Effect of linker insertion mutations in the human immunodeficiency virus type 1 gag gene on activation of viral protease expressed in bacteria.
We have expressed the human immunodeficiency virus type 1 (HIV-1) protease (PR) in bacteria as a Gag-PR polyprotein (J. Luban and S.P. Goff, J. Virol. 65:3203-3212, 1991). The protein displays enzymatic activity, cleaving the Gag polyprotein precursor Pr55gag to the expected products. The PR enzyme is only active as a dimer, and we hypothesized that PR activation might be used as an indicator of polyprotein multimerization. We constructed 25 linker insertion mutations throughout gag and assessed the PR activity of mutant Gag-PR polyproteins by the appearance of Gag cleavage products in bacterial lysates. All mutant constructs produced stable protein in bacteria. PR activity of the majority of the Gag-PR mutants was indistinguishable from that of the wild type. Six mutants, one with an insertion in the matrix (MA), four with insertions in the capsid (CA), and one with insertions in the nucleocapsid (NC), globally disrupted polyprotein processing. When PR was provided in trans on a separate plasmid, the Gag proteins were cleaved with wild-type efficiency. These results suggest that the gag mutations identified as disruptive of polyprotein processing did not conceal the scissile bonds of the polyprotein. Rather, the mutations prevented PR activation in the context of a Gag-PR polyprotein, perhaps by preventing polyprotein dimerization.
Luban
J
J
Department of Medicine, College of Physicians and Surgeons, Columbia University, New York 10032.
Lee
C
C
Goff
S P
SP
D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D015683:Gene Products, gag / Q000235:genetics*; D015750:Genes, gag / Q000235:genetics*; D016333:HIV Protease / Q000096:biosynthesis* / Q000235:genetics*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011498:Protein Precursors / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*
D015683:Gene Products, gag; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D016333:HIV Protease