Characterization of transcripts expressed from human herpesvirus 6A strain GS immediate-early region B U16-U17 open reading frames.

Human herpesvirus 6 (HHV-6) was first isolated from patients with AIDS and lymphoproliferative disorders (28). It was subsequently shown to be the causative agent of a common childhood disease, exanthem subitum (roseola) (1, 9, 10). HHV-6 isolates segregated into two closely related subgroups, A and B, based on their antigenic properties, T-cell line infectivities, restriction endonuclease cleavage, and genomic DNA sequences (1, 4, 7, 15, 31, 40). HHV-6A includes the prototype strains GS and U1102, and HHV-6B includes the prototype strain Z29 and isolates from roseola patients (1, 40). The genome of HHV-6 is approximately 160 kb long and consists of a unique long region of 140 kb flanked by direct-repeat regions of about 10 kb (15, 24, 25, 32). Sequence analyses of the HHV-6 genome show that it is closely related to human cytomegalovirus (HCMV), exhibiting a colinear arrangement of genes (6, 12, 24, 25). Several studies have described potential interactions between HHV-6 and human immunodeficiency virus type 1 (HIV-1) (3, 11, 13, 14, 16, 17, 18, 21, 42). HHV-6A(GS) and HHV-6B(Z29) transactivate the expression of the chloramphenicol acetyltransferase (CAT) reporter gene under the regulatory control of the HIV long terminal repeat (HIV LTR) in human peripheral blood mononuclear cells and in T-cell lines (14, 16, 17, 42). Our studies and others have identified at least six different cloned HHV-6 genomic fragments that can transactivate the HIV LTR (39). In our studies, the highest level of transactivation was seen with a 22-kb BamHI genomic fragment (pZVB70) of HHV-6A(GS) (14, 16, 17) and with 3.8- and 1.8-kb subfragments of pZVB70 (14). Within the 1.8-kb fragment, an open reading frame (ORF) encoding a 258-amino-acid (aa) protein mediated the transactivation (14). The first methionine encoded by this ORF was in the position corresponding to 115 aa downstream from the start of the ORF, and the region of the ORF corresponding to 143-aa carboxyl terminus of the product encoded a predicted protein of about 19-kDa. This ORF was designated B701, and its product was shown to activate CAT expression from an HIV-CAT plasmid in a cotransfection assay (14). The product of ORF B701 did not show any significant homology to the other viral proteins with transactivating functions, such as ICP0 and ICP4 of herpes simplex virus type 1, but did share some weak homology with the US22 family of immediate-early (IE) and early genes from HCMV. ORF B701 is positionally homologous to HCMV UL36 ORF exon 2 (15, 25). Comparison with the HHV-6A(U1102) published sequence shows that the HHV-6A(GS) ORF B701 corresponds to ORF U16. There are two potential TATA boxes upstream of the 143-aa coding region of ORF B701 (Fig. ​(Fig.1).1). Whether the mRNA for the putative B701 protein is transcribed from these promoter regions or is part of a larger protein generated by splicing is not known. In HCMV, the UL36 coding sequence has been shown to be generated by an in-frame splicing of the UL36 exons 1 and 2 (6, 12, 36, 37), and sequence analysis of the HHV-6 genome predicts a similar splicing site between the HHV-6A ORFs U17 and U16 (25). Using reverse transcriptase RT PCR, we show evidence of splicing in the transcripts encoded by the HHV-6A(GS) ORFs U16 and U17. Our data demonstrate that a family of transcripts is generated from the IE-B region of HHV-6, similar to the HCMV UL36-UL38 family of genes. Two cDNAs of 1.9 and 1.8 kb including ORF B701 were identified from an HHV-6A(GS) cDNA library. The 5′ rapid amplification of cDNA ends (5′ RACE) technique was used to identify the 5′ ends of transcripts generated from U17 and U16. Two different TATA boxes located upstream of ORF U17 were identified as potential transcriptional start sites. An additional cDNA generated with a unique splicing event was also identified by 5′ RACE. RT-PCR results suggest that some of the transcripts from this region are IE gene products and others are late gene products. Thus, while there are some similarities to the HCMV UL36-UL38 family, the U17/U16 family of transcripts has members that are unique to HHV-6. FIG. 1 Comparison of HHV-6A(GS) and HHV-6A(U1102) genomes encoding ORFs U15 to U17 and the relationships of the identified cDNAs. (A) Schematic representation of HHV-6 unique long region (UL). IE-A and IE-B, locations of the IE regions (16, 25). The locations ...