687 HIV Tat Down-regulates Host Nuclear Membrane Shuttling Protein Nucleoporin p62 in Astrocytes at Transcriptional and Translational Levels A. Chauhan*, A. Nath Johns Hopkins Univ, Baltimore, MD
Background: HIV-1 causes a brain infection and leads to a severe brain disorder known as HIV dementia. The exact pathogenetic mechanism is not yet known; however, secondary inflammatory mediators are implicated in neuronal damage. Astrocytes are infected with HIV, but replication is limited to initial viral cycle and then it undergoes dormant phase. HIV Tat and Rev are regulatory proteins, produced in the early phase of HIV infection. It has been shown in the previous studies that Rev function of HIV is impaired in astrocytes. The exact mechanism of Rev block in astrocyte infection is not yet known. However, nucleoporins are involved in mRNA and protein transport from the nucleus and shown to be involved in Rev function in non astrocytic cells. In this study, we investigated the effect of Tat on gene expression in astrocytes at transcriptional and translational levels.
Methods: HIV Tat was cloned downstream of CMV promoter in pCDNA-3 and pEGFP-N1 vectors. HIV LTR was cloned upstream of GFP gene in a CMV promoter deleted pEGFP-N1 vector. Stable Tat expressing SVGA (human astrocytes) cells were generated. LTR-gag-GFP from Quantum lab while Hela LTR-CAT, pNL4-3 and anti-Tat were obtained from NIAD reagent facility. Total RNA was extracted from SVGA-Tat as well as SVGA-neo cells and analyzed in triplicate by Affimatrix gene chip (22,000 human genes) array. Protein analysis in SVGA-Tat and neo cells was also performed using 800 different monoclonal antibodies by powerblot (www.translab.com/shtml) and mean up and down fold protein changes were calculated.
Results: Our initial studies revealed that transfection of molecular HIV pNL4-3 DNA into human astrocytes supported the viral transcription as revealed by Tat staining as well as Rev mediated LTR-gag-GFP flourescence. We did transfection studies with Tat plasmids in primary astrocytes and cell lines, and demonstrated the Tat expression using immunofluorescence and transactivation assays using HIV LTR-GFP reporter. Interestingly, mRNA profile using Affimatrix gene array ( 22,000 human genes) on constitutively Tat expressing SVGA as well as SVGA neo cells, revealed a statistically significant decrease in nucleoporin p62 (p < 0.0001) which was further confirmed in protein power blot.
Conclusions: Tat down-regulated nucleoporin p62 at transcriptional and translational levels in human astrocytes and may have effect on HIV replication in astrocytes.
