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1.
Retrovirology ; 15(1): 25, 2018 03 14.
Artículo en Inglés | MEDLINE | ID: mdl-29540207

RESUMEN

BACKGROUND: NSC260594, a quinolinium derivative from the NCI diversity set II compound library, was previously identified in a target-based assay as an inhibitor of the interaction between the HIV-1 (ψ) stem-loop 3 (SL3) RNA and Gag. This compound was shown to exhibit potent antiviral activity. Here, the effects of this compound on individual stages of the viral lifecycle were examined by qRT-PCR, ELISA and Western blot, to see if its actions were specific to the viral packaging stage. The structural effects of NSC260594 binding to the HIV-1 gRNA were also examined by SHAPE and dimerization assays. RESULTS: Treatment of cells with NSC260594 did not reduce the number of integration events of incoming virus, and treatment of virus producing cells did not affect the level of intracellular Gag protein or viral particle release as determined by immunoblot. However, NSC260594 reduced the incorporation of gRNA into virions by up to 82%, without affecting levels of gRNA inside the cell. This reduction in packaging correlated closely with the reduction in infectivity of the released viral particles. To establish the structural effects of NSC260594 on the HIV-1 gRNA, we performed SHAPE analyses to pinpoint RNA structural changes. NSC260594 had a stabilizing effect on the wild type RNA that was not confined to SL3, but that was propagated across the structure. A packaging mutant lacking SL3 did not show this effect. CONCLUSIONS: NSC260594 acts as a specific inhibitor of HIV-1 RNA packaging. No other viral functions are affected. Its action involves preventing the interaction of Gag with SL3 by stabilizing this small RNA stem-loop which then leads to stabilization of the global packaging signal region (psi or ψ). This confirms data, previously only shown in analyses of isolated SL3 oligonucleotides, that SL3 is structurally labile in the presence of Gag and that this is critical for the complete psi region to be able to adopt different conformations. Since replication is otherwise unaffected by NSC260594 the flexibility of SL3 appears to be a unique requirement for genome encapsidation and identifies this process as a highly specific drug target. This study is proof of principle that development of a new class of antiretroviral drugs that specifically target viral packaging by binding to the viral genomic RNA is achievable.


Asunto(s)
Genoma Viral , Infecciones por VIH/virología , VIH-1/fisiología , Conformación de Ácido Nucleico , ARN Viral/genética , Ensamble de Virus , Regiones no Traducidas 5' , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Inestabilidad Genómica , Humanos , Unión Proteica , Provirus/genética , ARN Viral/química , Reacción en Cadena en Tiempo Real de la Polimerasa , Carga Viral , Integración Viral , Liberación del Virus
3.
Environ Sci Pollut Res Int ; 30(15): 45369-45387, 2023 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-36705835

RESUMEN

Green innovation cooperation (GIC) has become an important measure for sustainable development of enterprises. Based on the knowledge-based theory, knowledge interaction is a necessary mode of GIC. Exploring the influencing factors of knowledge interaction in green innovation cooperation is an important aspect to improve the performance of GIC. From the perspective of ecological theory, taking ecological relations as the lens, this paper further analyzes the differences of different ecological relations (competitive relationship, mutualism relationship, commensalism relationship, and parasitism relationship) affecting knowledge interaction in GIC. The results show that there are significant differences between different ecological relationships and knowledge interaction in GIC. Mutualism and commensalism contribute to knowledge interaction, while competition and parasitism may lead to the weakening of knowledge interaction and even the breaking of knowledge connection. In addition, the willingness to cooperate in green innovation is conducive to the knowledge interaction of GIC. Therefore, the paper proposes that green innovation partners should be selected based on ecological relationship. This paper reveals the theoretical connection between ecological relationship and knowledge interaction, provides valuable insights for promoting knowledge interaction in GIC, and expands the influencing factors of knowledge interaction.


