infectivity: molecular mechanisms

The polypeptide compositions of single-shelled and double-shelled simian rotavirus particles were modified by exposure to proteolytic enzymes. Specifically, a major outer capsid polypeptide (VP3) having a molecular weight of 88,000 in double-shelled particles was cleaved by trypsin to yield two polypeptides, VP5* and VP8* (molecular weights, 60,000 and 28,000, respectively). The cleavage of VP3 by enzymes that enhanced infectivity (trypsin, elastase, and pancreatin) yielded different products compared to those detected when VP3 was cleaved by chymotrypsin, which did not enhance infectivity. The appearance of VP5* was correlated with an enhancement of infectivity. Cleavages of the major internal capsid polypeptide VP2 were also observed. The VP2 cleavage products had molecular weights similar to those of known structural and nonstructural rotavirus polypeptides. We confirmed the precursor-product relationships by comparing the peptide maps of the polypeptides generated by digestions with V-8 protease and chymotrypsin. The remaining rotavirus structural polypeptides, including the outer capsid glycoproteins (VP7 and 7a), were not altered by exposure to pancreatic enzymes. Cleavage of VP3 was not required for virus assembly, and specific cleavage of the polypeptides occurred only on assembled particles. We also discuss the role of cleavage activation in other virus-specific biological functions (e.g., hemagglutination and virulence).     

Receptor activity of rotavirus nonstructural glycoprotein NS28.

Rotavirus morphogenesis involves the budding of subviral particles through the rough endoplasmic reticulum (RER) membrane of infected cells. During this process, particles acquire the outer capsid proteins and a transient envelope. Previous immunocytochemical and biochemical studies have suggested that a rotavirus nonstructural glycoprotein, NS28, encoded by genome segment 10, is a transmembrane RER protein and that about 10,000 Mr of its carboxy terminus is exposed on the cytoplasmic side of the RER. We have used in vitro binding experiments to examine whether NS28 serves as a receptor that binds subviral particles and mediates the budding process. Specific binding was observed between purified simian rotavirus SA11 single-shelled particles and RER membranes from SA11-infected monkey kidney cells and from SA11 gene 10 baculovirus recombinant-infected insect cells. Membranes from insect cells synthesizing VP1, VP4, NS53, VP6, VP7, or NS26 did not possess binding activity. Comparison of the binding of single-shelled particles to microsomes from infected monkey kidney cells and from insect cells indicated that a membrane-associated component(s) from SA11-infected monkey kidney cells interfered with binding. Direct evidence showing the interaction of NS28 and its nonglycosylated 20,000-Mr precursor expressed in rabbit reticulocyte lysates and single-shelled particles was obtained by cosedimentation of preformed receptor-ligand complexes through sucrose gradients. The domain on NS28 responsible for binding also was characterized. Reduced binding of single-shelled particles to membranes was seen with membranes treated with (i) a monoclonal antibody previously shown to interact with the C terminus of NS28, (ii) proteases known to cleave the C terminus of NS28, and (iii) the Enzymobead reagent. VP6 on single-shelled particles was suggested to interact with NS28 because (i) a monoclonal antibody to the subgroup I epitope on VP6 reduced particle binding, (ii) a purified polyclonal antiserum raised against recombinant baculovirus-produced VP6 reduced ligand binding, and (iii) a monoclonal antibody to a conserved epitope on VP6 augmented ligand binding. These experimental data provide support for the hypothesized receptor role of NS28 before the budding stage of rotavirus morphogenesis.     

Synthesis and characterization of chimeric particles between epizootic hemorrhagic disease virus and bluetongue virus: functional domains are conserved on the VP3 protein.

