CYTOMEGALOVIRUS REPLICATION IN HUMAN RETINAL-PIGMENT EPITHELIAL-CELLS- ALTERED EXPRESSION OF VIRAL EARLY PROTEINS

Purpose. Cytomegalovirus (CMV) infections are frequent complications i n patients who have undergone kidney and bone marrow transplant and in patients with acquired immune deficiency syndrome. The mechanism by w hich CMV is activated and replicated within the retina is unknown. The authors evaluated the ability of human CMV to initiate replication in human retinal pigment epithelial (RPE) cells and compared this system with CMV replication in human fibroblasts (HEL-299, MRC-5) and human amnion epithelial (WISH) cells. Methods. Human RPE cells were obtained from donor eyes and propagated in vitro. Cells were infected, and CMV replication was evaluated in three ways: the detection of viral antig en by immunofluorescent, flow cytometry, and Western blot assays; the detection of virus-induced cytopathic effect (cpe), and the detection of infectious virus. Results, No evidence of viral replication in the epithelial (WISH) cells was found. Although CMV does not usually repli cate in vitro in epithelial cells, CMV replication was detected in RPE cells. There are a number of distinct differences in CMV replication in RPE cells compared to replication in human fibroblasts. Virus-induc ed cpe and the production of infectious virus by RPE cells were delaye d when compared to virus infection in either HEL or MRC 5 cells. At a multiplicity of infection of 0.1 and 1, cpe and infectious virus yield reached maximum levels at days 4 to 5 in fibroblasts and at days 19 t o 46 in RPE cells, respectively. Nevertheless, infectious virus produc ed by RPE cells (10(6.5) TCID50/0.1 ml) significantly surpassed levels produced by HEL cells (10(5.5) TCID50/0.1 ml). The permissive infecti on in RPE cells consisted of a prolonged period (5 to 6 days) of virus production in the absence of cytopathology. Virus protein expression evaluated by indirect immunofluorescence assays, Western blot analysis , and flow cytometry revealed a delay in viral protein expression in R PE cells compared to viral protein expression in fibroblasts. The patt ern of viral protein evaluated by flow cytometry was noticeably differ ent in the two cell types. At the middle phase of CMV replication in R PE cells, a low percentage of cells express immediate early (IE) prote in at a time when a high percentage of the cells express early (E) pro teins. This IE-1 protein is a stable protein found concurrently with E protein in fibroblasts. This difference in percentage of cells expres sing specific CMV proteins is transient, that is, it does not remain a pparent at 100% cpe. Conclusions. Retinal pigment epithelial cells app ear to demonstrate a distinct pattern of CMV infection. The low freque ncy of expression of IE viral protein in RPE cells, the subsequent slo w replication of CMV, and the altered expression of IE viral proteins may be critical variables that impact on their relationship to viral p ersistence and activation within the retina. Alterations in the IE gen e product may indicate the existence of positive or negative nuclear t ranscription factors within infected RPE cells.