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

  • Tao Zhou
    Footnotes
    Affiliations
    Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Harbin, 150001, China

    BEACON Center for the Study of Evolution in Action, Michigan State University, East Lansing, Michigan 48824

    Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824
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  • Dylan A. Frabutt
    Footnotes
    Affiliations
    BEACON Center for the Study of Evolution in Action, Michigan State University, East Lansing, Michigan 48824

    Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824
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  • Kelley W. Moremen
    Affiliations
    Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602
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  • Yong-Hui Zheng
    Correspondence
    Supported by a grant from the “948” plan, Ministry of Agriculture, China. To whom correspondence should be addressed: Rm. 2215, 567 Wilson Rd., East Lansing, MI 48824. Tel.: 517-884-5314; Fax: 517-353-8957.
    Affiliations
    Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Harbin, 150001, China

    BEACON Center for the Study of Evolution in Action, Michigan State University, East Lansing, Michigan 48824

    Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants AI063944 (to Y.-H. Z) and P41GM103390, GM47533, and DK075322 (to K. W. M.). The authors declare that they have no conflicts of interest with the contents of this article.
    1 Both authors contributed equally to this work.
Open AccessPublished:July 23, 2015DOI:https://doi.org/10.1074/jbc.M115.675207
      Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) α-mannosidases, ERManI, and ER-degradation enhancing α-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four α-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 α-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process.

