Madeleine Zufferey

Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome)

Hereditary major histocompatibility complex (MHC) class II deficiency (or bare lymphocyte syndrome) is a form of severe primary immunodeficiency with a total lack of MHC class II expression. It is due to a defect in the regulation of MHC class II genes. A novel gene was isolated by complementation cloning, using an MHC class II-negative mutant cell line. This gene (CIITA) functions as a transactivator of MHC class II gene expression and restores expression of all MHC class II isotypes in mutant cells. In addition, CIITA fully corrects the MHC class II regulatory defect of cells from patients with bare lymphocyte syndrome. In this disease we have identified a splicing mutation that results in a 24 amino acid deletion in CIITA, resulting in loss of function of the transactivator. Hence, the CIITA gene is essential for MHC class II gene expression and has been shown to be responsible for hereditary MHC class II deficiency.

A gene encoding a novel RFX-associated transactivator is mutated in the majority of MHC class II deficiency patients

Major histocompatibility class II (MHC-II) molecules are transmembrane proteins that have a central role in development and control of the immune system. They are encoded by a multi-gene family and their expression is tightly regulated. MHC-II deficiency (OMIM 209920) is an autosomal recessive immunodeficiency syndrome resulting from defects in trans-acting factors essential for transcription of MHC-II genes. There are four genetic complementation groups (A, B, C and D), reflecting the existence of four MHC-II regulators. The factors defective in groups A (CIITA), C (RFX5) and D (RFXAP) have been identified. CIITA is a non-DNA-binding co-activator that controls the cell-type specificity and inducibility of MHC-II expression. RFX5 and RFXAP are two subunits of RFX, a multi-protein complex that binds the X box motif of MHC-II promoters. Mutations in the genes encoding RFX5 (RFX5) or RFXAP (RFXAP) abolish binding of RFX (Refs 7,12). Similar to groups C and D, group B is characterized by a defect in RFX binding, and although it accounts for the majority of patients, the factor defective in group B has remained unknown. We report here the isolation of RFX by a novel single-step DNA-affinity purification approach and the identification of RFXANK, the gene encoding a third subunit of RFX. RFXANK restores MHC-II expression in cell lines from patients in group B and is mutated in these patients. RFXANK contains a protein-protein interaction region consisting of three ankyrin repeats. Its interaction with RFX5 and RFXAP is essential for binding of the RFX complex to MHC-II promoters.

HIV-1 Vpu neutralizes the antiviral factor tetherin/BST-2 by binding it and directing its Beta-TrCP2-dependent degradation

Host cells impose a broad range of obstacles to the replication of retroviruses. Tetherin (also known as CD317, BST-2 or HM1.24) impedes viral release by retaining newly budded HIV-1 virions on the surface of cells. HIV-1 Vpu efficiently counteracts this restriction. Here, we show that HIV-1 Vpu induces the depletion of tetherin from cells. We demonstrate that this phenomenon correlates with the ability of Vpu to counteract the antiviral activity of both overexpressed and interferon-induced endogenous tetherin. In addition, we show that Vpu co-immunoprecipitates with tetherin and β-TrCP in a tri-molecular complex. This interaction leads to Vpu-mediated proteasomal degradation of tetherin in a β-TrCP2-dependent manner. Accordingly, in conditions where Vpu-β-TrCP2-tetherin interplay was not operative, including cells stably knocked down for β-TrCP2 expression or cells expressing a dominant negative form of β-TrCP, the ability of Vpu to antagonize the antiviral activity of tetherin was severely impaired. Nevertheless, tetherin degradation did not account for the totality of Vpu-mediated counteraction against the antiviral factor, as binding of Vpu to tetherin was sufficient for a partial relief of the restriction. Finally, we show that the mechanism used by Vpu to induce tetherin depletion implicates the cellular ER-associated degradation (ERAD) pathway, which mediates the dislocation of ER membrane proteins into the cytosol for subsequent proteasomal degradation. In conclusion, we show that Vpu interacts with tetherin to direct its β-TrCP2-dependent proteasomal degradation, thereby alleviating the blockade to the release of infectious virions. Identification of tetherin binding to Vpu provides a potential novel target for the development of drugs aimed at inhibiting HIV-1 replication.

RFXAP, a novel subunit of the RFX DNA binding complex is mutated in MHC class II deficiency.