Asunto(s)
Ecología , Conocimiento , Desarrollo Sostenible , China , Simbiosis
4.
J Clin Virol ; 169: 105615, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-37948983

RESUMEN

BACKGROUND: The National Health Service (NHS) in England commissioned opt-out testing in London Emergency Departments (ED) in April 2022 to allow early identification and management of hepatitis B (HBV) and hepatitis C virus (HCV) infection in patients unaware of their infection status. METHODS: All adults over the age of 16 undergoing blood tests in the ED at the Royal Free Hospital were tested for HBV surface antigen and anti-HCV IgG unless they opted out. Data was collected between the 12th of April and 22nd of August 2022. OUTCOME: Of 11,215 patients tested for HCV, 164 patients were found to be anti-HCV IgG positive, giving a seroprevalence rate of 1.46 %. 52 of the anti-HCV IgG positive patients did not have any previous HCV serology result. 23 of the anti-HCV IgG positive patients were also HCV RNA positive giving an RNA seroprevalence of 0.21 %, and 17 of those were new diagnoses of HCV viraemia. For HBV testing, 82 (0.73 %) out of 11,192 patients tested were found to be HBsAg positive, including one patient who presented acutely with a positive HBV core IgM. 39 of the HBsAg positive patients were previously unknown to us; of these, 9 had an HBV viral load of more than 2000 IU/mL, including 3 patients with positive HBV e antigen and one patient with hepatitis D virus co-infection. CONCLUSION: Opt-out screening of HBV and HCV in ED is effective at identifying patients with previously undiagnosed viral hepatitis infection and providing an opportunity to engage them in specialist care.


Asunto(s)
Infecciones por VIH , Hepatitis B , Hepatitis C , Adulto , Humanos , Antígenos de Superficie de la Hepatitis B , Estudios Seroepidemiológicos , Londres/epidemiología , Medicina Estatal , Hepatitis B/diagnóstico , Hepatitis B/epidemiología , Hepatitis C/diagnóstico , Hepatitis C/epidemiología , Hepacivirus/genética , Virus de la Hepatitis B/genética , Servicio de Urgencia en Hospital , Hospitales de Enseñanza , Anticuerpos contra la Hepatitis C , ARN , Inmunoglobulina G
5.
Viruses ; 13(12)2021 11 29.
Artículo en Inglés | MEDLINE | ID: mdl-34960658

RESUMEN

HIV-1 packages two copies of its gRNA into virions via an interaction with the viral structural protein Gag. Both copies and their native RNA structure are essential for virion infectivity. The precise stepwise nature of the packaging process has not been resolved. This is largely due to a prior lack of structural techniques that follow RNA structural changes within an RNA-protein complex. Here, we apply the in-gel SHAPE (selective 2'OH acylation analysed by primer extension) technique to study the initiation of HIV-1 packaging, examining the interaction between the packaging signal RNA and the Gag polyprotein, and compare it with that of the NC domain of Gag alone. Our results imply interactions between Gag and monomeric packaging signal RNA in switching the RNA conformation into a dimerisation-competent structure, and show that the Gag-dimer complex then continues to stabilise. These data provide a novel insight into how HIV-1 regulates the translation and packaging of its genome.


Asunto(s)
Infecciones por VIH/virología , VIH-1/fisiología , Ensamble de Virus , Genoma Viral , VIH-1/química , VIH-1/genética , Humanos , Conformación de Ácido Nucleico , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo
6.
Nat Struct Mol Biol ; 28(3): 278-289, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33633400

RESUMEN

Trim-Away is a recently developed technology that exploits off-the-shelf antibodies and the RING E3 ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically activated upon target engagement, either during its normal immune function or when repurposed for targeted protein degradation, is unknown. Here we show that a mechanism of target-induced clustering triggers intermolecular dimerization of the RING domain to switch on the ubiquitination activity of TRIM21 and induce virus neutralization or drive Trim-Away. We harness this mechanism for selective degradation of disease-causing huntingtin protein containing long polyglutamine tracts and expand the Trim-Away toolbox with highly active TRIM21-nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has implications for targeted protein degradation technologies.


