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Systematic microRNA analysis identifies ATP6V0C as an essential host factor for human cytomegalovirus replication

Jon Pavelin1, Natalie Reynolds1, Stephen Chiweshe1

  • 1Division of Infection and Immunity, The Roslin Institute, University of Edinburgh, Easter Bush, Midlothian, United Kingdom.

Plos Pathogens
|January 4, 2014
PubMed

Insights

Researchers identified over 200 human cytomegalovirus (HCMV) microRNA targets crucial for virus replication. Knocking down ATP6V0C, a key target, nearly eliminated infectious virus production, highlighting essential host-virus interactions.

Area of Science:

  • Virology
  • Molecular Biology
  • Genetics

Background:

  • Viral microRNAs (miRNAs) regulate host gene expression, but identifying biologically relevant targets remains challenging.
  • Human cytomegalovirus (HCMV) encodes its own miRNAs that influence viral pathogenesis.
  • Previous methods identified numerous putative viral miRNA targets, necessitating validation of their functional significance.

Purpose of the Study:

  • To identify biologically relevant targets of HCMV-encoded miRNAs.
  • To investigate the role of these targets in HCMV replication and pathogenesis.
  • To validate specific HCMV miRNA-target interactions using functional assays.

Main Methods:

  • Combined RISC immunoprecipitation (RISC-IP) and focused siRNA screening.
  • Infection of fibroblast cells with laboratory and clinical HCMV strains.
  • Analysis of miRNA knockout viruses to resolve specific miRNA-target interactions.
  • Validation using Western blot, luciferase assays, and siRNA knockdown.

Main Results:

  • Identified over 200 putative targets of HCMV miRNAs in infected fibroblasts.
  • Resolved specific interactions between HCMV miRNAs and top candidate target transcripts.
  • Demonstrated that siRNA knockdown of miRNA target genes significantly impacts HCMV replication.
  • Observed a near-complete loss of infectious virus production upon knockdown of ATP6V0C, a crucial factor for endosomal acidification.

Conclusions:

  • HCMV miRNAs target essential cellular factors required for efficient virus replication.
  • The identified targets, particularly ATP6V0C, play critical roles in the HCMV life cycle.
  • Combined miRNA target identification and siRNA screening is an effective strategy for discovering novel host-virus interactions.

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