RESUMO
We developed a highly sensitive assay for detecting protein-protein interaction using chimeric receptors comprising two molecules of interest in the extracellular domain and interferon alpha and beta receptor subunit 1 or 2 (IFNAR1/2) in the intracellular domain. This intracellular IFNAR1/2 reconstitution system (IFNARRS) proved markedly more sensitive than the NanoBiT system, currently considered one of the best detection systems for protein interaction. Employing chimeric receptors with extracellular domains from the IFNγ or IL-2 receptor and the intracellular domains of IFNAR1/2, the IFNARRS system effectively identifies low IFNγ or IL-2 levels. Cells stably expressing these chimeric receptors responded to IFNγ secreted by activated T cells following various stimuli, including a specific peptide-antigen. The activation signals were further enhanced by the expression of relevant genes, such as costimulators, via IFN-stimulated response elements in the promoters. Besides IFNγ or IL-2, the IFNARRS system demonstrated the capability to detect other cytokines by using the corresponding extracellular domains from these target cytokine receptors.
Assuntos
Interferon gama , Interleucina-2 , Receptor de Interferon alfa e beta , Linfócitos T , Humanos , Receptor de Interferon alfa e beta/metabolismo , Receptor de Interferon alfa e beta/genética , Linfócitos T/metabolismo , Linfócitos T/imunologia , Interleucina-2/metabolismo , Interferon gama/metabolismo , Receptores de Interleucina-2/metabolismo , Receptores de Interleucina-2/genética , Ligação Proteica , Ativação Linfocitária , Células HEK293RESUMO
Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle, which is closely related to human T-cell leukemia viruses. BLV has spread worldwide and causes a serious problem for the cattle industry. The cellular receptor specifically binds with viral envelope glycoprotein (Env), and this attachment mediates cell fusion to lead virus entry. BLV Env reportedly binds to cationic amino acid transporter 1 (CAT1)/solute carrier family 7 member 1 (SLC7A1), but whether the CAT1/SLC7A1 is an actual receptor for BLV remains unknown. Here, we showed that CAT1 functioned as an infection receptor, interacting with BLV particles. Cells expressing undetectable CAT1 levels were resistant to BLV infection but became highly susceptible upon CAT1 overexpression. CAT1 exhibited specific binding to BLV particles on the cell surface and colocalized with the Env in endomembrane compartments and membrane. Knockdown of CAT1 in permissive cells significantly reduced binding to BLV particles and BLV infection. Expression of CAT1 from various species demonstrated no species specificity for BLV infection, implicating CAT1 as a functional BLV receptor responsible for its broad host range. These findings provide insights for BLV infection and for developing new strategies for treating BLV and preventing its spread.-Bai, L., Sato, H., Kubo, Y., Wada, S., Aida, Y. CAT1/SLC7A1 acts as a cellular receptor for bovine leukemia virus infection.
Assuntos
Transportador 1 de Aminoácidos Catiônicos/metabolismo , Leucose Enzoótica Bovina/metabolismo , Vírus da Leucemia Bovina/metabolismo , Animais , Células CHO , Células COS , Transportador 1 de Aminoácidos Catiônicos/genética , Gatos , Bovinos , Chlorocebus aethiops , Cricetinae , Cricetulus , Leucose Enzoótica Bovina/virologia , Células HEK293 , Células HeLa , Humanos , Vírus da Leucemia Bovina/patogenicidade , Ligação Proteica , Ovinos , Suínos , Proteínas do Envelope Viral/metabolismoRESUMO
In this study, we aimed to find chemicals from lower sea animals with defensive effects against human immunodeficiency virus type 1 (HIV-1). A library of marine natural products consisting of 80 compounds was screened for activity against HIV-1 infection using a luciferase-encoding HIV-1 vector. We identified five compounds that decreased luciferase activity in the vector-inoculated cells. In particular, portimine, isolated from the benthic dinoflagellate Vulcanodinium rugosum, exhibited significant anti-HIV-1 activity. Portimine inhibited viral infection with an 50% inhibitory concentration (IC50) value of 4.1 nM and had no cytotoxic effect on the host cells at concentrations less than 200 nM. Portimine also inhibited vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped HIV-1 vector infection. This result suggested that portimine mainly targeted HIV-1 Gag or Pol protein. To analyse which replication steps portimine affects, luciferase sequences were amplified by semi-quantitative PCR in total DNA. This analysis revealed that portimine inhibits HIV-1 vector infection before or at the reverse transcription step. Portimine has also been shown to have a direct effect on reverse transcriptase using an in vitro reverse transcriptase assay. Portimine efficiently inhibited HIV-1 replication and is a potent lead compound for developing novel therapeutic drugs against HIV-1-induced diseases.
