Repression of the alpha0 gene by ICP4 during a productive herpes simplex virus infection

Abstract

During a productive infection by herpes simplex virus type 1 (HSV-1), ICP4, the major regulatory protein encoded by the ␣4 gene, binds to its transcription initiation site and represses the accumulation of ␣4 RNA. Evidence suggests that the degree of repression by ICP4 is a function of the absolute distance of an ICP4 binding site 3 from a TATA box. However, repression of HSV-1 gene expression by ICP4 through binding sites located 5 of TATA boxes, as in the case of the ␣0 gene, has not been adequately addressed. To this end, recombinant ␣0 promoters with various arrays of ICP4 binding sites flanking the ␣0 TATA box were constructed and recombined into the HSV-1 genome. Our results demonstrate the following. (i) Destruction of the endogenous ␣0 ICP4 binding site, located 5 of the TATA box, results in derepression of ␣0 protein and RNA accumulation in infected Vero cells. (ii) The degree of ␣0 derepression is equivalent to that reported for the ␣4 gene following destruction of the ICP4 binding site at the ␣4 mRNA cap site in HSV-1. (iii) Introduction of an ICP4 binding site at the ␣0 mRNA cap site represses the accumulation of ␣0 RNA greater than threefold relative to the wild type. (iv) Changes in the abundance of ␣0 protein and RNA in infected cells do not affect replication or growth of HSV-1 in tissue culture. Our findings are consistent with the conclusion that ␣0 transcription is repressed by ICP4. These results demonstrate that repression by ICP4 can occur through binding sites located 5 of virus gene TATA boxes in HSV-1. Thus, models addressing repression of HSV-1 gene expression by ICP4 should incorporate the role of binding sites located 5, as well as 3, of virus gene TATA boxes.

Mutational analysis of the herpes simplex virus type 1 ICP0 C3HC4 zinc ring finger reveals a requirement for ICP0 in the expression of the essential alpha27 gene

“…ICP4 is an essential protein that functions as both a repressor and an activator of transcription. In concert with ICP0, ICP4 activates the expression of early and late genes while repressing its own expression, as well as that of the ␣0 gene (14,32,42,43,51,57,60). ICP27, another essential protein, is required for the expression of ␤ and ␥ genes during the HSV life cycle.…”

“…The proteins were separated by SDS-PAGE (41) and transferred to nitrocellulose membranes (72). Immunodetection of ICP0 was performed as previously described (54) with rabbit polyclonal antibody CLU7 (43). The relative levels of ICP0 were quantified by densitometric analysis using an AGFA ARCUS II scanner and NIH Image 1.51.…”

“…(ii) Infections. Western blot analysis of virus-specified proteins was performed as described previously (43), with one addition. HSV-1 VP5 was detected with rabbit polyclonal antibody NC-1 (provided by G. Cohen, Department of Microbiology, University of Pennsylvania).…”

“…(i) Transfections. Vero cells were transfected as previously described (43). Transfection mixtures contained 12.5 g of plasmid DNA (pEL0-C116G, pEL0-H126A, or pEL0-C116G/C156A), linearized with HindIII, and 100 PFU of IE-0:LacZ nucleocapsids (66).…”

“…(ii) Purification of recombinant viruses. Recombinant herpesviruses with mutations in the ␣0 gene were generated as previously described (43). The sitedirected mutations in the ␣0 C 3 HC 4 domain were verified by direct sequence analysis of PCR-amplified virus DNAs, as described below.…”