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1.
J Virol ; 97(11): e0104423, 2023 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-37905837

RESUMO

IMPORTANCE: HIV-infected host cells impose varied degrees of regulation on viral replication, from very high to abortive. Proliferation of HIV in astrocytes is limited when compared to immune cells, such as CD4+ T lymphocytes. Understanding such differential regulation is one of the key questions in the field as these cells permit HIV persistence and rebound viremia, challenging HIV treatment and clinical cure. This study focuses on understanding the molecular mechanism behind such cell-specific disparities. We show that one of the key mechanisms is the regulation of heterogenous nuclear ribonucleoprotein A2, a host factor involved in alternative splicing and RNA processing, by HIV-1 Tat in CD4+ T lymphocytes, not observed in astrocytes. This regulation causes an increase in the levels of unspliced/partially spliced viral RNA and nuclear export of Rev-RNA complexes which results in high viral propagation in CD4+ T lymphocytes. The study reveals a new mechanism imposed by HIV on host cells that determines the fate of infection.


Assuntos
Transporte Ativo do Núcleo Celular , Infecções por HIV , HIV-1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Humanos , Processamento Alternativo , Núcleo Celular/metabolismo , Produtos do Gene rev/genética , HIV-1/fisiologia , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Splicing de RNA , RNA Viral/genética , RNA Viral/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo
2.
J Neuroinflammation ; 20(1): 187, 2023 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-37580715

RESUMO

BACKGROUND: Neuroinflammation is a widely studied phenomenon underlying various neurodegenerative diseases. Earlier study demonstrated that pharmacological activation of GPR110 in both central and peripheral immune cells cooperatively ameliorates neuroinflammation caused by systemic lipopolysaccharide (LPS) administration. Ethanol consumption has been associated with exacerbation of neurodegenerative and systemic inflammatory conditions. The goal of this study is to determine the effects of single-dose acute ethanol exposure and GPR110 activation on the neuro-inflammation mechanisms. METHODS: For in vivo studies, GPR110 wild type (WT) and knockout (KO) mice at 10-12 weeks of age were given an oral gavage of ethanol (3 g/kg) or maltose (5.4 g/kg) at 1-4 h prior to the injection of LPS (1 mg/kg, i.p.) followed by the GPR110 ligand, synaptamide (5 mg/kg). After 2-24 h, brains were collected for the analysis of gene expression by RT-PCR or protein expression by western blotting and enzyme-linked immunosorbent assay (ELISA). Microglial activation was assessed by western blotting and immunohistochemistry. For in vitro studies, microglia and peritoneal macrophages were isolated from adult WT mice and treated with 25 mM ethanol for 4 h and then with LPS (100 ng/ml) followed by 10 nM synaptamide for 2 h for gene expression and 12 h for protein analysis. RESULTS: Single-dose exposure to ethanol by gavage before LPS injection upregulated pro-inflammatory cytokine expression in the brain and plasma. The LPS-induced Iba-1 expression in the brain was significantly higher after ethanol pretreatment in both WT and GPR110KO mice. GPR110 ligand decreased the mRNA and/or protein expression of these cytokines and Iba-1 in the WT but not in GPR110KO mice. In the isolated microglia and peritoneal macrophages, ethanol also exacerbated the LPS-induced expression of pro-inflammatory cytokines which was mitigated at least partially by synaptamide. The expression of an inflammasome marker NLRP3 upregulated by LPS was further elevated with prior exposure to ethanol, especially in the brains of GPR110KO mice. Both ethanol and LPS reduced adenylate cyclase 8 mRNA expression which was reversed by the activation of GPR110. PDE4B expression at both mRNA and protein level in the brain increased after ethanol and LPS treatment while synaptamide suppressed its expression in a GPR110-dependent manner. CONCLUSION: Single-dose ethanol exposure exacerbated LPS-induced inflammatory responses. The GPR110 ligand synaptamide ameliorated this effect of ethanol by counteracting on the cAMP system, the common target for synaptamide and ethanol, and by regulating NLRP3 inflammasome.


Assuntos
Etanol , Doenças Neuroinflamatórias , Receptores Acoplados a Proteínas G , Animais , Camundongos , Citocinas/metabolismo , Etanol/toxicidade , Inflamassomos/metabolismo , Ligantes , Lipopolissacarídeos/toxicidade , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , RNA Mensageiro/metabolismo
3.
Bioorg Chem ; 134: 106452, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36889201