Major Histocompatibility Complex class II (MHC‐II) deficiency is a disease of gene regulation that provides a unique opportunity for the genetic dissection of the molecular mechanisms controlling transcription of MHC‐II genes. Cell lines from MHC‐II deficiency patients have been assigned to three complementation groups (A, B and C) believed to reflect the existence of distinct essential MHC‐II regulatory genes. Groups B and C, as well as an in vitro generated regulatory mutant representing a fourth group (D), are characterized by a specific defect in the binding activity of RFX, a multimeric DNA binding complex that is essential for activation of MHC‐II promoters. RFX5, a subunit of RFX, was recently shown to be mutated in group C. We have now isolated a novel gene, RFXAP (RFX Associated Protein), that encodes a second subunit of the RFX complex. RFXAP is mutated in the 6.1.6 cell line (group D), as well as in an MHC‐II deficiency patient (DA). This establishes that group D is indeed a fourth MHC‐II deficiency complementation group. Complementation of the 6.1.6 and DA cell lines by transfection with RFXAP fully restores expression of all endogenous MHC‐II genes in vivo, demonstrating that RFXAP is a novel essential MHC‐II regulatory gene.

Expression of the Three Human Major Histocompatibility Complex Class II Isotypes Exhibits a Differential Dependence on the Transcription Factor RFXAP

ABSTRACT Major histocompatibility complex class II (MHCII) molecules play a pivotal role in the immune system because they direct the development and activation of CD4+ T cells. There are three human MHCII isotypes, HLA-DR, HLA-DQ, and HLA-DP. Key transcription factors controlling MHCII genes have been identified by virtue of the fact that they are mutated in a hereditary immunodeficiency resulting from a lack of MHCII expression. RFXAP—one of the factors affected in this disease—is a subunit of RFX, a DNA-binding complex that recognizes the X box present in all MHCII promoters. To facilitate identification of conserved regions in RFXAP, we isolated the mouse gene. We then delimited conserved domains required to restore endogenous MHCII expression in cell lines lacking a functionalRFXAP gene. Surprisingly, we found that 80% of RFXAP is dispensable for the reactivation of DR expression. Only a short C-terminal segment of the protein is essential for this isotype. In contrast, optimal expression of DQ and DP requires a larger C-terminal segment. These results define an RFXAP domain with an MHCII isotype-specific function. Expression of the three MHCII isotypes exhibits a differential requirement for this domain. We show that this is due to a differential dependence on this domain for promoter occupation and recruitment of the coactivator CIITA in vivo.

HIV-1 capsid is involved in post-nuclear entry steps

BackgroundHIV-1 capsid influences viral uncoating and nuclear import. Some capsid is detected in the nucleus but it is unclear if it has any function. We reported that the antibiotic Coumermycin-A1 (C-A1) inhibits HIV-1 integration and that a capsid mutation confers resistance to C-A1, suggesting that capsid might affect post-nuclear entry steps.ResultsHere we report that C-A1 inhibits HIV-1 integration in a capsid-dependent way. Using molecular docking, we identify an extended binding pocket delimited by two adjacent capsid monomers where C-A1 is predicted to bind. Isothermal titration calorimetry confirmed that C-A1 binds to hexameric capsid. Cyclosporine washout assays in Jurkat CD4+ T cells expressing engineered human TRIMCyp showed that C-A1 causes faster and greater escape from TRIMCyp restriction. Sub-cellular fractionation showed that small amounts of capsid accumulated in the nuclei of infected cells and C-A1 reduced the nuclear capsid. A105S and N74D capsid mutant viruses did not accumulate capsid in the nucleus, irrespective of C-A1 treatment. Depletion of Nup153, a nucleoporin located at the nuclear side of the nuclear pore that binds to HIV-1 capsid, made the virus less susceptible to TRIMCyp restriction, suggesting that Nup153 may help maintain some integrity of the viral core in the nucleus. Furthermore C-A1 increased binding of CPSF6, a nuclear protein, to capsid.ConclusionsOur results indicate that capsid is involved in post-nuclear entry steps preceding integration.

New functions of the major histocompatibility complex class II-specific transcription factor RFXANK revealed by a high-resolution mutagenesis study

ABSTRACT The transcription factors RFX and CIITA are major players in regulation of the expression of all classical and nonclassical major histocompatibility complex class II (MHC-II) genes. RFX nucleates the formation of a multiprotein complex, called the MHC-II enhanceosome, on MHC-II promoters. Assembly of this enhanceosome is an obligatory step for recruitment of the coactivator CIITA and thus for activation of MHC-II gene transcription. We have analyzed the function of the ankyrin repeat-containing protein RFXANK, which forms the heterotrimeric RFX complex together with RFX5 and RFXAP. We discovered that ANKRA2, the closest paralogue of RFXANK, can substitute for RFXANK in the activation of MHC-II genes and that this ability is mediated by its ankyrin repeat domain (ARD). This finding provided the basis for a high-resolution structure-function analysis of the ARD of RFXANK, which allowed us to map the RFX5 interaction domain and residues critical for assembly of the RFX complex. We also found that mutations in the fourth ankyrin repeat of RFXANK abolish assembly of the enhanceosome on MHC-II promoters in vivo but not in vitro, suggesting a new role of RFXANK in facilitating promoter occupation in the context of chromatin.

