A facile system for construction of HSV-1 variants: site directed mutation of the UL26 protease gene in HSV-1.
A 2-plasmid/4-cosmid-based system of mutagenesis is described for construction of herpes simplex virus type 1 (HSV-1) variants with point mutations in the protease gene. The system was used to reconstruct a mutant virus (V701) with Tyr30 to Phe and Ala48 to Val mutations in HSV-1 protease that exhibits the temperature sensitive phenotype of the previously characterized temperature sensitive HSV-1 mutant, ts1201. The 2-plasmid/4-cosmid system of mutagenesis was further validated by using it to construct a virus wherein the active site Ser129 of HSV-1 protease was mutated to Ala. The resulting virus mutant (V713) grew only on the Vero host range cell line PHS-23. In V713 infected Vero cells, the processing of Pra to N(O) was almost completely blocked, and B capsids accumulated in the nucleus in crystal-like aggregates, suggesting that protease activity is required for emergence of monodispersed capsids from these aggregates. Back mutation of Ala129 to Ser using the V713 viral DNA as template for PCR mutagenesis restored the wild-type phenotype verifying that the replicative incompetence of V713 reflected only the effect of the Ser to Ala mutation. The 2-plasmid/4-cosmid system of mutagenesis (and modifications thereof) should facilitate production of new mutant viruses for delineating interactions of domains of HSV-1 protease (as well as other HSV-1 proteins) important for virus replication.
Register
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RB
Department of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA.
Shafer
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JA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D004279:DNA, Viral; D005814:Genes, Viral; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D012697:Serine Endopeptidases / Q000235:genetics*; D014709:Vero Cells; D014764:Viral Proteins
D004279:DNA, Viral; D014764:Viral Proteins; C079287:scaffold protein, Herpes simplex virus-1; D012697:Serine Endopeptidases