The nucleocapsid region of HIV-1 Gag cooperates with the PTAP and LYPXnL late domains to recruit the cellular machinery necessary for viral budding

Abstract

HIV-1 release is mediated through two motifs in the p6 region of Gag, PTAP and LYPX(n)L, which recruit cellular proteins Tsg101 and Alix, respectively. The Nucleocapsid region of Gag (NC), which binds the Bro1 domain of Alix, also plays an important role in HIV-1 release, but the underlying mechanism remains unclear. Here we show that the first 202 residues of the Bro1 domain (Bro(i)) are sufficient to bind Gag. Bro(i) interferes with HIV-1 release in an NC-dependent manner and arrests viral budding at the plasma membrane. Similar interrupted budding structures are seen following over-expression of a fragment containing Bro1 with the adjacent V domain (Bro1-V). Although only Bro1-V contains binding determinants for CHMP4, both Bro(i) and Bro1-V inhibited release via both the PTAP/Tsg101 and the LYPX(n)L/Alix pathways, suggesting that they interfere with a key step in HIV-1 release. Remarkably, we found that over-expression of Bro1 rescued the release of HIV-1 lacking both L domains. This rescue required the N-terminal region of the NC domain in Gag and the CHMP4 binding site in Bro1. Interestingly, release defects due to mutations in NC that prevented Bro1 mediated rescue of virus egress were rescued by providing a link to the ESCRT machinery via Nedd4.2s over-expression. Our data support a model in which NC cooperates with PTAP in the recruitment of cellular proteins necessary for its L domain activity and binds the Bro1-CHMP4 complex required for LYPX(n)L-mediated budding.

Figure 1. Over-expression of Alix inhibits HIV-1 release.

(A) Schematic representation of the domain organization of Alix. Bro1: BCK1-like resistance to osmotic shock 1, V: V-shaped domain, PRD: Proline Rich Domain. Numbers indicate positions of amino acid residues. (B) Virus-release assay showing that over-expression of Alix inhibits virus release. 293T cells were transfected with either wild-type (wt) pNL4-3 plasmid alone or with increasing amounts of HA-Alix. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. Gag intermediate products are indicated with (*) symbols. (C) Relative release efficiency of HIV-1 virions upon over-expression of Alix. The release efficiency in presence of Alix (calculated at the highest amount shown in B, lane 4) is relative to the release efficiency of NL4-3 alone, which was arbitrarily set at 100. Error bars indicate the standard deviation from four separate experiments. (D) Transmission Electron Microscopy (TEM) images of thin-sectioned 293T cells transfected with pNL4-3 wt alone (upper left panel) or pNL4-3 wt + Alix (lower left panel and close-ups of regions of interest a, b, c) showing arrested budding particles.

Figure 2. The N-terminal region of Bro1 inhibits the release of HIV-1.

(A) Schematic representation of constructs used in the experiment. Numbers indicate positions of amino acid residues. (B, C, D) 293T cells were transfected with either pNL4-3 plasmid alone or with increasing amounts of the indicated dominant-negative fragments of Alix. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. (B) Over-expression of Bro1-V but not delBroV inhibited virus-release. (C) The Bro1-V_F676D mutant retains inhibitory effect on HIV-1 release. (D) Bro_i, but not Bro1, inhibits HIV-1 release. (E) Relative release efficiency of HIV-1 virions upon over-expression of each of the four N-terminal fragments of Alix. Error bars indicate the standard deviation from three separate experiments.

Figure 3. Bro_i and Bro1-V inhibit HIV-1 release mediated via both Tsg101 and Alix pathways.

(A) Inhibition of an HIV-1 mutant lacking the LYPX_nL motif. 293T cells were transfected with either pNL4-3 mutant lacking the LYPX_nL motif (pNL4-3 YP-) alone or with the indicated dominant-negative fragment of Alix. Pelleted virions and cell lysates were analyzed by western blot using the indicated antibodies. (B) Interference with Alix driven rescue of an HIV-1 mutant lacking the PTAP motif. 293T cells were transfected with either wt pNL4-3 alone, with pNL4-3 mutant lacking the PTAP motif (pNL4-3 PTAP-) alone or with Alix in the presence or absence of the indicated dominant-negative fragment of Alix. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies.

Figure 4. The NC domain of HIV-1 Gag is the primary target for Bro_i inhibition.

