Rubella virus capsid protein interacts with poly(a)-binding protein and inhibits translation

During virus assembly, the capsid proteins of RNA viruses bind to genomic RNA to form nucleocapsids. However, it is now evident that capsid proteins have additional functions that are unrelated to nucleocapsid formation. Specifically, their interactions with cellular proteins may influence signaling pathways or other events that affect virus replication. Here we report that the rubella virus (RV) capsid protein binds to poly(A)-binding protein (PABP), a host cell protein that enhances translational efficiency by circularizing mRNAs. Infection of cells with RV resulted in marked increases in the levels of PABP, much of which colocalized with capsid in the cytoplasm. Mapping studies revealed that capsid binds to the C-terminal half of PABP, which interestingly is the region that interacts with other translation regulators, including PABP-interacting protein 1 (Paip1) and Paip2. The addition of capsid to in vitro translation reaction mixtures inhibited protein synthesis in a dose-dependent manner; however, the capsid block was alleviated by excess PABP, indicating that inhibition of translation occurs through a stoichiometric mechanism. To our knowledge, this is the first report of a viral protein that inhibits protein translation by sequestration of PABP. We hypothesize that capsid-dependent inhibition of translation may facilitate the switch from viral translation to packaging RNA into nucleocapsids.     

Analyses of phosphorylation events in the rubella virus capsid protein: role in early replication events

The Rubella virus capsid protein is phosphorylated prior to virus assembly. Our previous data are consistent with a model in which dynamic phosphorylation of the capsid regulates its RNA binding activity and, in turn, nucleocapsid assembly. In the present study, the process of capsid phosphorylation was examined in further detail. We show that phosphorylation of serine 46 in the RNA binding region of the capsid is required to trigger phosphorylation of additional amino acid residues that include threonine 47. This residue likely plays a direct role in regulating the binding of genomic RNA to the capsid. We also provide evidence which suggests that the capsid is dephosphorylated prior to or during virus budding. Finally, whereas the phosphorylation state of the capsid does not directly influence the rate of synthesis of viral RNA and proteins or the assembly and secretion of virions, the presence of phosphate on the capsid is critical for early events in virus replication, most likely the uncoating of virions and/or disassembly of nucleocapsids.     

Phosphorylation of rubella virus capsid regulates its RNA binding activity and virus replication

Rubella virus is an enveloped positive-strand RNA virus of the family TOGAVIRIDAE: Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the RNA-binding activity of cell-associated capsids increased dramatically after treatment with phosphatase, suggesting that the capsid is dephosphorylated during virus assembly. In vitro assays indicate that the capsid may be a substrate for protein phosphatase 1A. As capsid is heavily phosphorylated under conditions where virus assembly does not occur, we propose that phosphorylation serves to negatively regulate binding of viral genomic RNA. This may delay the initiation of nucleocapsid assembly until sufficient amounts of virus glycoproteins accumulate at the budding site and/or prevent nonspecific binding to cellular RNA when levels of genomic RNA are low. It follows that at a late stage in replication, the capsid may undergo dephosphorylation before nucleocapsid assembly occurs.     

Rubella virus capsid associates with host cell protein p32 and localizes to mitochondria

Togavirus nucleocapsids have a characteristic icosahedral structure and are composed of multiple copies of a capsid protein complexed with genomic RNA. The assembly of rubella virus nucleocapsids is unique among togaviruses in that the process occurs late in virus assembly and in association with intracellular membranes. The goal of this study was to identify host cell proteins which may be involved in regulating rubella virus nucleocapsid assembly through their interactions with the capsid protein. Capsid was used as bait to screen a CV1 cDNA library using the yeast two-hybrid system. One protein that interacted strongly with capsid was p32, a cellular protein which is known to interact with other viral proteins. The interaction between capsid and p32 was confirmed using a number of different in vitro and in vivo methods, and the site of interaction between these two proteins was shown to be at the mitochondria. Interestingly, overexpression of the rubella virus structural proteins resulted in clustering of the mitochondria in the perinuclear region. The p32-binding site in capsid is a potentially phosphorylated region that overlaps the viral RNA-binding domain of capsid. Our results are consistent with the possibility that the interaction of p32 with capsid plays a role in the regulation of nucleocapsid assembly and/or virus-host interactions.     

Spike protein-nucleocapsid interactions drive the budding of alphaviruses.

