(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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI046459-02
Application #
6510939
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Beisel, Christopher E
Project Start
2001-07-01
Project End
2005-05-31
Budget Start
2002-06-01
Budget End
2003-05-31
Support Year
2
Fiscal Year
2002
Total Cost
$288,750
Indirect Cost
Name
Children's Research Institute
Department
Type
DUNS #
City
Washington
State
DC
Country
United States
Zip Code
20010
Gaddy, Charla E; Wong, Daniel S; Markowitz-Shulman, Ariel et al. (2010) Regulation of the subcellular distribution of key cellular RNA-processing factors during permissive human cytomegalovirus infection. J Gen Virol 91:1547-59
Easley, Rebecca; Carpio, Lawrence; Guendel, Irene et al. (2010) Human T-lymphotropic virus type 1 transcription and chromatin-remodeling complexes. J Virol 84:4755-68
Adair, Richard; Liebisch, Gregory W; Lerman, Bruce J et al. (2006) Human cytomegalovirus temporally regulated gene expression in differentiated, immortalized retinal pigment epithelial cells. J Clin Virol 35:478-84
Adair, Richard; Liebisch, Gregory W; Su, Yan et al. (2004) Alteration of cellular RNA splicing and polyadenylation machineries during productive human cytomegalovirus infection. J Gen Virol 85:3541-53
Su, Yan; Testaverde, James R; Davis, Candice N et al. (2003) Human cytomegalovirus UL37 immediate early target minigene RNAs are accurately spliced and polyadenylated. J Gen Virol 84:29-39
Adair, Richard; Liebisch, Gregory W; Colberg-Poley, Anamaris M (2003) Complex alternative processing of human cytomegalovirus UL37 pre-mRNA. J Gen Virol 84:3353-8
Su, Yan; Adair, Richard; Davis, Candice N et al. (2003) Convergence of RNA cis elements and cellular polyadenylation factors in the regulation of human cytomegalovirus UL37 exon 1 unspliced RNA production. J Virol 77:12729-41