SELF-ASSOCIATION OF HERPES-SIMPLEX VIRUS TYPE-1 ICP35 IS VIA COILED-COIL INTERACTIONS AND PROMOTES STABLE INTERACTION WITH THE MAJOR CAPSIDPROTEIN

The ordered copolymerization of viral proteins to form the herpes simp lex virus (HSV) capsid occurs within the nucleus of the infected cell and is a complex process involving the products of at least six viral genes, In common with capsid assembly in double-stranded DNA bacteriop hages, HSV capsid assembly proceeds via the assembly of an outer capsi d shell around an interior scaffold, This capsid intermediate matures through loss of the scaffold and packaging of the viral genomic DNA, T he interior of the HSV capsid intermediate contains the viral protease and assembly protein which compose the scaffold. Proteolytic processi ng of these proteins is essential for and accompanies capsid maturatio n, The assembly protein (ICP35) is the primary component of the scaffo ld, and previous studies have demonstrated it to be capable of intermo lecular association with itself and with the major capsid protein, VP5 , We have defined structural elements within ICP35 which are responsib le for intermolecular self-association and for interaction with VP5, Y east (Saccharomyces cerevisiae) two-hybrid assays and far-Western stud ies with purified recombinant ICP35 mapped a core self-association dom ain between Ser165 and His219. Site-directed mutations in this domain implicate a putative coiled coil in ICP35 self-association. This coile d-coil motif is highly conserved within the assembly proteins of other alpha herpesviruses. In the two-hybrid assay the core self-associatio n domain was sufficient to mediate stable self-association only in the presence of additional structural elements in either N- or C-terminal flanking regions, These regions also contain conserved sequences whic h exhibit a high propensity for cw helicity and may contribute to self -association by forming additional short coiled coils. Our data suppor ts a model in which ICP35 molecules have an extended conformation and associate in parallel orientation through homomeric coiled-coil intera ctions, In additional two-hybrid experiments we evaluated ICP35 mutant s for association with VP5. We discovered that in addition to the C-te rminal 25 amino acids of ICP35, previously shown to be required for VP 5 binding, an additional upstream region was required. This region is between Ser165 and His234 and contains the core self-association domai n, Site-directed mutations and construction of chimeric molecules in w hich the self-association domain of ICP35 was replaced by the GCN4 leu cine zipper indicated that this region contributes to VP5 binding thro ugh mediating self-association of ICP35 and not through direct binding interactions, Our results suggest that self-association of ICP35 stro ngly promotes stable association with VP5 in vivo and are consistent w ith capsid formation proceeding via formation of stable subassemblies of ICP35 and VP5 which subsequently assemble into capsid intermediates in the nucleus.