In the present work we report the cloning of a full-length cDNA encoding the immediate-early (IE) 2 protein from human herpesvirus 6 (HHV-6) variant A (GS strain). The transcript is 4690 nucleotides long and composed of 5 exons. Translation initiation occurs within the third exon and proceeds to the end of U86. Kinetic studies indicate that the 5.5-kb IE2 mRNA is expressed under IE condition, within 2-4 h of infection. IE2 transcripts from both variants A and B are expressed under similar kinetics with IE2 transcripts accumulating up to 96 h postinfection. Although several large transcripts (>5.5 kb) hybridized with the IE2 probe, suggesting multiple transcription initiation sites, a single form of the IE2 protein, in excess of 200 kDa, was detected by Western blot. Within cells, the IE2 protein was detected (8-48 h) as intranuclear granules while at later time points (72-120 h), the IE2 protein coalesced into a few large immunoreactive patches. Transfection of cells with an IE2 expression vector (pBK-IE2A) failed to reproduce the patch-like distribution, suggesting that other viral proteins are necessary for this process to occur. Last, IE2 was found to behave as a promiscuous transcriptional activator. Cotransfection experiments in T cells indicate that IE2 can induce the transcription of a complex promoter, such as the HIV-LTR, as well as simpler promoters, whose expression is driven by a unique set of responsive elements (CRE, NFAT, NF-kB). Moreover, minimal promoters having a single TATA box or no defined eukaryotic regulatory elements were significantly activated by IE2, suggesting that IE2 is likely to play an important role in initiating the expression of several HHV-6 genes. In all, the work presented represents the first report on the successful cloning, expression, and functional characterization of the major regulatory IE2 gene/protein of HHV-6.