UMLS:C0021051 - FACTA Search

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Query: UMLS:C0021051 (immunodeficiency)
71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nondenaturing gel electrophoresis was used to study the nucleotide substrate-induced conformational change in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). Dead-end complex was formed between HIV-1 RT, dideoxynucleotide chain-terminated primer, and DNA template in the presence of deoxynucleotide triphosphate (dNTP) complementary to the next position on the template. Complexes which form in the absence of the next complementary dNTP were disrupted by adding excess poly(rA)/oligo(dT) or heparin just prior to electrophoresis. Dead-end complex formation by noncomplementary dNTP's or ribonucleotides was at least 2000-fold less efficient than with the complementary nucleotide. When dA was the next nucleotide on the template, analogues of dTTP supported dead-end complex formation with increased apparent Kd (dTTP < dideoxy-TTP approximately alpha-thio-dTTP < dUTP < 3'-azidothymidine triphosphate). A similar relationship was observed for dGTP analogues across from dC on the template (dGTP < dideoxy-GTP < alpha-thio-dGTP << dITP < dideoxy-ITP). The optimal length of the primer/template duplex region for dead-end complex formation was between 20 and 32 base pairs. Primer-template with a mismatched primer terminus did not support dead-end complex formation, and primer terminated with 3'-azidothymidine formed dead-end complex with 25-fold elevated apparent Kd. By contrast, dead-end complex formation on primer terminated with dideoxy-IMP base paired with dC on the template was more efficient than on primer terminated with dideoxy-GMP. Implications for the mechanisms of discrimination between nucleotide analogues by HIV-1 RT are discussed.
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PMID:Nucleotide-induced stable complex formation by HIV-1 reverse transcriptase. 915 15

The human immunodeficiency virus type 1 (HIV-1) Rev protein binds to unspliced HIV-1 pre-mRNA and exports it from the nucleus. Rev itself can "shuttle" between the nucleus and cytoplasm. This bi-directional transport is mediated by two specific Rev sequences: a nuclear localisation signal (NLS), which overlaps the RNA-binding domain, and a distinct nuclear export signal (NES). In this study we characterised new monoclonal antibodies that bind different epitopes of Rev, including the import and export sequences. In RNA bandshift assays, we observed that formation of a multimeric complex between Rev and its target RNA completely masks the Rev NLS, whereas the NES remains readily accessible. We then tested for signal-mediated interactions between Rev and different nuclear transport receptors, using mutations in the Rev NES or NLS to control for specificity. Extensive biochemical analyses did not reveal any direct NES-dependent interaction between Rev (free or RNA-bound) and the previously proposed export co-factors, human RIP/Rab and eIF-5A. By contrast, similar tests showed that Rev binds directly via its arginine-rich NLS to the human nuclear import receptor, importin-beta. This interaction was highly specific and was abolished by mutation in the Rev NLS. Importin-beta did not bind to the RNA-bound form of Rev, providing a mechanism to ensure that Rev is imported only following release of its RNA cargo. Unlike many NLS-containing proteins that bind stably to an importin-alpha/beta heterodimer, the binding of Rev to importin-beta was actually blocked by importin-alpha receptor. Our findings suggest that Rev and importin-alpha bind (via an arginine-rich sequence) to a similar region on importin-beta. In addition, we show that the complex between Rev and importin-beta can be dissociated by the nuclear Ran GTPase, but only when Ran is in the GTP-bound form. The series of interactions we describe provide a novel pathway for the import of Rev across the nuclear pore complex, and a mechanism for its release into the nucleoplasm.
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PMID:Interactions between HIV Rev and nuclear import and export factors: the Rev nuclear localisation signal mediates specific binding to human importin-beta. 940 52

