(prepared by the applicant): Viruses provide valuable systems to study gene regulation since they exploit the cellular machinery in unconventional ways and can identify novel mechanisms. DNA replication of human cytomegalovirus (HCMV), a medically important herpesvirus, requires its UL36-38 gene products. Differential processing of UL36-3 8 RNAs generates four immediate early (IE) RNAs, which share sequences and an early RNA. The UL37 and UL37M spliced IE RNAs are expressed at very low abundance at IE times of HCMV infection, while the others are abundant throughout the viral life cycle. Our overall aim is to determine the mechanistic basis for this differential regulation. We hypothesize that the targets for differential regulation lie within UL37 RNA unique sequences and result from inefficient splicing, inhibition of transcription elongation, and/or RNA instability.
In Aim 1, we will determine if UL37 RNA splicing is reduced by efficient polyadenylation (PA). Intriguingly, UL37 Introns 1 and 2 have juxtaposed PA signals, branch points, and 3' splice sites. We hypothesize that abundance of UL37x1 unspliced RNA results from inhibition of RNA splicing by competition of factors for juxtaposed cis-elements. To test this, we will mutate the PA signals in UL37 Introns 1 and 2 and examine their effects on UL37 RNA splicing. We will characterize UL37 RNA splicing by defining the cis-elements controlling RNA processing and studying the effects of mutant cis-elements on splicing in cells and in vitro. As the abundance of UL37 spliced RNAs changes temporally, we will define how HCMV infection alters splicing and/or PA machineries at IE, early, or late times using in vitro extracts and gel shift assays.
In Aim 2, we will test whether transcription of the UL3 7 IE gene is blocked. We will test if thymidine (T)-rich regions in the UL3 7 Intron 1/Exon 2 boundary inhibit transcript elongation by measuring transcription through the UL37 gene using nuclear run-ons. We will identify and mutate the sequences responsible for inhibition of RNA elongation.
In Aim 3, we will test whether UL37 RNA sequences are selectively targeted for degradation. We will examine the stability of UL37 RNA in HCMVinfected cells and define its instability elements by selectively deleting UL37 sequences and measuring the stability of the encoded RNAs. A thorough understanding of how the UL36-38 essential gene locus is regulated in HCMV infected HFF cells is of fundamental importance to understanding the modulation of HCMV gene expression, ultimately understanding the viral life cycle and potentially identifying novel mechanisms of cellular gene regulation.
