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.

Summary

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.Rubella virus (RV) is the sole member of the genus Rubivirus within the family Togaviridae. In most cases, RV infection causes a mild self-limiting disease in humans known as rubella or German measles. However, in contrast to postnatal infection, which is relatively benign in nature, in utero infection can have serious consequences for the developing fetus (6). When contracted during the first trimester of pregnancy, RV infection results in a characteristic series of severe defects in the neonate known as congenital rubella syndrome. Despite the availability of an effective vaccine, RV remains a threat to public health in both developed and developing countries as a result of unvaccinated populations. The mechanisms by which RV causes developmental abnormalities are largely unknown.As with all togaviruses, the RV genome contains two open reading frames that encode the replicase proteins and the structural proteins (reviewed in reference 11). The structural proteins are translated as a polyprotein precursor from a subgenomic RNA. The precursor protein is then processed into three structural proteins: a capsid protein and two envelope glycoproteins, E2 and E1. Recently, our laboratory has focused largely on the processing and functional analyses of the capsid protein.During virus assembly, the capsid is thought to interact with viral genomic RNA and glycoproteins E2 and E1 to drive formation of the nucleocapsid and virus budding, respectively. Nucleocapsid formation is highly regulated in RV-infected cells and is coincident with virus budding (29, 32). The packaging signal within the RV genomic RNA and the capsid region that binds this sequence have been identified (24). Moreover, data from our lab suggest that phosphorylation of serine 46 within the RNA binding site of the capsid regulates interactions between the capsid and genomic RNA and, by extrapolation, nucleocapsid formation (22). Mutation of serine 46 to alanine results in capsids that are poorly phosphorylated and ...