| Vaccinia virus (VV), a member of the poxvirus family, is a large enveloped DNA virus that replicates exclusively in the cytoplasm of infected cells. Assembly of progeny virions is a complex process that requires coordination of several viral and cellular proteins. Although morphogenesis has been the subject of many studies, the molecular events involved in this process remain largely unknown. In our studies, we identified a previously uncharacterized protein, A30, which is required for the association of viral membranes with electron-dense masses called viroplasm. To investigate the role of A30 in VV replication, we generated a virus (vA30Li), containing an IPTG-inducible copy of A30. In the absence of IPTG, plaque formation was inhibited and virus yields were dramatically reduced. Inhibition of 30 expression led to the accumulation of crescent-shaped and circular membranes that were separated from the viroplasm.; Coimmunoprecipitation followed by mass spectrometry revealed that A30 interacts with the G7 protein, a component of VV cores. To investigate the role of G7 in replication, we constructed a virus (vG7Li), containing an IPTG-inducible G7. Interestingly, accumulation of A30 correlated with the levels of induced G7 and vice-versa, suggesting that the interaction of G7 and A30 is important for the stabilization of both proteins during infection. Not surprisingly, electron microscopy of cells infected with vG7Li revealed a phenotype identical to that described for vA30Li, indicating that A30 and G7 form a complex that is required for the attachment of viral membranes to the viroplasm.; We also found that A30 and G7 interact with the F10 kinase during infection. We demonstrated that this interaction is important for the stability of F10, since no F10 accumulated in the absence of G7 or A30 expression. Transient expression experiments revealed that coexpression of G7 and A30 with F10 increased the kinase activity of F10, suggesting that interaction of these proteins is required for the regulation of protein phosphorylation. Although the mechanisms by which A30 and G7 activate the F10 kinase remain unknown, evidence suggests that these proteins induce autophosphorylation of F10, a process that has been shown to regulate the activity of several protein kinases. |
