Analysis and mapping of a family of 3'-coterminal transcripts containing coding sequences for human cytomegalovirus open reading frames UL93 through UL99

Abstract

Human cytomegalovirus (HCMV) open reading frames (ORFs) UL93 through UL99 are contained within a region of viral genome that is well conserved 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 extremely complex and involves a number of large overlapping transcripts, none of which have been characterized. We have used an RNA-mapping approach consisting of Northern (RNA) hybridization, RNase protection, and primer extensions to determine the coding capacity of several large-molecular-weight transcripts which overlap the 1.3- and 1.6-kb UL99-specific transcripts. Our results suggest that six differentially regulated transcripts with sizes of 2.6, 4.7, 5.6, 7.3, 9.1, and 10.5 kb, and derived from the same strand of the viral genome overlap, are 3'-coterminal with the smaller UL99-specific transcripts. On the basis of 5'-end mapping via primer extension and RNase protection, we have determined that the 2.6- to 10.5-kb messages initiate upstream of each of the potential ORFs in this region, UL98, UL97, UL96, UL95, UL94, and UL93. By using cycloheximide and ganciclovir [9-(1,3-dihydroxy-2-propoxymethyl)guanine] to block de novo viral protein synthesis and viral DNA replication, respectively, we have determined that the 2.6-, 4.7-, 5.6-, and 7.3-kb messages have characteristics of early or early-late transcripts, whereas the 9.1- and 10.5-kb messages appear to be true late transcripts. The evolutionary conservation of ORFs UL93 through UL99 and their transcriptional regulation in other herpesviruses are discussed.