(A) Bro_i and HIV-1 Gag co-immunoprecipitate in 293T cell lysates. 293T cells were co-transfected with either Gag-Pol (left panel) or GagΔp6-Pol (right panel) constructs along with expression vectors for HA-Alix, HA-Bro1, HA-Bro_i, or HA-PRD. Cells were lysed in RIPA buffer and clear lysates were incubated with anti-HA antibody-conjugated beads. Both input and immunoprecipitated complexes were ran on SDS-PAGE for western blot analysis using the indicated antibodies. (B) The Bro1 domain of Alix binds NC in vitro. MBP and MBP-NC proteins were expressed in E.coli, immobilized on amylose resin and incubated with lysates from 293T cells expressing HA-Bro1. Captured Bro1 was detected with anti-HA antibody and MBP proteins were visualized by Coomassie blue staining. (C) The release of NL4-3 DelNC/PR- but not NL4-3 PR- is insensitive to the over-expression of Bro_i. 293T cells were transfected with either pNL4-3 DelNC/PR- (Left panel) or pNL4-3 PR- (right panel) in presence or absence of HA-Bro_i. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. (D) Alix but not Bro_i co-immunoprecipitated with HIV-1 Gag DelNC. 293T cells were co-transfected with HIV-1 Gag DelNC/PR- along with either HA-Alix or HA-Bro_i. Cells were lysed in RIPA buffer and clear lysates were incubated with anti-HA antibody-conjugated beads. Both input and immunoprecipitated complexes were ran on SDS-PAGE for western blot analysis using the indicated antibodies.

Figure 5. Bro_i is recruited by Gag to the plasma membrane and interferes with HIV-1 budding.

(A) First two upper left panels show a cell expressing HA-Bro_i (white). The next panel shows a reconstructed 3D view of a stack of Z sections from a cell expressing HA-Bro_i and Gag-GFP. Lower panels show a single Z section of the same cell showing colocalization of Bro_i and Gag-GFP at the plasma membrane. Bro_i (white) was stained with a mouse monoclonal anti-HA antibody and an Alexa 633-conjugated anti-mouse antibody. Nuclei were counterstained with DAPI (blue). F-actin was stained with Alexa 568-conjugated phalloidin (red) to delineate cells. The colocalization channel (yellow) of Bro_i and Gag-GFP was built using Imaris software. Scale bar = 5 µm. (B, C) Electron micrographs of 293T cells co-transfected with pNL4-3 wt and HA-Bro_i. (B) Arrested budding structures are indicated with black arrows. Two regions of interest in (a) and (b) show budding structures carrying electron-dense crescent-shaped material at a higher magnification. (C) HIV-1 budding structures tethered to the plasma membrane.

Figure 6. Over-expression of Bro_i causes the formation of Class E compartments but has no effect on MoMLV release.

(A) The two upper left panels show a Z section of a cell expressing HA-Bro_i (white). Inset shows, at a higher magnification, an area of the cell (white rectangle) where HA-Bro_i associated with membranes of intracytoplasmic vacuoles. The two upper right panels show a Z section of a cell expressing GFP-VPS4a (green). Lower panels show Z sections of cells expressing both HA-Bro_i and GFP-VPS4a. Bro_i was stained with a mouse monoclonal anti-HA antibody and an Alexa 633-conjugated anti-mouse antibody. Nuclei were counterstained with DAPI (blue). The colocalization channel (yellow) of Bro_i and GFP-VPS4a was built using Imaris software. Scale bar = 5 µm. (B) Over-expression of Bro_i does not inhibit the release of MoMLV. 293T cells were transfected with either pNCA plasmid alone or with Bro_i and Alix. Pelleted virions and cell lysates were analyzed by western blot using the indicated antibodies.

Figure 7. Over-expression of Bro1-V inhibits HIV-1 budding in a CHMP4–dependent manner.

(A) The inhibitory effect of Bro1-V is dependent on its CHMP4 binding site. 293T cells were transfected with either pNL4-3 plasmid alone or with increasing amounts of the indicated mutants of Bro1-V. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. (B) Bro1-V but not Bro1-V_I212D co-immunoprecipitates with HIV-1 GagΔp6. 293T cells were co-transfected with GagΔp6-Pol along with expression vectors for HA-Alix, HA-Bro1-V, HA-Bro1-V_I212D and HA-PRD. Cells were lysed in RIPA buffer and clear lysates were incubated with anti-HA antibody-conjugated beads. Both input and immunoprecipitated complexes were ran on SDS-PAGE for western blot analysis using the indicated antibodies. (C–D) Electron micrographs of 293T cells co-transfected with pNL4-3 wt and HA-Bro1-V. (C) The black boxes indicate two regions of interest shown in (a) and (b) at a greater magnification. (D) HIV-1 arrested budding structures. Arrows indicate the electron-dense “ring-like” structure visible at the budding neck of arrested particles.