Cyclophilin B Interacts with Sodium-Potassium ATPase and Is Required for Pump Activity in Proximal Tubule Cells of the Kidney

Cyclophilins (Cyps), the intracellular receptors for Cyclosporine A (CsA), are responsible for peptidyl-prolyl cis-trans isomerisation and for chaperoning several membrane proteins. Those functions are inhibited upon CsA binding. Albeit its great benefits as immunosuppressant, the use of CsA has been limited by undesirable nephrotoxic effects, including sodium retention, hypertension, hyperkalemia, interstial fibrosis and progressive renal failure in transplant recipients. In this report, we focused on the identification of novel CypB-interacting proteins to understand the role of CypB in kidney function and, in turn, to gain further insight into the molecular mechanisms of CsA-induced toxicity. By means of yeast two-hybrid screens with human kidney cDNA, we discovered a novel interaction between CypB and the membrane Na/K-ATPase β1 subunit protein (Na/K-β1) that was confirmed by pull-down, co-immunoprecipitation and confocal microscopy, in proximal tubule-derived HK-2 cells. The Na/K-ATPase pump, a key plasma membrane transporter, is responsible for maintenance of electrical Na+ and K+ gradients across the membrane. We showed that CypB silencing produced similar effects on Na/K-ATPase activity than CsA treatment in HK-2 cells. It was also observed an enrichment of both alpha and beta subunits in the ER, what suggested a possible failure on the maturation and routing of the pump from this compartment towards the plasma membrane. These data indicate that CypB through its interaction with Na/K-β1 might regulate maturation and trafficking of the pump through the secretory pathway, offering new insights into the relationship between cyclophilins and the nephrotoxic effects of CsA.

Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration

HIV-2 and SIVMAC are AIDS-causing, zoonotic lentiviruses that jumped to humans and rhesus macaques, respectively, from SIVSM-bearing sooty mangabey monkeys. Cross-species transmission events such as these sometimes necessitate virus adaptation to species-specific, host restriction factors such as TRIM5. Here, a new human restriction activity is described that blocks viruses of the SIVSM/SIVMAC/HIV-2 lineage. Human T, B, and myeloid cell lines, peripheral blood mononuclear cells and dendritic cells were 4 to >100-fold less transducible by VSV G-pseudotyped SIVMAC, HIV-2, or SIVSM than by HIV-1. In contrast, transduction of six epithelial cell lines was equivalent to that by HIV-1. Substitution of HIV-1 CA with the SIVMAC or HIV-2 CA was sufficient to reduce HIV-1 transduction to the level of the respective vectors. Among such CA chimeras there was a general trend such that CAs from epidemic HIV-2 Group A and B isolates were the most infectious on human T cells, CA from a 1° sooty mangabey isolate was the least infectious, and non-epidemic HIV-2 Group D, E, F, and G CAs were in the middle. The CA-specific decrease in infectivity was observed with either HIV-1, HIV-2, ecotropic MLV, or ALV Env pseudotypes, indicating that it was independent of the virus entry pathway. As2O3, a drug that suppresses TRIM5-mediated restriction, increased human blood cell transduction by SIVMAC but not by HIV-1. Nonetheless, elimination of TRIM5 restriction activity did not rescue SIVMAC transduction. Also, in contrast to TRIM5-mediated restriction, the SIVMAC CA-specific block occurred after completion of reverse transcription and the formation of 2-LTR circles, but before establishment of the provirus. Transduction efficiency in heterokaryons generated by fusing epithelial cells with T cells resembled that in the T cells, indicative of a dominant-acting SIVMAC restriction activity in the latter. These results suggest that the nucleus of human blood cells possesses a restriction factor specific for the CA of HIV-2/SIVMAC/SIVSM and that cross-species transmission of SIVSM to human T cells necessitated adaptation of HIV-2 to this putative restriction factor.

Cyclosporine blocks incorporation of HIV-1 envelope glycoprotein into virions.

ABSTRACT Cyclosporine (CsA) decreases HIV-1 infectivity by blocking HIV-1 capsid (CA) interaction with target cell cyclophilin A (CypA). Yet, HIV-1 virions produced in the presence of CsA also exhibit decreased infectivity that was previously shown to be independent of the well-characterized HIV-1 CA-CypA interaction. Here, we demonstrate that CsA decreases gp120 and gp41 incorporation into HIV-1 virions and that the fusion of these virions with susceptible target cells is impaired. This effect was not observed with HIV-1 virions pseudotyped with the vesicular stomatitis virus glycoprotein or with the amphotropic envelope protein of murine leukemia virus. It was independent of calcineurin signaling, the endoplasmic reticulum luminal protein cyclophilin B, and the long cytoplasmic tail of gp41. Thus, cyclosporine blocks HIV-1 infectivity via two independent mechanisms, the first involving HIV-1 CA in target cells and the second involving HIV-1 Env in producer cells.