RESUMO

The enzyme chorismate mutase (or CM that is vital for the survival of bacteria) is an interesting pharmacological target for the identification of new anti-tubercular agents. The 5,5-disibstituted pyrazolo[4,3-d]pyrimidinone derivatives containing the fragment based on 4-amino-1-methyl-3-propyl-1H-pyrazole-5-carboxamide were designed and explored as the potential inhibitors of chorismate mutase. Based on encouraging docking results of two representative molecules evaluated in silico against MtbCM (PDB: 2FP2) the Wang resin catalysed sonochemical synthesis of target N-heteroarenes were undertaken. The methodology involved the reaction of 4-amino-1-methyl-3-propyl-1H-pyrazole-5-carboxamide with the appropriate cyclic/acyclic ketones to afford the desired products in acceptable (51-94%) yields. The methodology was also extended successfully towards the synthesis of 2,2-disubstituted 2,3-dihydroquinazolin-4(1H)-ones in excellent (85-90%) yields. In vitro MTT assay against the RAW 264.7 cell line followed by enzymatic assay against MtbCM identified 3b and 3c as active compounds that showed two H-bonding via their NH (at position 6) and CO group with MtbCM in silico and encouraging (54-57%) inhibition at 30 µM in vitro. Notably, none of the 2,2-disubstituted 2,3-dihydroquinazolin-4(1H)-ones showed any significant inhibition of MtbCM suggesting the favourable role of the pyrazole moiety in case of pyrazolo[4,3-d]pyrimidinones. The favourable role of cyclopentyl ring attached to the pyrazolo[4,3-d]pyrimidinone moiety and that of two methyl groups in place of cyclopentyl ring was also indicated by the SAR study. Besides showing effects against MtbCM in the concentration response study, 3b and 3c showed little or no effects on mammalian cell viability up to 100 µM in an MTT assay but decreased the % Mtb cell viability at 10-30 µM with > 20% decrease at 30 µM in an Alamar Blue Assay. Moreover, no adverse effects were noted for these compounds when tested for teratogenicity and hepatotoxicity in zebrafish at various concentrations. Overall, being the only example of MtbCM inhibitors that showed effects on Mtb cell viability the compound 3b and 3c are of further interest form the view point of discovery and development of new anti-tubercular agents.


Assuntos
Mycobacterium tuberculosis , Animais , Estrutura Molecular , Pirimidinonas/química , Relação Estrutura-Atividade , Corismato Mutase , Sobrevivência Celular , Peixe-Zebra/metabolismo , Mamíferos/metabolismo
4.
Int J Med Microbiol ; 312(1): 151544, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34922100

RESUMO

Mycobacterium tuberculosis (M. tuberculosis) encodes an essential enzyme acetyl ornithine aminotransferase ArgD (Rv1655) of arginine biosynthetic pathway which plays crucial role in M. tuberculosis growth and survival. ArgD catalyzes the reversible conversion of N-acetylornithine and 2 oxoglutarate into glutamate-5-semialdehyde and L-glutamate. It also possesses succinyl diaminopimelate aminotransferase activity and can thus carry out the corresponding step in lysine biosynthesis. These essential roles played by ArgD in amino acid biosynthetic pathways highlight it as an important metabolic chokepoint thus an important drug target. We showed that M. tuberculosis ArgD rescues the growth of ΔargD E. coli grown in minimal media validating its functional importance. Phylogenetic analysis of M. tuberculosis ArgD showed homology with proteins in gram positive bacteria, pathogenic and non-pathogenic mycobacteria suggesting the essentiality of this protein. ArgD is a secretory protein that could be utilized by M. tuberculosis to modulate host innate immunity as its moonlighting function. In-silico analysis predicted it to be a highly antigenic protein. The recombinant ArgD protein when exposed to macrophage cells induced enhanced production of pro-inflammatory cytokines TNF, IL6 and IL12 in a dose dependent manner. ArgD also induced the increased production of innate immune effector molecule NOS2 and NO in macrophages. We also demonstrated ArgD mediated activation of the canonical NFkB pathway. Notably, we also show that ArgD is a specific TLR4 agonist involved in the activation of pro-inflammatory signaling for sustained production of effector cytokines. Intriguingly, ArgD protein treatment activated macrophages to acquire the M1 phenotype through the increased surface expression of MHCII and costimulatory molecules CD80 and CD86. ArgD induced robust B-cell response in immunized mice, validating its antigenicity potential as predicted by the in-silico analysis. These properties of M. tuberculosis ArgD signify its functional plasticity that could be exploited as a possible drug target to combat tuberculosis.


Assuntos
Mycobacterium tuberculosis , Animais , Proteínas de Bactérias/genética , Escherichia coli , Camundongos , Filogenia , Transaminases/genética
5.
J Nanobiotechnology ; 20(1): 317, 2022 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-35794557

