Development of an in-vitro Nuclear Import Assay Using Intact HIV Cores
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Nuclear import of the viral genome is essential for productive HIV infection. Uncoating and nuclear import of the viral genome is a poorly understood process. The purpose of this study is to determine whether HIV viral cores can directly bind cell nuclei and achieve nuclear import in permeabilized cells. HIV-1 was produced by transient transfection of 293T cells with the HIV-1 molecular clone, pNLX. Supernatants were collected every 24 h for a total of 72 h. Virions were concentrated by ultracentrifugation through a 20% (w/v) sucrose cushion and cores were isolated from virions using a modified equilibrium density gradient separation as follows: 20-70% sucrose gradients were overlayed with a thin layer of 15% sucrose containing TritionX-100 detergent, followed by a layer of 7.5% sucrose. Concentrated virus was added to the top of the tube and ultracentrifuged for 16 h at 4 C. Experiments included varying concentrations of detergent and multiple buffer solutions. The gradients were fractionated by positive displacement and the sedimentation of virions and cores determined by Western Blot for HIV Capsid. The sedimentation of viral RNA in fractions was determined by isolating RNA and measuring HIV-1 RNA by real-time RT-PCR. HIV-1 cores were labeled by co-transfecting pNLX with a green fluorescent fusion protein (GFP) – HIV-1 Vpr protein construct. Addition of 0.2% detergent resulted in the removal of viral envelope and matrix proteins and a concentrated band of intact viral cores at a higher density than intact virions. Detergent levels < 0.1% resulted in failure to effectively remove viral membrane. Preliminary experiments showed that a more physiologically relevant buffer that contained Mg2+ resulted in a loss of core stability compared to a buffer lacking divalent cations and containing EDTA. GFP-tagging of cores was demonstrated by the detection of GFP-Vpr fusion protein in core-containing fractions as well as the detection of fluorescent cores by epifluorescence microscopy. GFP-labeled HIV-1 cores were successfully isolated. The virions are highly unstable and have specific sensitivity to minute concentrations of detergent and divalent cations. Optimization of detergent concentration and buffer solution is required prior to nuclear binding and import experiments. Additional studies of detergent effects using decreasing concentrations and alternate formulations are planned. Investigation of the impacts of EDTA treatment on the stability of cores will also be performed.