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. 2019 Mar 19;116(12):5705-5714.
doi: 10.1073/pnas.1819475116. Epub 2019 Mar 6.

TIM-mediated Inhibition of HIV-1 Release Is Antagonized by Nef but Potentiated by SERINC Proteins

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Free PMC article

TIM-mediated Inhibition of HIV-1 Release Is Antagonized by Nef but Potentiated by SERINC Proteins

Minghua Li et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The T cell Ig and mucin domain (TIM) proteins inhibit release of HIV-1 and other enveloped viruses by interacting with cell- and virion-associated phosphatidylserine (PS). Here, we show that the Nef proteins of HIV-1 and other lentiviruses antagonize TIM-mediated restriction. TIM-1 more potently inhibits the release of Nef-deficient relative to Nef-expressing HIV-1, and ectopic expression of Nef relieves restriction. HIV-1 Nef does not down-regulate the overall level of TIM-1 expression, but promotes its internalization from the plasma membrane and sequesters its expression in intracellular compartments. Notably, Nef mutants defective in modulating membrane protein endocytic trafficking are incapable of antagonizing TIM-mediated inhibition of HIV-1 release. Intriguingly, depletion of SERINC3 or SERINC5 proteins in human peripheral blood mononuclear cells (PBMCs) attenuates TIM-1 restriction of HIV-1 release, in particular that of Nef-deficient viruses. In contrast, coexpression of SERINC3 or SERINC5 increases the expression of TIM-1 on the plasma membrane and potentiates TIM-mediated inhibition of HIV-1 production. Pulse-chase metabolic labeling reveals that the half-life of TIM-1 is extended by SERINC5 from <2 to ∼6 hours, suggesting that SERINC5 stabilizes the expression of TIM-1. Consistent with a role for SERINC protein in potentiating TIM-1 restriction, we find that MLV glycoGag and EIAV S2 proteins, which, like Nef, antagonize SERINC-mediated diminishment of HIV-1 infectivity, also effectively counteract TIM-mediated inhibition of HIV-1 release. Collectively, our work reveals a role of Nef in antagonizing TIM-1 and highlights the complex interplay between Nef and HIV-1 restriction by TIMs and SERINCs.