Semliki Forest virus (SFV) particles are released from infected cells by budding of nucleocapsids through plasma membrane regions that are modified by virus spike proteins. The budding process was studied with recombinant SFV genomes which lacked the nucleocapsid protein gene or, alternatively, the spike genes. No subviral particles were released from cells which expressed only the nucleocapsid protein or the spike proteins. Virus release was found to be strictly dependent on the coexpression of the nucleocapsid and the spike proteins. These results provide direct proof for the hypothesis that the alphavirus budding is driven by nucleocapsid-spike interactions. The importance of the viral 42S RNA for virus assembly and budding was investigated by using the heterologous vaccinia virus-T7 expression system for the synthesis of the SFV structural proteins. The results demonstrate that the viral genome is not absolutely required for formation of budding competent nucleocapsids, since small amounts of viruslike particles were assembled in the absence of 42S RNA.     

Form-determining functions in Sindbis virus nucleocapsids: nucleosomelike organization of the nucleocapsid.

Purified intact Sindbis virus nucleocapsids were treated at different pH values or with various concentrations of divalent cations, cation chelators, salt, or formamide. The resulting structures were examined by velocity sedimentation, electron microscopy, and protein-protein cross-linking. Changes in each of the test conditions led to alterations in the sedimentation profile of treated nucleocapsids. Appropriate concentrations of formamide or divalent cations generated beaded strandlike structures similar in morphology to those generated from adenovirus cores and nucleosomes. The capsid protein and RNA remained associated with each other at NaCl concentrations less than or equal to 1 M or after treatment of the structures with alkaline pH up to and including pH 10.7. Protein and RNA were dissociated by salt concentrations of greater than 1 M, suggesting that the arginine-rich, amino-terminal portion of the capsid protein is responsible for binding the RNA. Protein-protein cross-linking also indicated that the capsid proteins remained associated in small aggregates under some of the conditions that caused dissociation of the nucleocapsid and suggested the presence of more than one type of protein-protein interaction in the nucleocapsids. Collectively, these data suggest that, like histones and adenovirus core proteins, the Sindbis virus capsid protein serves to package segments of the genome into nucleoprotein beads which are capable of interacting with each other to form the nucleocapsid structure.     

Some properties of influenza virus nucleocapsids

Nucleocapsids released from influenza virions by sodium deoxycholate sedimented heterogeneously in sucrose gradients. Highly infectious virus (complete) preparations yielded nucleocapsids with peak distributions at 64 and 56S; von Magnus type virus (incomplete) lacked 64S nucleocapsids. Treatment of influenza virus nucleocapsids with pancreatic ribonuclease rendered the associated viral ribonucleic acid (RNA) molecules acid-soluble, indicating that capsid proteins do not completely surround the viral RNA's. However, the capsid proteins remained associated after enzymatic hydrolysis of the RNA, as judged by persistently high sedimentation rates. Sedimentation rates of viral nucleocapsids reflected the sedimentation rates of the associated RNA's: 64S nucleocapsids contained 18S RNA, whereas 56S nucleocapsids contained 15S RNA, although in both cases RNA's sedimenting at 4 to 13S were also recovered. Furthermore, just as incomplete virions lacked 64S nucleocapsids, they also lacked 18S RNA. These findings support the hypothesis that the influenza virus genome is divided among several distinct pieces of RNA.     

The movement and invasion of an insect baculovirus in tracheal cells of the armyworm, Pseudaletia unipuncta

The hypertrophy nuclear polyhedrosis virus of the armyworm, Pseudaletia unipuncta, causes a unique gradient of infected cells to form on the trachea. The movement and invasion of the virus apparently were not through adjacent intercellular membranes. The enveloped viruses emerged from the initially infected cell into an area between the cell plasma membrane and basal lamina, and then entered the uninfected tracheal cell either by lateral attachment and fusion of the viral envelope and the plasma membrane or by viropexis. The two methods of viral invasion into the cell suggest the presence of at least two phenotypically different enveloped viruses. Viropexis was initiated with an alignment of the peplomer spikes with regularly spaced, short radial striations on the inner coat of the plasma membrane. At a late state in viropexis, the viral envelope fused with the vacuole membrane, and an opening developed below the site of membrane fusion through which the nucleocapsid might enter the cytoplasm. Some nucleocapsids in membrane-lined vesicles resulting from viropexis appeared to be in a state of dissolution. Naked nucleocapsids were found along the nuclear envelope and within the nucleoplasm. No uncoating of the nucleocapsids was observed at the nucleopores, but uncoating seemed to occur in the nucleoplasm. Nucleocapsids were also found in the cytoplasm of nonsusceptible fat body cells, in which virus replication was not observed.     

Assembly of nucleocapsidlike structures in animal cells infected with a vaccinia virus recombinant encoding the measles virus nucleoprotein.