      Introduction

      Viral Env
      The abbreviations used are: Env
      envelope
      ER
      endoplasmic reticulum
      ERAD
      ER-associated protein degradation
      CAZy
      carbohydrate-active enZyme
      ERManI
      endoplasmic reticulum class I α-mannosidase
      GH47
      glycoside hydrolase family 47
      EDEM
      ER-degradation enhancing α-mannosidase-like
      NKR
      CEM.NKR
      TSPO
      mitochondrial translocator protein
      gRNA
      guide RNA
      KIF
      kifunensine
      CRISPR
      clustered, regularly interspaced, short palindromic repeat
      Cas9
      CRISPR-associated-9
      A3A
      APOBEC3A
      NHK
      null Hong Kong
      DPS
      decapeptide sequence.
      glycoproteins bind to receptors and mediate the entry of virions into cells to initiate infection. Unlike viral structural and enzymatic proteins, Env is produced through the host secretory pathway, where Env is folded into a natural conformation in the ER and delivered to the cell surface (
      • Checkley M.A.
      • Luttge B.G.
      • Freed E.O.
      HIV-1 envelope glycoprotein biosynthesis, trafficking, and incorporation.
      ). Notably, the efficiency of HIV-1 Env folding is very low: almost 85% Env proteins are retained in the ER and degraded (
      • Fennie C.
      • Lasky L.A.
      Model for intracellular folding of the human immunodeficiency virus type 1 gp120.
      ,
      • Hallenberger S.
      • Tucker S.P.
      • Owens R.J.
      • Bernstein H.B.
      • Compans R.W.
      Secretion of a truncated form of the human immunodeficiency virus type 1 envelope glycoprotein.
      ,
      • Willey R.L.
      • Bonifacino J.S.
      • Potts B.J.
      • Martin M.A.
      • Klausner R.D.
      Biosynthesis, cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160.
      ). The degradation mechanism remained unknown until we recently demonstrated that Env is targeted to the ERAD pathway for degradation (
      • Zhou T.
      • Dang Y.
      • Zheng Y.H.
      The mitochondrial translocator protein, TSPO, inhibits HIV-1 envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway.
      ). ERAD is a host quality control mechanism for protein folding (
      • Meusser B.
      • Hirsch C.
      • Jarosch E.
      • Sommer T.
      ERAD: the long road to destruction.
      ). It specifically delivers misfolded proteins to the SEL1L-containing translocon pore complex on the ER membrane and elicits their retro-translocation to the cytoplasm and subsequent degradation by the ubiquitin/proteasome system.
      Class I α-mannosidases belong to the carbohydrate-active enZymes (CAZy) GH47 (
      • Henrissat B.
      • Davies G.
      Structural and sequence-based classification of glycoside hydrolases.
      ), which consists of seven members: ERManI, EDEM1, EDEM2, EDEM3, and Golgi mannosidase IA, IB, and IC (
      • Mast S.W.
      • Moremen K.W.
      Family 47 α-mannosidases in N-glycan processing.
      ). Although the enzymatic activity of EDEM1, EDEM2, and EDEM3 has not been demonstrated in vitro, the others specifically cleave the α1,2-linked mannose residues during protein N-glycosylation. In addition, they also play an important role in the ERAD pathway.
      N-Glycosylation involves a number of enzymes and chaperones in the ER and requires the dedicated ERAD pathway to server as surveillance system. When nascent glycoprotein precursors enter the ER lumen, they are covalently modified with pre-assembled oligosaccharides on Asn residues in a consensus Asn-X-(Ser/Thr) motif (
      • Helenius A.
      • Aebi M.
      Roles of N-linked glycans in the endoplasmic reticulum.
      ). The N-linked oligosaccharides contain 14 sugars consisting of 2 N-acetylglucosamine (GlcNAc), 9 mannose (Man, 4 are α1,2-linked), and 3 terminal glucose (Glc) residues distributed on three extended Man branches A, B, and C (Fig. 1). The sequential removal of the two outermost Glc residues on branch A by glucosidases I and II allows client proteins to interact with ER chaperones calnexin and calreticulin. In conjunction with other chaperones and thiol-disulfide oxidoreductases, precursors are folded and oligomerized into native proteins. During this process, ERManI cleaves the outermost Man residue on branch B on native proteins (Fig. 1). After further removal of the last Glc residue on branch A by glucosidase II, native glycoproteins are released from calnexin/calreticulin and transported to their final destinations. Noticeably, the glycoprotein folding in the ER is error-prone. If glycoproteins display non-native conformation, they are then reglucosylated by the UDP-Glc:unfolded glycoprotein glucosyltransferase and subject to additional rounds of re-engagement with the chaperone machinery until folding is achieved. However, if proteins are terminally misfolded, accumulation of misfolded proteins activates the unfolded protein response. Misfolded proteins are then guided to the ERAD pathway for degradation.
      Figure thumbnail gr1
      FIGURE 1Schematic presentation of the N-linked core oligosaccharide structure. The core is composed of two N-acetylglucosamine (GlcNAc, blue squares), nine mannose (Man, green circles), and three glucose (Glc, red circles) residues. A, B, and C are three oligosaccharide branches. The ERManI preferred cleavage site is indicated.