The different classes of conventional nuclear localization sequences (NLSs) resemble one another in that NLS-dependent nuclear protein import is energy-dependent and mediated by the cytosolic NLS-binding importin/karyopherin subunits and monomeric GTP-binding protein Ran/TC4. Based on analysis of the nuclear import kinetics mediated by the NLS of the human immunodeficiency virus accessory protein Tat using in vivo and in vitro nuclear transport assays and confocal laser scanning microscopy, we report a novel nuclear import pathway. We demonstrate that the Tat-NLS, not recognized by importin 58/97 subunits as shown using an enzyme-linked immunosorbent assay-based binding assay, is sufficient to target the 476-kDa heterologous beta-galactosidase protein to the nucleus in ATP-dependent but cytosolic factor-independent fashion. Excess SV40 large tumor antigen (T-ag) NLS-containing peptide had no significant effect on the nuclear import kinetics implying that the Tat-NLS was able to confer nuclear accumulation through a pathway distinct from conventional NLS-dependent pathways. Nucleoplasmic accumulation of the Tat-NLS-beta-galactosidase fusion protein, in contrast to that of a T-ag-NLS-containing fusion protein, also occurred in the absence of an intact nuclear envelope, implying that the Tat-NLS conferred binding to nuclear components. This is in stark contrast to known NLSs such as those of T-ag which confer nuclear entry rather than retention. Significantly, the ability to accumulate in the nucleus in the absence of an intact nuclear envelope was blocked in the absence of ATP, as well as by nonhydrolyzable ATP and GTP analogs, demonstrating that ATP is required to effect release from a complex with insoluble cytoplasmic components. Taken together, the results demonstrate that, dependent on ATP for release from cytoplasmic retention, the Tat-NLS is able to confer nuclear entry and binding to nuclear components. These unique properties indicate that Tat accumulates in the nucleus through a novel import pathway.
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PMID:The HIV-1 Tat nuclear localization sequence confers novel nuclear import properties. 943 Jul 4

CGD is a rare inherited immunodeficiency syndrome, caused by the phagocytes' inability to produce (sufficient) reactive oxygen metabolites. This dysfunction is due to a defect in the NADPH oxidase, the enzyme responsible for the production of superoxide. It is composed of several subunits, two of which, gp91phox and p22phox, form the membrane-bound cytochrome b558, while its three cytosolic components, p47phox, p67phox and p40phox, have to translocate to the membrane upon activation. This is a tightly and intricately controlled process that involves, among others, several low-molecular weight GTP-binding proteins. Gp91phox is encoded on the X-chromosome and p22phox, p47phox and p67phox on different autosomal chromosomes, and a defect in one of these components leads to CGD. This explains the variable mode of inheritance seen in this syndrome. Clinically CGD manifests itself typically already at a very young age with recurrent and serious infections, most often caused by catalase-positive pathogens. Modern treatment options, including prophylaxis with trimethoprim-sulfamethoxazole and rIFN-gamma as well as early and aggressive anti-infection therapy, have improved the prognosis of this disease dramatically. CGD, as a very well-characterized inherited affection of the hematopoietic stem cells, is predestined to be among the first diseases to profit from the advances in cutting-edge therapeutics, such as gene therapy and in utero stem cell transplantation.
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PMID:The molecular basis of chronic granulomatous disease. 961 66

The hypothesis that the cellular protein Crm1 mediates human immunodeficiency virus type 1 (HIV-1) Rev-dependent nuclear export posits that Crm1 can directly interact both with the Rev nuclear export signal (NES) and with cellular nucleoporins. Here, we demonstrate that Crm1 is indeed able to interact with active but not defective forms of the HIV-1 Rev NES and of NESs found in other retroviral nuclear export factors. In addition, we demonstrate that Crm1 can bind the Rev NES when Rev is assembled onto the Rev response element RNA target and that Crm1, like Rev, is a nucleocytoplasmic shuttle protein. Crm1 also specifically binds the Rev NES in vitro, although this latter interaction is detectable only in the presence of added Ran . GTP. Overexpression of a truncated, defective form of the nucleoporin Nup214/CAN, termed DeltaCAN, that retains Crm1 binding ability resulted in the effective inhibition of HIV-1 Rev or human T-cell leukemia virus Rex-dependent gene expression. In contrast, DeltaCAN had no significant affect on Mason-Pfizer monkey virus constitutive transport element (MPMV CTE)-dependent nuclear RNA export or on the expression of RNAs dependent on the cellular mRNA export pathway. As a result, DeltaCAN specifically blocked late, but not early, HIV-1 gene expression in HIV-1-infected cells. These data strongly validate Crm1 as a cellular cofactor for HIV-1 Rev and demonstrate that the MPMV CTE nuclear RNA export pathway uses a distinct, Crm1-independent mechanism. In addition, these data identify a novel and highly potent inhibitor of leucine-rich NES-dependent nuclear export.
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PMID:Inhibition of human immunodeficiency virus Rev and human T-cell leukemia virus Rex function, but not Mason-Pfizer monkey virus constitutive transport element activity, by a mutant human nucleoporin targeted to Crm1. 976 2

