The mitochondrial translocator protein, TSPO, inhibits HIV-1 envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway

Abstract

The HIV-1 Env glycoprotein is folded in the endoplasmic reticulum (ER), which is necessary for viral entry and replication. Currently, it is still unclear how this process is regulated. The glycoprotein folding in the ER is controlled by the ER-associated protein degradation (ERAD) pathway, which specifically targets misfolded proteins for degradation. Previously, we reported that HIV-1 replication is restricted in the human CD4(+) T cell line CEM.NKR (NKR). To understand this mechanism, we first analyzed cellular protein expression in NKR cells and discovered that levels of the mitochondrial translocator protein TSPO were upregulated by ∼64-fold. Notably, when NKR cells were treated with TSPO antagonist PK-11195, Ro5-4864, or diazepam, HIV restriction was completely disrupted, and TSPO knockdown by short hairpin RNAs (shRNAs) achieved a similar effect. We next analyzed viral protein expression, and, interestingly, we discovered that Env expression was specifically inhibited. Both TSPO knockdown and treatment with TSPO antagonist could restore Env expression in NKR cells. We further discovered that Env proteins were rapidly degraded and that kifunensine, an ERAD pathway inhibitor, could restore Env expression and viral replication, indicating that Env proteins were misfolded and degraded through the ERAD pathway in NKR cells. We also knocked out the TSPO gene in 293T cells using CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat [CRISPR]/CRISPR-associated-9) technology and found that TSPO could similarly inhibit Env expression in these cells. Taken together, these results demonstrate that TSPO inhibits Env protein expression through the ERAD pathway and suggest that mitochondria play an important role in regulating the Env folding process.

Importance: The HIV-1 Env glycoprotein is absolutely required for viral infection, and an understanding of its expression pathway in infected cells will identify new targets for antiretroviral therapies. Env proteins are folded in the ER and secreted through the classical secretory pathway. The Env folding process involves extensive cross-linking of 10 Cys residues by disulfide bond formation and heavy N-glycosylation on ∼30 Asn residues. Currently, it is still unclear how this process is regulated. Here, we studied this mechanism in the HIV nonpermissive human CD4(+) T cell line CEM.NKR. We found that Env proteins were rapidly degraded through a cellular pathway that specifically targets misfolded proteins, resulting in inhibition of Env expression. Importantly, we have identified a mitochondrial translocator protein, TSPO, which could trigger this degradation by interfering with the Env folding process. Further characterization of TSPO antiviral activity will reveal a novel antiretroviral mechanism that targets the Env protein.

FIG 1

NKR cell resistance to apoptosis induction. (A) Resistance to cell death. Indicated cells were treated with 1 μM As₂O₃, and cell viability was detected by conversion of resazurin into resorufin (see Materials and Methods). The viability of untreated cells (control, Ctrl) was considered as 100% and that of treated cells was calculated and presented as relative values. (B) Resistance to apoptosis. Indicated cells were treated with 1 μM As₂O₃, and cells were stained with JC-1. Cell fluorescence was then measured on a flow cytometer using the FL1 and FL2 channels. Each of these experiments was repeated at least twice, and consistent results were obtained.

FIG 2

High levels of TSPO expression in NKR cells. (A) Microarray determination of mitochondrial protein expression. mRNA expression profiles in CEM.NKR (NKR), CEM-SS (SS), and CEM-T4 (T4) cells were analyzed by Human Genome U133 Plus 2.0 Array (Affymetrix). After that, a total of 246 mitochondrial proteins were selected, and their levels of expression were compared as relative ratios, as indicated. (B) TSPO protein expression in CEM-derived cells. The indicated cell lysate was prepared from a total of 1 × 10⁷ cells, and one-sixth of the sample was analyzed by Western blotting using the indicated antibodies. (C) To compare the difference between TSPO expression levels in N2-NP and N5-P cells, the lysate from N2-NP cells was serially diluted and then compared to that of an undiluted N5-P cell-derived sample by Western blotting. Each of these experiments was repeated at least twice, and consistent results were obtained.

