Recent biochemical and genetic studies have demonstrated that an essential step of the herpes simplex virus type 1 capsid assembly pathway involves the interaction of the major capsid protein (VP5) with either the C terminus of the scaffolding protein (VP22a, ICP35) or that of the protease (Pra, product of UL26). To better understand the nature of the interaction and to further map the sequence motif, we expressed the C-terminal 30-amino-acid peptide of ICP35 in Escherichia coli as a glutathione S-transferase fusion protein (GST/CT). Purified GST/CT fusion proteins were then incubated with 35 S-labeled herpes simplex virus type 1-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. Deletion analysis of the GST/CT binding domain mapped the interaction to a minimal 12-amino-acid motif. Substitution mutations further revealed that the replacement of hydrophobic residues with charged residues in the core region of the motif abolished the interaction, suggesting that the interaction is a hydrophobic one. A chaotropic detergent, 0.1% Nonidet P-40, also abolished the interaction, further supporting the hydrophobic nature of the interaction. Computer analysis predicted that the minimal binding motif could form a strong ␣-helix structure. Most interestingly, the ␣-helix model maximizes the hydropathicity of the minimal domain so that all of the hydrophobic residues are centered around a Phe residue on one side of the ␣-helix. Mutation analysis revealed that the Phe residue is absolutely critical for the binding, since changes to Ala, Tyr, or Trp abrogated the interaction. Finally, in a peptide competition experiment, the C-terminal 25-amino-acid peptide, as well as a minimal peptide derived from the binding motif, competed with GST/CT for interaction with VP5. In addition, a cyclic analog of the minimal peptide which is designed to stabilize an ␣-helical structure competed more efficiently than the minimal peptide. The evidence suggests that the C-terminal end of ICP35 forms an ␣-helical secondary structure, which may bind specifically to a hydrophobic pocket in VP5.

Multiple alignments of hepatitis C virus (HCV) polyproteins from six different genotypes identified a total of 22 nonconsensus mutations in a clone derived from the Hutchinson (H77) isolate. These mutations, collectively, may have contributed to the failure in generating a "functionally correct" or "infectious" clone in earlier attempts. A consensus clone was constructed after systematic repair of these mutations, which yielded infectious virions in a chimpanzee after direct intrahepatic inoculation of in vitro transcribed RNAs. This RNA-infected chimpanzee has developed hepatitis and remained HCV positive for more than 11 months. To further verify this RNA-derived infectivity, a second naive chimpanzee was injected intravenously with serum collected from the first chimpanzee. Infectivity analysis of the second chimpanzee demonstrated that the HCV infection was successfully transmitted, which validated unequivocally the infectivity of our repaired molecular clone. Amino acid sequence comparisons revealed that our repaired infectious clone had 4 mismatches with the isogenic clone reported by Kolykhalov et al. (1997, Science 277, 570-574) and 8 mismatches with that reported by Yanagi et al. (1997, Proc. Natl. Acad. Sci. USA 94, 8738-8743). At the RNA level, more mismatches (43 and 67, respectively) were identified; most of them were synonymous substitutions. Further comparisons with 16 isolates from different genotypes demonstrated that our repaired clone shares greater consensus than the reported isogenic clones. This approach of generating infectious HCV RNA validates the importance of amino acid sequence consensus in relation to the biology of HCV.

We previously identified a minimal 12-amino-acid domain in the C terminus of the herpes simplex virus type 1 (HSV-1) scaffolding protein which is required for interaction with the HSV-1 major capsid protein. An ␣-helical structure which maximizes the hydropathicity of the minimal domain is required for the interaction. To address whether cytomegalovirus (CMV) utilizes the same strategy for capsid assembly, several glutathione S-transferase fusion proteins to the C terminus of the CMV assembly protein precursor were produced and purified from bacterial cells. The study showed that the glutathione S-transferase fusion containing 16 amino acids near the C-terminal end was sufficient to interact with the major capsid protein. Interestingly, no cross-interaction between HSV-1 and CMV could be detected. Mutation analysis revealed that a three-aminoacid region at the N-terminal side of the central Phe residue of the CMV interaction domain played a role in determining the viral specificity of the interaction. When this region was converted so as to correspond to that of HSV-1, the CMV assembly protein domain lost its ability to interact with the CMV major capsid protein but gained full interaction with the HSV-1 major capsid protein. To address whether the minimal interaction domain of the CMV assembly protein forms an ␣-helical structure similar to that in HSV-1, peptide competition experiments were carried out. The results showed that a cyclic peptide derived from the interaction domain with a constrained ␣-helical structure competed for interaction with the major capsid protein much more efficiently than the unconstrained linear peptide. In contrast, a cyclic peptide containing an Ala substitution for the critical Phe residue did not compete for the interaction at all. The results of this study suggest that (i) CMV may have developed a strategy similar to that of HSV-1 for capsid assembly; (ii) the minimal interaction motif in the CMV assembly protein requires an ␣-helix for efficient interaction with the major capsid protein; and (iii) the Phe residue in the CMV minimal interaction domain is critical for interaction with the major capsid protein.