The Nef proteins of Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) have been shown to associate with several cellular kinases. Further, the ability of SIVmac239 Nef to associate with a p21-activated kinase (PAK)-related kinase has been correlated with pathogenic progression to AIDS in rhesus macaques. Because the ability of Nef to associate with the PAK-related kinase is viral isolate dependent, we reasoned that viral isolates derived from distinct physiological locations may encode Nef proteins that exhibit distinct kinase association profiles. In this study, we compared kinase activities associated with Nef proteins derived from the prototypic lymphocyte-tropic SIVmac239 and a macrophage-tropic, neurovirulent clone, SIV/17E-Fr. Our findings not only support previous studies that have documented the association of SIVmac239 Nef with a PAK-related kinase and a Nef-associated kinase complex (NAKC) but describe a novel serine kinase activity detectable only in conjunction with the Nef protein derived from the neurovirulent clone, SIV/17E-Fr. The latter Nef protein does not associate with PAK, and unlike PAK or NAKC, this novel kinase activity is enhanced in association with nonmyristoylated forms of Nef and can utilize both ATP and GTP as phosphodonors. We also show that at least one substrate for the kinase is Nef itself and demonstrate that the SIV/17E-Fr Nef protein is phosphorylated in SIV-infected cells. These results suggest that the ability to associate with cellular kinases in general may be a conserved feature of Nef, but particular kinase/Nef associations may evolve with changes in the host environment concomitant with viral spread.
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PMID:A novel kinase activity associated with Nef derived from neurovirulent simian immunodeficiency virus. 981 12

Nuclear export of intron-containing human immunodeficiency virus type 1 (HIV-1) RNA is mediated by the viral Rev protein that contains both an RNA binding domain specific for the viral Rev response element (RRE) and a nuclear export signal (NES). The cellular CRM1 (Exportin1) protein functions as a nuclear export receptor for proteins carrying a Rev-like NES in a process that also requires the GTP bound form of the Ran GTPase. Using purified recombinant factors, we show by co-precipitation, gel mobility shift and protein footprinting assays that full-length Rev protein interacts directly with CRM1 in vitro independently of both the integrity of the characteristic leucine residues of the NES and the presence of the cytotoxin leptomycin B (LMB). Addition of RanGTP induces the formation of an RRE-Rev-CRM1-RanGTP complex that is sensitive to LMB, NES mutations, and Ran being charged with GTP. Within this complex, CRM1 is readily cross-linked to Cys89 near the NES of Rev. By protein footprinting, we demonstrate that the NES of Rev and two regions in CRM1 become inaccessible to endoproteinases upon binding suggesting that these regions are involved in protein-protein interactions. Our data are consistent with a model in which CRM1 is the nuclear export receptor for the Rev-RRE ribonucleoprotein complex and that RanGTP binds to a preformed Rev-CRM1 complex and specifies a functional interaction with the NES.
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PMID:The specificity of the CRM1-Rev nuclear export signal interaction is mediated by RanGTP. 983 18

We discovered several novel interactions between proteins involved in Crm1-mediated nuclear export of the nuclear export signal containing human immunodeficiency virus type 1 protein Rev. First, a Rev/Crm1/RanGTP complex (where Ran is Ras-related nuclear protein) reacts with some nucleoporins (Nup42 and Nup159) but not others (NSP1, Nup116, and Nup1), forming a Nup/Crm1/RanGTP complex and concomitantly releasing Rev. Second, RanBP1 (or homologous proteins) can displace Nup and form a ternary RanBP1/RanGTP/Crm1 complex that can be disassembled by RanGAP via GTP hydrolysis. Third, and most surprisingly, RanBP1/RanGTP/Crm1 can be disassembled without GTP hydrolysis by the nucleotide exchange factor RanGEF. Recycling of a Ran/RanGEF complex by GTP and Mg2+ is stimulated by both Crm1 and Rev, allowing reformation of a Rev/Crm1/RanGTP complex. Based on these reactions we propose a model for Crm1-mediated export.
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PMID:Putative reaction intermediates in Crm1-mediated nuclear protein export. 1034 84

