Mutations in capsid major homology region affect assembly and membrane affinity of HIV-1 Gag |
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Authors: | Chang Yu-Fen Wang Shiu-Mei Huang Kuo-Jung Wang Chin-Tien |
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Institution: | Institute of Public Health, National Yang-Ming University School of Medicine, Taipei, Taiwan. |
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Abstract: | We introduced mutations into the HIV-1 major homology region (MHR; capsids 153-172) and adjacent C-terminal region to analyze their effects on virus-like particle (VLP) assembly, membrane affinity, and the multimerization of the Gag structural protein. Results indicate that alanine substitutions at K158, F168 or E175 significantly diminished VLP production. All assembly-defective Gag mutants had markedly reduced membrane-binding capacities, but results from a velocity sedimentation analysis suggest that most of the membrane-bound Gag proteins were present, primarily in a higher-order multimerized form. The membrane-binding capacity of the K158A, F168A, and E175A Gag proteins increased sharply upon removal of the MA globular domain. While demonstrating improved multimerization capability, the two MA-deleted versions of F168A and E175A did not show marked improvement in VLP production, presumably due to a defect in association with the raft-like membrane domain. However, K158A bound to detergent-resistant raft-like membrane; this was accompanied by noticeably improved VLP production following MA removal. Our results suggest that the HIV-1 MHR and adjacent downstream region facilitate multimerization and tight Gag packing. Enhanced Gag multimerization may help expose the membrane-binding domain and thus improve Gag membrane binding, thereby promoting Gag multimerization into higher-order assembly products. |
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Keywords: | HIV-1 human immunodeficiency virus type 1 MA matrix CA capsid NC nucleocapsid VLP virus-like particle MHR major homology region GFP green fluorescent protein wt wild-type |
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