      ERManI and EDEM1 play an indispensable role in ERAD. Genetic knock-out of the ERManI gene MAN1B1 orthologue Mns1p and EDEM1 orthologue Htm1p in Saccharomyces cerevisiae showed a clear involvement of these two genes in this pathway (
      • Jakob C.A.
      • Bodmer D.
      • Spirig U.
      • Battig P.
      • Marcil A.
      • Dignard D.
      • Bergeron J.J.
      • Thomas D.Y.
      • Aebi M.
      Htm1p, a mannosidase-like protein, is involved in glycoprotein degradation in yeast.
      ,
      • Jakob C.A.
      • Burda P.
      • Roth J.
      • Aebi M.
      Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure.
      ). In mammalian cells an inhibition of ERAD is achieved by inhibiting the CAZy GH47 α-mannosidase activity with kifunensine or by small interfering RNA-mediated gene knockdown (
      • Avezov E.
      • Frenkel Z.
      • Ehrlich M.
      • Herscovics A.
      • Lederkremer G.Z.
      Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5–6GlcNAc2 in glycoprotein ER-associated degradation.
      ,
      • Hosokawa N.
      • Tremblay L.O.
      • You Z.
      • Herscovics A.
      • Wada I.
      • Nagata K.
      Enhancement of endoplasmic reticulum (ER) degradation of misfolded null Hong Kong α1-antitrypsin by human ER mannosidase I.
      ,
      • Wu Y.
      • Swulius M.T.
      • Moremen K.W.
      • Sifers R.N.
      Elucidation of the molecular logic by which misfolded α1-antitrypsin is preferentially selected for degradation.
      ). In addition, both ERManI and EDEM1 accelerate misfolded glycoprotein degradation in a dose-dependent manner (
      • Hosokawa N.
      • Tremblay L.O.
      • You Z.
      • Herscovics A.
      • Wada I.
      • Nagata K.
      Enhancement of endoplasmic reticulum (ER) degradation of misfolded null Hong Kong α1-antitrypsin by human ER mannosidase I.
      ,
      • Wu Y.
      • Swulius M.T.
      • Moremen K.W.
      • Sifers R.N.
      Elucidation of the molecular logic by which misfolded α1-antitrypsin is preferentially selected for degradation.
      ,
      • Hosokawa N.
      • Wada I.
      • Hasegawa K.
      • Yorihuzi T.
      • Tremblay L.O.
      • Herscovics A.
      • Nagata K.
      A novel ER α-mannosidase-like protein accelerates ER-associated degradation.
      ). It has been suggested that EDEM1 extracts misfolded proteins from the calnexin/calreticulin cycle (
      • Molinari M.
      • Calanca V.
      • Galli C.
      • Lucca P.
      • Paganetti P.
      Role of EDEM in the release of misfolded glycoproteins from the calnexin cycle.
      ,
      • Oda Y.
      • Hosokawa N.
      • Wada I.
      • Nagata K.
      EDEM as an acceptor of terminally misfolded glycoproteins released from calnexin.
      ), and misfolded proteins are targeted to the ER-derived quality control compartment where ERManI is enriched (
      • Avezov E.
      • Frenkel Z.
      • Ehrlich M.
      • Herscovics A.
      • Lederkremer G.Z.
      Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5–6GlcNAc2 in glycoprotein ER-associated degradation.
      ,
      • Benyair R.
      • Ogen-Shtern N.
      • Mazkereth N.
      • Shai B.
      • Ehrlich M.
      • Lederkremer G.Z.
      Mammalian ER mannosidase I resides in quality control vesicles, where it encounters its glycoprotein substrates.
      ). Although ERManI prefers to cleave the outermost Man residue on branch B, it may continue to cleave the other α1,2-linked Man residues on branches A and C under conditions of overexpression (
      • Herscovics A.
      • Romero P.A.
      • Tremblay L.O.
      The specificity of the yeast and human class I ER α1,2-mannosidases involved in ER quality control is not as strict previously reported.
      ). Thus, ERManI and possibly the EDEM proteins may catalyze more extensive demannosylation, which constitutes a signal of protein misfolding, resulting in misfolded proteins being degraded via ERAD.
      Recently, we reported that the mitochondrial translocator protein TSPO induces HIV-1 Env glycoprotein degradation via ERAD in the human CD4+ T cell line CEM.NKR (NKR), resulting in a potent HIV-1 restriction (
      • Zhou T.
      • Dang Y.
      • Zheng Y.H.
      The mitochondrial translocator protein, TSPO, inhibits HIV-1 envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway.
      ). TSPO associates with the mitochondrial permeability transition pore complex by interacting with one of its component, the voltage-dependent anion channel protein (
      • McEnery M.W.
      • Snowman A.M.
      • Trifiletti R.R.
      • Snyder S.H.
      Isolation of the mitochondrial benzodiazepine receptor: association with the voltage-dependent anion channel and the adenine nucleotide carrier.
      ). Mitochondrial permeability transition pore establishes the mitochondrial transmembrane potential (Δψm), which allows carrier proteins to exchange small molecules between the mitochondrial matrix and cytoplasm for energy production and controls the integrity of the mitochondrial membrane (
      • Zamzami N.
      • Kroemer G.
      The mitochondrion in apoptosis: how Pandora's box opens.
      ). The goal of this study was to elucidate how HIV-1 Env is degraded via the ERAD pathway, and we identified ERManI as a critical initiator for the Env degradation, resulting in inhibition of HIV-1 replication.