Asunto(s)
Proteolisis , Ribonucleoproteínas/metabolismo , Ubiquitinación , Animales , Biocatálisis , Línea Celular , Drosophila melanogaster/citología , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Ratones , Modelos Moleculares , Optogenética , Péptidos/metabolismo , Unión Proteica , Multimerización de Proteína , Ribonucleoproteínas/química , Ubiquitina-Proteína Ligasas/metabolismo
7.
Elife ; 82019 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-31613747

RESUMEN

The genetic basis of most human disease cannot be explained by common variants. One solution to this 'missing heritability problem' may be rare missense variants, which are individually scarce but collectively abundant. However, the phenotypic impact of rare variants is under-appreciated as gene function is normally studied in the context of a single 'wild-type' sequence. Here, we explore the impact of naturally occurring missense variants in the human population on the cytosolic antibody receptor TRIM21, using volunteer cells with variant haplotypes, CRISPR gene editing and functional reconstitution. In combination with data from a panel of computational predictors, the results suggest that protein robustness and purifying selection ensure that function is remarkably well-maintained despite coding variation.


Asunto(s)
Adenoviridae/química , Anticuerpos Antivirales/química , Mutación Missense , Polimorfismo Genético , Ribonucleoproteínas/química , Adenoviridae/genética , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Sitios de Unión , Biología Computacional/métodos , Secuencia Conservada , Expresión Génica , Células HEK293 , Haplotipos , Células HeLa , Voluntarios Sanos , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Ribonucleoproteínas/genética , Ribonucleoproteínas/inmunología , Selección Genética
8.
Nat Commun ; 10(1): 4502, 2019 10 03.
Artículo en Inglés | MEDLINE | ID: mdl-31582740

RESUMEN

The cytosolic antibody receptor TRIM21 possesses unique ubiquitination activity that drives broad-spectrum anti-pathogen targeting and underpins the protein depletion technology Trim-Away. This activity is dependent on formation of self-anchored, K63-linked ubiquitin chains by the heterodimeric E2 enzyme Ube2N/Ube2V2. Here we reveal how TRIM21 facilitates ubiquitin transfer and differentiates this E2 from other closely related enzymes. A tri-ionic motif provides optimally distributed anchor points that allow TRIM21 to wrap an Ube2N~Ub complex around its RING domain, locking the closed conformation and promoting ubiquitin discharge. Mutation of these anchor points inhibits ubiquitination with Ube2N/Ube2V2, viral neutralization and immune signalling. We show that the same mechanism is employed by the anti-HIV restriction factor TRIM5 and identify spatially conserved ionic anchor points in other Ube2N-recruiting RING E3s. The tri-ionic motif is exclusively required for Ube2N but not Ube2D1 activity and provides a generic E2-specific catalysis mechanism for RING E3s.


Asunto(s)
Lisina/metabolismo , Ribonucleoproteínas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación/fisiología , Secuencias de Aminoácidos/genética , Factores de Restricción Antivirales , Biocatálisis , Cristalografía por Rayos X , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Mutación , Resonancia Magnética Nuclear Biomolecular , Unión Proteica/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitina-Proteína Ligasas/metabolismo
9.
Cell Host Microbe ; 24(6): 761-775.e6, 2018 12 12.
Artículo en Inglés | MEDLINE | ID: mdl-30503508

RESUMEN

TRIM5 is a RING domain E3 ubiquitin ligase with potent antiretroviral function. TRIM5 assembles into a hexagonal lattice on retroviral capsids, causing envelopment of the infectious core. Concomitantly, TRIM5 initiates innate immune signaling and orchestrates disassembly of the viral particle, yet how these antiviral responses are regulated by capsid recognition is unclear. We show that hexagonal assembly triggers N-terminal polyubiquitination of TRIM5 that collectively drives antiviral responses. In uninfected cells, N-terminal monoubiquitination triggers non-productive TRIM5 turnover. Upon TRIM5 assembly on virus, a trivalent RING arrangement allows elongation of N-terminally anchored K63-linked ubiquitin chains (N-K63-Ub). N-K63-Ub drives TRIM5 innate immune stimulation and proteasomal degradation. Inducing ubiquitination before TRIM5 assembly triggers premature degradation and ablates antiviral restriction. Conversely, driving N-K63 ubiquitination after TRIM5 assembly enhances innate immune signaling. Thus, the hexagonal geometry of TRIM5's antiviral lattice converts a capsid-binding protein into a multifunctional antiviral platform.


