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.


OBJECTIVE
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.


METHODS
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.
Adair
Richard
R
Center for Cancer and Immunology Research, Children's Research Institute, Children's National Medical Center, Washington, DC 20010, USA.
Liebisch
Gregory W
GW
Lerman
Bruce J
BJ
Colberg-Poley
Anamaris M
AM
D017398:Alternative Splicing; D002454:Cell Differentiation; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism / Q000472:pathogenicity*; D004847:Epithelial Cells / Q000821:virology; D015967:Gene Expression Regulation, Viral; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000821:virology*; D012367:RNA, Viral / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D017874:Immediate-Early Proteins; D012367:RNA, Viral; C420323:UL37 protein, Human herpesvirus 5; D014764:Viral Proteins