Alteration of cellular RNA splicing and polyadenylation machineries during productive human cytomegalovirus infection.
Alternative processing of human cytomegalovirus (HCMV) UL37 pre-mRNA predominantly produces the unspliced UL37 exon 1 (UL37x1) RNA and multiple, lower abundance, alternatively spliced UL37 RNAs. The relative abundance of UL37x1 unspliced RNA is surprising because it requires the favoured use of a polyadenylation signal within UL37 intron 1, just upstream of the UL37 exon 2 (UL37x2) acceptor. Here, it was shown that a downstream element (DSE) in UL37x2 strongly enhanced processing at the UL37x1 polyadenylation site, but did not influence UL37x1-x2 splicing. There was a potential binding site (UCUU) for polypyrimidine tract-binding protein (PTB) at the UL37x1 polyadenylation/cleavage site and its mutation to UGGG reduced both polyadenylation and splicing of UL37x1-x2 minigene pre-mRNA, suggesting a role in both RNA processing events. To determine whether lytic HCMV infection altered the balance of RNA processing factors, which bind to UL37 pre-mRNA cis elements, these were investigated in permissively infected primary and immortalized human diploid fibroblasts (HFFs) and epithelial cells. Induction of polyadenylation factors in HCMV-infected, serum-starved (G(0)) HFFs was also investigated. Permissive HCMV infection consistently increased, albeit with different kinetics, the abundance of cleavage stimulation factor 64 (CstF-64) and PTB, and altered hypo-phosphorylated SF2 in different cell types. Moreover, the preponderance of UL37x1 RNA increased during infection and correlated with CstF-64 induction, whereas the complexity of the lower abundance UL37 spliced RNAs transiently increased following reduction of hypo-phosphorylated SF2. Collectively, multiple UL37 RNA polyadenylation cis elements and induced cellular factors in HCMV-infected cells strongly favoured the production of UL37x1 unspliced RNA.
Adair
Richard
R
Center for Cancer and Immunology Research, Children's Research Institute, Children's National Medical Center, Room 5720, 111 Michigan Avenue NW, Washington, DC 20010, USA.
Liebisch
Gregory W
GW
Center for Cancer and Immunology Research, Children's Research Institute, Children's National Medical Center, Room 5720, 111 Michigan Avenue NW, Washington, DC 20010, USA.
Su
Yan
Y
Center for Cancer and Immunology Research, Children's Research Institute, Children's National Medical Center, Room 5720, 111 Michigan Avenue NW, Washington, DC 20010, USA.
Colberg-Poley
Anamaris M
AM
Department of Pediatrics, George Washington University School of Medicine and Health Sciences, Washington, DC 20010, USA.|Center for Cancer and Immunology Research, Children's Research Institute, Children's National Medical Center, Room 5720, 111 Michigan Avenue NW, Washington, DC 20010, USA.
D001665:Binding Sites; D003587:Cytomegalovirus / Q000502:physiology*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics; D026723:Polyadenylation; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D014764:Viral Proteins / Q000235:genetics
D017874:Immediate-Early Proteins; D012322:RNA Precursors; C420323:UL37 protein, Human herpesvirus 5; D014764:Viral Proteins