Transcript Mapping in Human Cytomegalovirus Strain AD169

Human cytomegalovirus (HCMV) is of considerable medical importance, with infection in utero being a major health risk for the developing foetus, causing a variety of neonatal abnormalities including deafness, physical abnormality and mental retardation. HCMV also poses a life-threatening risk to immunosuppressed individuals such as allograft recipients and HIV-infected people. HCMV is responsible for the blindness due to retinitis that can affect some AIDS patients. The gene content of HCMV is less well understood than that of any other human herpesvirus. This reflects the large size and complexity of the genome, and also the lack of a laboratory strain with the full genetic complement of wild type virus. The complete DNA sequence (229,354 bp) of HCMV strain AD 169 was published in 1990, and the genome was predicted to contain 208 protein-coding open reading frames (ORFs). This is not likely to be an accurate estimate of the actual number of genes, as the criteria employed to identify coding regions were necessarily arbitrary and applied without the benefit of comparisons with other betaherpesviruses. Moreover, HCMV strain Toledo and other low passage isolates were later found to possess a 15 kbp genome segment absent from AD 169. Recently, the gene content of HCMV has been revised by comparison to the chimpanzee cytomegalovirus (CCMV) sequence (241,087 bp), and the number of protein-coding genes in AD 169 is now estimated at 145, several of which are novel. It is anticipated that this picture of the gene content of HCMV will be improved further. The HCMV genome contains a set of 41 conserved herpesvirus-common 'core' genes, which are arranged in blocks that maintain relative position and orientation in different herpesviruses and reflect evolution from a common ancestor. The majority of genes are not spliced and overall the genome has relatively few polyadenylation signals. At the outset of this project, 12 HCMV genes had been shown experimentally to be spliced, and more spliced genes probably remained to be identified. Ten different families of related genes (RL11, US6, US22, OCR, UL25, UL82, UL146, US1, US12 and US22) have been recognised in HCMV that appear to have been generated by gene duplication events. The US22 gene family contains 13 distantly related members (UL23, UL24, UL26, UL36, UL43, US22, US23, US24, US26, TRS1 and IRS1) sharing one or more of four conserved amino acid sequence motifs. Three members of this family (UL36, TRS1 and IRS1) have been reported as exhibiting transcriptional trans-activating properties in transient transfection assays, indicating that US22 genes are likely to be regulatory proteins. Moreover, since each of the sequenced betaherpesviruses contains a similar number of US22 genes, it is anticipated that these genes provide important functions during virus replication. Although the AD 169 genome was sequenced over ten years ago, the products of a large number of HCMV genes have not been identified, and the assignment of gene function is largely based on sequence similarity to homologous genes in herpes simplex virus type 1 (HSV-1). Transcript mapping data are also fragmentary. The purpose of this study was to evaluate transcription of a selection of AD 169 genes, including several that are conserved in CCMV and some that appear unlikely to encode functional proteins because the HCMV ORFs are not conserved in CCMV. Primary use was made of northern blotting, RT-PCR and RACE techniques, employing RNA isolated from infected human fibroblasts. Three groups of genes were analysed: the 13 members of the US22 gene family; the 14 ORFs in TRL and 30 adjacent ORFs at the left end of UL; and the novel spliced genes UL128 and UL131A. Transcripts were detected by northern blotting for nine members of the US22 family, and 5'- and 3'-ends were identified for eight. Failure to obtain data for the other members analysed was probably due to transcription at low levels. RNAs were identified for most ORFs in TRL and the adjacent part of UL. Most of the 5'-ends are located 20-30 bp downstream from TATA elements, and all the 3'-ends are located 20-24 bp downstream from polyadenylation signals. The 5'-ends of two genes (UL18 and US24) appeared to be located downstream from the first ATG codon in the relevant ORF. Transcripts were detected for five ORFs in TRL and one in UL that appear unlikely to encode proteins. Certain ORFs in TRL and UL have more than one 5'-end, suggesting that they are transcribed in a complex manner