Human immunodeficiency virus type 2 preintegration complexes: activities in vitro and response to 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.

HIV integrase: a target for drug discovery

“…In that respect, the use of partially purified PICs may be more relevant. The use of PIC-based assays for drug discovery has been discussed previously [120,121]. In contrast with the oligonucleotide assay, in vitro integration assays based on PICs resemble more the in vivo integration process, because PICs are able to perform double-ended integration [13].…”

Conserved amino acids of the human immunodeficiency virus type 2 Vpx nuclear localization signal are critical for nuclear targeting of the viral preintegration complex in non-dividing cells

“…Among those identified, only a few, including barrier to auto integration factor (BAF) and HMG Y(I), have been shown to be functionally relevant (Chen and Engelman, 1998, Harris and Engelman, 2000, Hindmarsh et al, 1999, Li et al, 2000, and Lin and Engelman, 2003. The viral components include the dsDNA, reverse transcriptase, matrix, nucleocapsid, integrase, and Vpr (HIV-1) or Vpx (HIV-2/SIV) proteins (Bukrinsky et al, 1993, Farnet and Haseltine, 1991, Hansen and Bushman, 1997, Karageorgos et al, 1993, and Miller et al, 1997. HIV-2 contains both the vpr and vpx genes, each with discrete functions (Fletcher et al, 1996).…”

Identification of the nuclear localization signal of human immunodeficiency virus type 2 Vpx

“…This task is accomplished through the viral preintegration complex (PIC), a nucleoprotein complex composed of cellular and viral components (reviewed in Fouchier and Malim 1999). The viral components include the dsDNA, reverse transcriptase, matrix, nucleocapsid, integrase, and Vpr (HIV-1) or Vpx (HIV-2/SIV) proteins (Bukrinsky et al 1993b, Farnet and Haseltine 1991, Hansen and Bushman 1997, Heinzinger et al 1994, and Miller et al 1997. The matrix, integrase, Vpr, and Vpx proteins have each been identified as having karyophilic properties (Bukrinsky et al 1993a, Gallay et al 1997, Heinzinger et al 1994, and Pancio et al 2000.…”

“…HIV-2 Vpx is necessary for efficient replication in nondividing cells (Pancio et al 2000). As a component of the HIV-2 PIC, Vpx, similar to HIV-1 Vpr, has been shown to be necessary and sufficient for nuclear translocation of the PIC, but plays no role in integration (Hansen and Bushman 1997). As such, Vpx plays an important role in the early steps of virus replication.…”