A functional assay has been developed to determine the conservative nature of the interacting sites of various structural proteins of orbiviruses by using baculovirus expression vectors. For this investigation, proteins of two serologically related orbiviruses, bluetongue virus (BTV) and the less studied epizootic hemorrhagic disease virus (EHDV), were used to synthesize chimeric particles. The results demonstrate that the inner capsid protein VP3 of EHDV-1 can replace VP3 protein of BTV in formation of the single-shelled corelike particles and the double-shelled viruslike particles. Moreover, we have demonstrated that all three minor core proteins (VP1, VP4, and VP6) can be incorporated into the homologous and chimeric corelike and viruslike particles, indicating that the functional epitopes of the VP3 protein are conserved for the morphological events of the virus. This is the first evidence of assembly of seven structural proteins of the virus by a baculovirus expression system. Confirmation at the molecular level was obtained by determining the EHDV-1 L3 gene nucleic sequence and by comparing it with sequences available for BTV. The analysis revealed a high degree homology between the two proteins: 20% difference, 50% of which is conservative. The consequences for Orbivirus phylogeny and the possibility of gene reassortments are discussed.     

Rotavirus protein rearrangements in purified membrane-enveloped intermediate particles.

Rotavirus, a double-shelled nonenveloped member of the REoviridae family, becomes transiently membrane enveloped during its maturation process, as single-shelled particles bud from cytoplasmic viroplasm structures into the adjacent endoplasmic reticulum. The present study describes the isolation of these membrane-enveloped viral intermediates from rotavirus SA11-infected Ma104 cells. The enveloped intermediates comprised the proteins VP1, VP2, VP4, VP6, VP7, and NS28 and small amounts of NS35 and NS34. VP7 in the intermediate particles was recognized by either a polyclonal antibody to VP7, which previous studies had shown recognizes the membrane-associated form of VP7, or a monoclonal antibody which recognizes VP7 on mature virus. NS28, VP7, and VP4 could be complexed to a higher-molecular-weight form when the membrane-permeable cross-linker dithiobis(succinimidylproprionate) was used. However, when an impermeable cross-linker was used, the structural proteins, including VP7, were not accessible to cross-linking. Velocity sedimentation of cross-linked immunoisolated enveloped virus particles showed that VP7 and VP4 were located in the same fractions only when the membrane-permeable cross-linker was used, implying their heterooligomeric association during outer capsid formation. When intermediate enveloped virus particles were treated with protease, VP6 and VP7 were protected, but not in the presence of detergent. Taken together, these results support the idea that in the membrane-enveloped intermediate, VP7 is repositioned from its location in the endoplasmic reticulum lumen back across the viral membrane envelope to the inferior of the virus particle during the maturation process.     

Expression of the major capsid protein VP6 of group C rotavirus and synthesis of chimeric single-shelled particles by using recombinant baculoviruses.

VP6 of group C (Cowden strain) rotavirus was expressed in the baculovirus system. The recombinant protein, expressed to a high level in insect cells, was purified by ion-exchange chromatography. The purified protein was proven to be trimeric. The effect of pH on the trimer's stability was investigated. Coexpression of VP6 from group A (bovine strain RF) and VP6 from group C in the baculovirus system did not result in the formation of chimeric trimers. Coexpression of VP2 from group A rotavirus (bovine strain RF) and VP6 from group C in the baculovirus system led to the formation of chimeric, empty, single-shelled particles. These results demonstrate conservation in the domains necessary for binding to VP2 in different serogroups of VP6. The locations of the domains involved in trimerization and in the interaction with VP2 are discussed.     

Rotavirus VP1 alone specifically binds to the 3' end of viral mRNA, but the interaction is not sufficient to initiate minus-strand synthesis.

