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, 90 (19), 8509-19

Molecular Determinants Directing HIV-1 Gag Assembly to Virus-Containing Compartments in Primary Macrophages

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Molecular Determinants Directing HIV-1 Gag Assembly to Virus-Containing Compartments in Primary Macrophages

Jingga Inlora et al. J Virol.

Abstract

The subcellular sites of HIV-1 assembly, determined by the localization of the structural protein Gag, vary in a cell-type-dependent manner. In T cells and transformed cell lines used as model systems, HIV-1 assembles at the plasma membrane (PM). The binding and localization of HIV-1 Gag to the PM are mediated by the interaction between the matrix (MA) domain, specifically the highly basic region, and a PM-specific acidic phospholipid, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. In primary macrophages, prominent accumulation of assembling or assembled particles is found in the virus-containing compartments (VCCs), which largely consist of convoluted invaginations of the PM. To elucidate the molecular mechanism of HIV-1 Gag targeting to the VCCs, we examined the impact of overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P2, in primary macrophages. We found that the VCC localization and virus release of HIV-1 are severely impaired upon 5ptaseIV overexpression, suggesting an important role for the MA-PI(4,5)P2 interaction in HIV-1 assembly in primary macrophages. However, our analysis of HIV-1 Gag derivatives with MA changes showed that this interaction contributes to Gag membrane binding but is dispensable for specific targeting of Gag to the VCCs per se We further determined that deletion of the NC domain abolishes VCC-specific localization of HIV-1 Gag. Notably, HIV-1 Gag localized efficiently to the VCCs when the NC domain was replaced with a leucine zipper dimerization motif that promotes Gag multimerization. Altogether, our data revealed that targeting of HIV-1 Gag to the VCCs requires NC-dependent multimerization.

Importance: In T cells and model cell lines, HIV-1 Gag localizes to the PM in a manner dependent on the MA-PI(4,5)P2 interaction. On the other hand, in primary macrophages, HIV-1 Gag localizes to convoluted intracellular membrane structures termed virus-containing compartments (VCCs). Although these compartments have been known for decades, and despite the implication of viruses in VCCs being involved in virus reservoir maintenance and spread, the viral determinant(s) that promotes Gag targeting to VCCs is unknown. In this study, we found that the MA-PI(4,5)P2 interaction facilitates efficient Gag membrane binding in macrophages but is not essential for Gag targeting to VCCs. Rather, our results revealed that NC-dependent multimerization promotes VCC targeting. Our findings highlight the differential roles played by MA and NC in HIV-1 Gag membrane binding and targeting and suggest a multimerization-dependent mechanism for Gag trafficking in primary macrophages similar to that for Gag localization to uropods in polarized T cells.

