ANALYSIS AND MAPPING OF A FAMILY OF 3'-COTERMINAL TRANSCRIPTS CONTAINING CODING SEQUENCES FOR HUMAN CYTOMEGALOVIRUS OPEN READING FRAMES UL93 THROUGH UL99

Human cytomegalovirus (HCMV) open reading frames (ORFs) UL93 through U L99 are contained within a region of viral genome that is well conserv ed in all herpesviruses. Previous reports detailing the expression of ORF UL99 (also referred to as the 28-kDa virion phosphoprotein or pp28 ) indicated that the pattern of transcription proximal to pp28 is extr emely complex and involves a number of large overlapping transcripts, none of which have been characterized. We have used an RNA-mapping app roach consisting of Northern (RNA) hybridization, RNase protection, an d primer extensions to determine the coding capacity of several large- molecular-weight transcripts which overlap the 1.3- and 1.6-kb UL99-sp ecific transcripts. Our results suggest that six differentially regula ted transcripts with sizes of 2.6, 4.7, 5.6, 7.3, 9.1, and 10.5 kb, an d derived from the same strand of the viral genome overlap, are 3' cot erminal with the smaller UL99-specific transcripts. On. the basis of 5 '-end mapping via primer extension and RNase protection, we have deter mined that the 2.6 to 10.5-kb messages initiate upstream of each of th e potential ORFs in this region, UL98, UL97, UL96, UL95, UL94 and UL93 . By using cycloheximide and ganciclovir [9-(1,3-dihydroxy-2-propoxyme thyl)guanine] to block de novo viral protein synthesis and viral DNA r eplication, respectively, we have determined that the 2.6- 4.7-, 5.6-, and 7.3-kb messages have characteristics of early or early-late trans cripts, whereas the 9.1- and 10.5-kb messages appear to be true late t ranscripts. The evolutionary conservation of ORFs UL93 through UL99 an d their transcriptional regulation in other herpesviruses are discusse d.