Analysis of HCF, the cellular cofactor of VP16, in herpes simplex virus-infected cells

Herpes simplex virus (HSV) immediate-early (IE) gene expression is initiated via the recruitment of the structural protein VP16 onto specific sites upstream of each IE gene promoter in a multicomponent complex (TRF.C) that also includes the cellular proteins Oct-1 and HCF. In vitro results have shown that HCF binds directly to VP16 and stabilizes TRF.C. Results from transfection assays have also indicated that HCF is involved in the nuclear import of VP16. However, there have been no reports on the role or the fate of HCF during HSV type 1 (HSV-1) infection. Here we show that the intracellular distribution of HCF is dramatically altered during HSV-1 infection and that the protein interacts with and colocalizes with VP16. Moreover, viral protein synthesis and replication were significantly reduced after infection of a BHK-21-derived temperature-sensitive cell line (tsBN67) which contains a mutant HCF unable to associate with VP16 at the nonpermissive temperature. Intracellular distribution of HCF and of newly synthesized VP16 in tsBN67-infected cells was similar to that observed in Vero cells, suggesting that late in infection the trafficking of both proteins was not dependent on their association. We constructed a stable cell line (tsBN67r) in which the temperature-sensitive phenotype was rescued by using an epitope-tagged wild-type HCF. In HSV-1-infected tsBN67r cells at the nonpermissive temperature, direct binding of HCF to VP16 was observed, but virus protein synthesis and replication were not restored to levels observed at the permissive temperature or in wild-type BHK cells. Together these results indicate that the factors involved in compartmentalization of VP16 alter during infection and that late in infection, VP16 and HCF may have additional roles reflected in their colocalization in replication compartments.     

Mechanisms for flexibility in DNA sequence recognition and VP16-induced complex formation by the Oct-1 POU domain.

DNA binding by the Oct-1 protein is directed by its POU domain, a bipartite DNA-binding domain made up of a POU-specific (POUS) domain and a POU-homeo (POUH) domain, two helix-turn-helix-containing DNA-binding modules that cooperate in DNA recognition. Although the best-characterized DNA target for Oct-1 binding is the octamer sequence ATGCAAAT, Oct-1 also binds a number of different DNA sequence elements. For example, Oct-1 recognizes a form of the herpes simplex virus VP16-responsive TAATGARAT element, called the (OCTA-)TAATGARAT site, that lacks octamer site similarity. Our studies suggest two mechanisms by which Oct-1 achieves flexible DNA sequence recognition. First, an important arginine found in the Oct-1 POUS domain tolerates substitutions of its base contacts within the octamer site. Second, on the (OCTA-)TAATGARAT site, the POUS domain is located on the side of the POUH domain opposite from where it is located on an octamer site. This flexibility of the Oct-1 POU domain in DNA binding also has an impact on its participation in a multiprotein-DNA complex with VP16. We show that Oct-1 POUS domain residues that contact DNA have different effects on VP16-induced complex formation depending on whether the VP16-responsive element involved has overlapping octamer similarity or not.     

Role of alpha-transinducing factor (VP16) in the induction of alpha genes within the context of viral genomes.

In herpes simplex virus 1, the five alpha genes are induced by alpha-transinducing factor (alpha TIF; VP16), a virion protein, acting in concert with Oct-1 and other cellular proteins on a cis-acting site in the promoter domain of alpha genes. Because alpha TIF is an essential virion protein, its function as an inducer can best be evaluated only by mutating the cis-acting site. Earlier we reported on a series of 17 mutations in and around the cis-acting site of a 275-bp alpha 27 promoter fused to a reporter gene and recombined into the viral genome. These recombinant viruses were tested in Vero cells in the presence of cycloheximide, and we demonstrated that mutations in the sequence required for Oct-1 binding abolished transactivation whereas mutations in the alpha TIF-dependent GARAT sequence decreased but did not abolish transactivation. We now report that (i) in limited-passage human embryonic lung cells, alpha gene expression from promoters mutated in the GARAT sequences is often higher and more variable than in Vero cells, (ii) in the absence of cycloheximide, the mutant viruses show less significant impairment of reporter gene expression, (iii) Oct-1 can bind either to the overlapping octamer element or to various TAATGARAT sequences with differing degrees of binding strength and these relative binding levels correlate well with levels of gene expression observed in infected cells, (iv) in the cis-acting site upstream of the alpha 4 gene, no degenerate overlapping Oct-1 sequence exists, and therefore in this instance Oct-1 must be binding directly to the TAATGARAT sequence, (v) extension of the alpha 27 promoter by an additional 1,334 bp results in much higher expression of the reporter gene as a result of additional upstream cis-acting sites, and (vi) obliteration of the most proximal Oct-1 binding element within the 275-bp promoter dramatically reduces gene expression even in the presence of the additional upstream cis-acting sites.