Biochemical evidence of a role for matrix trimerization in HIV-1 envelope glycoprotein incorporation

Abstract

The matrix (MA) domain of HIV Gag has important functions in directing the trafficking of Gag to sites of assembly and mediating the incorporation of the envelope glycoprotein (Env) into assembling particles. HIV-1 MA has been shown to form trimers in vitro; however, neither the presence nor the role of MA trimers has been documented in HIV-1 virions. We developed a cross-linking strategy to reveal MA trimers in virions of replication-competent HIV-1. By mutagenesis of trimer interface residues, we demonstrated a correlation between loss of MA trimerization and loss of Env incorporation. Additionally, we found that truncating the long cytoplasmic tail of Env restores incorporation of Env into MA trimer-defective particles, thus rescuing infectivity. We therefore propose a model whereby MA trimerization is required to form a lattice capable of accommodating the long cytoplasmic tail of HIV-1 Env; in the absence of MA trimerization, Env is sterically excluded from the assembling particle. These findings establish MA trimerization as an obligatory step in the assembly of infectious HIV-1 virions. As such, the MA trimer interface may represent a novel drug target for the development of antiretrovirals.

Keywords: HIV; envelope; matrix; retrovirus; trimerization.

Fig. 1.

Visualization of trimeric MA in HIV-1 particles. (A) Two residues near the trimer interface, Gln62 and Ser66, were mutated to lysines to permit cross-linking with glutaraldehyde. 62QK, dark blue; 66SK, light blue. (B) Jurkat cells were transfected with the pNL4-3 molecular clone or a mutant bearing the double-lysine substitution at positions 62 and 66. At 2-d intervals, the cells were split and samples of media were assayed for reverse transcriptase (RT) activity. (C) HeLa cells were transfected with the pNL4-3 molecular clone or a mutant bearing the double-lysine substitution. After 48 h, supernatants were filtered and virions were pelleted by ultracentrifugation at 76,000 × g. Virions were resuspended in PBS and treated with glutaraldehyde. Cross-linking was stopped by the addition of Tris, and then samples were boiled in Laemmli buffer and analyzed by SDS/PAGE and Western blotting for MA. Positions of monomeric MA and MA dimers and trimers are indicated. Positions of molecular mass markers are shown (Left). The asterisk indicates the position of a band presumed to be p55Gag. (D) 293T cells were transfected with the pNL4-3 molecular clone or a mutant bearing the double-lysine substitution and a plasmid expressing vesicular stomatitis virus G glycoprotein (VSV-G). After 48 h, supernatants were filtered and infectious particles were titered on TZM-bl cells. These supernatants were used to infect Jurkat and MT4 cells. At 48 h postinfection, supernatants were filtered and virions were pelleted by ultracentrifugation. Virions were resuspended in PBS and treated with glutaraldehyde. Cross-linking was stopped by the addition of Tris, and then samples were boiled in Laemmli buffer and analyzed by SDS/PAGE and Western blotting for MA. Positions of monomeric MA and MA dimers and trimers are indicated. The asterisk indicates the position of a band presumed to be p55Gag. (E) Mature particles were produced as described in C; immature particles were generated using clones with an inactive PR. Before treatment with glutaraldehyde, the resuspended particles were treated with PBS (control), Triton X-100 (Tx100), or SDS. Detergents were prepared as 10× solutions in water before use. Cross-linking and analysis were then performed as described above. Conditions under which cross-linking is possible are indicated by a red box.

Fig. S1.

Trimer cross-linking requires two lysine residues. HeLa cells were transfected with the HIV-1 mutants indicated. After 48 h, supernatants were filtered and virions were pelleted by ultracentrifugation. Virions were resuspended in PBS and treated with glutaraldehyde. Cross-linking was stopped by the addition of Tris, and then samples were boiled in Laemmli buffer and analyzed by SDS/PAGE and Western blotting (WB) for MA and Gag. At 0.01% glutaraldehyde, nonspecific cross-linking is apparent for both MA and CA.

Fig. 2.

Mutation of Thr69 can inhibit MA trimerization and Env incorporation. (A) Model of the MA trimer interface showing threonine (WT) and arginine side chains in red at position 69 on chain A. Side chains of Gln58, Ile59, and Gln62 on chain B are shown in dark gray. (B) Jurkat cells were transfected with the pNL4-3 molecular clone or mutants bearing substitutions at position 69. At 2-d intervals, the cells were split and samples of media were assayed for RT activity. (C) HeLa cells were transfected with the HIV-1 mutants indicated. At 48 h posttransfection, virus- and cell-associated samples were collected, separated by SDS/PAGE, and analyzed by Western blotting with anti-gp41 Ab and then HIV Ig to detect Gag. Positions of the Gag precursor Pr55Gag (p55), the Gag processing intermediate Pr41Gag (p41), the mature CA protein, and gp41 are indicated. Virus release was calculated as the amount of virion CA relative to total Gag levels, and Env incorporation was expressed as the amount of virion gp41 per virion CA; both were expressed relative to WT. Virus-containing supernatants were used to infect TZM-bl cells; the resulting luciferase signal was normalized to the corresponding RT values to provide a measure of specific infectivity. Averages from four independent experiments are shown, ± SEM. (D) HeLa cells were transfected with the HIV-1 mutants indicated. After 48 h, supernatants were filtered and virions were pelleted by ultracentrifugation. Virions were resuspended in PBS and treated with glutaraldehyde. Cross-linking was stopped by the addition of Tris, and then samples were boiled in Laemmli buffer and analyzed by SDS/PAGE and Western blotting for MA. Band volumes were measured, and the amount of trimeric MA was determined as a percentage of the total MA for that lane and expressed relative to WT. Averages from three independent experiments are shown, ± SEM.

