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

We report on the properties of a temperature-sensitive mutant produced by transfection of cells with intact DNA and a specific DNA fragment mutagenized with low levels of hydroxylamine. The plating efficiency of the mutant at 390C relative to that at 33.50C was 5 x 10'. The pattern of polypeptides produced at the nonpermissive temperature was similar to that seen with wild-type virus in infected cells treated with inhibitory concentrations of phosphonoacetic acid in that a and ,B polypeptides were produced, whereas most y polypeptides were either 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 and 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 molecular weight), which was previously identified as a /8 polypeptide with high affinity for viral DNA, and a polypeptide (25,000 molecular weight) not previously identified in lysates of labeled cells. ucts have been mapped (21, 23). Several /3 genes have been shown to cluster in the region of the genome between 0.30 and 0.42 map units on the 191 on September 29, 2020 by guest http://jvi.asm.org/ Downloaded from 192 CONLEY ET AL. physical map of the HSV-1 genome (23). To delineate the function of some of these genes, we have selected for mutagenesis a DNA fragment mapping between 0.31 and 0.41 map units. MATERIALS AND METHODS Viruses and cells. The isolation and relevant properties of HSV-1(F) (8), HSV-1(imiP) (11), and HSV-2(G) (8) strains were published elsewhere. Virus stocks were prepared and titrated on Vero or HEp-2 cells as previously described (30). Rabbit skin cells were originally obtained from J. McLaren. Human embryonic lung (HEL) cells were originally obtained from Flow Laboratories, Rockville, Md. Enzynmes and radioisotopes. The restriction endonucleases BamHI and KpnI were obtained from

Intrinsic Innate Immunity Fails To Control Herpes Simplex Virus and Vesicular Stomatitis Virus Replication in Sensory Neurons and Fibroblasts

“…Having shown that these cell types could all respond robustly to IFN-␤, we next measured STAT1 relocalization upon HSV infection. Cells were infected with wild-type HSV-1 strain 17 and stained for both STAT1 and the abundant HSV-1 DNA replication protein ICP8 as a marker for infected cells (67). In these infected cultures, BMDCs exhibiting both ICP8-positive and ICP8-negative staining showed significantly increased nuclear STAT1 staining 5 h postinfection ( Fig.…”

Serum antibodies to the major HSV‐2‐ specified DNA‐binding protein in patients with an acute HSV infection or cervical neoplasia

“…Surprisingly, the ICSP 11/12 was obtained under these conditions practically free of contaminating viral or cellular proteins (subsequent elutions with 0.15 M, 0.3 M, and 0.5 M NaCl without carrier protein). The expression of the HSV-specified major DNA-binding protein is needed for the replication of viral DNA [Conley et al, 1981;Littler et al, 19831, and in a nonproductive in vitro infection it inhibits the production of late viral antigens from parental virus DNA [Godowsh and Knipe, 19831. In this respect, the markedly increased yield of the ICSP 11 / 12 obtained from the PFA-treated cells is of particular interest.…”

Immune response to the herpes simplex type 1 regulatory proteins ICP8 and VP16 in infected persons

“…This report demonstrates that the major tegument protein VP16 and the major DNA binding protein ICP8 of HSV-1 are immunogenic in humans, even though a T cell recall response with blood monocytes serving as antigen presenting cells could not be demonstrated in HSV-1 seropositive healthy individuals. This was surprising at first as both proteins are produced in large amounts during infection compared to other proteins [Zhang and McKnight, 1993] and have been shown to be essential for productive viral replication in vivo [Conley et al, 1981;Ace et al, 1989]. It seems likely that if infected cells acted as facultative antigen presenting cells this presentation obviously does not induce long-lasting T cell memory for these antigens.…”