title: High-Throughput Small Interfering RNA Screening Identifies Phosphatidylinositol 3-Kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions.
abstract: 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.
  
  
  
  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.
authors.affiliation: Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: William S
authors.initials: WS
authors.lastname: Polachek
authors.affiliation: Institute of Infection & Immunity, St. George's, University of London, London, United Kingdom.
authors.forename: Hanan F
authors.initials: HF
authors.lastname: Moshrif
authors.affiliation: Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Franti
authors.affiliation: Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Donald M
authors.initials: DM
authors.lastname: Coen
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.
authors.forename: Vattipally B
authors.initials: VB
authors.lastname: Sreenu
authors.affiliation: Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA Institute of Infection & Immunity, St. George's, University of London, London, United Kingdom bstrang@sgul.ac.uk.
authors.forename: Blair L
authors.initials: BL
authors.lastname: Strang
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000502:physiology*; D005347:Fibroblasts / Q000821:virology; D005820:Genetic Testing / Q000379:methods; D057166:High-Throughput Screening Assays; D054884:Host-Pathogen Interactions; D006801:Humans; D008854:Microscopy, Electron; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014779:Virus Replication
chemical_list: D034741:RNA, Small Interfering; D019869:Phosphatidylinositol 3-Kinases; C546621:PIK3C2A protein, human