RESUMO

BACKGROUND: Exosomes are nano-sized vesicles secreted by various cells into the intra and extracellular space and hence is an integral part of biological fluids including milk. In the last few decades, many research groups have proved the potential of milk exosomes as a sustainable, economical and non-immunogenic drug delivery and therapeutic agent against different pathological conditions. However, its anti-viral properties still remain to be unearthed. METHODS: Here, we have been able to isolate, purify and characterize the milk derived exosomes from Cow (CME) and Goat (GME) and further studied its antiviral properties against Dengue virus (DENV), Newcastle Disease Virus strain Komarov (NDV-K) and Human Immunodeficiency Virus (HIV-1) using an in-vitro infection system. RESULTS: TEM, NTA and DLS analysis validated the appropriate size of the isolated cow and goat milk exosomes (30-150 nm). Real-time PCR and immunoblotting results confirmed the presence of several milk exosomal miRNAs and protein markers. Our findings suggest that GME significantly decreased the infectivity of DENV. In addition, we confirmed that GME significantly reduces DENV replication and reduced the secretion of mature virions. Furthermore, heat inactivation of GME did not show any inhibition on DENV infection, replication, and secretion of mature virions. RNase treatment of GME abrogates the anti-viral properties indicating direct role of exosomes in DENV inhibition. In addition GME inhibited the infectivity of NDV-K, but not HIV-1, suggesting that the GME mediated antiviral activity might be virus specific. CONCLUSION: This study demonstrates the anti-viral properties of milk exosomes and opens new avenues for the development of exosome-based therapies to treat viral diseases.


Assuntos
Vírus da Dengue , Exossomos , Animais , Antivirais/farmacologia , Bovinos , Exossomos/metabolismo , Feminino , Leite , Vírus da Doença de Newcastle
6.
Crit Rev Microbiol ; 47(4): 499-516, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33900141

RESUMO

Recognition of cell-surface receptors and co-receptors is a crucial molecular event towards the establishment of HIV infection. HIV exists as several variants that differentially recognize the principal co-receptors, CCR5 and CXCR4, in different cell types, known as HIV co-receptor-tropism. The relative levels of these variants dynamically adjust to the changing host selection pressures to infect a vast repertoire of cells in a stage-specific manner. HIV infection sets in through immune cells such as dendritic cells, macrophages, and T-lymphocytes in the acute stage, while a wide range of other cells, including astrocytes, glial cells, B-lymphocytes, and epithelial cells, are infected during chronic stages. A change in tropism occurs during the transition from acute to a chronic phase, termed as co-receptor switching marked by a change in disease severity. The cellular and molecular events leading to co-receptor switching are poorly understood. This review aims to collate our present understanding of the dynamics of HIV co-receptor-tropism vis-à-vis host and viral factors, highlighting the cellular and molecular events involved therein. We present the possible correlations between virus entry, cell tropism, and co-receptor switching, speculating its consequences on disease progression, and proposing new scientific pursuits to help in an in-depth understanding of HIV biology.


Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Receptores Virais/metabolismo , Tropismo Viral , Animais , Infecções por HIV/genética , HIV-1/genética , Humanos , Receptores CCR4/genética , Receptores CCR4/metabolismo , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores Virais/genética , Internalização do Vírus
7.
Arch Toxicol ; 94(7): 2293-2317, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32524152

RESUMO

The immune system plays a pivotal role in maintaining the defense mechanism against external agents and also internal danger signals. Metabolic programming of immune cells is required for functioning of different subsets of immune cells under different physiological conditions. The field of immunometabolism has gained ground because of its immense importance in coordination and balance of immune responses. Metabolism is very much related with production of energy and certain by-products. Reactive oxygen species (ROS) are generated as one of the by-products of various metabolic pathways. The amount, localization of ROS and redox status determine transcription of genes, and also influences the metabolism of immune cells. This review discusses ROS, metabolism of immune cells at different cellular conditions and sheds some light on how ROS might regulate immunometabolism.


Assuntos
Metabolismo Energético , Sistema Imunitário/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Animais , Humanos , Sistema Imunitário/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Doenças do Sistema Nervoso/imunologia , Doenças do Sistema Nervoso/metabolismo , Neuroimunomodulação , Oxirredução , Espécies Reativas de Oxigênio/imunologia
8.
J Bacteriol ; 201(15)2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31138627

RESUMO

Mycobacterium smegmatis, the saprophytic soil mycobacterium, is routinely used as a surrogate system to study the human pathogen Mycobacterium tuberculosis It has also been reported as an opportunistic pathogen in immunocompromised hosts. In addition, it can exist in several ecological setups, thereby suggesting its capacity to adapt to a variety of environmental cues. In this study, we employed untargeted proton nuclear magnetic resonance (1H-NMR)-based metabolomics to identify metabolites and metabolic pathways critical for early adaptive responses to acidic stress, oxidative stress, and nutrient starvation in Mycobacterium smegmatis We identified 31, 20, and 46 metabolites that showed significant changes in levels in response to acidic, oxidative, and nutrient starvation stresses, respectively. Pathway analyses showed significant perturbations in purine-pyrimidine, amino-acid, nicotinate-nicotinamide, and energy metabolism pathways. Besides these, differential levels of intermediary metabolites involved in α-glucan biosynthesis pathway were observed. We also detected high levels of organic osmolytes, methylamine, and betaine during nutrient starvation and oxidative stress. Further, tracing the differential levels of these osmolytes through computational search tools, gene expression studies (using reverse transcription-PCR [RT-PCR]), and enzyme assays, we detected the presence of a putative pathway of biosynthesis of betaine, methylamine, and dimethylamine previously unreported in Mycobacterium smegmatisIMPORTANCE Alterations in metabolite levels provide fast and direct means to regulate enzymatic reactions and, therefore, metabolic pathways. This study documents, for the first time, the metabolic changes that occur in Mycobacterium smegmatis as a response to three stresses, namely, acidic stress, oxidative stress, and nutrient starvation. These stresses are also faced by intracellular mycobacteria during infection and therefore may be extended to frame therapeutic interventions for pathogenic mycobacteria. In addition to the purine-pyrimidine, amino acid, nicotinate-nicotinamide, and energy metabolism pathways that were found to be affected in response to different stresses, a novel putative methylamine biosynthesis pathway was identified to be present in Mycobacterium smegmatis.