Recent studies have shown that disrupted (open) rotavirus cores have an associated replicase activity which supports the synthesis of dsRNA from viral mRNA in a cell-free system (D. Chen, C. Q.-Y. Zeng, M. J. Wentz, M. Gorziglia, M. K. Estes, and R. F. Ramig, J. Virol. 68:7030-7039, 1994). To determine which of the core proteins, VP1, VP2, or VP3, recognizes the template mRNA during RNA replication, SA11 open cores were incubated with 32P-labeled RNA probes of viral and nonviral origin and the reaction mixtures were analyzed for the formation of RNA-protein complexes by gel mobility shift assay. In mixtures containing a probe representing the 3' end of SA11 gene 8 mRNA, two closely migrating RNA-protein complexes, designated s and f, were detected. The interaction between the RNA and protein of the s and f complexes was shown to be specific by competitive binding assay with tRNA and brome mosaic virus RNA. By electrophoretic analysis of RNA-protein complexes recovered from gels, VP1 was shown to be the only viral protein component of the complexes, thereby indicating that VP1 specifically recognizes the 3' end of gene 8 mRNA. Analysis of VP1 purified from open cores by glycerol gradient centrifugation verified that VP1 recognizes the 3' end of viral mRNA but also showed that in the absence of other viral proteins, VP1 lacks replicase activity. When reconstituted with VP2-rich portions of the gradient, VP1 stimulated levels of replicase activity severalfold. These data indicate that VP1 can bind to viral mRNA in the absence of any other viral proteins and suggest that VP2 must interact with the RNA-protein complex before VP1 gains replicase activity.     

Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of BTV.

The L3 and M7 genes of bluetongue virus (BTV), which encode the two major core proteins of the virus (VP3 and VP7, respectively), were inserted into a baculovirus dual-expression transfer vector and a recombinant baculovirus expressing both foreign genes isolated following in vivo recombination with wild-type Autographa californica nuclear polyhedrosis virus DNA. Spodoptera frugiperda insect cells infected with the recombinant synthesized large amounts of BTV corelike particles. These particles have been shown to be similar to authentic BTV cores in terms of size, appearance, stoichiometric arrangement of VP3 to VP7 (ratio, 2:15), and the predominance of VP7 on the surface of the particles. In infected insect cells, the corelike particles were observed in paracrystalline arrays. The formation of these structures indicates that neither the BTV double-stranded viral RNA species nor the associated minor core proteins are necessary for assembly of cores in insect cells. Furthermore, the three BTV nonstructural proteins NS1, NS2, and NS3, are not required to assist or direct the formation of empty corelike particles from VP3 and VP7.     

Steady-state infection by echovirus 6 associated with nonlytic viral RNA and an unprocessed capsid polypeptide.

We established a human cell line which was persistently infected (PI) by the normally cytolytic echovirus 6. All of the cultured PI cells contained genome-size viral RNA which was synthesized continuously and incorporated into virus particles. This steady-state infection has been maintained for more than 6 years. In contrast to RNA of wild-type echovirus 6, the viral RNA from PI cells was not lytic when transfected into uninfected, susceptible cells. The capsid polypeptides of the virus particles produced during lytic infections were compared with those of virus particles from PI cells. Wild-type virions contained five polypeptides with molecular masses of 31.5, 27, 25.8, 21.2, and 9.5 kilodaltons. Comparison of polypeptide profiles of virions and empty immature capsids along with peptide analyses by immunoblotting and partial proteolysis of isolated viral proteins identified the cleavage products of the 31.5-kilodalton polypeptide (VP0) as the two smaller polypeptides (VP2 and VP4). The virus particles produced by PI cells as well as cellular extracts of PI cells contained only the three largest proteins (VP0, VP1, and VP3), indicating that VP0 was not processed during persistent infection. The lack of VP2 and VP4 in the defective virus particles coincided with their inability to attach to uninfected, susceptible cells. The maintenance of the steady-state infection of echovirus 6 was not dependent upon the release of virus particles from PI cells.     

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants.

