The phenotype in vitro and in infected cells of herpes simplex virus 1 alpha trans-inducing factor (VP16) carrying temperature-sensitive mutations introduced by substitution of cysteines.
alpha trans-inducing factor (alpha TIF, VP16, Vmw65) is an essential structural protein of herpes simplex virus, being required for virion assembly. The protein also forms complexes with host proteins and a response element and transactivates the alpha genes which carry this element. The protein contains an acidic carboxyl terminus required for transactivation and a much larger amino-terminal domain required for promoter recognition. We report the first set of temperature-sensitive (ts) mutations deliberately introduced into the protein by substitution of the cysteine codons with those specifying glycine at positions 78, 102, and 176, either singly or in combinations. We report the following results. (i) All mutated proteins synthesized in vitro formed complexes with the DNA response element at room temperature. However, the mutant with the triple substitution and two mutants with substitutions in two of the three cysteines exhibited a ts phenotype at 33 and 37 degrees C, and one exhibited a ts phenotype only at 37 degrees C. (ii) Replacement of wild-type alpha TIF with genes carrying substitutions in any two cysteines conferred a ts phenotype for replication at 39.5 degrees C. Shift-down experiments indicated that the 10(4)- to 10(5)-fold reduction in virus yield at the nonpermissive temperature was due to the disfunction of alpha TIF late in infection, presumably in virion maturation. (iii) The alpha TIF expressed in cells infected with mutant viruses exhibited the same ts phenotype in protein-DNA complex formation as those expressed in vitro from mutated plasmids. Although the virus carrying the alpha TIF substitutions at Cys-102 and Cys-176 failed to induce a reporter gene linked to the alpha 4 promoter at 39.5 degrees C, it replicated as well as the parent virus in cells maintained for the first 10 h of infection at 39.5 degrees C. We conclude the following. (i) Formation of DNA-protein complexes containing alpha TIF is a poor prognosticator of alpha TIF function. (ii) The data presented here and in the literature strongly support the hypothesis that the secondary structure of the alpha TIF is very sensitive to deletions or insertions which probably affect the interaction of alpha TIF with both viral proteins in the virion and cellular proteins during infection. As a consequence, deletion-insertion mutagenesis may not shed useful information on the role of transactivating function of alpha TIF in infection. (iii) Since cysteines may play a role in stabilizing the secondary structure of proteins, substitutions of cysteines may be a powerful technique for site-specific construction of ts mutants in essential viral proteins.
D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D002478:Cells, Cultured; D002645:Chickens; D002522:Chlorocebus aethiops; D003545:Cysteine; D017405:Herpes Simplex Virus Protein Vmw65 / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010047:Ovalbumin / Q000096:biosynthesis; D010641:Phenotype; D010957:Plasmids; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011817:Rabbits; D012867:Skin; D013462:Sulfur Radioisotopes; D013696:Temperature; D015534:Trans-Activators / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014709:Vero Cells; D014779:Virus Replication
D017405:Herpes Simplex Virus Protein Vmw65; D013462:Sulfur Radioisotopes; D015534:Trans-Activators; D010047:Ovalbumin; D008715:Methionine; D003545:Cysteine