Keywords: HIV; Nef; SERINC; TIM.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
HIV-1 Nef counteracts the inhibitory effect of TIM-1 on HIV-1 production. (A) Effect of TIM-1 on viral production of different proviral HIV-1 NL4-3 clones. HEK293T cells were transfected with HIV-1 proviral DNA plasmids encoding wild-type (WT) NL4-3 or nef, vpu, vif, vpr, or env-deficient NL4-3, along with indicated amounts of TIM-1 expression plasmid. Forty-eight hours posttransfection, Western blotting was performed to measure cell-associated Gag (Cells) and cell-free viral particles (VPs) by using an anti–HIV-1 p24 antibody. TIM-1 expression in cell lysates was determined by using an anti–TIM-1 antibody. The values for NL4-3 Δnef are boxed to highlight the differences. Virion-associated p24 and cellular Gag (Pr55, p41, and p24) were quantified by using Quantity One (Bio-Rad) software. Viral release efficiency was determined as the ratio of virion-associated p24 vs. the total Gag (i.e., virion p24 plus cellular Pr55, p41, and p24). Relative virion release (indicated as p24/total Gag) was calculated by setting the value of WT in the absence of TIM-1 to 1.00. (B) Relative release efficiency of HIV-1 and mutants was determined by averaging results of three independent experiments, with means and SDs shown. *P < 0.05; **P < 0.01; ****P < 0.0001. (C) Effect of ectopic expression of an exogenous Nef on the production of HIV-1 WT and Δnef particles. HEK293T cells were cotransfected with HIV-1 NL4-3 WT or Δnef proviral DNA along with increasing amounts of NL4-3 Nef expression plasmid (0.5 or 1.5 μg) in the presence or absence of TIM-1. Viral release was determined as described for A. Note that an anti-Nef antibody was used to detect both the endogenous Nef (En) encoded by the provirus and an exogenous Nef (Ex) expressed in trans from an expression plasmid. (D) Relative release efficiency of HIV-1 WT and Δnef in the presence and absence of TIM-1 and exogenous Nef was determined by averaging results of three independent experiments, with means and SDs shown. **P < 0.01; ****P < 0.0001. (EH) Effect of TIM-3 knockdown on HIV-1 release in MDMs derived from three donors. Viral release was measured by quantifying the viral p24 level of the cell supernatants harvested at 48-h postinfection. The fold increase of HIV-1 release caused by TIM-3 shRNA knockdown relative to that of shRNA control was indicated. H shows a summary result of all three donors. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 2.
Fig. 2.
The ability of Nef to antagonize TIM-1 is conserved among primate lentiviruses. (A) Effect of Nef proteins derived from different HIV-1 groups and SIV on HIV-1 production. HEK293T cells were transiently transfected with NL4-3 Δnef and TIM-1 plasmids, along with expression vectors encoding the Nef alleles derived from HIV-1 group M (NL4-3, JRCSF), N (YBF30, N-2693), O (O-13127, O-HJ162), P (P-14788, P-RBF168), as well as SIVgor (CP2139, CR8757) and SIVcpz (EK505, MB897). HIV-1 production was determined as described for Fig. 1A. (B) Effect of lentivirus Nef proteins on release of NL4-3 Δnef as determined by averaging results of three independent experiments. (C) Effect of SIVmac Nef on TIM-1 inhibition of SIVmac production. HEK293T cells were transfected with proviral DNAs encoding SIVmac239 WT or SIVmac239 Δnef, along with increasing amounts of TIM-1 expression plasmids (200 or 500 ng). Levels of cell-associated and virion-associated SIVmac239 Gag proteins were determined by Western blotting using anti-SIV p27 antibody. (D) Relative RT activities from experiments described in C. Viral particles released from transfected cells described in C were quantified by RT assay. Data are means ± SDs of three independent experiments. ***P < 0.001; ****P < 0.0001. (E) Effect of HIV-2 Nef on the production of HIV-2 particles in the presence or absence of TIM-1. Western blotting was conducted to measure the expression of HIV-2 Gag in the cell lysates and in the purified virions. (F) Relative RT activities from experiments described in E. Shown are means ± SDs of three independent experiments by comparing with the RT activity of HIV-2 WT alone. ****P < 0.0001.
Fig. 3.
Fig. 3.
HIV-1 Nef promotes internalization of TIM-1 from the plasma membrane and sequesters TIM-1 in intracellular compartments. (A and B) HIV-1 Nef modestly down-regulates TIM-1 expression on the plasma membrane. HEK293T cells were transfected with a FLAG-tagged TIM-1 plasmid in the presence or absence of NL4-3 Nef (an equal DNA input for Nef and TIM-1 vector), and the expression of TIM-1 on the plasma membrane was determined by using an anti-FLAG antibody. A summary plot of the relative geometric means of TIM-1 from three independent experiments is shown in A. *P < 0.05. A representative flow cytometry profile demonstrating a modest decrease of TIM-1 in the presence of Nef is shown in B. (C and D) HIV-1 Nef sequesters TIM-1 in intracellular compartments. HEK293T cells cultured in chamber slides were transfected with pNL4-3ΔNef and the human TIM-1 expression vector in the absence or presence of a vector expressing HA-tagged Nef. At 24-h posttransfection, cells were permeabilized and incubated with primary and secondary antibodies to detect TIM-1 (green) and Nef (anti-HA, red). Cells were mounted with Vectashield mounting media with DAPI and examined with a Delta-Vision RT deconvolution microscope (C). Colocalization was quantified by calculating the Pearson correlation coefficients (R values) using the softWoRx colocalization module (D). (EG) COS-7 cells cultured in chamber slides were transfected with a vector expressing HA-tagged Nef; cells were fixed, permeabilized, and