title: Cytomegalovirus-mediated induction of antisense mRNA expression to UL44 inhibits virus replication in an astrocytoma cell line: identification of an essential gene.
abstract: We have used an antisense RNA approach in the analysis of gene function in human cytomegalovirus (HCMV). An astrocytoma cell line (U373-MG) that is permissive for virus replication was permanently transfected with a construct bearing sequence from HCMV UL44 (coding for the major late DNA-binding protein, ppUL44, also known as pp52 or ICP36) in an antisense orientation and under the control of the immediate-early enhancer-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 of 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 products such as pUS22 and pUL32 were also inhibited, pUL123 and pUL82 accumulated in the infected cells over time. Our data suggest an essential role for the UL44 family of proteins in HCMV replication and represent a model of virus inhibition by virus-induced antisense RNA synthesis in genetically modified cells.
authors.affiliation: Istituto di Microbiologia, Facoltà di Medicina e Chirurgia, Policlinico S. Orsola, Bologna, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Ripalti
authors.forename: M C
authors.initials: MC
authors.lastname: Boccuni
authors.forename: F
authors.initials: F
authors.lastname: Campanini
authors.forename: M P
authors.initials: MP
authors.lastname: Landini
mesh_terms: D000818:Animals; D001254:Astrocytoma; D001483:Base Sequence; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003587:Cytomegalovirus / Q000235:genetics / Q000502:physiology*; D003588:Cytopathogenic Effect, Viral; D004279:DNA, Viral / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D018259:Herpesvirus 1, Human / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D016372:RNA, Antisense / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D014407:Tumor Cells, Cultured; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics*; D014779:Virus Replication / Q000502:physiology*
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; C072217:ICP36 protein, Cytomegalovirus; D016372:RNA, Antisense; D012333:RNA, Messenger; D014764:Viral Proteins