Fig. S2.

Visualization of MA dimers by disulfide-bridge formation. (A) Jurkat cells were transfected with the pNL4-3 molecular clone or a mutant bearing a double-cysteine substitution at positions 62 and 66. At 2-d intervals, the cells were split and samples of media were assayed for RT activity. (B) HeLa cells were transfected with the HIV-1 mutants indicated. After 48 h, supernatants were filtered and virions were pelleted by ultracentrifugation. Virions were resuspended in PBS and treated with glutaraldehyde. Cross-linking was stopped by the addition of Tris, and then samples were boiled in nonreducing Laemmli buffer and analyzed by SDS/PAGE and Western blotting for MA. Band volumes were measured and the amount of trimeric MA was expressed as a percentage of the total MA for that lane. Averages from two independent experiments are shown, ± SEM.

Fig. 3.

Identification of MA trimer interface residues. (A) HeLa cells were transfected with the HIV-1 mutants indicated. After 48 h, supernatants were filtered and virions were pelleted by ultracentrifugation. Virions were resuspended in PBS and treated with glutaraldehyde. Cross-linking was stopped by the addition of Tris, and then samples were boiled in Laemmli buffer and analyzed by SDS/PAGE and Western blotting for MA. Band volumes were measured, and the amount of trimeric MA was determined as a percentage of the total MA for that lane and expressed relative to WT. Averages from three to five independent experiments are shown, ± SEM. (B) HeLa cells were transfected with the HIV-1 mutants indicated. At 48 h posttransfection, virus- and cell-associated samples were collected, separated by SDS/PAGE, and analyzed by Western blotting with anti-gp41 Ab and then HIV Ig to detect Gag. Positions of the Gag precursor Pr55Gag (p55), the mature CA protein, and gp41 are indicated. Virus release was calculated as the amount of virion CA relative to total Gag levels, and Env incorporation was expressed as the amount of virion gp41 per virion CA; both were expressed relative to WT. Virus-containing supernatants were used to infect TZM-bl cells; the resulting luciferase signal was normalized to the corresponding RT values to provide a measure of specific infectivity. Averages from three to six independent experiments are shown, ± SEM. (C and D) Top-down (C) and side-view (D) models of the MA trimer interface, indicating in red positions at which mutations inhibited trimerization (labeled) and in green positions at which mutations did not affect MA trimerization.

Fig. 4.

Replication of MA trimer-defective mutants. Jurkat cells were transfected with the HIV-1 mutants indicated. At 2-d intervals, the cells were split and samples of media were assayed for RT activity. Mutations of (A) Ala44, (B) Ser71, and (C) Leu74 were analyzed.

Fig. 5.

MA trimer-defective mutants block MA trimerization in immature particles. 293T cells were transfected with the HIV-1 mutants indicated. After 48 h, supernatants were filtered and virions were pelleted by ultracentrifugation. Virions were resuspended in PBS and treated with glutaraldehyde. Cross-linking was stopped by the addition of Tris, and then samples were boiled in Laemmli buffer and analyzed by SDS/PAGE and Western blotting for MA. Band volumes were measured and the amount of trimeric Gag was determined as a percentage of the total Gag for that lane and expressed relative to WT. Positions of molecular mass markers are shown. Averages from four independent experiments are shown, ± SEM.

Fig. 6.

Rescue of MA trimer-defective mutants by truncated Env. (A) HeLa cells were transfected with the HIV-1 mutants indicated. At 48 h posttransfection, virus- and cell-associated samples were collected, separated by SDS/PAGE, and analyzed by Western blotting with anti-gp41 Ab and then HIV Ig to detect Gag. Positions of the Gag precursor Pr55Gag (p55), the Gag processing intermediate Pr41Gag (p41), the mature CA protein, and gp41 (full-length or CTdel144) are indicated. Virus release was calculated as the amount of virion CA relative to total Gag levels, and Env incorporation was expressed as the amount of virion gp41 per virion CA; both were expressed relative to WT. Virus-containing supernatants were used to infect TZM-bl cells; the resulting luciferase signal was normalized to the corresponding RT values to provide a measure of specific infectivity. Averages from four independent experiments are shown, ± SEM. (B) MT4 cells were transfected with the HIV-1 mutants indicated. At 2-d intervals, the cells were split and samples of media were assayed for RT activity.

Fig. 7.

Schematic illustrating the role of MA trimerization in Env incorporation. Adapted from Tedbury and Freed (27). (A) Combining the models proposed by Alfadhli et al. (21) and Hill et al. (19) reveals a hexamer-of-trimers arrangement of MA, with a large (∼45-nm) central aperture ringed by residues where mutations are known to be able to block Env incorporation (indicated in blue). Hypothetical positions of the gp41 CT are depicted in green. (B) An earlier model by Alfadhli et al. (51) showed a hexameric configuration for MA, without trimers; in this model, the MA lattice has a much smaller central aperture (∼30 nm). The narrowed central aperture could cause the steric exclusion of the HIV-1 Env CT. (C) The truncation of the Env CT rescues Env incorporation into MA trimer-defective particles, possibly by relieving the steric clash between MA and the Env CT.