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. 2019 Mar 21;93(7):e02128-18.
doi: 10.1128/JVI.02128-18. Print 2019 Apr 1.

The Polar Region of the HIV-1 Envelope Protein Determines Viral Fusion and Infectivity by Stabilizing the gp120-gp41 Association

Wuxun Lu #  1 , Shuliang Chen #  1 , Jingyou Yu  1 , Ryan Behrens  2 , Joshua Wiggins  3 , Nathan Sherer  2 , Shan-Lu Liu  1   4 , Yong Xiong  5 , Shi-Hua Xiang  3 , Li Wu  6   4
Affiliations

The Polar Region of the HIV-1 Envelope Protein Determines Viral Fusion and Infectivity by Stabilizing the gp120-gp41 Association

Wuxun Lu et al. J Virol. .

Abstract

HIV-1 enters cells through binding between viral envelope glycoprotein (Env) and cellular receptors to initiate virus and cell fusion. HIV-1 Env precursor (gp160) is cleaved into two units noncovalently bound to form a trimer on virions, including a surface unit (gp120) and a transmembrane unit (gp41) responsible for virus binding and membrane fusion, respectively. The polar region (PR) at the N terminus of gp41 comprises 17 residues, including 7 polar amino acids. Previous studies suggested that the PR contributes to HIV-1 membrane fusion and infectivity; however, the precise role of the PR in Env-mediated viral entry and the underlying mechanisms remain unknown. Here, we show that the PR is critical for HIV-1 fusion and infectivity by stabilizing Env trimers. Through analyzing the PR sequences of 57,645 HIV-1 isolates, we performed targeted mutagenesis and functional studies of three highly conserved polar residues in the PR (S532P, T534A, and T536A) which have not been characterized previously. We found that single or combined mutations of these three residues abolished or significantly decreased HIV-1 infectivity without affecting viral production. These PR mutations abolished or significantly reduced HIV-1 fusion with target cells and also Env-mediated cell-cell fusion. Three PR mutations containing S532P substantially reduced gp120 and gp41 association, Env trimer stability, and increased gp120 shedding. Furthermore, S532A mutation significantly reduced HIV-1 infectivity and fusogenicity but not Env expression and cleavage. Our findings suggest that the PR of gp41, particularly the key residue S532, is structurally essential for maintaining HIV-1 Env trimer, viral fusogenicity, and infectivity.IMPORTANCE Although extensive studies of the transmembrane unit (gp41) of HIV-1 Env have led to a fusion inhibitor clinically used to block viral entry, the functions of different domains of gp41 in HIV-1 fusion and infectivity are not fully elucidated. The polar region (PR) of gp41 has been proposed to participate in HIV-1 membrane fusion in biochemical analyses, but its role in viral entry and infectivity remain unclear. In our effort to characterize three nucleotide mutations of an HIV-1 RNA element that partially overlaps the PR coding sequence, we identified a novel function of the PR that determines viral fusion and infectivity. We further demonstrated the structural and functional impact of six PR mutations on HIV-1 Env stability, viral fusion, and infectivity. Our findings reveal the previously unappreciated function of the PR and the underlying mechanisms, highlighting the important role of the PR in regulating HIV-1 fusion and infectivity.

Keywords: HIV-1; entry; envelope glycoprotein; fusion; gp160; gp41; infectivity; polar region; trimer.

