CYTOMEGALOVIRUS-MEDIATED INDUCTION OF ANTISENSE MESSENGER-RNA EXPRESSION TO UL44 INHIBITS VIRUS-REPLICATION IN AN ASTROCYTOMA CELL FINE - IDENTIFICATION OF AN ESSENTIAL GENE

We have used an antisense RNA approach in the analysis of gene functio n in human cytomegalovirus (HCMV). An astrocytoma cell line (U373-MG) that is permissive for virus replication was permanently transfected w ith a construct bearing sequence from HCMV UL44 (coding for the major late DNA-binding protein, ppUL44, also known as pp52 or ICP36) in an a ntisense orientation and under the control of the immediate-early enha ncer-promoter element. Upon HCMV infection at a high multiplicity, we found a marked reduction in UL44 protein products (the ICP36 family of proteins) in established cell transfectants and a strong inhibition o f virus yield in infected-cell supernatants at two weeks postinfection , while herpes simplex virus replication was not affected. In infected cells, viral DNA replication was strongly inhibited. While gene produ cts such as pUS22 and pUL32 were also inhibited, pUL123 and pUL82 accu mulated in the infected cells over time. Our data suggest an essential role for the UL44 family of proteins in HCMV replication and represen t a model of virus inhibition by virus-induced antisense RNA synthesis in genetically modified cells.