incubated for 1 h with antibodies specific for TIM-1, HA, p62, or TGN46 appropriately diluted in 3% BSA–PBS. In this experiment, the primary antibodies were directly labeled with either Zenon Alexa Fluor 488 (TIM-1, green), Zenon Alexa Fluor 594 (p62 or TGN46, red), or Zenon Alexa Fluor 647 (anti-HA, blue) using the Zenon antibody labeling kit (Thermo Fisher). Colocalization between TIM-1 and p62 and between TIM-1 and Nef was performed; no colocalization between TIM-1 and TGN46 was detected. (H) Comparison of the internalization kinetics of TIM-1 in the presence of WT and Nef mutants. HEK293T cells stably expressing TIM-1 were transfected with plasmids encoding Nef-GFP (WT), Nef-G2A-GFP, Nef-D123A-GFP, or Vpr-YFP (negative control, Mock). The geometric means of the fluorescence intensity of TIM-1 at each time points were recorded, compared, and their relative values were plotted against time by setting the value of time point 0 for each Nef construct or mock as zero. Results are averaged percentages ± SDs of the internalized TIM-1 protein from three to five independent experiments. ***P < 0.001. (I) Effect of Nef mutants on TIM-1–mediated inhibition of HIV-1 release. HEK293T cells were cotransfected with NL4-3 Δnef proviral DNA plus plasmids encoding TIM-1, WT Nef, or Nef mutants. The relative viral release efficiency was determined as described in Fig. 1 and as indicated. A summary plot of the effect of Nef mutants on antagonizing TIM-1–mediated restriction of HIV-1 Δnef virus release from four independent experiments (SI Appendix, Fig. S4I).
Fig. 4.
Fig. 4.
SERINC3 and SERINC5 proteins potentiate TIM-mediated inhibition of HIV-1 production. (A) Effect of knockdown of SERINC3 on HIV-1 production. HEK293T cells stably expressing control shRNA or SERINC3 shRNAs (two clones) were transfected with proviral DNA plasmids encoding NL4-3 WT or Δnef together with TIM-1 plasmid. Viral production was evaluated by examining RT activity. Shown are means and SDs of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. (B) Effect of SERINC3 knockdown on HIV-1 infectivity as determined by infecting HeLa-TZM cells. *P < 0.05; ***P < 0.001. (C) Effect of coexpression of SERINC3 or SERINC5 with TIM-1 on HIV-1 production. HEK293T cells were transfected with plasmids encoding NL4-3 WT or ΔNef and a TIM-1 plasmid plus increasing amounts (200 and 500 ng) of pBJ-SERINC3-HA or SERINC5-HA plasmids. Forty-eight hours posttransfection, Western blotting was performed to examine Gag and SERINC (HA-tagged) expression in the cell lysates and purified virions using specific primary antibodies. (D) Effect of SERINC3 or SERINC5 coexpression with TIM-1 on HIV-1 release, as quantified by viral p24 vs. the total cellular Gag. Data are means ± SDs of three independent experiments relative to that of WT NL4-3 alone (“Vector”). (E) Effect of SERINC3 and SERINC5 on the expression of TIM-1 on the cell surface as determined by flow cytometry using an anti–TIM-1 antibody. (FJ) Effect of SERINC3 and SERINC5 knockdown on HIV-1 WT and ΔNef release in PBMCs and CD4+ T cells. The experimental design is shown in F. PBMCs (G and H) or CD4+ T cells (I and J) were transduced with VSV-G–pseudotyped lentiviral vectors encoding either control shRNA or shRNA targeting SERINC3 or SERINC5. Cells were then infected with HIV-1 LAI Δenv (WT) or LAI ΔenvΔnef (ΔNef) bearing VSV-G. The RT activities of newly produced HIV-1 virions were determined. Shown are means ± SDs of three independent experiments relative to the RT activity from PBMCs or CD4+ T cells transduced with control shRNA and infected with WT HIV-1. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant.
Fig. 5.
Fig. 5.
SERINC5 stabilizes TIM-1 as determined by pulse-chase labeling assay. HEK293T cells were transfected with pCIneo-FLAG-TIM-1 in the presence or absence of pBJ-SERINC5-HA plasmid. Twenty-four hours after transfection, cells were subjected to pulse-labeling for 1 h and chased for indicated periods of time. Cell were lysed at 24-h posttransfection, and lysates were immunoprecipitated using an anti-FLAG antibody and resolved by SDS/PAGE. (A) A representative image of the pulse-chase labeling experiment. Proteins were detected by PhosphorImager analysis, and the TIM intensity in the presence or absence of SERINC5 was quantified by using Quantity One (Bio-Rad) software. Relative TIM-1 intensity was determined by setting the level of TIM-1 (time point 0) without SERINC5 to 1.00. (B) A summary plot of the half-life of TIM-1 in the absence or presence of SERINC5. Results are from five independent experiments.
Fig. 6.
Fig. 6.
MLV glycoGag and EIAV S2 antagonize TIM-1–mediated inhibition of HIV-1 production. (A) Effect of MLV glycoGag on HIV-1 production. HEK293T cells were transiently transfected with plasmids expressing WT or Δnef NL4-3 (1 μg each) and TIM-1 (200 ng), together with increasing amounts of glycoGag plasmid (200 or 500 ng). Viral release was determined as described in Fig. 1C. (B) Averaged HIV-1 release efficiency was determined from results of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Effect of EIAV S2 on HIV-1 production. The experimental procedure was the same as described for MLV glycoGag, except that 200 ng of an EIAV S2 plasmid was used. (D) Effect of EIAV S2 on TIM-1–mediated inhibition of HIV-1 release was determined from results of three independent experiments. *P < 0.05; ****P < 0.0001. (E and F) MLV glycoGag and EIAV S2 alone do not inhibit HIV-1 release. The experiments were performed as described in A and C, except that no TIM-1 plasmid was transfected; results from one representative experiment are shown.

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