High-Throughput Small Interfering RNA Screening Identifies Phosphatidylinositol 3-Kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions

J Virol. 2016 Aug 26;90(18):8360-71. doi: 10.1128/JVI.01134-16. Print 2016 Sep 15.


High-throughput small interfering RNA (siRNA) screening is a useful methodology to identify cellular factors required for virus replication. Here we utilized a high-throughput siRNA screen based on detection of a viral antigen by microscopy to interrogate cellular protein kinases and phosphatases for their importance during human cytomegalovirus (HCMV) replication and identified the class II phosphatidylinositol 3-kinase class II alpha (PI3K-C2A) as being involved in HCMV replication. Confirming this observation, infected cells treated with either pooled or individual siRNAs targeting PI3K-C2A mRNA produced approximately 10-fold less infectious virus than the controls. Western blotting and quantitative PCR analysis of infected cells treated with siRNAs indicated that depletion of PI3K-C2A slightly reduced the accumulation of late but not immediate early or early viral antigens and had no appreciable effect on viral DNA synthesis. Analysis of siRNA-treated cells by electron microscopy and Western blotting indicated that PI3K-C2A was not required for the production of viral capsids but did lead to increased numbers of enveloped capsids in the cytoplasm that had undergone secondary envelopment and a reduction in the amount of viral particles exiting the cell. Therefore, PI3K-C2A is a factor important for HCMV replication and has a role in the production of HCMV virions.

Importance: There is limited information about the cellular factors required for human cytomegalovirus (HCMV) replication. Therefore, to identify proteins involved in HCMV replication, we developed a methodology to conduct a high-throughput siRNA screen of HCMV-infected cells. From our screening data, we focused our studies on the top hit from our screen, the lipid kinase phosphatidylinositol 3-kinase class II alpha (PI3K-C2A), as its role in HCMV replication was unknown. Interestingly, we found that PI3K-C2A is important for the production of HCMV virions and is involved in virion production after secondary envelopment of viral capsids, the encapsidation of HCMV capsids by a lipid bilayer that occurs before virions exit the cell.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Cells, Cultured
  • Cytomegalovirus / physiology*
  • Fibroblasts / virology
  • Genetic Testing / methods
  • High-Throughput Screening Assays
  • Host-Pathogen Interactions*
  • Humans
  • Microscopy, Electron
  • Phosphatidylinositol 3-Kinases / genetics
  • Phosphatidylinositol 3-Kinases / metabolism*
  • RNA, Small Interfering / genetics
  • Real-Time Polymerase Chain Reaction
  • Virus Replication*


  • RNA, Small Interfering
  • Phosphatidylinositol 3-Kinases
  • PIK3C2A protein, human