title: Human cytomegalovirus replication is strictly inhibited by siRNAs targeting UL54, UL97 or UL122/123 gene transcripts.
abstract: Human cytomegalovirus (HCMV) causes severe sequelae in immunocompromised hosts. Current antiviral therapies have serious adverse effects, with treatment in many clinical settings problematic, making new therapeutic approaches necessary. We examined the in vitro efficacy of small interfering RNAs (siRNAs) targeting the HCMV gene transcripts UL54 (DNA polymerase), UL97 (protein kinase) and UL122/123 (immediate-early proteins) as inhibitors of viral protein expression and virus replication in cell cultures. Two siRNAs for each HCMV target (designated A and B) were assessed for inhibition efficacy using western blot and standard plaque assays. Continuous human embryonic kidney 293T cells were treated with HCMV or non-specific scrambled (siSc) siRNA followed by transfection with plasmids expressing the target transcripts. Human MRC-5 fibroblasts were HCMV-siRNA or siSc treated, infected with HCMV strain AD169 (1 pfu/cell) and HCMV immediate-early (IE1p72 and IE2p86), early (pp65), early-late (pUL97) and true late (MCP) protein and virus progeny production measured during a single round of replication. Concordant results showed siUL54B, siUL97A and siUL122B displayed the most potent inhibitory effects with a reduction of 92.7%, 99.6% and 93.7% in plasmid protein expression, 65.9%, 58.1% and 64.8% in total HCMV protein expression and 97.2%, 96.2% and 94.3% (p<0.0001) in viral progeny production respectively. Analysing the siRNA inhibitory effects during multiple rounds of HCMV replication at a multiplicity of infection of 0.001 pfu/cell, siUL54B, siUL97A and siUL122B treatment resulted in a reduction of 80.0%, 59.6% and 84.5% in total HCMV protein expression, 52.9%, 49.2% and 58.3% in number of cells infected and 98.5%, 91.4% and 99.1% (p<0.0001) in viral progeny production at 7 dpi respectively. These results suggest potential in vivo siRNA therapies targeting the HCMV gene transcripts UL54, UL97 and UL122/123 would be highly effective, however, the antiviral efficacy of siRNAs targeting UL97 may be more highly dependent on viral load and methods of administration.
authors.affiliation: Virology Division, SEALS Microbiology, Prince of Wales Hospital, Sydney, Australia; School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia.
authors.forename: Stuart T
authors.initials: ST
authors.lastname: Hamilton
authors.affiliation: Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, Erlangen, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Milbradt
authors.affiliation: Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, Erlangen, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Marschall
authors.affiliation: Virology Division, SEALS Microbiology, Prince of Wales Hospital, Sydney, Australia; School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia; School of Medical Sciences, University of New South Wales, Sydney, Australia.
authors.forename: William D
authors.initials: WD
authors.lastname: Rawlinson
mesh_terms: D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics* / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics*
chemical_list: C088645:IE1 protein, cytomegalovirus; C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D015534:Trans-Activators; C109367:UL54 protein, Human herpesvirus 5; D014764:Viral Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase; D004259:DNA-Directed DNA Polymerase