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

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

Author information

1
Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
2
Institute of Infection & Immunity, St. George's, University of London, London, United Kingdom.
3
Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut, USA.
4
MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.
5
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.

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.

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.

PMID:
27412598
PMCID:
PMC5008103
[Available on 2017-02-26]
DOI:
10.1128/JVI.01134-16
[Indexed for MEDLINE]
Free PMC Article
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