title: Mutations in the N-terminus of VP5 alter its interaction with the scaffold proteins of herpes simplex virus type 1.
abstract: During the assembly process of herpes simplex virus type 1 capsids, there is an essential interaction between the C-terminal tail of the scaffold proteins (22a and 21) and the major capsid protein (VP5). Recent studies of spontaneous revertant viruses that overcome a blocked maturation cleavage site of the scaffold proteins have shown that the N-terminus of VP5 is important for this interaction. One of the revertant viruses, PR7, encodes a second-site mutation at residue 69 of VP5 which unlike wild-type VP5 fails to interact with 22a and thus gives white colonies in the yeast two-hybrid assay. In the present study a small DNA fragment, encoding residues 1 to 85 of wild-type and PR7 VP5, was mutagenized using error-prone PCR. Mutagenized DNA was used in the yeast two-hybrid assay to identify mutations in wild-type VP5 that resulted in loss of 22a binding (white colonies), or in PR7 VP5 that resulted in a gain of function (blue colonies). For the loss of function experiments, using KOS VP5, a row of eight thymidine nucleotides (codons 37-40) resulted in many frameshift mutations, which led us to terminate the study without reaching a statistically significant result. For the PR7 experiment, 30 clones were identified that had single amino acid substitutions, and these mutations were localized to amino acids 27-45 and 63-84 of VP5. The most frequent mutation was a reversion back to wild-type. The next most frequent were E28K and N63S, and these gave the highest beta-galactosidase enzyme activities (indicative of PR7VP5-22a interaction), 30 and 20% of wild-type, respectively. When E28K and N63S were transferred into the wild-type VP5 background, that is, in the absence of the PR7 mutation, they gave rise to different phenotypes. The E28K mutation lost its ability to interact with the scaffold proteins as judged by this assay. Therefore, it may be acting as a compensatory mutation whose phenotype is only expressed in the presence of the original PR7 mutation. However, the N63S mutation in the wild-type VP5 background increased the interaction, as judged by the beta-galactosidase activity, by a factor of 9 relative to when the PR7 mutation was present. Even more surprising, in the absence of the PR7 mutation the enzyme activity was still greater, by a factor of 2, than that observed for wild-type VP5. This study provides further evidence that the N-terminus of VP5 is in intimate association with the C-terminus of the scaffold proteins.
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: S C
authors.initials: SC
authors.lastname: Warner
authors.forename: G
authors.initials: G
authors.lastname: Chytrova
authors.forename: P
authors.initials: P
authors.lastname: Desai
authors.forename: S
authors.initials: S
authors.lastname: Person
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002213:Capsid / Q000235:genetics* / Q000378:metabolism; D036022:Capsid Proteins; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D019065:Virus Assembly
chemical_list: D036022:Capsid Proteins; C442523:VP5 protein, Herpes simplex virus type 1