Human cytomegalovirus temporally regulated gene expression in differentiated, immortalized retinal pigment epithelial cells.

BACKGROUND: Human cytomegalovirus (HCMV) replication in epithelial cells is crucial for its pathogenesis. To date, HCMV gene expression has been primarily studied in human foreskin fibroblasts (HFFs), although their importance for HCMV pathogenesis remains unclear. Primary retinal pigment epithelial (RPE) cells are permissive for HCMV. OBJECTIVES: Our objectives were to determine the production of alternatively processed HCMV major immediate-early (MIE) and UL37 RNAs and their essential products in infected, terminally differentiated immortalized RPE (hTERT-RPE) cells. STUDY DESIGN: hTERT-RPE cells were studied because of their notable similarities with primary RPE cells, and because they overcome key limitations of primary cells. hTERT-RPE cells were terminally differentiated in vitro and infected with HCMV. Total RNA or cell proteins were analyzed at various times post-infection. RESULTS: We show for the first time that HCMV-infected, differentiated hTERT-RPE cells produce IE1, IE2, UL37 exon 1 (UL37x1) and UL37 alternatively spliced RNAs, albeit with abundances and kinetics distinct from those observed in HCMV-infected HFFs. IE1-72 was produced in HCMV-infected, differentiated hTERT-RPEs within 24h post-infection (hpi); whereas, IE2-86 and pUL37x1 were produced within 72 hpi. IE2-86 was detected after IE1-72 even though its transcript appeared first. Early/late (pp65) and late (pp28) proteins were produced within 96-120 hpi. CONCLUSIONS: The temporal cascade of HCMV gene expression was observed in infected, differentiated hTERT-RPE cells. Moreover, HCMV IE RNAs are alternatively and accurately processed in differentiated hTERT-RPE cells. However, the delayed temporal expression suggests further regulation of HCMV gene expression at post-transcriptional/translational levels in differentiated hTERT-RPE cells.

Complex alternative processing of human cytomegalovirus UL37 pre-mRNA.

Differentially processed human cytomegalovirus (HCMV) UL37 RNAs encode biologically significant proteins. Due to the recent discovery of alternative UL37 exon 3 (UL37x3) splice donors, permissively infected cells were thoroughly examined for additional alternatively spliced UL37 RNAs. Newly described donors within UL37 exon 1 (nt 52520) and intron 1 (nt 52209) as well as UL37x3 di (nt 50770) and dii (nt 50782) were differentially spliced to known downstream UL37 acceptors. The alternatively spliced UL37(S), UL37(L), UL37(di) and UL37d(ii) RNAs predictably encode proteins of 83, 163, 217 and 213 residues, respectively, which share UL37x1 N-terminal sequences but differ downstream in their C termini. Moreover, temporal expression of the alternatively spliced UL37 RNAs differs during HCMV infection. The complexity of UL37 pre-mRNA processing is evidenced by the detection of 11 UL37 spliced and unspliced UL37x1 RNAs in HCMV-infected cells. Based upon these data, a revised HCMV UL37 gene map is presented, which incorporates all RNA species detected during permissive infection.

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.