Modulation of human immunodeficiency virus type 1 protease autoprocessing by charge properties of surface residue 69

Abstract

Mature, fully active human immunodeficiency virus protease (PR) is liberated from the Gag-Pol precursor via regulated autoprocessing. A chimeric protease precursor, glutathione S-transferase-transframe region (TFR)-PR-FLAG, also undergoes N-terminal autocatalytic maturation when it is expressed in Escherichia coli. Mutation of the surface residue H69 to glutamic acid, but not to several neutral or basic amino acids, impedes protease autoprocessing in bacteria and mammalian cells. Only a fraction of mature PR with an H69E mutation (PR(H69E)) folds into active enzymes, and it does so with an apparent Kd (dissociation constant) significantly higher than that of the wild-type protease, corroborating the marked retardation of the in vitro N-terminal autocatalytic processing of TFR-PR(H69E) and suggesting a folding defect in the precursor.

FIG. 1.

Structural organization of Gag and Gag-Pol polyproteins in HIV-1 and tube representation of the three-dimensional structure of the mature protease dimer in complex with the inhibitor DRV (Protein Data Bank accession number 2IEN [20]). Straight arrows indicate the specific sites of cleavage by the viral protease. The TFR, N-terminal to the protease, consists of the transframe octapeptide TFP and 48 amino acids of p6^pol. The nomenclature of HIV-1 proteins is according to Leis at al. (9), as follows: CA, capsid; NC, nucleocapsid; RT, reverse transcriptase; RN, RNase H; and IN, integrase. Residues H69, F99, and I93 (proximal to H69) and DRV are shown as stick and surface representations. The protease precursor constructs used for monitoring the autocatalytic maturation reaction in vitro (TFR-PR) and in E. coli (GST-TFR-PR-FLAG) as well as the proviral construct expressed in 293T cells in vivo (Gag-TFR-PR) are all drawn to scale. The position of the frameshift (FS) that produces Gag-Pol is indicated as an orange dot. Calculated molecular weights of the domains indicated for the precursors are 26,583, 1,010, 5,357, 10,727, and 1,112 for GST, TFP, p6^pol, PR, and FLAG, respectively. The proviral DNA construct contains the region for the expression of Gag and Gag-TFR-PR.

FIG. 2.

Protease autocatalytic maturation in E. coli and transfected 293T cells. (A) E. coli BL21(DE3) cells expressing GST-TFR-PR-FLAG were collected and divided into soluble and insoluble fractions. PR-FLAG (lane 1) and GST-containing proteins (lane 2) associated with the soluble fraction were affinity purified with anti-FLAG and anti-GST matrices (Sigma, St. Louis, MO) by following the manufacturer's instructions. They were then resolved by 13.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Coomassie brilliant blue R-250 staining. (B) Lysates derived from equal volumes of cultured bacteria expressing the indicated mutations were resolved by 15% SDS-PAGE and immunoblotted with mouse anti-FLAG M2 (Sigma) as the primary antibody and IR800 goat anti-mouse as the secondary antibody. M denotes the molecular mass standards in kDa. (All secondary antibodies used in Fig. 2 were obtained from Rockland Immunochemicals, Inc., Gilbertsville, PA). wt, wild type. (C) pNL4-3-derived proviral constructs expressing pseudo-wild-type or mutant PRs were transfected into 293T cells (4). At 48 h posttransfection, VLPs and postnuclear cell lysates were subjected to SDS-PAGE and Western blot analysis as described previously (2, 10). About 10% of cell lysates and 25% of VLPs derived from one well of a six-well plate were analyzed. Virus-specific Gag proteins were detected with human anti-HIV immunoglobulins and mouse anti-HA (Sigma) as primary antibodies and IR800 goat anti-human and IR700 goat anti-mouse as secondary antibodies. The blot was stripped and reprobed for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (clone 6C5; Fisher Scientific, Pittsburgh, PA) as a loading control. (D) Relative protease activity in VLPs was expressed as the ratio of MA to the sum of the band intensities in each lane and normalized relative to the ratio observed for the wild type (set to 100%). The graph presents averages of results from two independent experiments, with standard deviations. (E) The mature and precursor proteases in the VLPs produced by the indicated constructs were detected by polyclonal rabbit anti-PR antibodies and IR800 goat anti-rabbit antibodies.

FIG. 3.

Time course of autocatalytic maturation of protease precursors in vitro and kinetic characterization of mature PR_H69E. Processing reactions for the wild-type and H69E precursor proteins were conducted at final concentrations of 3.1 and 8.8 μM (A and B, respectively) in 50 mM sodium acetate, pH 5, at room temperature. Reactions were terminated as indicated (in min [A] or h [B]) and subjected to SDS-PAGE on 10 to 20% Tris-Tricine premade gels. M and Δ denote the molecular mass standards in kDa and “truncated,” respectively. The four arrows in panel B indicate TFP-p6^pol-PR, p6^pol-PR, PR, and p6^pol, from top to bottom. The intermediate cleavage that occurs within p6^pol to generate Δp6^pol-PR in the wild-type precursor (A) was not observed in the H69E mutant precursor (B). (C) Initial rates of cleavage of substrate IV (0.4 mM) were measured at 28°C as a function of protein concentration (expressed as monomers) in 50 mM sodium acetate buffer, pH 5, containing 250 mM NaCl, and an apparent K_d of 30 nM was obtained by curve fitting an equation for the fraction of dimeric protease as a function of protein concentration (21) to the data. Filled and open symbols represent data from two separate experiments. (D) Kinetic parameters (K_m and k_cat) for hydrolysis of the chromogenic peptide, substrate IV, by 0.11 μM PR_H69E (expressed as dimers) were determined under the same conditions. The inset shows a titration with the active-site-directed inhibitor DRV indicating that only ∼50% of the refolded PR_H69E population possessed a functional active site.