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...