Molecular genetics of herpes simplex virus. VII. Characterization of a temperature-sensitive mutant produced by in vitro mutagenesis and defective in DNA synthesis and accumulation of gamma polypeptides

Abstract

The properties of a temperature-sensitive mutant produced by transfection of [rabbit skin] cells with intact DNA and a specific DNA fragment mutagenized with low levels of hydroxylamine are reported. The plating efficiency of the mutant at 39.degree. C relative to that at 33.5.degree. C was 5 .times. 10-6. The pattern of polypeptides produced at the nonpermissive temperature was similar to that seen with wild-type virus in infected [human epidermoid carcinoma HEp-2] cells treated with inhibitory concentrations of phosphonoacetic acid in that .alpha. and .beta. polypeptides were produced, whereas most .gamma. polypeptides were reduced or absent. Consistently, the mutant did not make viral DNA, although temperature sensitivity of the viral DNA polymerase could not be demonstrated. Marker rescue studies with herpes simplex virus type 2 (HSV-2) DNA mapped the mutant in the L component within map positions 0.385 and 0.402 in the prototype (P) arrangement of the HSV-1 genome. Analysis of the recombinants permitted the mapping of the genes specifying infected cell polypeptides 36, 35, 37, 19.5, 11, 8, 2, 43 and 44, but only the infected cell polypeptide 8 of HSV-2 was consistently made by all recombinants containing demonstrable HSV-2 sequences. Marker rescue studies with cloned HSV-1 DNA fragments mapped the temperature-sensitive lesion within less than 103 base pairs between 0.383-0.388 map units. Translation of the RNA hybridizing to cloned HSV-1 DNA, encompassing the smallest region containing the mutation, revealed polypeptide 8 (128,000 MW), which was previously identified as a .beta. polypeptide with high affinity for viral DNA, and a polypeptide (25,000 MW) not previously identified in lysates of labeled cells.