Assuntos
Fármacos Anti-HIV/farmacologia , Organismos Aquáticos/química , Dinoflagellida/química , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Iminas/farmacologia , Compostos de Espiro/farmacologia , Fármacos Anti-HIV/isolamento & purificação , Fármacos Anti-HIV/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , HIV-1/fisiologia , Células HeLa , Humanos , Iminas/isolamento & purificação , Iminas/uso terapêutico , Concentração Inibidora 50 , Compostos de Espiro/isolamento & purificação , Compostos de Espiro/uso terapêutico , Replicação Viral/efeitos dos fármacosRESUMO
Interferon regulatory factor (IRF) 4 and the proto-oncogene c-Rel cooperate in growth and antiviral drug resistance of adult T-cell leukemia/lymphoma (ATLL). To elucidate the target of IRF4 and c-Rel in ATLL, we determined the simultaneous binding sites of IRF4 and c-Rel using ChIP-seq technology. Nine genes were identified within 2â¯kb of binding sites, including MIR3662. Expression of miR-3662 was regulated by IRF4, and to a lesser extent by c-Rel. Cell proliferation was inhibited by knockdown of miR-3662 and expression of miR-3662 was correlated with antiviral drug resistance in ATLL cell lines. Thus, miR-3662 represents a target for therapies against ATLL.
Assuntos
Farmacorresistência Viral/genética , Regulação Leucêmica da Expressão Gênica , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/virologia , MicroRNAs/genética , Adulto , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Fatores Reguladores de Interferon/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , MicroRNAs/metabolismo , Ligação Proteica/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-rel/metabolismoRESUMO
Xenotropic murine leukemia virus-related virus (XMRV) is a novel gammaretrovirus that was originally isolated from human prostate cancer. It is now believed that XMRV is not the etiologic agent of prostate cancer. An analysis of murine leukemia virus (MLV) infection in various human cell lines revealed that prostate cancer cell lines are preferentially infected by XMRV, and this suggested that XMRV infection may confer some sort of growth advantage to prostate cancer cell lines. To examine this hypothesis, androgen-dependent LNCaP cells were infected with XMRV and tested for changes in certain cell growth properties. We found that XMRV-infected LNCaP cells can proliferate in the absence of the androgen dihydrotestosterone. Moreover, androgen receptor expression is significantly reduced in XMRV-infected LNCaP cells. Such alterations were not observed in uninfected and amphotropic MLV-infected LNCaP cells. This finding explains why prostate cancer cell lines are preferentially infected with XMRV.
Assuntos
Androgênios/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias da Próstata/virologia , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/fisiologia , Antagonistas de Receptores de Andrógenos/farmacologia , Anilidas/farmacologia , Animais , Linhagem Celular Tumoral , Di-Hidrotestosterona/farmacologia , Humanos , Masculino , Camundongos , Nitrilas/farmacologia , Ratos , Receptores Androgênicos/biossíntese , Receptores Androgênicos/efeitos dos fármacos , Compostos de Tosil/farmacologiaRESUMO
Ecotropic murine leukemia viruses (Eco-MLVs) infect mouse and rat, but not other mammalian cells, and gain access for infection through binding the cationic amino acid transporter 1 (CAT1). Glycosylation of the rat and hamster CAT1s inhibits Eco-MLV infection, and treatment of rat and hamster cells with a glycosylation inhibitor, tunicamycin, enhances Eco-MLV infection. Although the mouse CAT1 is also glycosylated, it does not inhibit Eco-MLV infection. Comparison of amino acid sequences between the rat and mouse CAT1s shows amino acid insertions in the rat protein near the Eco-MLV-binding motif. In addition to the insertion present in the rat CAT1, the hamster CAT1 has additional amino acid insertions. In contrast, tunicamycin treatment of mink and human cells does not elevate the infection, because their CAT1s do not have the Eco-MLV-binding motif. To define the evolutionary pathway of the Eco-MLV receptor, we analyzed CAT1 sequences and susceptibility to Eco-MLV infection of other several murinae animals, including the southern vole (Microtus rossiaemeridionalis), large Japanese field mouse (Apodemus speciosus), and Eurasian harvest mouse (Micromys minutus). Eco-MLV infection was enhanced by tunicamycin in these cells, and their CAT1 sequences have the insertions like the hamster CAT1. Phylogenetic analysis of mammalian CAT1s suggested that the ancestral CAT1 does not have the Eco-MLV-binding motif, like the human CAT1, and the mouse CAT1 is thought to be generated by the amino acid deletions in the third extracellular loop of CAT1.