Assuntos
Aminas/metabolismo , Mycobacterium smegmatis/metabolismo , Aminas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Regulação Bacteriana da Expressão Gênica , Metabolômica , Metilação , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Estresse Oxidativo
9.
Biochem J ; 474(12): 2009-2026, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28476776

RESUMO

The enigmatic methyltransferase, DNMT2 (DNA methyltransferase 2), structurally resembles a DNA methyltransferase, but has been shown to be a tRNA methyltransferase targeting cytosine within a specific CpG in different tRNA molecules. We had previously shown that, during environmental stress conditions, DNMT2 is re-localized from the nucleus to the cytoplasmic stress granules (SGs) and is associated with RNA-processing proteins. In the present study, we show that DNMT2 binds and methylates various mRNA species in a sequence-independent manner and gets re-localized to SGs in a phosphorylation-dependent manner. Importantly, our results indicate that HIV-1 enhances its survivability in the host cell by utilizing this RNA methylation capability of DNMT2 to increase the stability of its own genome. Upon infection, DNMT2 re-localizes from the nucleus to the SGs and methylates HIV-1 RNA. This DNMT2-dependent methylation provided post-transcriptional stability to the HIV-1 RNA. Furthermore, DNMT2 overexpression increased the HIV-1 viral titre. This would suggest that HIV hijacks the RNA-processing machinery within the SGs to ensure its own survival in the host cell. Thus, our findings provide for a novel mechanism by which virus tries to modulate the host cell machinery to its own advantage.


Assuntos
Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Grânulos Citoplasmáticos/enzimologia , Grânulos Citoplasmáticos/virologia , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , Células HEK293 , HIV-1/crescimento & desenvolvimento , Humanos , Metilação , Viabilidade Microbiana , Fosforilação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Interferência de RNA , Estabilidade de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Regulação para Cima , Replicação Viral
10.
Cell Microbiol ; 18(3): 355-68, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26332641

RESUMO

Environmental mycobacteria, highly prevalent in natural and artificial (including chlorinated municipal water) niches, are emerging as new threat to human health, especially to HIV-infected population. These seemingly harmless non-pathogenic mycobacteria, which are otherwise cleared, establish as opportunistic infections adding to HIV-associated complications. Although immune-evading strategies of pathogenic mycobacteria are known, the mechanisms underlying the early events by which opportunistic mycobacteria establish infection in macrophages and influencing HIV infection are unclear. Proteomics of phagosome-enriched fractions from Mycobacterium bovis Bacillus Calmette-Guérin (BCG) mono-infected and HIV-M. bovis BCG co-infected THP-1 cells by LC-MALDI-MS/MS revealed differential distribution of 260 proteins. Validation of the proteomics data showed that HIV co-infection helped the survival of non-pathogenic mycobacteria by obstructing phagosome maturation, promoting lipid biogenesis and increasing intracellular ATP equivalents. In turn, mycobacterial co-infection up-regulated purinergic receptors in macrophages that are known to support HIV entry, explaining increased viral titers during co-infection. The mutualism was reconfirmed using clinically relevant opportunistic mycobacteria, Mycobacterium avium, Mycobacterium kansasii and Mycobacterium phlei that exhibited increased survival during co-infection, together with increase in HIV titers. Additionally, the catalogued proteins in the study provide new leads that will significantly add to the understanding of the biology of opportunistic mycobacteria and HIV coalition.