A vaccinia virus recombinant containing the measles virus nucleoprotein gene was shown to induce the synthesis of a 60 kDa phosphorylated nucleoprotein similar to authentic measles virus nucleoprotein. Mammalian or avian cells infected with the recombinant virus displayed tubular structures reminiscent of viral nucleocapsids both in the cytoplasm and in the nucleus. Such structures could be labelled in situ by using an immunogold detection method specific for measles virus proteins. Electron microscopic examination of tubular structures purified from cells infected with the vaccinia virus recombinant indicated that they displayed most of the features of measles virus nucleocapsids, although their length was on the average shorter. These results demonstrate the spontaneous assembly of measles virus nucleocapsids in the absence of viral leader RNA and provide a means for a detailed molecular analysis of the requirements for nucleocapsid assembly. Furthermore, these findings raise the possibility of achieving complete assembly of measles virus particles, devoid of infectious RNA, by using a vaccinia virus vector.     

Ribonucleic Acid Species of Intracellular Nucleocapsids and Released Virions of Vesicular Stomatitis Virus

Infection of chicken embryo cells with vesicular stomatitis (VS) virus resulted in variable production of three classes of intracellular viral ribonucleocapsids with sedimentation coefficients of approximately 140S, 110S, and 80S, as well as three corresponding classes of released virions designated B, LT, and T. Intracellular nucleocapsids of each class contained three proteins of which the major N protein was firmly bound, and the minor L and NS1 proteins were readily dissociated with 0.5 m NaCl. The ribonucleic acid (RNA) species extracted from B, LT, and T virions, and from corresponding intracellular nucleocapsids, contained RNA species with approximate molecular weights of 3.2 x 10(6), 2.0 x 10(6), and 10(6), respectively, as determined by polyacrylamide gel electrophoresis. These values are roughly equivalent to sedimentation coefficients of 42S, 28S, and 23S for each of the virion and nucleocapsid RNA species. Cells infected at high multiplicity with undiluted passage VS virus gave rise primarily to virions and nucleocapsids containing 23S RNA, whereas cells productively infected with purified B virions produced predominantly B and LT virions and nucleocapsids. At late stages in the productive cycle of infection, more virions containing 42S RNA were produced, but the intracellular pool of nucleocapsids containing 28S and 23S RNA remained relatively constant. Additional studies by more refined techniques are required to test the hypothesis that nucleocapsids containing 28S and 23S RNA are precursors of the 42S RNA in infectious VS-B virions and that production of defective T and LT virions results from failure of ligation of the RNA precursors.     

Early steps in the assembly of vesicular stomatitis virus nucleocapsids in infected cells.

The assembly of nucleocapsids is an essential step in the replicative cycle of vesicular stomatitis virus (VSV). In this study, we have examined the early events of vesicular stomatitis virus nucleocapsid assembly in BHK-21 cells. Nuclease-resistant intracellular nucleocapsids were isolated at various stages of assembly and analyzed for RNA and protein contents. The smallest ribonucleoprotein complex formed during nucleocapsid assembly contains the 5'-terminal 65 nucleotides of nascent viral RNA complexed with the viral proteins N and NS. Elongation of the assembling nucleocapsids proceeds unidirectionally towards the 3' terminus by the sequential addition of viral proteins which incrementally protect short stretches of the growing RNA chain. Pulse-chase studies show that the assembling nucleocapsids can be chased into full-length nucleocapsids which are incorporated into mature virions. Our results also suggest an involvement of the cytoskeletal framework during nucleocapsid assembly.     

Organization of the Sindbis virus nucleocapsid as revealed by bifunctional cross-linking agents

Purified Sindbis virus nucleocapsids were reacted with a variety of bifunctional protein-specific cross-linking agents. The products were analyzed in concentration-gradient polyacrylamide gels and amounts of various products determined. These studies indicated that available lysine residues within adjacent capsid proteins in purified intact nucleocapsids are separated by 6 A. The capsid proteins in intact nucleocapsids are cross-linked in a pattern predicted for discrete monomeric entities, rather than in dimeric or trimeric aggregates. Purified, soluble capsid protein exists in a conformation that differs from the arrangement of protein within nucleocapsids. These conformational differences suggest that topological changes may occur in the capsid protein during virus maturation. Cross-linked nucleocapsids that were treated with RNases resulted in the generation of RNA-free protein shells that retained hexagonal morphology, indicating that, together, the RNA and protein form the outer surface of the nucleocapsid. These data are used to produce a model of the Sindbis virus nucleocapsid in which the proteins are arranged quasi-equivalently in a T = 4 icosahedral shell.     

