Expression of herpes simplex virus type 1 major DNA-binding protein, ICP8, in transformed cell lines: complementation of deletion mutants and inhibition of wild-type virus

Abstract

To minimize the contribution of residual activity associated with the temperature-sensitive (ts) form of ICP8 specified by available ts mutants, deletion mutations in this gene were constructed. Cells permissive for the generation and propagation of ICP8 deletion mutants were first obtained. Vero cells were cotransfected with pKEF-P4, which contains the gene for ICP8, and pSV2neo or a hybrid plasmid containing the G418 resistance gene linked to pKEF-P4. Of the 48 G418-resistant cell lines, 21 complemented ICP8 ts mutants in plaque asssays at the nonpermissive temperature. Four of these were examined by Southern blot analysis and shown to contain 1 to 3 copies of the ICP8 gene per haploid genome equivalent. Cell line U-47 was used as the permissive host for construction of ICP8 deletion mutants. In addition to cell lines which complemented ts mutants, two lines, U-27 and U-35, significantly inhibited plaque formation by wild-type virus, contained 30 and 100 copies of the ICP8 gene per haploid genome equivalent, respectively, and expressed large amounts of ICP8 after infection with wild-type virus. At low but not high multiplicities of infection, this inhibition was accompanied by underproduction of viral polypeptides of the early, delayed-early, and late kinetic classes. For construction of deletion mutants, a 780-base-pair XhoI fragment was deleted from pSG18-SalIA, a plasmid which contains the gene for ICP8, to yield pDX. U-47 cells were then cotransfected with pDX and infectious wild-type DNA. Mutant d61, isolated from the progeny of cotransfection, was found to contain both the engineered deletion in the ICP8 gene and an oriL-associated deletion of approximately 55 base pairs. Because d61 contained two mutations, a second mutant, d21, which carried the engineered ICP8 deletion but an intact oriL, was constructed by cotransfection of U-47 cells with wild-type DNA and an SalI-KpnI fragment purified from pDX. Phenotypic analysis of d21 and d61 revealed that they were similar in all properties examined: both exhibited efficient growth in U-47 cells but not in Vero cells; both induced the synthesis of an ICP8 polypeptide which was smaller than the wild-type form of the protein and which, unlike the wild-type protein, was found in the cytoplasm and not the nucleus of infected Vero cells; and nonpermissive Vero cells infected with either mutant failed to express late viral polypeptides.

Characterization of a major DNA-binding domain in the herpes simplex virus type 1 DNA-binding protein (ICP8)

“…To obtain large quantities of ICP8 for analysis, we used U-35 cells which had been infected with HSV-1 as a source of protein. U-35 cells contain multiple copies of the ICP8 gene integrated into their genomes (42). Upon superinfection, ICP8 protein is overproduced.…”

“…Cells and viruses. The U-35 cell line was obtained from P. Schaffer (Harvard Medical School) (42). It was derived from Vero cells (an established line of African green monkey kidney cells) and contains multiple integrated copies of the ICP8 gene.…”

A Dominant Mutant Form of the Herpes Simplex Virus ICP8 Protein Decreases Viral Late Gene Transcription

“…Transcription of the g1 gD gene was Levels of wild-type and d105 ICP8 in infected Vero reduced to a lesser extent (5-to 6-fold) in infected V2.6 cells and V2.6 cells compared with the reduction in the g2 genes, gC or UL47. Transcription of the pol gene was reduced in infected Vero It had been reported previously that overexpression of ICP8 may cause decreased viral DNA replication and cells by 25 and 29% with the addition of 25 and 50 mg/ viral gene expression (Orberg and Schaffer, 1987). To labeling infected cells with [ 35 S]methionine for 30 min at 3, 6, and 12 hpi.…”

“…transcription from parental viral genomes (Godowski andKnipe, 1985, 1986). In the latter studies, a negative effect Because viral DNA replication is absolutely required for viral late gene expression and ICP8 is one of the of ICP8 on gene expression from parental viral genomes was demonstrated by analyzing cells infected with an essential proteins required for viral DNA replication, most ICP8 mutants are defective for viral DNA replication and ICP8 temperature-sensitive mutant virus, which expressed increased levels of late proteins and mRNAs at show reduced expression of viral late genes (Conley et al, 1981;Orberg and Schaffer, 1987;Gao et al, 1988; the nonpermissive temperature under conditions in which viral DNA replication was inhibited (Godowski and Gao and Knipe, 1989). Nevertheless, it seems likely that viral DNA synthesis and activation of late gene expres- Knipe, 1985).…”

Regulated Expression of a Sindbis Virus Replicon by Herpesvirus Promoters

“…This type of mutant is equivalent to wild-type HSV-1 in the presence of acyclovir and would provide another means of activating SINrep/LacZ in the absence of HSV-1 replication. One such mutant of HSV-1, d21, which has a large deletion in the ICP8 gene (28), was able to induce ␤-galactosidase in V3-45N cells to essentially the same levels as those obtained with wild-type HSV-1 (data not shown). We also examined the distribution of ␤-galactosidase-positive cells in both V3-45N cells and the parental cells (V3-45-21) that were not capable of packaging the replicon.…”