| Summary: | 碩士 === 國立陽明大學 === 公共衛生研究所 === 94 === The human immunodeficiency virus type 1 (HIV-1) gag encodes a polyprotein precursor Pr55, which can self-assemble into virus-like particles (VLPs). During or after virus budding, the Pr55gag is cleaved into four major proteins, namely matrix (MA), capsid (CA), nucleocapsid (NC), and p6. The N-terminus of Pr55gag contains a myristate (myr) moiety that is required for both membrane association of Gag and virus budding. It has been proposed that the myr signal is masked at low concentration of Gag proteins; however, a conformation change occurs during Gag multimerization, leading to exposure of the myr signal. This results in enhanced membrane binding of Gag and hence promotes VLP assembly. Domains involved in VLP assembly reside mainly in the conserved major homology region (MHR) and the adjacent C-terminal region of CA. Some point mutations in MHR have been shown to markedly diminish VLP production. It remains to be determined whether the MHR has any contribution to membrane association and whether the assembly defect induced by MHR mutation correlates with reduced membrane binding capacity of Gag. To address this issue, we constructed a series of substitution mutations in the MHR and the adjacent C-terminal CA. Analyses indicate that membrane association of most of the mutants has been affected to a certain degree. All of the assembly-defective mutants were impaired in Gag multimerization and showed significantly reduced membrane binding capacity. Interestingly, membrane binding capacity of the assembly-defective mutants K158A, F168A, and E175A could be markedly improved after removal most of MA codones. This also leads to a significant improvement in oligomerization status. Velocity sedimentation analysis indicates that the membrane-binding defective myr-Gag is markedly defective in multimerization. In contrast, all of the membrane-bound Gag mutants display a multimerization pattern similar to that of wild-type. Our results suggested that both MHR and the adjacent C-terminal CA could help the binding of Gag to cell membrane, possibly via facilitating Gag multimerization through Gag-Gag interaction.
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