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 (RV) is a member of the family Togaviridae, a group of positive-strand RNA viruses with relatively simple virion structures and replication schemes (10,15,44). The virus is highly teratogenic and causes devastating malformations in human fetuses when in utero infection occurs during the first trimester of pregnancy. Although congenital rubella syndrome is rare in western countries, it remains endemic in the developing world and parts of Eastern Europe (14, 40). Despite its medical importance and the widespread use of live RV vaccines, most aspects of virus replication and pathogenesis remain poorly understood.Virions are comprised of three structural proteins: two membrane-spanning glycoproteins, E2 and E1, and a capsid protein (15, 34). The membrane glycoproteins are required for binding to host cells and membrane fusion during infection (10). During virus assembly, they are thought to play a major role in facilitating intracellular budding at the Golgi complex (17). Recently, we have focused our efforts on studying the roles of the capsid protein in virus replication. Our data suggest that the capsid is a multifunctional protein that has roles in virus-host interactions as well as replication. For example, the capsid interacts with multiple host...

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.Rubella virus (RV) is a positive-strand RNA virus of the family Togaviridae. Despite routine vaccination programs which have been in place for 30 years, the virus persists in the human population and remains an important human pathogen (11). The most serious medical consequences of RV infection occur when seronegative women contract the virus during the first trimester of pregnancy. RV is highly teratogenic and causes a characteristic pattern of defects in the fetus which are collectively known as congenital rubella syndrome. The molecular basis of RV pathology remains poorly understood, but several recent reports have shown that the virus induces apoptosis in a cell type-dependent manner (9,19,33,45). This pattern of apoptosis could potentially explain the organ-specific malformations observed in congenital rubella syndrome.Virions contain three structural proteins that are translated from a 24S subgenomic RNA; two membrane-spanning glycoproteins (E2 and E1) and a capsid protein (38). The capsid protein is multifunctional and is involved in several different types of intermolecular interactions. First, it contains an RNAbinding domain and is responsible for packaging the genomic RNA into nucleocapsids (11,28). Second, by analogy with other togaviruses, capsid must engage in homo-oligomeric (capsid-capsid) interactions during nucleocapsid formation. Finally, it must also interact with E2 and/or E1 during budding (37, 40).The nucleocapsids of togaviruses have a characteristic icosahedral structure which has been extensively studied in alphaviruses (41). Although the overall structures of RV and alphavirus capsids are similar, their assembly pathways are quite different. Wh...

The distribution and morphology of mitochondria are dramatically affected during infection with rubella virus (RV). Expression of the capsid, in the absence of other viral proteins, was found to induce both perinuclear clustering of mitochondria and the formation of electron-dense intermitochondrial plaques, both hallmarks of RV-infected cells. We previously identified p32, a host cell mitochondrial matrix protein, as a capsid-binding protein. Here, we show that two clusters of arginine residues within capsid are required for stable binding to p32. Mutagenic ablation of the p32-binding site in capsid resulted in decreased mitochondrial clustering, indicating that interactions with this cellular protein are required for capsid-dependent reorganization of mitochondria. Recombinant viruses encoding arginine-to-alanine mutations in the p32-binding region of capsid exhibited altered plaque morphology and replicated to lower titers. Further analysis indicated that disruption of stable interactions between capsid and p32 was associated with decreased production of subgenomic RNA and, consequently, infected cells produced significantly lower amounts of viral structural proteins under these conditions. Together, these results suggest that capsid-p32 interactions are important for nonstructural functions of capsid that include regulation of virus RNA replication and reorganization of mitochondria during infection.

