IDENTIFICATION OF A MINIMAL HYDROPHOBIC DOMAIN IN THE HERPES-SIMPLEX VIRUS TYPE-1 SCAFFOLDING PROTEIN WHICH IS REQUIRED FOR INTERACTION WITH THE MAJOR CAPSID PROTEIN

Recent biochemical and genetic studies have demonstrated that an essen tial step of the herpes simplex virus type 1 capsid assembly pathway i nvolves the interaction of the major capsid protein (VP5) with either the C terminus of the scaffolding protein (VP22a, ICP35) or that of th e protease; (Pra, product of UL26). To better understand the nature of the interaction and to further map the sequence motif, we expressed t he C-terminal 30-amino-acid peptide of ICP35 in Escherichia coli as a glutathione S-transferase fusion protein (GST/CT). Purified GST/CT fus ion proteins were then incubated with S-35-labeled herpes simplex viru s type I-infected cell lysates containing VP5. The interaction between GST/CT and VP5 was determined by coprecipitation of the two proteins with glutathione Sepharose beads. Our results revealed that the GST/CT fusion protein specifically interacts with VP5, suggesting that the C -terminal domain alone is sufficient for interaction with VP5. Deletio n analysis of the GST/CT binding domain mapped the interaction to a mi nimal 12-amino-acid motif. Substitution mutations further revealed tha t the replacement of hydrophobic residues with charged residues in the core region of the motif abolished the interaction, suggesting that t he interaction is a hydrophobic one. A chaotropic detergent, 0.1% Noni det P-40, also abolished the interaction, further supporting the hydro phobic nature of the interaction. Computer analysis predicted that the minimal binding motif could form a strong alpha-helix structure. Most interestingly, the alpha-helix model maximizes the hydropathicity of the minimal domain so that all of the hydrophobic residues are centere d around a Phe residue on one side of the alpha-helix. Mutation analys is revealed that the Phe residue is absolutely critical for the bindin g, since changes to Ala, Tyr, or Trp abrogated the interaction. Finall y, in a peptide competition experiment, the C-terminal 25-amino-acid p eptide, as well as a minimal peptide derived from the binding motif, c ompeted with GST/CT for interaction,vith VP5. In addition, a cyclic an alog of the minimal peptide which is designed to stabilize an ex-helic al structure competed more efficiently than the minimal peptide. The e vidence suggests that the C-terminal end of ICP35 forms an a-helical s econdary structure, which may bind specifically to a hydrophobic pocke t in VP5.