Assuntos
Coinfecção/microbiologia , Coinfecção/virologia , Infecções por HIV/microbiologia , Infecções por Mycobacterium/virologia , Trifosfato de Adenosina/metabolismo , Linhagem Celular , Coinfecção/metabolismo , Citocinas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/microbiologia , Macrófagos/virologia , Mycobacterium/patogenicidade , Mycobacterium bovis/patogenicidade , Fagossomos/microbiologia , Fagossomos/virologia , Proteômica/métodos , Simbiose , Carga Viral
11.
Proc Natl Acad Sci U S A ; 110(51): 20467-72, 2013 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-24282299

RESUMO

Resistin, a cysteine-rich adipocytokine, proposed as a link between obesity and diabetes in mice, was shown as a proinflammatory molecule in humans. We earlier reported that human resistin (hRes), a trimer, was resistant to heat and urea denaturation, existed in an oligomeric polydispersed state, and showed a concentration-dependent conformational change. These properties and an intimate correlation of hRes expression with cellular stress prompted us to investigate hRes as a possible chaperone. Here, we show that recombinant human resistin was able to protect the heat-labile enzymes citrate synthase and Nde1 from thermal aggregation and inactivation and was able to refold and restore their enzymatic activities after heat/guanidinium chloride denaturation. Furthermore, recombinant human resistin could bind misfolded proteins only. Molecular dynamics-based association-dissociation kinetics of hRes subunits pointed to resistin being a molecular chaperone. Bis-ANS, which blocks surface hydrophobicity, abrogated the chaperone activity of hRes, establishing the importance of surface hydrophobicity for chaperone activity. Replacement of Phe49 with Tyr (F49YhRes), a critical residue within the hydrophobic patch of hRes, although it could prevent thermal aggregation of citrate synthase and Nde1, was unable to refold and restore their activities. Treatment of U937 cells with tunicamycin/thapsigargin resulted in reduced hRes secretion and concomitant localization in the endoplasmic reticulum. Escherichia coli transformants expressing hRes could be rescued from thermal stress, pointing to hRes's chaperone-like function in vivo. HeLa cells transfected with hRes showed protection from thapsigargin-induced apoptosis. In conclusion, hRes, an inflammatory protein, additionally exhibited chaperone-like properties, suggesting a possible link between inflammation and cellular stress.


Assuntos
Citocinas/metabolismo , Resposta ao Choque Térmico/fisiologia , Mediadores da Inflamação/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Chaperonas Moleculares/metabolismo , Resistina/metabolismo , Animais , Antibacterianos/farmacologia , Citocinas/genética , Inibidores Enzimáticos/farmacologia , Células HeLa , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Inflamação/genética , Inflamação/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Chaperonas Moleculares/genética , Resistina/genética , Tapsigargina/farmacologia , Tunicamicina/farmacologia , Células U937
12.
Retrovirology ; 11: 18, 2014 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-24520823

RESUMO

BACKGROUND: The export of intron containing viral RNAs from the nucleus to the cytoplasm is an essential step in the life cycle of Human Immunodeficiency Virus-1 (HIV-1). As the eukaryotic system does not permit the transport of intron containing RNA out of the nucleus, HIV-1 makes a regulatory protein, Rev, that mediates the transportation of unspliced and partially spliced viral mRNA from the nucleus to the cytoplasm, thereby playing a decisive role in the generation of new infectious virus particles. Therefore, the host factors modulating the RNA export activity of Rev can be major determinants of virus production in an infected cell. RESULTS: In this study, human Staufen-2 (hStau-2) was identified as a host factor interacting with HIV-1 Rev through affinity chromatography followed by MALDI analyses. Our experiments involving transient expressions, siRNA mediated knockdowns and infection assays conclusively established that hStau-2 is a positive regulator of HIV-1 pathogenesis. We demonstrated that Rev-hStau-2 interactions positively regulated the RNA export activity of Rev and promoted progeny virus synthesis. The Rev-hStau-2 interaction was independent of RNA despite both being RNA binding proteins. hStau-2 mutant, with mutations at Q314R-A318F-K319E, deficient of binding Rev, failed to promote hStau-2 dependent Rev activity and viral production, validating the essentiality of this protein-protein interaction. The expression of this positive regulator was elevated upon HIV-1 infection in both human T-lymphocyte and astrocyte cell lines. CONCLUSIONS: With this study, we establish that human Staufen-2, a host factor which is up-regulated upon HIV-1 infection, interacts with HIV-1 Rev, thereby promoting its RNA export activity and progeny virus formation. Altogether, our study provides new insights into the emerging role of the Staufen family of mRNA transporters in host-pathogen interaction and supports the notion that obliterating interactions between viral and host proteins that positively regulate HIV-1 proliferation can significantly contribute to anti-retroviral treatments.


Assuntos
Núcleo Celular/metabolismo , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Proteínas do Tecido Nervoso/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Transporte Ativo do Núcleo Celular , Cromatografia de Afinidade , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
13.
Front Immunol ; 15: 1330738, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38449868

