Myristate exposure in the human immunodeficiency virus type 1 matrix protein is modulated by pH

Abstract

Human immunodeficiency virus type 1 (HIV-1) encodes a polypeptide called Gag that is capable of forming virus-like particles (VLPs) in vitro in the absence of other cellular or viral constituents. During the late phase of HIV-1 infection, Gag polyproteins are transported to the plasma membrane (PM) for assembly. A combination of in vivo, in vitro, and structural studies have shown that Gag targeting and assembly on the PM are mediated by specific interactions between the myristoylated matrix [myr(+)MA] domain of Gag and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Exposure of the MA myristyl (myr) group is triggered by PI(4,5)P2 binding and is enhanced by factors that promote protein self-association. In the studies reported here, we demonstrate that myr exposure in MA is modulated by pH. Our data show that deprotonation of the His89 imidazole ring in myr(+)MA destabilizes the salt bridge formed between His89(Hδ2) and Glu12(COO-), leading to tight sequestration of the myr group and a shift in the equilibrium from trimer to monomer. Furthermore, we show that oligomerization of a Gag-like construct containing matrix-capsid is also pH-dependent. Disruption of the His−Glu salt bridge by single-amino acid substitutions greatly altered the myr-sequestered−myr-exposed equilibrium. In vivo intracellular localization data revealed that the H89G mutation retargets Gag to intracellular compartments and severely inhibits virus production. Our findings reveal that the MA domain acts as a “pH sensor” in vitro, suggesting that the effect of pH on HIV-1 Gag targeting and binding to the PM warrants investigation.

Figure 1

Comparison of HIV-1 and HIV-2 MA protein structures. The imidazole ring of His89 forms a salt bridge with Glul2(COO) in HIV-1 myr(−)MA but not in the myr(+)MA protein. For HIV-2, Glul2 is substituted with Lys, which can form a hydrogen bond with deprotonated His89 in myr(−)MA.

Figure 2

(A) Overlay of 2D ¹H-¹⁵N HSQC spectra collected for HIV-1 myr(+)MA and myr(−)MA at pH 5.5 (Black), 6.0 (Blue), 6.5 (Red), 7.0 (Magenta), 7.5 (Green) and 8.0 (Cyan) (150 µM 35 °C). (B) A histogram showing chemical shift changes extracted from the 2D HSQC data collected for myr(+)MA (black) and myr(−)MA (red) upon increasing the pH from 5.5 to 8.0. (C) Chemical shift changes are shown for the ¹H-¹⁵N resonances of Leu8 and Ser9 of myr(+)MA (black) and myr(−)MA (red).

Figure 3

3D ¹³C-edited/¹²C-double-half-filtered NOE data obtained for HIV-1 myr(+)MA at pH 7.2 showing unambiguously assigned NOE cross-peaks between the myr group and key residues of a Relabeled protein sample (myr group is unlabeled). Solid lines denote ¹H-¹²C breakthrough doublets NOE peaks.

Figure 4

Sedimentation coefficient distributions, c(s), obtained from the sedimentation profiles for myr(+)MA and myr(−)MA as a function of pH.

Figure 5

Sedimentation coefficient distributions, c(s), obtained from the sedimentation profiles for myr(+)MACA and myr(−)MACA at different pH values. The small peaks marked with asterisk indicate uncharacterized minor species likely to be impurity.

Figure 6

A structural view of the HIV-1 myr(+)MA protein in the myr-sequestered (green) and myr-exposed (brown) forms. Notice the conformational change involving Leu8, which acts as a “NMR reporter” for the movement of the myr group.

Figure 7

(A) Subcellular localization of WT HIV-1 Gag compared to that of the H89G and K30E/K32E mutants. HeLa cells were transfected with the indicated molecular clones. Cells were fixed and stained with an antibody specific for p17 (MA) or were costained with antibodies specific for p17 (MA) and the MVB marker CD63. (B) Virus release efficiency data for WT and mutant Gag (calculated as follows: virus release efficiency = virion p24/(cell Gag + virion Gag)). (C) The MA H89G mutant induces defects in virus replication in T cells. Jurkat or MT-4 T cells were transfected with WT pNL4-3 or the indicated MA-mutant derivatives. Cells were split every two days; virus replication was measured by RT assay. For comparison, virus replication data for V7R and K30E/K32E mutants are also shown.

Figure 8

Schematic representation showing multimerization events of Gag and MA proteins as a function of pH.