Rotavirus has a capsid composed of three concentric protein layers. We coexpressed various combinations of the rotavirus structural proteins of single-layered (core) and double-layered (single-shelled) capsids from baculovirus vectors in insect cells and determined the ability of the various combinations to assemble into viruslike particles (VLPs). VLPs were purified by centrifugation, their structure was examined by negative-stain electron microscopy, their protein content was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and GTP binding assays, and their ability to support synthesis of negative-strand RNAs on positive-sense template RNAs was determined in an in vitro replication system. Coexpression of all possible combinations of VP1, VP2, VP3, and VP6, the proteins of double-layered capsids, resulted in the formation of VP1/2/3/6, VP1/2/6, VP2/3/6, and VP2/6 double-layered VLPs. These VLPs had the structural characteristics of empty rotavirus double-layered particles and contained the indicated protein species. Only VPI/2/3/6 and VP1/2/6 particles supported RNA replication. Coexpression of all possible combinations of VPl, VP2, and VP3, the proteins of single-layered capsids, resulted in the formation of VP1/2/3, VP1/2, VP2/3, and VP2 single-layered VLPs. These VLPs had the structural characteristics of empty single-layered rotavirus particles and contained the indicated protein species. Only VP1/2/3 and VP1/2 VLPs supported RNA replication. We conclude that (i) the assembly of VP1 and VP3 into VLPs requires the presence of VP2, (ii) the role of VP2 in the assembly of VP1 and VP3 and in replicase activity is most likely structural, (iii) VP1 is required and VP3 is not required for replicase activity of VLPs, and (iv) VP1/2 VLPs constitute the minimal replicase particle in the in vitro replication system.     

Polypeptides of the surface projections and the ribonucleoprotein of avian infectious bronchitis virus.

Purified avian infectious bronchitis virus was digested with bromelain (0.7 mg/ml), and the surface projections were removed. Polyacrylamide gel electrophoresis of the polypeptides from these bromelain-treated particles showed that VP1, VP2, and VP5 were missing from the seven polypeptides. VP1 to VP7, that were present in untreated virus preparations. Milder bromelain treatment (0.07 mg/ml) left visible surface projections and polypeptides comprising VP1 and VP2 intact, but removed VP5. Thus, there are apparently two types of surface projections on the virus particle. The ribonucleoprotein complex was released from virus particles disrupted with 1% Nonidet P-40. The proportion of VP6 in such preparations was greatly reduced, implying that VP6 is the structural polypeptide of the ribonucleoprotein. Polypeptides VP1, VP2, VP4, and VP5 are glycosylated, but none of the polypeptides contains lipid.     

Expression of rotavirus VP2 produces empty corelike particles.

The complete VP2 gene of bovine rotavirus strain RF has been inserted into the baculovirus transfer vector pVL941 under the control of the polyhedrin promoter. Cotransfection of Spodoptera frugiperda 9 cells with wild-type baculovirus DNA and transfer vector DNA led to the formation of recombinant baculoviruses which contain bovine rotavirus gene 2. Infection of S. frugiperda cells with this recombinant virus resulted in the production of a protein similar in size and antigenic properties to the authentic rotavirus VP2. The protein binds double-stranded RNA and DNA in an overlay protein blot assay. Expressed VP2 assembles in the cytoplasm of infected cells in corelike particles 45 nm in diameter. These corelike particles were purified by sucrose gradient centrifugation and found to be devoid of nucleic acid. Coexpression of VP2 and VP6 from heterologous rotavirus strains (bovine and simian) resulted in the formation of single-shelled particles. These results definitively show the existence of an innermost protein shell in rotavirus which is formed independently of other rotavirus proteins. These results have implications for schemes of rotavirus morphogenesis.     

Crystal Structure of Marburg Virus VP24

The VP24 protein plays an essential, albeit poorly understood role in the filovirus life cycle. VP24 is only 30% identical between Marburg virus and the ebolaviruses. Furthermore, VP24 from the ebolaviruses is immunosuppressive, while that of Marburg virus is not. The crystal structure of Marburg virus VP24, presented here, reveals that although the core is similar between the viral genera, Marburg VP24 is distinguished by a projecting β-shelf and an alternate conformation of the N-terminal polypeptide.