|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥1,800,000 (Direct Cost: ¥1,800,000)
Human immunodeficiency virus (HIV) Gag proteins are assembled underneath the plasma membrane to form the budding virus particles. During or after budding, the particles undergo the process termed maturation in which Gag is cleaved by virion-containing HIV protease to yield the N-terminal matrix (MA), the central capsid (CA), the nucleocapsid (NC), the C-terminal p6 proteins. Concomitant with the processing, doughnut-like HIV particles (the immature form) are converted to particles containing condensed cores (the mature form). As the immature particles are non-infectious, the maturation process are essential for HIV infectivity. However, it is difficult to undestand nature of Gag processing in cell-based experiments as the process of virus particle budding and that of Gag processing are interlinked and neither process is not synchronized. To understand this process, we carried out the in vitro processing of immature HIV Gag virus-like particle (VLP) by exogenously added HIV protease. Fo
llowing delipidization with Triton X-100, sequential processing of immature VLP was carried out in acidic or neutral buffers with different concentrations of salt and confirmed the optimum activity for HIV PR (mildly acidic pH and low salt concentration). Under all conditions tested, the MA/CA junction was cleaved faster than the CA/NC junction, an altered order of processing when compared with authentic processing. When the in vitro- processed VLP was analyzed on sucrose density gradients, most of MA, CA-p15 intermediate, and NC were detected as a highly multimeric form, equivalent to the unprocessed VLP.Reverse transcriptase, a processing product of the Pot region, was also found associated to the highly multimeric complex. In contrast, CA was found as a monomer dissociated from the multimeric CA-p15 following cleavage of the CA/NC junction. Electron microscopy revealed that the in vitro processing was accompanied by conversion of the doughnut-like particles to particles containing outer shells (which may correspond to a multimer of MA) and condensed cores (likely corresponding to a complex of NC and RNA). Chracteristic of most of the cores was the absence of core shells. These results imply that the processing order in VLP may be important for core shell formation but not outer shell or core formation. To examine whether in vitro processing of the immature particles leads to produce infectious particles, we initially explored a method by which the lipid bilayer of VLP was permeabilized without a loss of viral envelope protein.
Immature pseudotype particles was prepared by co-transfection with HIV proviral DNA clone whose PR was inactive and env gene was replaced with the GFP gene and expression plasmid of vesicular stomatitis virus G protein. Following in vitro processing, the processed pseudotype particles were inoculated on 239T or HeLa cells. The level of GFP expression will clarify whether or not the in vitro-processed particles are capable to produce integration-competent replication complexes. Less