RESUMO

Nucleoporins (NUPs) are cellular effectors of human immunodeficiency virus-1 (HIV-1) replication that support nucleocytoplasmic trafficking of viral components. However, these also non-canonically function as positive effectors, promoting proviral DNA integration into the host genome and viral gene transcription, or as negative effectors by associating with HIV-1 restriction factors, such as MX2, inhibiting the replication of HIV-1. Here, we investigated the regulatory role of NUP98 on HIV-1 as we observed a lowering of its endogenous levels upon HIV-1 infection in CD4+ T cells. Using complementary experiments in NUP98 overexpression and knockdown backgrounds, we deciphered that NUP98 negatively affected HIV-1 long terminal repeat (LTR) promoter activity and lowered released virus levels. The negative effect on promoter activity was independent of HIV-1 Tat, suggesting that NUP98 prevents the basal viral gene expression. ChIP-qPCR showed NUP98 to be associated with HIV-1 LTR, with the negative regulatory element (NRE) of HIV-1 LTR playing a dominant role in NUP98-mediated lowering of viral gene transcription. Truncated mutants of NUP98 showed that the attenuation of HIV-1 LTR-driven transcription is primarily contributed by its N-terminal region. Interestingly, the virus generated from the producer cells transiently expressing NUP98 showed lower infectivity, while the virus generated from NUP98 knockdown CD4+ T cells showed higher infectivity as assayed in TZM-bl cells, corroborating the anti-HIV-1 properties of NUP98. Collectively, we show a new non-canonical function of a nucleoporin adding to the list of moonlighting host factors regulating viral infections. Downregulation of NUP98 in a host cell upon HIV-1 infection supports the concept of evolutionary conflicts between viruses and host antiviral factors.


Assuntos
HIV-1 , Complexo de Proteínas Formadoras de Poros Nucleares , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Poro Nuclear/genética , Repetição Terminal Longa de HIV/genética , Expressão Gênica
14.
ACS Infect Dis ; 10(6): 2288-2302, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38717380

RESUMO

The current tuberculosis (TB) treatment is challenged by a complex first-line treatment for drug-sensitive (DS) TB. Additionally, the prevalence of multidrug (MDR)- and extensively drug (XDR)-resistant TB necessitates the search for new drug prototypes. We synthesized and screened 30 hybrid compounds containing aminopyridine and 2-chloro-3-formyl quinoline to arrive at a compound with potent antimycobacterial activity, UH-NIP-16. Subsequently, antimycobacterial activity against DS and MDR Mycobacterium tuberculosis (M.tb) strains were performed. It demonstrated an MIC50 value of 1.86 ± 0.21 µM for laboratory pathogenic M.tb strain H37Rv and 3.045 ± 0.813 µM for a clinical M.tb strain CDC1551. UH-NIP-16 also decreased the MIC50 values of streptomycin, isoniazid, ethambutol, and bedaquiline to about 45, 55, 68, and 76%, respectively, when used in combination, potentiating their activities. The molecule was active against a clinical MDR M.tb strain. Cytotoxicity on PBMCs from healthy donors and on human cell lines was found to be negligible. Further, blind docking of UH-NIP-16 using Auto Dock Vina and MGL tools onto diverse M.tb proteins showed high binding affinities with multiple M.tb proteins, the top five targets being metabolically critical proteins CelA1, DevS, MmaA4, lysine acetyltransferase, and immunity factor for tuberculosis necrotizing toxin. These bindings were confirmed by fluorescence spectroscopy using a representative protein, MmaA4. Envisaging that a pathogen will have a lower probability of developing resistance to a hybrid molecule with multiple targets, we propose that UH-NIP-16 can be further developed as a lead molecule with the bacteriostatic potential against M.tb, both alone and in combination with first-line drugs.


Assuntos
Antituberculosos , Ácidos Isonicotínicos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis , Quinolinas , Mycobacterium tuberculosis/efeitos dos fármacos , Antituberculosos/farmacologia , Antituberculosos/síntese química , Antituberculosos/química , Humanos , Quinolinas/farmacologia , Quinolinas/química , Quinolinas/síntese química , Ácidos Isonicotínicos/farmacologia , Ácidos Isonicotínicos/química , Ácidos Isonicotínicos/síntese química , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
15.
Mitochondrion ; 78: 101931, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38986924

RESUMO

Mycobacterium tuberculosis (Mtb) successfully thrives in the host by adjusting its metabolism and manipulating the host environment. In this study, we investigated the role of Rv0547c, a protein that carries mitochondria-targeting sequence (MTS), in mycobacterial persistence. We show that Rv0547c is a functional oxidoreductase that targets host-cell mitochondria. Interestingly, the localization of Rv0547c to mitochondria was independent of the predicted MTS but depended on specific arginine residues at the N- and C-terminals. As compared to the mitochondria-localization defective mutant, Rv0547c-2SDM, wild-type Rv0547c increased mitochondrial membrane fluidity and spare respiratory capacity. To comprehend the possible reason, comparative lipidomics was performed that revealed a reduced variability of long-chain and very long-chain fatty acids as well as altered levels of phosphatidylcholine and phosphatidylinositol class of lipids upon expression of Rv0547c, explaining the increased membrane fluidity. Additionally, the over representation of propionate metabolism and ß-oxidation intermediates in Rv0547c-targeted mitochondrial fractions indicated altered fatty acid metabolism, which corroborated with changes in oxygen consumption rate (OCR) upon etomoxir treatment in HEK293T cells transiently expressing Rv0547c, resulting in enhanced mitochondrial fatty acid oxidation capacity. Furthermore, Mycobacterium smegmatis over expressing Rv0547c showed increased persistence during infection of THP-1 macrophages, which correlated with its increased expression in Mtb during oxidative and nutrient starvation stresses. This study identified for the first time an Mtb protein that alters mitochondrial metabolism and aids in survival in host macrophages by altering fatty acid metabolism to its benefit and, at the same time increases mitochondrial spare respiratory capacity to mitigate infection stresses and maintain cell viability.

