Human cytomegalovirus replication is strictly inhibited by siRNAs targeting UL54, UL97 or UL122/123 gene transcripts

PLoS One. 2014 Jun 2;9(6):e97231. doi: 10.1371/journal.pone.0097231. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Cytomegalovirus / genetics*
  • Cytomegalovirus / physiology*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Gene Expression Regulation, Viral
  • Genes, Viral*
  • Humans
  • Immediate-Early Proteins / genetics
  • Immediate-Early Proteins / metabolism
  • Intracellular Space / metabolism
  • Phosphotransferases (Alcohol Group Acceptor) / genetics
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism
  • Plasmids / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / metabolism*
  • Trans-Activators / genetics
  • Viral Proteins / genetics
  • Viral Proteins / metabolism
  • Virus Replication / genetics*

Substances

  • IE1 protein, cytomegalovirus
  • IE2 protein, Cytomegalovirus
  • Immediate-Early Proteins
  • RNA, Messenger
  • RNA, Small Interfering
  • Trans-Activators
  • UL54 protein, Human herpesvirus 5
  • Viral Proteins
  • Phosphotransferases (Alcohol Group Acceptor)
  • ganciclovir kinase
  • DNA-Directed DNA Polymerase

Grants and funding

This work was supported by grants from the Stillbirth Foundation Australia (http://www.stillbirthfoundation.org.au), the NHMRC (APP1045989), the Deutsche Forschungsgemeinschaft SFB796 (grant C3) and DAAD/Go8 (grant 54390135), support which is gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.