Roy Persson

Activation and inhibition of expression of the 72,000-Da early protein of adenovirus type 5 in mouse cells constitutively expressing an immediate early protein of herpes simplex virus type 1

It has been previously reported that immediate early proteins of pseudorabies and cytomegalo viruses can substitute for the products of the human adenovirus type 5 (Ad5) E1A gene in the activation of early Ad5 transcription. In the present report the effect of one of the herpes simplex virus type 1 (HSV-1) immediate early genes, ICP4, on Ad5 early gene expression has been examined using mouse cell lines that constitutively express ICP4. These lines as well as nonproducers were infected with wild-type (wt) Ad5 or with various Ad5 E1A mutants and the levels of expression of the Ad5 E2A 72K DNA binding protein were measured by immunoprecipitation with a monoclonal antibody specific for 72K. With dl 312, which lacks E1A, some 72K expression was seen in nonproducer lines but levels were considerably higher in the producer lines. A similar result was also obtained using dl 312-infected nonproducer cells that were superinfected with HSV-1 virions. These data suggest that HSV-1 ICP4 can substitute for E1A in the activation of expression of early Ad5 proteins. With wt Ad5, 72K was also expressed at high levels in nonproducer mouse cells, however, in the ICP4 producer cell lines, a marked inhibition of 72K expression was observed and this inhibition correlated with the amount of ICP4 present. Using the E1A mutants pm 975 and hr 1, this inhibition was found to be specific for the products of the 1.1-kb E1A mRNA. These data suggest that ICP4 and E1A proteins either directly inhibit each other, or more likely, operate independently and competitively on factors required for viral gene activation.

In cell lines constitutively synthesizing a temperature-sensitive ICP4 protein of herpes simplex virus type 1, amount and function of ICP4 are both regulated by temperature

We have established two cell lines that constitutively synthesize a temperature-sensitive form of ICP4, the herpes simplex virus immediate-early protein that activates early and late transcription. ICP4 in both cell lines was confirmed to be functionally temperature sensitive when tested by complementation of an ICP4 deletion mutant virus for expression of viral early and late genes. When grown at the permissive temperature the two cell lines contained approximately 5 and 25%, respectively, of the ICP4 present in control HSV-infected cells. If the cells were grown at the nonpermissive temperature, ICP4 levels were reduced by approximately fourfold; a twofold reduction was observed in control cells synthesizing the wild-type protein. The lower levels of ICP4 at the nonpermissive temperature were the result of two effects: a decrease in mRNA which was similar in cells producing the mutant or wild-type form of ICP4 and a more rapid turnover of the protein which was greater for the mutant than for the wild-type form. Our observations of lower levels of ICP4 in producer cells differ from published reports of overproduction of immediate-early proteins at the nonpermissive temperature in human or hamster cells infected with ICP4 temperature-sensitive mutant viruses. This discrepancy may be related to cell species differences since we observed only a modest twofold overproduction of immediate-early proteins at the nonpermissive temperature in infections of mouse cell lines with an ICP4 temperature-sensitive mutant virus.