Physical and Functional Interactions between Vaccinia Virus F10 Protein Kinase and Virion Assembly Proteins A30 and G7

Abstract

An early step in vaccinia virus morphogenesis, the association of crescent membranes with electron-dense granular material, is perturbed when expression of the viral protein encoded by the A30L or G7L open reading frame is repressed. Under these conditions, we found that phosphorylation of the A17 membrane protein, which is mediated by the F10 kinase, was severely reduced. Furthermore, A30 and G7 stimulated F10-dependent phosphorylation of A17 in the absence of other viral late proteins. Evidence for physical interactions between A30, G7, and F10 was obtained by their coimmunoprecipitation with antibody against A30 or F10. In addition, phosphorylation of A30 was dependent on the F10 kinase and autophosphorylation of F10 was stimulated by A30 and G7. Nevertheless, the association of A30, G7, and F10 occurred even with mutated, catalytically inactive forms of F10. Just as A30 and G7 are mutually dependent on each other for stability, F10 was nearly undetectable in the absence of A30 and G7. The reverse is not true, however, as repression of F10 did not diminish A30 or G7. Interaction of F10 with A30 and G7 presumably occurred within the virus factory areas of the cytoplasm, where each was concentrated. F10 localized predominantly in the cortical region of immature virions, beneath the membrane where A17 is located. F10 remained associated with the particulate core fraction of mature virions after treatment with a nonionic detergent and reducing agent. The formation of protein complexes such as the one involving A30, G7, and F10 may be a mechanism for the regulated packaging and processing of virion components.The assembly of vaccinia virus (VV), the prototype poxvirus, occurs within cytoplasmic viral factory areas that appear largely cleared of cellular organelles. Although studied intensively by electron microscopy, little is known about the molecular events involved in viral morphogenesis. Several viral proteins required for early stages of virus assembly have been identified through the characterization of temperature-sensitive and inducible conditional lethal mutants. Few recognizable membrane structures can be discerned in cells infected under nonpermissive conditions with mutants that map to the F10L open reading frame (ORF) (14,18,20). The product of the F10L ORF has been characterized as a dual-specificity protein kinase that phosphorylates tyrosine as well as serine and threonine residues of target proteins (1,3,8,20). Mutated forms of F10, with single amino acid substitutions in the active site, were unable to transcomplement an inducible conditional lethal F10 mutant, confirming the importance of phosphorylation in virion assembly (14). Studies with F10 conditional lethal mutants suggest that the membrane components A17 (1, 3) and A14 (1) are substrates of the kinase. A17 is needed for an early step in viral membrane formation (11, 21) and A14 is needed at a slightly later stage of assembly (12,19). The precursor of mature A17 is phosphorylated at serine, threonine, and tyrosine residues (1, 3) and is c...

Role of the I7 Protein in Proteolytic Processing of Vaccinia Virus Membrane and Core Components

“…There was, however, a slight difference in the mobility of the upper A17 band which was likely due to differences in phosphorylation (20). Taken together, our results suggest that although I7 is required for the processing of membrane and core proteins, the two events are not coupled.…”

“…7B, the cells were transfected with a plasmid expressing wild-type A17 and with the vector plasmid. As expected, the V5 antibody detected the 23-kDa form of A17 and also a slightly retarded incompletely resolved band that represents a phosphorylated species (20). The V5 antibody also detected a 21-kDa band that must represent a product that is cleaved only at the C terminus when I7 was also expressed by transfection (Fig.…”

Viral Serine/Threonine Protein Kinases

“…F10 phosphorylates two viral membrane proteins that are critical in early morphogenesis, A14 and A17, which is thought to be required to form the membranes associated with immature virions (20,51,138). Together with viral proteins A30 and G7, F10 makes part of a viral assembly complex, which stimulates F10-mediated A17 phosphorylation (203). Additional components of this viral assembly complex were subsequently identified and include J1, A15, D2, and D3 (37,203).…”

Vaccinia Virus Nonstructural Protein Encoded by the A11R Gene Is Required for Formation of the Virion Membrane

“…Early blocks in morphogenesis can alter the levels and posttranslational modifications of membrane proteins (12,36). To determine whether the absence of A11 had such effects, cells were infected with vA11Ri in the presence of 0 to 100 M IPTG, and several proteins were analyzed by SDS-PAGE and Western blotting of whole-cell extracts prepared 24 h after infection.…”