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Figures

FIG 1
FIG 1
RRE mutations overlapping the gp41 coding sequence do not affect RRE function. (A) Alignment of nucleotide (nt) and amino acid (aa) sequences of overlapped HIV-1 RRE and Env in wild-type (WT) and mutants (M1 to M5) based on HIV-1NL4-3 (GenBank accession number M19921.2). (B) Secondary structure of the hairpin loop IIB of the RRE of HIV-1NL4-3. The mutation frequencies of the indicated nucleotides shown in parentheses are based on analysis of 63,612 RRE sequences in the HIV Sequence Database. Three nucleotides that were mutated are shown in red, which are equivalent to U7871, A7877, and A7883 in the HIV-1LAI strain reported by Lichinchi et al. (10). (C) HIV-1 gp41 domains (residue numbers are based on HIV-1NL4-3 strain). FP, fusion peptide; PR, polar region; NHR, N-terminal heptad repeat; CHR, C-terminal heptad repeat; MPER, membrane-proximal external region; TM, transmembrane domain; CT, cytoplasmic tail. (D) Mutation frequency of the PR amino acids in HIV-1 gp41. A total of 57,645 aligned HIV-1 Env sequences from the HIV database were analyzed. Frequency of each residue (total, 17 aa) in the PR was calculated to generate sequence logo using WebLogo. (E and F) Single-cycle HIV-1 pseudotyped with VSV-G was generated by transfecting pNL4-3 E-R-Rev-/YFP (WT or M1 to M5 separately) and plasmids encoding VSV-G or Rev fused with mApple (Rev-mApple). Plasmid encoding mApple alone was used as a negative control. Cell lysates were used for immunoblotting, and the results are shown below the bar figures (one representative from three independent experiments is shown); viruses, as indicated, were used to infect HeLa cells to quantify HIV-1 infectivity. HSP90 was used as a loading control. All experiments were performed with triplicate samples and repeated at least three times, and means ± standard errors of the means are shown. No statistical difference in levels of infectivity was observed between WT and each mutant.
FIG 2
FIG 2
PR mutations significantly decrease HIV-1 infectivity without affecting viral production. (A) HIV-1 infectivity of equal amounts of p24 of WT and mutants was quantified with TZM-bl cells. (B) Lysates of transfected HEK293T cells were analyzed for HIV-1 Gag, CA, and gp160 expression by immunoblotting. GAPDH was a loading control. (C) HIV-1 p24 levels in supernatants of transfected HEK293T cells were quantified by ELISA. All experiments were performed with triplicate samples and repeated at least three times, and means ± standard errors of the means are shown. Dunnett's multiple-comparison test was used for statistical analysis. ***, P < 0.0001, for the comparison of the result with an individual mutant to that with WT HIV-1.
FIG 3
FIG 3
PR mutations decrease gp120 association and reduce HIV-1 fusion and infectivity. HEK293T cells were transfected with full-length WT or mutant (M1 to M5) proviral constructs without (control) or with (+Env) a WT gp160 expression plasmid. (A) Cell lysates were used for immunoblotting of the indicated HIV-1 proteins. GAPDH was a loading control of cell lysates. (B) Purified HIV-1 virions were used for immunoblotting of the indicated HIV-1 proteins. (C and D) Densitometry quantification of gp160, gp120, and gp41 bands in immunoblotting of cell lysates (control samples in panel A) and purified HIV-1 (control samples in panel B), respectively. Relative levels of gp120/gp160 or gp41/gp160 ratios were calculated from three independent experiments. The values of WT HIV-1 were set as 100%, and relative levels are shown as means ± standard errors of the means (n = 3). *, P < 0.05; **, P < 0.005; ***, P < 0.0001, for the comparison of the result with an individual mutant to that with WT HIV-1 (E) WT or mutant HIV-1 generated from transfected HEK293T cells without (control) or with Env overexpression (+Env) were quantified for viral infectivity using TZM-bl cells. (F) Virion-cell fusion was determined by flow cytometry-based BlaM-Vpr assays using TZM-bl cells (10 or 50 ng of p24 for HIV-1 with or without Env trans-supplementation, respectively). All experiments were performed with triplicate samples for panels E and F and repeated at least three times, and means ± standard errors of the means are shown. Dunnett's multiple-comparison test was used for statistical analysis. ***, P < 0.0001, for the comparison of the result with an individual mutant to that with WT HIV-1.
FIG 4
FIG 4
PR mutations abolish or significantly decrease Env-mediated cell-cell fusion. HEK293T cells were separately transfected with pRK empty vector, WT, or mutant Env (gp160) expression construct together with pCMV-Rev. HEK293T cells transfected without plasmid DNA were used as a mock control. (A) At 24 h posttransfection, HEK293T cells were collected and lysed for immunoblotting analysis with antibodies to gp160/gp120, gp41, or GAPDH. (B and C) At 24 h posttransfection, the cells were stained with gp120 monoclonal antibody (2G12) at 4°C to measure the cell surface gp120 expression using flow cytometry. Average percentages (B) and the mean fluorescence intensity (C) of gp120-positive cells from four independent experiments were determined. (D) Transfected HEK293T cells were cocultured with TZM-bl cells for 24 h and then lysed for firefly luciferase activity measurement of Env-mediated cell-cell fusion. Average percentages of HIV-1 Env-mediated cell-cell fusion from three independent experiments are shown, with the value for the WT set as 100%. All experiments were performed with triplicate samples and repeated at least three times, and means ± standard errors of the means are shown. Dunnett's multiple-comparison test was used for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.0001, for the comparison of the result with an individual mutant Env to that with WT Env.
