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.
LaBoissière
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S
Marie Curie Research Institute, Oxted, Surrey RH8 OTL, United Kingdom.
O'Hare
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P
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D017781:Genes, Immediate-Early; D017405:Herpes Simplex Virus Protein Vmw65 / Q000378:metabolism*; D006358:Hot Temperature; D011485:Protein Binding; D018139:Simplexvirus / Q000302:isolation & purification* / Q000502:physiology; D014709:Vero Cells; D019065:Virus Assembly
D017405:Herpes Simplex Virus Protein Vmw65