Adair Richard, Liebisch Gregory W, Lerman Bruce J, Colberg-Poley Anamaris M
Center for Cancer and Immunology Research, Children's Research Institute, Children's National Medical Center, Washington, DC 20010, USA.
J Clin Virol. 2006 Apr;35(4):478-84. doi: 10.1016/j.jcv.2005.10.015. Epub 2006 Jan 4.
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.
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.
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.
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.
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.
人巨细胞病毒（HCMV）在上皮细胞中的复制对其发病机制至关重要。迄今为止，HCMV基因表达主要是在人包皮成纤维细胞（HFFs）中进行研究，尽管其对HCMV发病机制的重要性仍不清楚。原代视网膜色素上皮（RPE）细胞对HCMV易感。
我们的目的是确定在感染的、终末分化的永生化RPE（hTERT-RPE）细胞中可变加工的HCMV主要立即早期（MIE）和UL37 RNA及其重要产物的产生情况。
研究hTERT-RPE细胞是因为它们与原代RPE细胞有显著相似性，并且因为它们克服了原代细胞的关键局限性。hTERT-RPE细胞在体外进行终末分化并感染HCMV。在感染后的不同时间分析总RNA或细胞蛋白。
我们首次表明，HCMV感染的、分化的hTERT-RPE细胞产生IE1、IE2、UL37外显子1（UL37x1）和UL37可变剪接RNA，尽管其丰度和动力学与在HCMV感染的HFFs中观察到的不同。IE1-72在感染后24小时（hpi）内在HCMV感染的、分化的hTERT-RPE细胞中产生；而IE2-86和pUL37x1在72 hpi内产生。尽管IE2-86的转录本最先出现，但在IE1-72之后才检测到IE2-86。早期/晚期（pp65）和晚期（pp28）蛋白在96-120 hpi内产生。
在感染的、分化的hTERT-RPE细胞中观察到HCMV基因表达的时间级联。此外，HCMV IE RNA在分化的hTERT-RPE细胞中进行可变且精确的加工。然而，延迟的时间表达表明在分化的hTERT-RPE细胞中，HCMV基因表达在转录后/翻译水平受到进一步调控。