Replication of Measles Virus: Distinct Species of Short Nucleocapsids in Cytoplasmic Extracts of Infected Cells

Cytoplasmic extracts of Vero cells infected with wild-strain Edmonston measles virus were found to contain two and probably three distinct species of nucleocapsids. Species sedimenting at 200 and 110S contained RNA which sedimented at 50 and 16 to 18S, respectively. The third nucleocapsid species which sedimented at 170S was not present in all experiments and was not characterized in detail. Essentially all 200 and 170S, as well as a portion of the 110S, nucleocapsids were membrane associated and probably present in part in cell-associated virions. Five of six plaque purified strains derived from wild-type Edmonston virus produced only 200S nucleocapsids. One of these five plaque-purified strains subsequently produced both 200 and 110S nucleocapsids after being passaged by using undiluted inocula. These results suggest that measles virus may produce distinct classes of defective virus containing short nucleocapsids and subgenomic viral RNA.     

The ESCRT-I subunit TSG101 controls endosome-to-cytosol release of viral RNA

Like other enveloped viruses, vesicular stomatitis virus infects cells through endosomes. There, the viral envelope undergoes fusion with endosomal membranes, thereby releasing the nucleocapsid into the cytoplasm and allowing infection to proceed. Previously, we reported that the viral envelope fuses preferentially with the membrane of vesicles present within multivesicular endosomes. Then, these intra-endosomal vesicles (containing nucleocapsids) are transported to late endosomes, where back-fusion with the endosome limiting membrane delivers the nucleocapsid into the cytoplasm. In this study, we show that the tumor susceptibility gene 101 (Tsg101) subunit of the endosomal sorting complexes required for transport (ESCRT)-I complex, which mediates receptor sorting into multivesicular endosomes, is dispensable for viral envelope fusion with endosomal membranes and viral RNA transport to late endosomes but is necessary for infection. Our data indicate that Tsg101, in contrast to the ESCRT-0 component Hrs, plays a direct role in nucleocapsid release from within multivesicular endosomes to the cytoplasm, presumably by controlling the back-fusion process. We conclude that Tsg101, through selective interactions with its partners including Hrs and Alix, may link receptor sorting and lysosome targeting to the back-fusion process involved in viral capsid release.     

Replication of Sendai Virus: II. Steps in Virus Assembly

Chick embryo fibroblast cultures infected with Sendai virus were incubated with (3)H-uridine in the presence of actinomycin D beginning at 18 hr after infection. The 35 and 18S virus-specific ribonucleic acid (RNA) components were found in a ribonuclease-sensitive form in the cell and appeared to be associated with polyribosomes. Newly synthesized 57S viral RNA was rapidly coated with protein to form intracellular viral nucleocapsid, and no 57S RNA was found "free" (ribonucleasesensitive) in the 2,000 x g supernatant fraction of disrupted cells. The nucleocapsid from detergent-disrupted Sendai virus and that from disrupted cells were indistinguishable in ultrastructure and buoyant density, and neither was found to be infectious or have hemagglutinating activity. Kinetic studies of nucleocapsid and virus formation indicated a relative block in conversion of viral nucleocapsid to complete enveloped virus in these cells, resulting in accumulation of large amounts of nucleocapsid in the cell cytoplasm.     

Cell-free translation of simian virus 40 16S and 19S L-strand-specific mRNA classes to simian virus 40 major VP-1 and minor VP-2 and VP-3 capsid proteins.

Simian virus 40 capsid proteins VP-1, VP-2, and VP-3 have been synthesized in wheat germ and reticulocyte cell-free systems in response to either poly(A)-containing mRNA from the cytoplasm of infected cells or viral RNA purified by hybridization to simian virus 40 DNA linked to Sepharose. All three viral polypeptides synthesized in vitro are specifically immunoprecipitated with anti-simian virus 40 capsid serum. VP-2 and VP-3 are related by tryptic peptide mapping to each other but not to VP-1. The most abundant class of L-strand-specific viral mRNA, the 16S species, codes for the major capsid protein. The relatively minor 19S class directs the cell-free synthesis of VP-1, VP-2, and VP-3. Whether the 19S RNA represents more than one distinct species of mRNA is not yet clear. VP-1 mRNA can be isolated from the cytoplasm, detergent-washed nuclei, and the nuclear wash fraction. The mRNA from the nuclear wash fraction is enriched for VP-2 mRNA when compared to other viral or cellular polypeptides.