Figures

FIG 1
FIG 1
HIV-1 release from MDMs is sensitive to 5ptaseIV overexpression. (A) MDMs expressing HIV-1 along with full-length 5ptaseIV (FL) or its Δ1 mutant were metabolically labeled for 4 h. Cell- and virus-associated Gag proteins were recovered by immunoprecipitation and analyzed by SDS-PAGE. (B) Relative virus release efficiencies of HIV-1 were calculated. Data for 7 donors are shown as means and standard deviations. The average virus release efficiency of MDMs that expressed HIV-1 along with 5ptaseIV Δ1 was 18.4%. P values were determined using Student's t test, using raw data. **, P < 0.001.
FIG 2
FIG 2
HIV-1 Gag failed to localize to VCCs upon 5ptaseIV overexpression. (A) MDMs were infected with pseudotyped HIV-1 encoding Gag-YFP along with 5ptaseIV (FL) or 5ptaseIV Δ1. At 48 h postinfection, cells were stained with ConA labeled with Alexa Fluor 594 (a PM marker), fixed, immunostained with a mouse monoclonal anti-CD81 antibody and anti-mouse IgG conjugated with Alexa Fluor 647, and analyzed using a confocal microscope. (B) Pearson's correlation coefficients for colocalization of Gag-YFP with CD81 are shown as means and standard errors of the means (SEM). Twenty to 30 cells were analyzed per condition. **, P < 0.001.
FIG 3
FIG 3
The myristate moiety but not the intact HIV-1 MA sequence is required for VCC localization. (A) MDMs were infected with pseudotyped HIV-1 encoding WT Gag-YFP or Gag-YFP derivatives containing the indicated MA substitutions. At 48 h postinfection, cells were stained with ConA labeled with Alexa Fluor 594, fixed, immunostained with a mouse monoclonal anti-CD81 antibody and anti-mouse IgG conjugated with Alexa Fluor 647, and analyzed using a confocal microscope. (B) Pearson's correlation coefficients for colocalization of Gag-YFP with CD81 are shown as means and SEM. Twenty to 30 cells were analyzed per condition. *, P < 0.005; n.s., not significant.
FIG 4
FIG 4
Heterologous membrane binding sequences can replace HIV-1 MA without affecting VCC localization. (A) Schematic illustrations of WT, PH/ΔMA, Kmyr/ΔMA, and Fyn(10)/ΔMA Gag-YFP. (B) MDMs were infected with pseudotyped HIV-1 encoding WT Gag-YFP or Gag-YFP derivatives in which MA was replaced by a heterologous membrane binding sequence. At 48 h postinfection, cells were stained with ConA labeled with Alexa Fluor 594, fixed, immunostained with a mouse monoclonal anti-CD81 antibody and anti-mouse IgG conjugated with Alexa Fluor 647, and analyzed using a confocal microscope. (C) Pearson's correlation coefficients for colocalization of Gag-YFP with CD81 are shown as means and SEM. Twenty to 30 cells were analyzed per condition. n.s., not significant.
FIG 5
FIG 5
HIV-1 Gag localization to VCCs requires higher-order multimerization. (A) Schematic illustrations of WT, EE75,76AA, delNC, LZ, and LZ4 Gag-YFP. LZ4 Gag-YFP contains the WM184,185AA CA mutation, which disrupts Gag dimerization, in addition to replacement of NC with a tetramer-forming LZ sequence. (B) MDMs were infected with pseudotyped HIV-1 encoding WT Gag-YFP or Gag-YFP derivatives containing the substitutions shown in panel A. At 48 h postinfection, cells were stained with ConA labeled with Alexa Fluor 594, fixed, immunostained with a mouse monoclonal anti-CD81 antibody and anti-mouse IgG conjugated with Alexa Fluor 647, and analyzed using a confocal microscope. Note that for cells expressing delNC Gag-YFP or LZ4 Gag-YFP, prominent YFP signals were found at the cell surface as detected by ConA (white arrowheads), whereas such surface YFP signals were nearly undetectable in WT Gag-YFP-expressing cells. (C) Pearson's correlation coefficients for colocalization of Gag-YFP with ConA are shown as means and SEM. Twenty to 40 cells were analyzed per condition. *, P < 0.005; ***, P < 0.0001. Note that while the EE75,76AA and LZ constructs showed higher PCC values than that of the WT, the values are less than 0.3, consistent with the lack of obvious PM localization of these Gag derivatives. (D) Pearson's correlation coefficients for colocalization of Gag-YFP with CD81 are shown as means and SEM. Twenty to 40 cells were analyzed per condition. n.s., not significant. (E) Examples of delNC Gag-YFP distribution to internal tubular structures. In the left panel, the boxed area from panel B is magnified. The YFP intensity was enhanced in this panel relative to that in panel B.
FIG 6
FIG 6
Working model for HIV-1 Gag targeting to VCCs. HIV-1 Gag proteins are synthesized in the cytosol and bind to membranes containing PI(4,5)P2, which include the PM and VCCs as well as the tubular network connecting these locations. These membranes may also receive Gag from vesicles of endosomal pathways in cases where Gag lacks specificity for PI(4,5)P2 (e.g., HTMA Gag or some ΔMA Gag derivatives). Higher-order multimers of Gag accumulate in VCCs, potentially via lateral movement along membranous connections to the VCCs, where assembly continues to progress.

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