Figure 8. The Bro1 domain of Alix links Gag to ESCRT-III via NC.

(A) The isolated Bro1 domain, but not the full-length Alix, rescues the release of the NL4-3 PTAP-/YP- virus. 293T cells were transfected with either pNL4-3 PTAP-/YP- plasmid alone or with HA-Bro1 or HA-Alix. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. The asterisk indicates a longer exposure showing details of p24/p25 processing in the cells. (B) The rescue of the NL4-3 PTAP-/YP- double mutant virus depends on an intact NC domain in Gag. HA-Bro1 was co-expressed in 293T cells with either pNL4-3 DelNC PTAP-/YP-/PR-, which lacks NC (DelNC) and carries an inactive viral protease (PR-) (lane 4) or pNL4-3 PTAP-/YP- (lane 6) as a control. Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies. (C) The rescue of NL4-3 PTAP-/YP- release by Bro1 requires an intact CHMP4 binding site. 293T cells were transfected with either pNL4-3 PTAP-/YP- plasmid alone or with increasing amounts of HA-Bro1 (lanes 3–4) or HA-Bro1_I212D mutant (lanes 5–6). Pelleted virions and cell lysates were analyzed by western blot using the indicated antibodies.

Figure 9. Mapping of regions in NC important for HIV-1 release.

(A) Schematic representation of NC residues (1–55) with the two zinc fingers (Zf). Cysteine residues (C) changed to serine in the 2Zf- mutant are circled whereas arginine and lysine residues (R, K) were substituted to alanine in RKI (K3 to K26) and RKII (K26 to K52) mutants are underlined. (B, C) Zinc fingers and basic residues in the N-terminal portion of NC are necessary for the rescue of HIV-1 PTAP-/YP- (here named PY-) release by Bro1. (B) 293T cells were transfected with either pNL4-3 2Zf-, which carries disrupted zinc fingers (lane 1), or pNL4-3 2Zf-/PY- (lanes 2–3). pNL4-3 PY- is used (lanes 4–5) as a control for Bro1 rescue. In lane 3, co-transfection with pNL4-3 2Zf-/PY- and HA-Bro1 partially rescues viral release. (C) 293T cells were transfected with either pNL4-3 RKI (lanes 1-3) or pNL4-3 RKII (lanes 4–6) carrying either wt p6 (lanes 1 and 4) or a (PY-) p6 (lanes 2–3 and 5–6) and HA-Bro1 to examine the ability of Bro1 to rescue viral release. The asterisk indicates a longer exposure showing p24/p25 doublets in cell extracts. (D, E) Release defect of 2Zf-, RKI, RKII mutants is rescued by over-expression of Nedd4.2s. (D) 293T cells were transfected either with pNL4-3 (lane 1) or 2Zf-, RKI, RKII, and PTAP- mutant constructs (lanes 2–5). (E) 293T cells were transfected with either 2Zf-, RKI, RKII (lanes 1, 4 and 7) or their (PY-) mutant counterparts alone (lanes 2, 5, and 8) or with Nedd4.2s (lanes 3, 6, and 9). Pelleted virions and cell lysates were analyzed by SDS-PAGE and western blot using the indicated antibodies.

Figure 10. A model for NC cooperation with the PTAP/Tsg101 and LYPX_nL/Alix budding pathways.

(A) Role of NC in the LYPX_nL/Alix pathway: NC interacts with the Bro1 domain of Alix through its N-terminal basic residues and zinc fingers to recruit the essential downstream budding machinery components, ESCRT-III and VPS4, to promote virus egress. (B) Role of NC in the PTAP/Tsg101 pathway: basic residues throughout NC as well as zinc fingers may be required for HIV-1 budding via the PTAP motif, because they participate in the recruitment of members of the cellular budding machinery that include a Bro1-containing protein (Bro1? or Alix) and possible additional host factor(s) (X?) that cooperate with PTAP-bound complexes to facilitate virus release. (+) symbols represent basic residues in NC, (Zf): zinc finger.