      Discussion

      In this report we studied the molecular mechanism of TSPO-induced HIV-1 Env degradation via ERAD and identified ERManI as a critical initiator for the degradation. Env is expressed through the classical secretory pathway, in which it needs to be properly folded in the ER (
      • Checkley M.A.
      • Luttge B.G.
      • Freed E.O.
      HIV-1 envelope glycoprotein biosynthesis, trafficking, and incorporation.
      ). The Env folding involves cross-linking of 20 cysteine residues, which is dependent on heavy N-glycosylation and the most oxidizing redox status in the ER (
      • Feige M.J.
      • Hendershot L.M.
      Disulfide bonds in ER protein folding and homeostasis.
      ). It has been suggested that the oxidative protein folding in the ER is controlled by mitochondria, likely via regulating the ER redox status through releasing reactive oxygen species (
      • Yang Y.
      • Song Y.
      • Loscalzo J.
      Regulation of the protein disulfide proteome by mitochondria in mammalian cells.
      ). Intracellular reactive oxygen species is mainly produced by mitochondria as a byproduct from energy production. Indeed, ER contains a specialized subcompartment that is called the mitochondrial-associated ER membrane, which physically connects ER to mitochondria (
      • Simmen T.
      • Lynes E.M.
      • Gesson K.
      • Thomas G.
      Oxidative protein folding in the endoplasmic reticulum: tight links to the mitochondria-associated membrane (MAM).
      ). In mammalian cells, mitochondrial-associated ER membrane is supported by a protein complex consisting of voltage-dependent anion channel and several other proteins (
      • Kornmann B.
      • Walter P.
      ERMES-mediated ER-mitochondria contacts: molecular hubs for the regulation of mitochondrial biology.
      ). As introduced earlier, TSPO is a mitochondrial protein (
      • Dellisanti C.
      TSPO through the crystal looking glass.
      ) that interacts with voltage-dependent anion channel (
      • McEnery M.W.
      • Snowman A.M.
      • Trifiletti R.R.
      • Snyder S.H.
      Isolation of the mitochondrial benzodiazepine receptor: association with the voltage-dependent anion channel and the adenine nucleotide carrier.
      ). We speculate that TSPO overexpression reduces the oxidative redox status in the ER, likely by blocking the mitochondria-ER communication, to interfere with HIV-1 Env folding. Accumulation of misfolded Env then activates unfolded protein response, resulting in recognition of these misfolded Env proteins by ERManI and their degradation via ERAD.
      We found that the catalytic domain of ERManI plays an indispensible role in inhibition of HIV-1 Env expression. The structure of this domain shows an (αα)7-barrel composed of 14 consecutive helices, and Glu-330, Asp-463, and Glu-599 were proposed as potential catalytic residues (
      • Vallee F.
      • Karaveg K.
      • Herscovics A.
      • Moremen K.W.
      • Howell P.L.
      Structural basis for catalysis and inhibition of N-glycan processing class I α1,2-mannosidases.
      ). Mutations of Glu-330, Asp-463, and Glu-599 caused 96.5%, 99.9%, or ∼100% reduction in enzyme efficiency (kcat/Km), respectively (
      • Karaveg K.
      • Moremen K.W.
      Energetics of substrate binding and catalysis by class 1 (glycosylhydrolase family 47) α-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.
      ). In addition, ERManI has two highly conserved cysteine residues Cys-527 and Cys-556, which are also conserved in three other Golgi CAZy GH47 α1,2-mannosidases, IA, IB, and IC, but not in EDEM proteins (
      • Vallee F.
      • Karaveg K.
      • Herscovics A.
      • Moremen K.W.
      • Howell P.L.
      Structural basis for catalysis and inhibition of N-glycan processing class I α1,2-mannosidases.
      ). The formation of a disulfide bond between these residues was demonstrated in the yeast Mns1, which was proposed to stabilize the protein (
      • Lipari F.
      • Herscovics A.
      Role of the cysteine residues in the α1,2-mannosidase involved in N-glycan biosynthesis in Saccharomyces cerevisiae. The conserved Cys-340 and Cys-385 residues form an essential disulfide bond.
      ). Moreover, R334C and E397K mutations are identified in nonsyndromic autosomal-recessive intellectual disability (NS-ARID) patients (
      • Rafiq M.A.
      • Kuss A.W.
      • Puettmann L.
      • Noor A.
      • Ramiah A.
      • Ali G.
      • Hu H.
      • Kerio N.A.
      • Xiang Y.
      • Garshasbi M.
      • Khan M.A.
      • Ishak G.E.
      • Weksberg R.
      • Ullmann R.
      • Tzschach A.
      • Kahrizi K.
      • Mahmood K.
      • Naeem F.
      • Ayub M.
      • Moremen K.W.
      • Vincent J.B.
      • Ropers H.H.
      • Ansar M.
      • Najmabadi H.
      Mutations in the α1,2-mannosidase gene, MAN1B1, cause autosomal-recessive intellectual disability.
      ), and the R334C mutation is also found in the congenital disorders of glycosylation (
      • Rymen D.
      • Peanne R.
      • Millón M.B.
      • Race V.
      • Sturiale L.
      • Garozzo D.
      • Mills P.
      • Clayton P.
      • Asteggiano C.G.
      • Quelhas D.
      • Cansu A.
      • Martins E.
      • Nassogne M.C.
