Human immunodeficiency virus type 1 protease triggers a myristoyl switch that modulates membrane binding of Pr55(gag) and p17MA

Abstract

The human immunodeficiency virus type 1 (HIV-1) Pr55(gag) gene product directs the assembly of virions at the inner surface of the cell plasma membrane. The specificity of plasma membrane binding by Pr55(gag) is conferred by a combination of an N-terminal myristoyl moiety and a basic residue-rich domain. Although the myristate plus basic domain is also present in the p17MA proteolytic product formed upon Pr55(gag) maturation, the ability of p17MA to bind to membranes is significantly reduced. It was previously reported that the reduced membrane binding of p17MA was due to sequestration of the myristate moiety by a myristoyl switch (W. Zhou and M. D. Resh, J. Virol. 70:8540-8548, 1996). Here we demonstrate directly that treatment of membrane-bound Pr55(gag) in situ with HIV-1 protease generates p17MA, which is then released from the membrane. Pr55(gag) was synthesized in reticulocyte lysates, bound to membranes, and incubated with purified HIV-1 protease. The p17MA product in the membrane-bound and soluble fractions was analyzed following proteolysis. Newly generated p17MA initially was membrane bound but then displayed a slow, time-dependent dissociation resulting in 65% solubilization. Residual p17MA could be extracted from the membranes with either high pH or high salt. Treatment of membranes from transfected COS-1 cells with protease revealed that Pr55(gag) was present within sealed membrane vesicles and that the release of p17MA occurred only when detergent and salt were added. We present a model proposing that the HIV-1 protease is the "trigger" for a myristoyl switch mechanism that modulates the membrane associations of Pr55(gag) and p17MA in virions and membranes.

FIG. 1

Proteolysis of reticulocyte lysate-synthesized Pr55^gag with HIV-1 protease. (A) Pr55^gag was synthesized in reticulocyte lysates as described in Materials and Methods, bound to COS-1 plasma membranes, and incubated with purified HIV-1 protease for 0, 5, or 120 min. The proteolytic products were fractionated by ultracentrifugation into soluble (S) and membrane bound (P), analyzed by SDS gel electrophoresis, and visualized with a PhosphorImager. Note the presence of p17MA in the membrane fraction and that of the p24-p2 intermediate in the soluble fraction at 5 min. After 2 h, 65% of p17MA has shifted to the soluble fraction. t, time. (B) Generation of p17MA and p24CA after treatment of Pr55^gag with HIV-1 protease as a function of time. The time dependence of p24CA and p17MA generation follows the same kinetics, since they arise from a common first cleavage intermediate of Pr55^gag, p39-p41. (C) Time course of p17MA and p24CA release from the membrane. The membrane dissociation of p17MA is slow in comparison to the release of p24CA.

FIG. 2

Myristoylation of Pr55^gag and p17MA. Synthesis of pp60^v-src, Pr55^gag, and p17MA in reticulocyte lysates was carried out in the presence of ³⁵S-cysteine to normalize for protein levels (left panel) and with ³H-myristic acid to determine relative myristoylation levels (right panel). Lane 1, unprogrammed lysate; lane 2, v-Src; lane 3, Pr55^gag; lane 4, p17MA. A nonspecific myristoylated band present in the reticulocyte lysates was evident in all lanes (arrowhead) and migrated just below Src and Pr55^gag.

FIG. 3

Gag69-DHFR is not released from the membrane by HIV protease. Pr55gag and Gag69-DHFR, a p17MA mutant containing the first 69 amino acids of Gag fused to dihydrofolate reductase (DHFR), were synthesized in reticulocyte lysates, bound to membranes, and treated (+ PR) or not treated (C) with HIV-1 protease for 2 h. Membranes were reisolated, and membrane-bound (P) and soluble (S) fractions were analyzed by SDS-PAGE and autoradiography. Reticulocyte lysate-synthesized ³⁵S-p17MA was used as a molecular weight marker (p17, left lane).

FIG. 4

Extraction of p17MA from membranes. (A) Pr55^gag was synthesized in reticulocyte lysates, incubated with COS-1 cell membranes, and treated or not treated (C lanes) with HIV-1 protease for 10 min to generate membrane-bound p17MA. The products were fractionated by ultracentrifugation into soluble (S) and membrane bound. The membrane fraction was subsequently extracted with 0.1 M Na₂CO₃ (pH 9) (CO₃⁼ lanes) or with buffer containing 1 M NaCl (1 M NaCl lanes). The extraction mixtures were then refractionated into soluble (S′) and membrane bound (P′), and the products were resolved by SDS-PAGE and visualized by PhosphorImager analysis. Note that under these conditions, Pr55^gag was not completely digested and could be detected in the membrane fraction. A marker for ³⁵S-labeled p17MA is included in the last lane. (B) Membranes containing Pr55^gag or Gag69-DHFR were treated with HIV-1 protease and either carbonate or salt as described in panel A. Note that Gag69-DHFR did not undergo proteolysis and was not released from the membrane fraction (P′).

FIG. 5

Proteolytic cleavage of Pr55^gag in transfected COS-1 cell membranes. COS-1 cells were transfected with pHXB2gtpΔBal-D25S, and the plasma membrane-enriched fraction was isolated as described in Materials and Methods. Membranes were incubated (+ PR) or not incubated (− PR) with HIV-1 protease in the presence of 0.1% Triton X-100. The reactions were performed in the absence of salt addition or with 0.5 M NaCl added during or immediately after (last two lanes) proteolysis. The products were fractionated into soluble (S) and membrane bound (P) and resolved by SDS gel electrophoresis. p17MA was visualized by Western blotting (WB) with anti-p17MA monoclonal antibody (bottom panel). The blot was stripped and reprobed with anti-p24CA antibody to detect Pr55^gag and p24CA (top panel).