Expression of human herpesvirus 6B rep within infected cells and binding of its gene product to the TATA-binding protein in vitro and in vivo

We have characterized the human herpesvirus 6B (HHV-6B) rep gene, which is a homologue of the adeno-associated virus type 2 rep and is unique in the herpesvirus family. Three transcripts, 9.0, 5.0, and 2.7 kb, (the major transcript), were detected by Northern blotting using an HHV-6B rep probe under late conditions. We investigated the expression kinetics of the rep gene using cycloheximide (CHX) and phosphonoformic acid (PFA), which are inhibitors of protein synthesis and viral DNA synthesis, respectively. The 5.2-kb transcript was mainly detected in the absence of protein biosynthesis upon infection, and none of the 9.0-, 5.0-, and 2.7-kb transcripts detected under the late conditions were detected in the presence of CHX and PFA. Sequences obtained from a cDNA library showed that the 5.0- and 2.7-kb transcripts were spliced from two and three exons, respectively, and the 2.7-kb transcript was more abundant. Immunohistochemistry using an antibody raised against the HHV-6 rep gene product (REP) revealed that REP was mainly present in the nucleus of MT-4 cells within 24 h after infection with HHV-6B. Using pull-down assays, coimmunoprecipitation, and a mammalian two hybrid system, we showed that HHV-6 REP binds to a transcription factor, human TATA-binding protein, through its N-terminal region.