Binding of human immunodeficiency virus type 1 Gag to membrane: role of the matrix amino terminus

Abstract

Binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein precursor, Pr55(Gag), to membrane is an indispensable step in virus assembly. Previously, we reported that a matrix (MA) residue 6 substitution (6VR) imposed a virus assembly defect similar to that observed with myristylation-defective mutants, suggesting that the 6VR change impaired membrane binding. Intriguingly, the 6VR mutation had no effect on Gag myristylation. The defective phenotype imposed by 6VR was reversed by changes at other positions in MA, including residue 97. In this study, we use several biochemical methods to demonstrate that the residue 6 mutation, as well as additional substitutions in MA amino acids 7 and 8, reduce membrane binding without affecting N-terminal myristylation. This effect is observed in the context of Pr55(Gag), a truncated Gag containing only MA and CA, and in MA itself. The membrane binding defect imposed by the 6VR mutation is reversed by second-site changes in MA residues 20 and 97, both of which, when present alone, increase membrane binding to levels greater than those for the wild type. Both reduced and enhanced membrane binding imposed by the MA substitutions depend upon the presence of the N-terminal myristate. The results support the myristyl switch model recently proposed for the regulation of Gag membrane binding, according to which membrane binding is determined by the degree of exposure or sequestration of the N-terminal myristate moiety. Alternatively, insertion of the myristate into the lipid bilayer might be a prerequisite event for the function of other distinct MA-encoded membrane binding domains.

FIG. 1

HIV-1 MA mutations analyzed for their effect on Gag membrane binding. At the top is indicated the linear organization of the Gag precursor Pr55^Gag showing MA, CA, NC, and p6 domains. MA mutations were analyzed in the contexts of full-length Pr55^Gag, a truncated Gag containing MA-CA (p41stop), and MA alone (MAstop). The locations of the five major α-helices in MA (H1 to H5) are indicated; the N terminus of MA and the sequences surrounding residues 20 and 97 are enlarged. The positions of the mutations are indicated. Dashes denote sequence identity with wild type (w.t.). The myristylation consensus sequence is underlined.

FIG. 2

Effect of N-terminal MA mutations on Gag processing and virus production. HeLa cells transfected with wild-type (WT) pNL4-3 or derivatives containing the indicated MA mutations were metabolically labeled with [³⁵S]Cys. Virions were pelleted in an ultracentrifuge. Cell (left panel)- and virion (right panel)-associated material was immunoprecipitated with sera from patients with AIDS (Materials and Methods). The relative levels of virion-associated p24 (normalized for cell-associated gp120) are indicated under each lane of the virion panel. The positions of the Pr160^Gag-Pol (Pr160) and Pr55^Gag (Pr55) precursors, the Env precursor gp160, the mature surface Env glycoprotein gp120, the Gag processing intermediates p41 and p39, and p24 (CA) and p17 (MA) are shown.

FIG. 3

Effect of N-terminal MA mutations on Gag myristylation. HeLa cells were transfected with pNL4-3/PR⁻ or derivatives containing the indicated MA mutations. Cells were metabolically labeled with either [³⁵S]Met (top panel) or [³H]Myr (lower panel). Cell lysates were prepared and immunoprecipitated with sera from patients with AIDS. The Pr55^Gag band is shown.

FIG. 4

Cell fractionation of MA-mutant Pr55^Gag. HeLa cells were transfected with pNL4-3/PR⁻ or derivatives containing the indicated MA mutations. Postnuclear supernatants were treated with no salt (top panel) or high salt (1 M NaCl; lower panel) and fractionated (Materials and Methods). Pr55^Gag and the transmembrane Env glycoprotein gp41 were detected by Western blotting. The percentage of Pr55^Gag in the pellet fraction, normalized to the wild-type (WT) value, is indicated under each lane. Approximately 70% of wild-type Pr55^Gag was detected in the pellet under either no- or high-salt conditions.

FIG. 5

Analysis of MA mutants by membrane flotation centrifugation. HeLa cells were transfected with pNL4-3/PR⁻ or derivatives containing the indicated MA mutations. Postnuclear supernatants were prepared and subjected to membrane flotation centrifugation (Materials and Methods), during which membrane-bound material floats to the interface between 10 and 65% sucrose (fractions 3 and 4). The transmembrane Env glycoprotein gp41, which is found almost exclusively in fractions 3 and 4, serves as a control membrane-bound protein. Pr55^Gag and gp41 were detected by Western blotting.

FIG. 6

Membrane flotation centrifugation with p41 (MA-CA) and p17 (MA). HeLa cells were transfected with pNL4-3/p41stop (which expresses a truncated Gag protein lacking sequences C-terminal to CA; panel A) or pNL4-3/MAstop (which expresses MA; panel B) or derivatives containing the 1GA or 6VR MA mutations. Postnuclear supernatants were prepared and subjected to membrane flotation centrifugation; p41 (MA-CA) was detected by Western blotting, and p17 (MA) was detected by immunoprecipitation (Materials and Methods).

FIG. 7

Opposing effect of MA mutations on membrane binding. HeLa cells were transfected with pNL4-3/PR⁻ or derivatives containing the indicated MA mutations. Postnuclear supernatants were prepared and subjected to membrane flotation centrifugation (Materials and Methods). The amount of Gag in each fraction was determined by Western blotting.

FIG. 8

Membrane flotation centrifugation of 1GA/6VR and 1GA/20LK double mutants. HeLa cells were transfected with pNL4-3/PR⁻ or derivatives containing the indicated MA mutations. Postnuclear supernatants were prepared and subjected to membrane flotation centrifugation (Materials and Methods). The amount of Gag in each fraction was determined by Western blotting.