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 ϳ64-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]/CRISPRassociated-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. IMPORTANCEThe 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 ϳ30 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.

TSPO: kaleidoscopic 18-kDa amid biochemical pharmacology, control and targeting of mitochondria

“…Conversely, there are increased levels of Zbtb7b, which is associated with T-cell maturation (115) and regulates the development of Natural Killer T (NKT) cells (116) and ties in the Tu et al study (110) with the recent work by Banati et al (32), who observed an increase in NKT cells present during haematological analysis in female Tspo -/-mice. TSPO has previously been reported as having antiretroviral activity by inhibiting Env protein expression (117). In support of this study, decreased levels of Trim12a, coding for protein within the anti-retroviral TRIM family, and Pydc4, coding for an AIM2-like receptor that activates STING-dependent interferon (IFN) production as part of the antiviral response (118),…”

Enhanced photocytotoxicity of curcumin delivered by solid lipid nanoparticles

“…42 High levels of ROS can disrupt the mitochondrial permeability transition pore and destroy the integrity of the mitochondrial membrane, resulting in immediate dissipation of mitochondrial transmembrane potential and osmotic swelling of the mitochondrial matrix. 43,44 Once …”

ERManI (Endoplasmic Reticulum Class I α-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway

“…The human CEM-T4 T cell line and HIV-1 luciferase reporter GHOST cells were obtained from the NIH AIDS Research and Reference Reagent Program. The TSPO-KO 293T cell line A3 was reported before (5). The human CEM.NKR T cell line subclones N2-NP and N5-P were described before (22).…”

“…Knock-out of MAN1B1 in 293T Cells by CRISPR/Cas9 -A detailed protocol was described before (5). Briefly, 293T cells were transfected with a Cas9 expression vector and a MAN1B1 gRNA expression vector, and cloned by limiting dilution.…”