The Rho-family GTP-hydrolysing proteins (GTPases), Cdc42, Rac and Rho, act as molecular switches in signalling pathways that regulate cytoskeletal architecture, gene expression and progression of the cell cycle. Cdc42 and Rac transmit many signals through GTP-dependent binding to effector proteins containing a Cdc42/Rac-interactive-binding (CRIB) motif. One such effector, the Wiskott-Aldrich syndrome protein (WASP), is postulated to link activation of Cdc42 directly to the rearrangement of actin. Human mutations in WASP cause severe defects in haematopoletic cell function, leading to clinical symptoms of thrombocytopenia, immunodeficiency and eczema. Here we report the solution structure of a complex between activated Cdc42 and a minimal GTPase-binding domain (GBD) from WASP. An extended amino-terminal GBD peptide that includes the CRIB motif contacts the switch I, beta2 and alpha5 regions of Cdc42. A carboxy-terminal beta-hairpin and alpha-helix pack against switch II. The Phe-X-His-X2-His portion of the CRIB motif and the alpha-helix appear to mediate sensitivity to the nucleotide switch through contacts to residues 36-40 of Cdc42. Discrimination between the Rho-family members is likely to be governed by GBD contacts to the switch I and alpha5 regions of the GTPases. Structural and biochemical data suggest that GBD-sequence divergence outside the CRIB motif may reflect additional regulatory interactions with functional domains that are specific to individual effectors.
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PMID:Structure of Cdc42 in complex with the GTPase-binding domain of the 'Wiskott-Aldrich syndrome' protein. 1036 May 78

Mycophenolate mofetil (MMF), the morpholinoethyl ester of mycophenolic acid (MPA), is currently used as an immunosuppressive agent in kidney transplant recipients. After oral administration, MMF is hydrolysed to MPA, the active compound, which is a potent inhibitor of inosine monophosphate dehydrogenase (IMP-DH). Inhibition of this enzyme results in a depletion of the intracellular GTP and dGTP pools. MPA has been shown to inhibit the replication of a number of viruses, including arena viruses (Junin and Tacaribe), yellow fever virus, reovirus-1, parainfluenza-3 virus, Coxsackie B4 virus, Epstein-Barr virus and human immunodeficiency virus. To examine whether MPA also has an inhibitory effect on HBV replication, experiments were performed using cultures of primary human hepatocytes and HBV-transfected, HepG2 2.2.15 cells. After in vitro infection with HBV in human hepatocytes, HBV covalently-closed-circular (ccc) DNA and HBV mRNAs were detectable in the cells during the 10 days following infection. HBV DNA and hepatitis B surface antigen (HBsAg) were also secreted into the culture medium. In the presence of 10 microg ml-1 MPA (the therapeutic serum level of MPA as an immunosuppressive agent) in culture medium, HBV ccc DNA and HBV mRNAs became undetectable 5 days after treatment was started. The secretion of HBV DNA and HBsAg into the medium was also markedly reduced. No cytotoxic effect of the drug was noted during the experiments. The effect of MPA on HBV replication was abolished by the presence of guanosine (50 microg ml-1). In HepG2 2.2.15 cells (which contain an integrated tandem dimer of the HBV genome), MPA treatment had no significant inhibitory effect on the secretion of HBV DNA and HBsAg into the culture medium. HBV ccc DNA and HBV mRNAs in HepG2 2.2.15 cells were also not affected. The observed effect of MPA on HBV replication in primary human hepatocyte cultures may involve only episomal replication and may have clinical implications, especially before integration of HBV DNA into the host genome.
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PMID:Mycophenolic acid, an immunosuppressive agent, inhibits HBV replication in vitro. 1060 35

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