title: Placement of leucine zipper motifs at the carboxyl terminus of HIV-1 protease significantly reduces virion production.
abstract: Natural HIV-1 protease (PR) is homodimeric. Some researchers believe that interactions between HIV-1 Gag-Pol molecules trigger the activation of embedded PR (which mediates Gag and Gag-Pol cleavage), and that Gag-Pol assembly domains outside of PR may contribute to PR activation by influencing PR dimer interaction in a Gag-Pol context. To determine if the enhancement of PR dimer interaction facilitates PR activation, we placed single or tandem repeat leucine zippers (LZ) at the PR C-terminus, and looked for a correlation between enhanced Gag processing efficiency and increased Gag-PR-LZ multimerization capacity. We found significant reductions in virus-like particles (VLPs) produced by HIV-1 mutants, with LZ fused to the end of PR as a result of enhanced Gag cleavage efficiency. Since VLP production can be restored to wt levels following PR activity inhibition, this assembly defect is considered PR activity-dependent. We also found a correlation between the LZ enhancement effect on Gag cleavage and enhanced Gag-PR multimerization. The results suggest that PR dimer interactions facilitated by forced Gag-PR multimerization lead to premature Gag cleavage, likely a result of premature PR activation. Our conclusion is that placement of a heterologous dimerization domain downstream of PR enhances PR-mediated Gag cleavage efficiency, implying that structural conformation, rather than the primary sequence outside of PR, is a major determinant of HIV-1 PR activation.
authors.affiliation: Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.
authors.forename: Yen-Yu
authors.initials: YY
authors.lastname: Pan
authors.forename: Shiu-Mei
authors.initials: SM
authors.lastname: Wang
authors.forename: Kuo-Jung
authors.initials: KJ
authors.lastname: Huang
authors.forename: Chien-Cheng
authors.initials: CC
authors.lastname: Chiang
authors.forename: Chin-Tien
authors.initials: CT
authors.lastname: Wang
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D015683:Gene Products, gag / Q000737:chemistry; D005821:Genetic Techniques; D057809:HEK293 Cells; D016333:HIV Protease / Q000737:chemistry*; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D016350:Leucine Zippers; D008854:Microscopy, Electron / Q000379:methods; D008954:Models, Biological; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D014162:Transfection
chemical_list: D015683:Gene Products, gag; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1