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Background:  Active nuclear import of the HIV reverse transcription (RT) complex is essential for infection of nondividing macrophages. Importin 7 (IPO7) has recently been described to play a role in nuclear uptake of the RT complex in vitro, while HIV integrase nuclear import has been proposed to be mediated by association with LEDGFp75. We hypothesized that if IPO7 or LEDGFp75 are integral to nuclear import of the RT complex in vivo, their loss in primary macrophages should result in a nuclear import defect and a decrease in infection efficiency by HIV.

Methods:  We used RNAi technology to knock down IPO7 or LEDGFp75 in human primary macrophages. Following knockdown, cells were infected with VSV-G pseudotyped or wild type enveloped HIV and the formation of viral 2LTR circles (indicating nuclear import) and late viral cDNA was monitored using real time quantitative PCR. A GFP-expressing HIV clone was used to analyze infection efficiency.

Results:  We evaluated infection of macrophages under RNAi conditions which resulted in 80 to 95% IPO7 knockdown. At 20 hours post-infection, no differences in 2LTR circle or late cDNA formation was observed using VSV-G pseudotype or wild type HIV infection. In non-specific siRNA (NS-control) transfected macrophages infected by a GFP-expressing HIV, 5 to 9% were GFP⁺ while 11 to 19% of IPO7 knockdown macrophages were infected. LEDGFp75 RNAi in macrophages resulted in at least 80% knockdown of protein. Macrophages were infected with VSV-G pseudotyped HIV and DNA isolated after 20 hours. Analysis of 2LTR circles showed similar numbers per cell in LEDGFp75 knockdown cells vs NS-control cells. Late cDNA formation was comparable between LEDGF-deficient and control cells.

Conclusions:  Using primary macrophages as biologically relevant targets of HIV and infection, and under a natural infection process, we found no evidence to support IPO7 or LEDGFp75 as mediators of RT complex nuclear import. Neither did loss of IPO7 impair infection efficiency.

Keywords: LEDGF; IPO7; importin