FIG 5
FIG 5
PR mutations decrease gp120 association and increase gp120 shedding. (A) Representative results of gp120 shedding of 35S-labeled WT and mutant Env (M1 to M5) from one of four separate experiments. HEK293T cells were transfected with WT or mutant Env expression constructs for the gp120 shedding assay. The gp120 processing and the association index were calculated as described in Materials and Methods and are shown below the shedding gel. Values are representative of the average of four separate experiments. (B) Summary results of four independent experiments of gp120 shedding. Dunnett's multiple-comparison test was used for statistical analysis. ***, P < 0.001, for a comparison of the association index of mutant M1, M3, or M4 with that of WT Env.
FIG 6
FIG 6
S532A mutant of the PR reduces HIV-1 infectivity and fusogenicity. (A) Alignment of nucleotide (nt) and amino acid (aa) sequences of overlapped HIV-1 RRE and Env in wild-type (WT) and S532A mutants (M6) based on HIV-1NL4-3 (GenBank accession number M19921.2). (B) HIV-1 p24 levels in supernatants of transfected HEK293T cells were quantified by ELISA (n = 6). (C to E) HEK293T cells were transfected with full-length WT or M6 mutant proviral constructs without (control) or with (+Env) a WT gp160 expression plasmid. HIV-1 infectivity of equal amounts of p24 of WT and mutants was quantified with TZM-bl cells (C). (D) Lysates of transfected HEK293T cells or purified HIV-1 were analyzed for the indicated HIV-1 proteins by immunoblotting. GAPDH was a loading control (D). Virion-cell fusion was determined by flow cytometry-based BlaM-Vpr assays using TZM-bl cells (10 ng p24 for HIV-1 with or without Env trans-supplementation) (E). (F to H) HEK293T cells were separately transfected with a pRK empty vector, WT, or M6 Env (gp160) expression construct together with pCMV-Rev. HEK293T cells transfected without plasmid DNA were used as a mock control. At 24 h posttransfection, HEK293T cells were collected and lysed for immunoblotting analysis with antibodies to gp160/gp120, gp41, or GAPDH (loading control). Relative gp160 levels were quantified and normalized to the level of GAPDH (F). At 24 h posttransfection, the cells were stained with gp120 monoclonal antibody (2G12) at 4°C to measure the cell surface gp120 expression using flow cytometry. Average percentages (G) and the mean fluorescence intensity (H) of gp120-positive cells from three independent experiments were determined. (I) Transfected HEK293T cells were cocultured with TZM-bl cells for 24 h and then lysed for firefly luciferase activity measurement of Env-mediated cell-cell fusion. Average percentages of HIV-1 Env-mediated cell-cell fusion from four independent experiments are shown, with the value for the WT set as 100%. All experiments were performed with triplicate samples and repeated at least three times, and means ± standard errors of the means are shown. Dunnett's multiple-comparison test was used for statistical analysis. *, P < 0.05; ***, P < 0.0001, for results with M6 mutant compared to those with WT HIV-1.
FIG 7
FIG 7
Structural models of the PR in the Env trimer of prefusion state. (A) PR sequence alignment between HIV-1BG505 and HIV-1NL4-3. (B) PR location at the trimer junction of gp120 (tan) and gp41 (blue). One protomer is shown in ribbon representation. The neighboring protomers are in surface representation and are shown in gray and teal blue. (C) PR (blue) interaction with gp41 C-terminal heptad repeat (CHR) of a neighboring protomer (gray). Fusion peptide (FP) is shown in red. The hydrogen bonds between residues are shown in dashed lines. Residue numbers and the structure prediction in panels B and C are based on the HIV-1BG505 strain (19, 20). Of note, residue numbers of HIV-1BG505 shown in panels B and C are two numbers greater than those of HIV-1NL4-3.
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References

    1. Melikyan GB. 2017. How entry inhibitors synergize to fight HIV. J Biol Chem 292:16511–16512. doi:10.1074/jbc.H117.791731. - DOI - PMC - PubMed
    1. Checkley MA, Luttge BG, Freed EO. 2011. HIV-1 envelope glycoprotein biosynthesis, trafficking, and incorporation. J Mol Biol 410:582–608. doi:10.1016/j.jmb.2011.04.042. - DOI - PMC - PubMed
    1. Kwong PD, Mascola JR. 2018. HIV-1 vaccines based on antibody identification, B cell ontogeny, and epitope structure. Immunity 48:855–871. doi:10.1016/j.immuni.2018.04.029. - DOI - PubMed
    1. Peisajovich SG, Epand RF, Pritsker M, Shai Y, Epand RM. 2000. The polar region consecutive to the HIV fusion peptide participates in membrane fusion. Biochemistry 39:1826–1833. - PubMed
    1. Sackett K, Wexler-Cohen Y, Shai Y. 2006. Characterization of the HIV N-terminal fusion peptide-containing region in context of key gp41 fusion conformations. J Biol Chem 281:21755–21762. doi:10.1074/jbc.M603135200. - DOI - PubMed

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