The complete cDNA sequence for canine ZO-2, a tight junction-specific protein, is presented. A single open reading frame encodes a polypeptide of 1,174 amino acids with a predicted molecular mass of 132,085 daltons. As noted previously (1), ZO-2 is a member of the membraneassociated guanylate kinase-containing (MAGUK) protein family, a family which includes an additional tight junction-associated protein, ZO-1. These proteins contain a region homologous to guanylate kinase, an SH3 domain, and variable numbers of PSD-95/discs-large/ ZO-1 (PDZ) domains, shown to be involved in proteinprotein interactions. ZO-2 and ZO-1 contain three PDZ domains in the N-terminal half of the molecule. Between the first and second PDZ domains, ZO-2 displays a basic region (pI ‫؍‬ 10.27) containing 22% arginine residues. Both ZO-1 and ZO-2 have proline-rich C-terminal regions that are not homologous to other MAGUK family members. Sequence analysis of multiple ZO-2 cDNAs reveals a 36-amino acid domain in this C-terminal region present in only some of the cDNAs. Overall, ZO-2 is highly homologous to ZO-1, showing 51% amino acid identity; however, the C-terminal ends of the molecules show only 25% amino acid identity. This suggests that the C-terminal ends of ZO-1 and ZO-2 have different functions.Several molecular constituents of the tight junction have been identified. These include ZO-1 (2), cingulin (3), ZO-2 (1, 4), 7H6 (5), Rab3B (6), and occludin (7). The presence of other proteins at the tight junction, including spectrin (8, 9), another Rab protein (Rab13) (10), and p130 (11), has been implicated but not confirmed.The complete mouse (12) and human (13) protein and cDNA sequences of ZO-1 have been published. A partial sequence for ZO-2 has also been characterized (1). Analyses of these sequences indicate that ZO-1 and ZO-2 share significant homology with each other and with several other proteins, including the lethal(1)discs-large-1 (dlg) tumor suppressor gene product (dlg-A) of Drosophila (14), erythrocyte membrane-associated p55 (15), and PSD-95/SAP90, a protein found at brain presynaptic membranes (16,17). These proteins share several conserved regions, including a region homologous to guanylate kinase (GUK), 1 an enzyme which converts GMP to GDP, a single src homology (SH3) domain, hypothesized to be involved in protein-protein interactions necessary for signal transduction (18,19), and a variable number of N-terminal repeats termed PDZ domains (from PSD-95, discs-large, ZO-1), shown to bind integral plasma membrane proteins (20 -23). As all proteins in this group are associated with the plasma membrane, they have collectively been termed the MAGUK family (membrane-associated guanylate kinase-containing) (24).ZO-1 and ZO-2 are different from other family members but similar to each other in that they display C-terminal acidic and proline-rich regions (1, 13). ZO-1 also contains an alternatively spliced region, termed the ␣ motif, in the C-terminal region (25,26). The exact function of this splice domain remains unknown. Her...

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 PABPinteracting 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.The rubella virus (RV) capsid is an RNA-binding phosphoprotein (40). During virus assembly, the capsid engages in homotypic and heterotypic binding interactions to package the viral genome into a compact nucleocapsid structure (reviewed in reference 18). Assembly and disassembly of the nucleocapsid appear to be regulated by dynamic phosphorylation of serine/threonine residues in the RNA-binding motif of the capsid (34, 35). Nucleocapsid assembly occurs on membranes of the Golgi complex, and association of the capsid with this organelle presumably reflects its role in virus budding (5,20). Similar to alphavirus budding (53), interactions between the capsid and viral glycoproteins E2 and E1 are thought to drive virus assembly. As well as being targeting to the virus budding site, the RV capsid associates with other intracellular membranes, including endocytic vacuoles (13) and mitochondria (7, 37). These organelles have no obvious link to virus assembly, and the presence of the capsid at these sites is indicative of its nonstructural roles.Recent studies revealed the unexpected finding that capsid modulates the synthesis of viral RNAs (8, 55-57). It is not clear how the capsid protein affects viral transcription, but the fact that it binds to the nonstructural protein p150 (57) may indicate that it regulates the activity of the replicase complex. Interactions between capsid and host proteins may also influence viral transcription. For example, capsid binds to the mitochondrial matrix protein p32 (7, 44), and indirect evidence indicates that this interaction is important for virus replication (6). Specifically, ablation o...