Title

Expression of HLA class I heavy chain and TcR gamma-chain homologs in human cytomegalovirus-infected human cells

Date of Completion

January 1998

Keywords

Biology, Genetics|Biology, Cell|Health Sciences, Pathology|Health Sciences, Immunology

Degree

Ph.D.

Abstract

Identification of human cytomegalovirus (HCMV) homologs of the HLA class I heavy chain (designated UL18) and the T cell receptor (TcR) $\gamma$-chain (designated UL20) has raised the possibility that these viral products modulate the MHC class I- and TcR-restricted immune response to HCMV-infected cells. In order to study the expression of these genes, HCMV-infected human foreskin fibroblasts (HFF) and U373 human astrocytoma/glioblastoma cells were analyzed. In HFF, UL18 and UL20 RNA became detectable by 24 hours following HCMV infection and peaked at 96 hours post-infection. In U373 cells, steady state RNA levels for UL18 and UL20 were already maximal by 24 hours post-infection. The UL18 and UL20 transcripts were expressed as several prominent species of RNA in both cell lines. Transcription of UL18 and UL20 in both cell lines was inversely correlated with cell surface HLA class I expression. As determined by surface immunofluorescence, HLA class I and $\beta$2 microglobulin ($\beta$2m) levels dropped to 20-30% of those in uninfected cells by 96 hours post-infection. However, steady-state RNA levels for HLA class I and $\beta$2m were not reduced, indicating that class I downregulation by HCMV is a posttranscriptional phenomenon. UL18 and UL20 RNA in HFF was largely nonadenylated, while the majority of UL18 and UL20 RNA was polyadenylated in U373 cells. Treatment of HFF and U373 cells with cycloheximide plus actinomycin D, acyclovir, or phosphonoformic acid after infection indicated that both UL18 and UL20 were transcribed at the $\gamma$1-late stage of HCMV gene expression. By Western blotting, a mouse anti-UL18-GST antiserum detected a 47 kD protein species unique to lysates of infected cells, a size which approximated that proposed for the nonglycosylated form of UL18. This protein appeared by 72-96 hours post-infection in cells infected with the AD169 laboratory strain and by 48 hours in cells infected with a clinical isolate. Failure of peptide N-glycosidase F to reduce its molecular weight indicated that it lacked N-linked carbohydrate residues. Drug-blocking studies indicated that this protein was synthesized during the late stage of viral replication, mirroring transcription of the UL18 gene, while viral early gene expression was responsible for HLA class I downregulation. ^