      • Gonçalves-Rocha M.
      • Topaloglu H.
      • Jaeken J.
      • Foulquier F.
      • Matthijs G.
      MAN1B1 deficiency: an unexpected CDG-II.
      ). The E397K mutation was found to reduce the ERManI expression, and the R334C mutation was found to reduce the enzyme efficiency by ∼100% (
      • Rafiq M.A.
      • Kuss A.W.
      • Puettmann L.
      • Noor A.
      • Ramiah A.
      • Ali G.
      • Hu H.
      • Kerio N.A.
      • Xiang Y.
      • Garshasbi M.
      • Khan M.A.
      • Ishak G.E.
      • Weksberg R.
      • Ullmann R.
      • Tzschach A.
      • Kahrizi K.
      • Mahmood K.
      • Naeem F.
      • Ayub M.
      • Moremen K.W.
      • Vincent J.B.
      • Ropers H.H.
      • Ansar M.
      • Najmabadi H.
      Mutations in the α1,2-mannosidase gene, MAN1B1, cause autosomal-recessive intellectual disability.
      ). We created seven ERManI mutants, E330A, R334C, E397K, D463A, C527A, C556A, and E599A, to inactivate these critical residues, and found that they all lost the Env inhibitory activity (Fig. 6B). In addition, we tested the activity of two previously reported catalytic domain deletion mutants, FL-1–240 and FL-1–240/ΔDPS. Although the FL-1–240 mutant still has the activity to trigger NHK degradation, the FL-1–240/ΔDPS mutant does not (
      • Iannotti M.J.
      • Figard L.
      • Sokac A.M.
      • Sifers R.N.
      A Golgi-localized mannosidase (MAN1B1) plays a non-enzymatic gatekeeper role in protein biosynthetic quality control.
      ). Nevertheless, we found that they all lost the Env inhibitory activity (Fig. 6C). Together, these results demonstrate that the catalytic activity and the catalytic domain are required for the ERManI activity. The importance of the catalytic domain was further underscored from our investigation on Env-ERManI interaction. We found that WT ERManI could pull down HIV-1 Env, whereas both FL-1–240 and FL-1–240/ΔDPS mutants could not, suggesting that ERManI interacts with Env, and this interaction is dependent on the catalytic domain (Fig. 6D). Therefore, it is likely that Env cycles between the ER and Golgi and interacts with ERManI in a post-ER compartment, resulting in Env degradation.
      Results from this report point out two remarkable differences in ERAD-mediated degradation of HIV-1 Env and misfolded host glycoproteins. First, although ectopic expression of EDEM proteins is able to accelerate the degradation of NHK and/or misfolded β-secretase (
      • Hosokawa N.
      • Wada I.
      • Hasegawa K.
      • Yorihuzi T.
      • Tremblay L.O.
      • Herscovics A.
      • Nagata K.
      A novel ER α-mannosidase-like protein accelerates ER-associated degradation.
      ,
      • Hirao K.
      • Natsuka Y.
      • Tamura T.
      • Wada I.
      • Morito D.
      • Natsuka S.
      • Romero P.
      • Sleno B.
      • Tremblay L.O.
      • Herscovics A.
      • Nagata K.
      • Hosokawa N.
      EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming.
      ,
      • Mast S.W.
      • Diekman K.
      • Karaveg K.
      • Davis A.
      • Sifers R.N.
      • Moremen K.W.
      Human EDEM2, a novel homolog of family 47 glycosidases, is involved in ER-associated degradation of glycoproteins.
      ,
      • Olivari S.
      • Galli C.
      • Alanen H.
      • Ruddock L.
      • Molinari M.
      A novel stress-induced EDEM variant regulating endoplasmic reticulum-associated glycoprotein degradation.
      ), it is unable to inhibit HIV-1 Env expression (Fig. 4, A and B). Second, although the ERManI catalytic domain is not required for NHK degradation, it is required for the Env degradation. Because the FL-1–240 mutant still triggers the NHK degradation but the FL-1–240/ΔDPS mutant does not, it is suggested that instead of the catalytic domain, the conserved DPS in the stem domain is critical for the NHK degradation (
      • Iannotti M.J.
      • Figard L.
      • Sokac A.M.
      • Sifers R.N.
      A Golgi-localized mannosidase (MAN1B1) plays a non-enzymatic gatekeeper role in protein biosynthetic quality control.
      ). However, because both FL-1–240 and FL-1–240/ΔDPS mutants fail to inhibit HIV-1 Env expression, it is suggested that the catalytic domain is critical for the Env degradation (Fig. 6C). Thus, although both HIV-1 Env and NHK are degraded via ERAD, different downstream signaling cascades could be involved in their degradation. A further understanding of these differences may identify a specific pathway for inhibition of the Env expression and HIV-1 replication.

      Author Contributions

      Y.-H. Z. conceived and coordinated the study and wrote the paper. T. Z. designed, performed, and analyzed the experiments shown in FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5. D. A. F. designed, performed, and analyzed the experiments shown in Fig. 6. Y.-H. Z. and K. W. M. edited the paper. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank Ying Zhang, Changqing Yu, Jiajun Zhou and Matthew A. Wexler for experimental supports. We thank the Hosokawa, Suzuki, and Sifers laboratories for providing expression vectors. We also thank the NIH AIDS Research and Reference Reagent Program for providing the other reagents.

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