Background:  Human lens epithelium-derived growth factor p75 (LEDGF/p75) is a nuclear protein that binds and directs the intracellular trafficking of ectopically expressed HIV integrase, and can stimulate integrase-mediated integration in vitro. Although clearly interacting with HIV integrase both in vivo and in vitro, genetic evidence using siRNA-based strategies for a role for p75 in HIV infection is lacking. However, a region highly homologous to the p75 integrase-binding domain was recently been identified in a second protein, hepatoma-derived growth factor-related protein 2 (HRP-2). HRP-2 binds HIV integrase in in vitro pull-down assays and strongly stimulates integrase-mediated integration in vitro. We have used siRNA to investigate whether HIV replication is affected in cells reduced for both p75 and HRP-2, and whether HIV’s ability to insert its genome preferentially within genes is altered in these cells.

Methods:  Single-round infectivity assays were conducted in HeLa-P4 cells at 48 hours post infection. For integration site cloning, DNA was extracted at 24 hours post infection (MOI of 10), digested with Nla III, ligated to linkers, digested with Bgl II, and then subjected to nested PCR amplification before TOPO TA cloning and sequencing. Cellular chromosomal sequences were mapped within the human genome using the BLAT search program.

Results:  Transient siRNA transfection of HeLa-P4 cells generated a 10-fold knock-down in p75 protein levels. Under these conditions, HIV infection was not affected as measured by single-round infection. Similarly, reducing HRP-2 protein levels, either alone, or in combination with p75, did not adversely affect HIV infection. Similar to previous reports, we found that HIV preferred integrating into genes in control siRNA-treated cells (85.7% integration into AceView-defined genes; 119 events). The gene-targeting frequency of HIV was largely unaltered in cells depleted for both p75 and HRP-2 (78.4% events into AceView-defined genes; 125 events).

Conclusions:  HIV infection appears unaffected under p75/HRP-2 knock-down conditions generated by siRNA transfection.

Keywords: Cofactors; Integrase; Replication