16.
Int Rev Cell Mol Biol ; 377: 87-119, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37268352

RESUMO

The ability of Mycobacterium tuberculosis (M. tb) to hijack host mitochondria and control host immune signaling is the key to its successful infection. Infection of M. tb causes distinct changes in mitochondrial morphology, metabolism, disruption of innate signaling, and cell fate. The alterations in mitochondria are intricately linked to the immunometabolism of host immune cells such as macrophages, dendritic cells, and T cells. Different immune cells are tuned to diverse immunometabolic states that decide their immune response. These changes could be attributed to the several proteins targeted to host mitochondria by M. tb. Bioinformatic analyses and experimental evidence revealed the potential localization of secreted mycobacterial proteins in host mitochondria. Given the central role of mitochondria in the host metabolism, innate signaling, and cell fate, its manipulation by M. tb renders it susceptible to infection. Restoring mitochondrial health can override M. tb-mediated manipulation and thus clear infection. Several reviews are available on the role of different immune cells in tuberculosis infection and M. tb evasion of immune responses; in the present chapter, we discuss the mitochondrial functional alterations in the innate immune signaling of various immune cells driven by differential mitochondrial immunometabolism during M. tb infection and the role of M. tb proteins, which are directly targeted to the host mitochondria and compromise its innate signaling system. Further studies would help in uncovering the molecular mechanisms of M. tb-directed proteins in host mitochondria to conceptualize both host- directed and pathogen- directed interventions in TB disease management.


Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Tuberculose/metabolismo , Tuberculose/microbiologia , Mycobacterium tuberculosis/fisiologia , Macrófagos , Transdução de Sinais , Mitocôndrias/metabolismo
17.
Front Immunol ; 14: 1276817, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37928551

RESUMO

Mycobacterium tuberculosis (Mtb) and HIV are known to mutually support each other during co-infection by multiple mechanisms. This synergistic influence could be either by direct interactions or indirectly through secreted host or pathogen factors that work in trans. Mtb secretes several virulence factors to modulate the host cellular environment for its persistence and escaping cell-intrinsic immune responses. We hypothesized that secreted Mtb transcription factors that target the host nucleus can directly interact with host DNA element(s) or HIV LTR during co-infection, thereby modulating immune gene expression, or driving HIV transcription, helping the synergistic existence of Mtb and HIV. Here, we show that the Mtb-secreted protein, EspR, a transcription regulator, increased mycobacterial persistence and HIV propagation during co-infection. Mechanistically, EspR localizes to the nucleus of the host cells during infection, binds to its putative cognate motif on the promoter region of the host IL-4 gene, activating IL-4 gene expression, causing high IL-4 titers that induce a Th2-type microenvironment, shifting the macrophage polarization to an M2 state as evident from CD206 dominant population over CD64. This compromised the clearance of the intracellular mycobacteria and enhanced HIV propagation. It was interesting to note that EspR did not bind to HIV LTR, although its transient expression increased viral propagation. This is the first report of an Mtb transcription factor directly regulating a host cytokine gene. This augments our understanding of the evolution of Mtb immune evasion strategies and unveils how Mtb aggravates comorbidities, such as HIV co-infection, by modulating the immune microenvironment.


Assuntos
Coinfecção , Infecções por HIV , Mycobacterium tuberculosis , Humanos , Interleucina-4/genética , Interleucina-4/metabolismo , Coinfecção/metabolismo , Macrófagos , Infecções por HIV/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Virulência/metabolismo
18.
FEBS J ; 289(21): 6731-6751, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35653259