FIG 3

Disruption of HIV-1 restriction in NKR cells by TSPO ligands. (A) Effects of PK11195 and Ro5-4864 on HIV-1 restriction. N2-NP cells were infected with HIV-1 (pNL4-3) and treated with increasing amounts of PK11195 or Ro5-4864. Viral replication was determined by measuring the p24^Gag in the culture supernatant using ELISAs. (B) Effects of other TSPO ligands on HIV-1 restriction. N2-NP and N5-P cells were treated with 50 μM FGIN-1-27, 65 μM PK11195, 50 μM Ro5-4864, or 5 μM diazepam during HIV-1 infection, and viral replication was determined. The concentration of these compounds used in this experiment was the highest that did not affect cell viability (data now shown). (C) Effects of cholesterol on HIV-1 restriction. NKR, N2-NP, N5-P, and SS cells were treated with 0.2 μM As₂O₃, 50 μM cholesterol (Chol), or 25 μM water-soluble cholesterol (β-CD Chol) or remained untreated during HIV-1 infection, and viral replication was determined. Each of these experiments was repeated at least twice, and consistent results were obtained.

FIG 4

(A) HIV-1 replication in the original NKR, nonpermissive subclone N2-NP, semipermissive subclone N8-SP, and permissive subclone N5-P cells. A total of 2 × 10⁵ cells were incubated with equal amounts of HIV-1 (pNL4-3), and viral replication was determined every other day by p24^Gag ELISA. (B and C) As₂O₃ and PK11195 increase Env expression. Indicated cells were spinoculated with VSV-G pseudotyped HIV-1 and cultured in the presence or absence of 0.25 μM As₂O₃ or 50 μM PK11195. Virions were purified from culture supernatants by ultracentrifugation. Viral protein expression in virions (B) and virus-producing cells (C) was detected by Western blotting as indicated. Each of these experiments was repeated at least twice, and consistent results were obtained.

FIG 5

TSPO knockdown increases HIV-1 replication. (A) Comparison of TSPO expression in TSPO knockdown (KD) and control cell lines by Western blotting. These cell lines were created by stable transduction of two different TSPO-specific shRNAs or a control (Ctrl) shRNA, as described in Materials and Methods. (B) Indicated cells were infected with wild-type (WT) or ΔVpr HIV-1, and viral replication was determined by p24^Gag ELISA. (C) Comparison of Env expression levels in TSPO knockdown and control cell lines. Indicated cells were infected with HIV-1, and the Env and Gag expression were determined by Western blotting.

FIG 6

Env proteins are rapidly degraded through the ERAD pathway. (A) Detection of rapid Env degradation. N5-P and N2-NP cells were infected with HIV-1 (pNL4-3) for 24 h. Cells were pulse labeled with [³⁵S]Met-Cys for 1 h and then incubated for the indicated length of time. Env proteins were immunoprecipitated with an anti-gp120 antibody and analyzed by autoradiography, followed by quantification using the Image-Quant TL program. (B) Kifunensine (KIF) rescues HIV-1 replication in N2-NP cells. HIV-infected N2-NP and N5-P cells were treated with increasing amounts of KIF, and viral replication was determined by p24^Gag ELISA. (C) KIF rescues Env expression in NKR and N2-NP cells. Indicated cells were spinoculated with VSV-G pseudotyped HIV-1 and cultured in the absence or presence of 40 μM KIF for 48 h. Cells were lysed, and Env and Gag expression levels were determined by Western blotting. (D) Activation of the ERAD pathway inhibits Env expression. N5-P and T4 cells were treated with brefeldin A (10 μg/ml), tunicamycin (5 μg/ml), or thapsigargin (5 μg/ml) for 48 h during HIV-1 infection or remain untreated, and viral protein expression was determined by Western blotting.

FIG 7

TSPO inhibits HIV-1 Env expression in 293T cells. (A) Schematic illustrating Cas9 inactivation of the human TSPO locus. Numbers indicate the nucleotide positions in the TSPO open reading frame. The 19-bp guide RNA target sequence is shown in green, and the protospacer-adjacent motif (PAM) is shown in red. The sense primer TSPO-ko-S and antisense primer TSPO-ko-A sequences that were used to amplify this gene locus are underlined. (B) Analysis of TSPO protein expression in three clones (A2, A3, and A4) isolated from 293T cells transfected with Cas9 and TSPO guide RNA expression vectors by Western blotting. (C) An 88-bp DNA fragment was PCR amplified from the TSPO locus of the A3 clone and wild-type (WT) 293T cells using primers TSPO-ko-S and TSPO-ko-A and analyzed by 10% TBE-polyacrylamide gel. M, marker. (D) Wild-type and A3 cells were transfected with the HIV-1 proviral clone pNL4-3, and Gag and Env protein expression levels were analyzed by Western blotting. (E) Indicated amounts of pNL4-3 and TSPO expression vectors were transfected into A3 cells, and TSPO and HIV-1 viral protein expression levels were analyzed by Western blotting.