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10:34
Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes
Published on: February 22, 2017
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GCN2 phosphorylates HIV-1 integrase and decreases HIV-1 replication by limiting viral integration
A Jaspart1,2, C Calmels1,2, O Cosnefroy1,2
1Université de Bordeaux, CNRS UMR 5234, MFP, 146 rue Léo Saignat, 33076, Bordeaux cedex, France.
Scientific Reports
|May 25, 2017
Summary
The GCN2 kinase phosphorylates HIV-1 integrase at S255, impacting viral replication. This interaction suggests GCN2 acts as a cellular guardian against foreign DNA integration.
Area of Science:
- Molecular Biology
- Virology
- Cellular Stress Response
Background:
- GCN2 (general control nonderepressible 2) is a kinase crucial for cellular stress response, particularly amino acid starvation.
- Previous research established an interaction between GCN2 and HIV-1 integrase, with GCN2 activation during HIV-1 infection.
Purpose of the Study:
- To identify HIV-1 integrase as a substrate of GCN2.
- To elucidate the mechanism of GCN2-mediated phosphorylation of HIV-1 integrase.
- To investigate the role of this interaction in viral replication and genome stability.
Main Methods:
- In vitro kinase assays to identify and characterize GCN2 phosphorylation sites on HIV-1 integrase.
- Site-directed mutagenesis to investigate the functional significance of S255 phosphorylation.
- Viral replication assays in GCN2-knockout cells and with mutated viral integrases.
- Analysis of viral DNA integration rates.
Main Results:
- HIV-1 integrase was identified as a novel in vitro substrate for GCN2, with S255 in the C-terminal domain being a major phosphorylation site.
- GCN2 targeting of S255 requires the integrase active site.
- GCN2 also phosphorylates integrases from other retroviruses like MLV and ASV.
- Mutations at integrase S255 in HIV-1 impaired viral replication, while preventing phosphorylation increased infectivity and viral DNA integration.
- MLV infectivity was higher in GCN2-knockout cells, indicating a conserved regulatory mechanism.
Conclusions:
- GCN2 directly regulates HIV-1 integrase activity through phosphorylation at S255.
- This GCN2-mediated phosphorylation controls viral replication and DNA integration.
- The findings suggest GCN2 plays a conserved role in maintaining genome stability by acting as a cellular defense against foreign DNA integration.
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