Asunto(s)
Proteínas Portadoras/metabolismo , Inmunidad Innata/inmunología , Infecciones por Retroviridae/inmunología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Factores de Restricción Antivirales , Cápside/química , Cápside/metabolismo , Proteínas Portadoras/genética , Células HEK293 , Humanos , Virus de la Leucemia Murina/enzimología , Virus de la Leucemia Murina/genética , Virus de la Leucemia Murina/inmunología , Ratones , Ratones Endogámicos C57BL , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/genética , Polisacáridos Bacterianos/metabolismo , Infecciones por Retroviridae/metabolismo , Infecciones por Retroviridae/virología , Células THP-1 , Proteínas de Motivos Tripartitos , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética
10.
Elife ; 72018 04 18.
Artículo en Inglés | MEDLINE | ID: mdl-29667579

RESUMEN

Cell surface Fc receptors activate inflammation and are tightly controlled to prevent autoimmunity. Antibodies also simulate potent immune signalling from inside the cell via the cytosolic antibody receptor TRIM21, but how this is regulated is unknown. Here we show that TRIM21 signalling is constitutively repressed by its B-Box domain and activated by phosphorylation. The B-Box occupies an E2 binding site on the catalytic RING domain by mimicking E2-E3 interactions, inhibiting TRIM21 ubiquitination and preventing immune activation. TRIM21 is derepressed by IKKß and TBK1 phosphorylation of an LxxIS motif in the RING domain, at the interface with the B-Box. Incorporation of phosphoserine or a phosphomimetic within this motif relieves B-Box inhibition, promoting E2 binding, RING catalysis, NF-κB activation and cytokine transcription upon infection with DNA or RNA viruses. These data explain how intracellular antibody signalling is regulated and reveal that the B-Box is a critical regulator of RING E3 ligase activity.


Asunto(s)
Regulación de la Expresión Génica , Procesamiento Proteico-Postraduccional , Receptores Fc/metabolismo , Ribonucleoproteínas/metabolismo , Transducción de Señal , Animales , Línea Celular , Humanos , Quinasa I-kappa B/metabolismo , Ratones , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo
11.
Atherosclerosis ; 211(1): 216-23, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20303493

RESUMEN

OBJECTIVE: Mitofusin 2 (Mfn2) is an important suppressor of vascular smooth muscle cell (VSMC) proliferation. It contains a protein kinase A (PKA) phosphorylation site at serine 442 (S442) and can be phosphorylated by PKA. This study examined the role of phosphorylating specific sites on the regulation of Mfn2 protein activity in vitro and in vivo. METHODS AND RESULTS: We introduced two mutations at S442 in rat Mfn2, and investigated their effects using cultured rat VSMCs and the balloon injury model. Our results indicated that, in VSMCs, Mfn2 expression and mitochondrial morphology are affected by adenoviral-mediated overexpression of the two Mfn2 mutant proteins in the same way as the wild-type Mfn2 protein. Specifically, overexpression of the protein harboring the phospho-deficient mutation Mfn2-S442A (serine replaced by alanine at residue 442) increased the inhibitory effects of Mfn2 on proliferation of VSMCs in culture, and neointimal hyperplasia and restenosis in the rat carotid artery balloon injury model at days 14 after injury. On the other hand, the phospho-mimetic mutation Mfn2-S442D (serine replaced by aspartic acid at residue 442) led to loss of growth suppressor activity. CONCLUSIONS: These results suggest that this specific PKA phosphorylation site plays a key role in Mfn2-mediated suppression of VSMC growth, which is independent of its effects on modulation of mitochondrial morphology.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Músculo Liso Vascular/metabolismo , Sustitución de Aminoácidos , Animales , Cateterismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , GTP Fosfohidrolasas , Masculino , Proteínas de la Membrana/fisiología , Mitocondrias/genética , Mitocondrias/ultraestructura , Proteínas Mitocondriales/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Ratas , Ratas Endogámicas WKY , Transducción de Señal/genética , Túnica Íntima/efectos de los fármacos , Túnica Íntima/patología
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