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Human immunodeficiency virus type 2 preintegration complexes: activities in vitro and response to inhibitors
1Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Insights
Researchers developed an assay for human immunodeficiency virus type 2 (HIV-2) preintegration complexes (PICs) to study viral integration. The assay confirmed authentic integration and found no role for Vpx protein, aiding inhibitor screening.
Area of Science:
- Virology
- Molecular Biology
- Drug Discovery
Background:
- Preintegration complexes (PICs) are crucial for retroviral DNA integration.
- Understanding human immunodeficiency virus type 2 (HIV-2) integration is key to developing effective therapies.
- The role of accessory proteins like Vpx in HIV-2 replication remains an area of investigation.
Purpose of the Study:
- To establish a functional assay for HIV-2 PICs.
- To investigate the mechanism of HIV-2 integration.
- To explore the role of Vpx in HIV-2 integration and evaluate PICs for inhibitor screening.
Main Methods:
- Partial purification of HIV-2 PICs.
- In vitro integration assays using viral cDNA and target DNA.
- Analysis of integration products via Southern blotting and junction sequencing.
- Comparison of PICs from wild-type and Vpx-deficient HIV-2 virions.
Main Results:
- HIV-2 PICs were competent for in vitro integration, producing authentic integration products with characteristic 5-bp target sequence duplication.
- The accessory protein Vpx showed no discernible role in the integration process.
- HIV-2 PIC assays were less sensitive to small molecule inhibitors compared to purified integrase assays.
Conclusions:
- A functional assay for HIV-2 PICs was successfully established, enabling detailed mechanistic studies.
- Vpx is not essential for HIV-2 integration.
- Comparing HIV-1 and HIV-2 PIC assays may identify broadly effective antiviral inhibitors.
Abstract:
We have established an assay for the function of preintegration complexes (PICs) of human immunodeficiency virus type 2 (HIV-2) to investigate the integration mechanism and to develop additional methods for screening candidate integration inhibitors. We partially purified HIV-2 PICs and found that they were competent to integrate viral cDNA into target DNA in vitro. Analysis of the structure of integration products on Southern blots revealed forms consistent with those expected for authentic integration products and circular forms containing one and two long terminal repeats. To determine whether in vitro products had the detailed structure expected of integration products formed in vivo, we recovered product molecules and analyzed junctions between viral DNA and target DNA. In the integration junctions of all nine molecules examined, we observed the 5-bp duplication of target sequence characteristic of integration in vivo. We investigated the possible role in integration of Vpx, a protein present in HIV-2 but not HIV-1 and known to be present in viral cores. Although association of Vpx with viral cDNA was detectable, our studies revealed no obvious role of Vpx in integration since the activities of PICs from Vpx- virions were indistinguishable from those of wild type. We have also investigated the use of HIV-2 PICs as tools to screen candidate HIV inhibitors. Assays with HIV-2 PICs, like assays with HIV-1 PICs, were less sensitive to many small molecule inhibitors than were reactions with purified integrase only. Comparing results of assays with PICs from HIV-1 and HIV-2 may be particularly useful, since inhibitors active against both may be more widely useful and less vulnerable to escape mutants.