title: Self-association of herpes simplex virus type 1 ICP35 is via coiled-coil interactions and promotes stable interaction with the major capsid protein.
abstract: The ordered copolymerization of viral proteins to form the herpes simplex 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 bacteriophages, HSV capsid assembly proceeds via the assembly of an outer capsid shell around an interior scaffold. This capsid intermediate matures through loss of the scaffold and packaging of the viral genomic DNA. The interior of the HSV capsid intermediate contains the viral protease and assembly protein which compose the scaffold. Proteolytic processing of these proteins is essential for and accompanies capsid maturation. The assembly protein (ICP35) is the primary component of the scaffold, and previous studies have demonstrated it to be capable of intermolecular association with itself and with the major capsid protein, VP5. We have defined structural elements within ICP35 which are responsible for intermolecular self-association and for interaction with VP5. Yeast (Saccharomyces cerevisiae) two-hybrid assays and far-Western studies with purified recombinant ICP35 mapped a core self-association domain between Ser165 and His219. Site-directed mutations in this domain implicate a putative coiled coil in ICP35 self-association. This coiled-coil motif is highly conserved within the assembly proteins of other alpha herpesviruses. In the two-hybrid assay the core self-association 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 which exhibit a high propensity for alpha helicity and may contribute to self-association by forming additional short coiled coils. Our data supports a model in which ICP35 molecules have an extended conformation and associate in parallel orientation through homomeric coiled-coil interactions. In additional two-hybrid experiments we evaluated ICP35 mutants for association with VP5. We discovered that in addition to the C-terminal 25 amino acids of ICP35, previously shown to be required for VP5 binding, an additional upstream region was required. This region is between Ser165 and His234 and contains the core self-association domain. Site-directed mutations and construction of chimeric molecules in which the self-association domain of ICP35 was replaced by the GCN4 leucine zipper indicated that this region contributes to VP5 binding through mediating self-association of ICP35 and not through direct binding interactions. Our results suggest that self-association of ICP35 strongly promotes stable association with VP5 in vivo and are consistent with capsid formation proceeding via formation of stable subassemblies of ICP35 and VP5 which subsequently assemble into capsid intermediates in the nucleus.
authors.affiliation: Department of Biological Sciences, Bio-Méga Research Division, Boehringer Ingelheim, (Canada) Ltd., Laval, Quebec.
authors.forename: A
authors.initials: A
authors.lastname: Pelletier
authors.forename: F
authors.initials: F
authors.lastname: Dô
authors.forename: J J
authors.initials: JJ
authors.lastname: Brisebois
authors.forename: L
authors.initials: L
authors.lastname: Lagacé
authors.forename: M G
authors.initials: MG
authors.lastname: Cordingley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D036022:Capsid Proteins; C054582:ICP35 protein, Human herpesvirus 1; C442523:VP5 protein, Herpes simplex virus type 1; D014764:Viral Proteins