RESUMO

Nucleocytoplasmic shuttling of viral elements, supported by several host factors, is essential for the replication of the human immunodeficiency virus (HIV). HIV-1 uses a nuclear RNA export pathway mediated by viral protein Rev to transport its Rev response element (RRE)-containing partially spliced and unspliced transcripts aided by the host nuclear RNA export protein CRM1. The factor(s) interacting with the CRM1-Rev complex are potential antiretroviral target(s) and could serve as a retroviral model system to study nuclear export machinery adapted by these viruses. We earlier reported that cellular Staufen-2 interacts with Rev, facilitating viral-RNA export. Here, we identified the formation of a complex between Staufen-2, CRM1 and Rev. Molecular docking and simulations mapped the interacting residues in the RNA-binding Domain 4 of Staufen-2 as R336 and R337, which were experimentally verified to be critical for interactions among Staufen-2, CRM1 and Rev by mutational analysis. Staufen-2 mutants defective in interaction with CRM1 or Rev failed to supplement the Rev-RNA export activity and viral production, demonstrating the importance of these interactions. Rev-dependent reporter assays and proviral DNA-construct transfection-based studies in Staufen-2 knockout cells in the presence of leptomycin-B (LMB) revealed a significant reduction in CRM1-mediated Rev-dependent RNA export with decreased virus production as compared to Staufen-2 knockout background or LMB treatment alone, suggesting the relevance of these interactions in augmenting RNA export activity of Rev. Our observations provide further insights into the mechanistic intricacies of unspliced viral-RNA export to the cytoplasm and support the notion that abrogating such interactions can reduce HIV-1 proliferation.


Assuntos
HIV-1 , Humanos , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Genômica , HIV-1/fisiologia , Carioferinas/genética , Carioferinas/metabolismo , Simulação de Acoplamento Molecular , Proteínas Nucleares/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , RNA Nuclear/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo
19.
Immunol Lett ; 242: 27-36, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35007662

RESUMO

Mycobacterium tuberculosis (M.tb) is a multifaceted bacterial pathogen known to infect more than 2 billion people globally. However, a majority of the individuals (>90%) show no overt clinical symptoms of active Tuberculosis (TB) and, it is reported that M.tb in these individuals resides in the latent form. Therefore, a huge burden of latently infected population poses serious threat to the human health. Inconsistent efficacy of BCG vaccine and poor understanding of latency-associated determinants contribute to the failure of combating M.tb. The discovery of DosR as the master regulator of dormancy, opened new avenues to understand the pathophysiology of the bacterium. Though the specific functions of various DosR genes are yet to be discovered, they have been reported as potent T-cell activators and could elicit strong protective immune responses. Rv0569 is a DosR-encoded conserved hypothetical protein overexpressed during dormancy. However, it is not clearly understood how this protein modulates the host immune response. In the present study, we have demonstrated that Rv0569 has a high antigenic index and induces enhanced secretion of Th1 cytokines IL-12p40 and TNF-α as compared to Th2 cytokine IL-10 in macrophages. Mechanistically, Rv0569 induced the transcription of these pro-inflammatory signatures through the activation of NF-κB pathway. Further, immunization of mice with DosR protein Rv0569 switched the immune response towards Th1-biased cytokine pattern, characterized by the enhanced production of IFN-γ, IL-12p40, and TNF-α. Rv0569 augmented the expansion of antigen-specific IFN-γ and IL-2 producing effector CD4+and CD8+ T-cells which are hallmarks of Th1 biased protective immunity. Additionally, IgG2a/IgG1 and IgG2b/IgG1 ratio in the serum of immunized mice further confirmed the ability of Rv0569 to skew Th1 biased immune response. In conclusion, we emphasize that Rv0569 has the ability to generate signals to switch on Th1-dominated responses and further suggest that it could be a potential vaccine candidate against latent M.tb infection.


Assuntos
Mycobacterium tuberculosis , Animais , Antígenos de Bactérias , Proteínas de Bactérias/genética , Linfócitos T CD8-Positivos , Citocinas/metabolismo , Humanos , Imunoglobulina G/metabolismo , Subunidade p40 da Interleucina-12 , Camundongos , Células Th1 , Fator de Necrose Tumoral alfa/metabolismo
20.
Viruses ; 14(11)2022 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-36423112

RESUMO

Human immunodeficiency virus-1 (HIV-1) is a retrovirus that integrates its reverse-transcribed genome as proviral DNA into the host genome to establish a successful infection. The viral genome integration requires safeguarding the subviral complexes, reverse transcription complex (RTC) and preintegration complex (PIC), in the cytosol from degradation, presumably effectively secured by the capsid surrounding these complexes. An intact capsid, however, is a large structure, which raises concerns about its translocation from cytoplasm to nucleus crossing the nuclear membrane, guarded by complex nuclear pore structures, which do not allow non-specific transport of large molecules. In addition, the generation of new virions requires the export of incompletely processed viral RNA from the nucleus to the cytoplasm, an event conventionally not permitted through mammalian nuclear membranes. HIV-1 has evolved multiple mechanisms involving redundant host pathways by liaison with the cell's nucleocytoplasmic trafficking system, failure of which would lead to the collapse of the infection cycle. This review aims to assemble the current developments in temporal and spatial events governing nucleocytoplasmic transport of HIV-1 factors. Discoveries are anticipated to serve as the foundation for devising host-directed therapies involving selective abolishment of the critical interactomes between viral proteins and their host equivalents.


Assuntos
Infecções por HIV , HIV-1 , Humanos , Transporte Ativo do Núcleo Celular , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , HIV-1/genética , HIV-1/metabolismo , Integração Viral
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