Assuntos
Transportador 1 de Aminoácidos Catiônicos/genética , Evolução Molecular , Vírus da Leucemia Murina/fisiologia , Muridae/genética , Receptores Virais/genética , Infecções por Retroviridae/virologia , Doenças dos Roedores/virologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Arvicolinae , Transportador 1 de Aminoácidos Catiônicos/química , Transportador 1 de Aminoácidos Catiônicos/metabolismo , Linhagem Celular , Cricetinae , Gerbillinae , Humanos , Camundongos , Dados de Sequência Molecular , Muridae/classificação , Muridae/virologia , Filogenia , Ratos , Receptores Virais/química , Receptores Virais/metabolismoRESUMO
Mice deficient for interferon regulatory factor (Irf)2 (Irf2(-/-) mice) exhibit immunological abnormalities and cannot survive lymphocytic choriomeningitis virus infection. The pancreas of these animals is highly inflamed, a phenotype replicated by treatment with poly(I:C), a synthetic double-stranded RNA. Trypsinogen5 mRNA was constitutively up-regulated about 1,000-fold in Irf2(-/-) mice compared with controls as assessed by quantitative RT-PCR. Further knockout of IFNα/ß receptor 1(Ifnar1) abolished poly(I:C)-induced pancreatitis but had no effect on the constitutive up-regulation of trypsinogen5 gene, indicating crucial type I IFN signaling to elicit the inflammation. Analysis of Ifnar1(-/-) mice confirmed type I IFN-dependent transcriptional activation of dsRNA-sensing pattern recognition receptor genes MDA5, RIG-I, and TLR3, which induced poly(I:C)-dependent cell death in acinar cells in the absence of IRF2. We speculate that Trypsin5, the trypsinogen5 gene product, leaking from dead acinar cells triggers a chain reaction leading to lethal pancreatitis in Irf2(-/-) mice because it is resistant to a major endogenous trypsin inhibitor, Spink3.
Assuntos
Fator Regulador 2 de Interferon/metabolismo , Pancreatite/metabolismo , RNA de Cadeia Dupla/metabolismo , Transcrição Gênica , Tripsinogênio/genética , Células Acinares/metabolismo , Animais , Catepsina B/metabolismo , Glicoproteínas/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Pancreatite/genética , Poli I-C/genética , Proteínas Secretadas pela Próstata/metabolismo , Inibidor da Tripsina Pancreática de Kazal , Inibidores da Tripsina/farmacologiaRESUMO
BACKGROUND: This study aimed to evaluate VE of primary, first, and second booster ancestral-strain monovalent mRNA COVID-19 vaccination against symptomatic infections and severe diseases in Japan. METHODS: We conducted a test-negative case-control study. We included medically attended episodes and hospitalizations involving individuals aged ≥16 with signs and symptoms from July to November 2022, when Omicron BA.5 was dominant nationwide. To evaluate VE, we calculated adjusted ORs of vaccination among test-positive versus test-negative individuals using a mixed-effects logistic regression. RESULTS: For VE against symptomatic infections among individuals aged 16 to 59, VE of primary vaccination at > 180 days was 26.1% (95% CI: 10.6-38.8%); VE of the first booster was 58.5% (48.4-66.7%) at ≤90 days, decreasing to 41.1% (29.5-50.8%) at 91 to 180 days. For individuals aged ≥60, VE of the first booster was 42.8% (1.7-66.7%) at ≤90 days, dropping to 15.4% (-25.9-43.2%) at 91 to 180 days, and then increasing to 44.0% (16.4-62.5%) after the second booster. For VE against severe diseases, VE of the first and second booster was 77.3% (61.2-86.7%) at ≤90 days and 55.9% (23.4-74.6%) afterward. CONCLUSION: mRNA booster vaccination provided moderate protection against symptomatic infections and high-level protection against severe diseases during the BA.5 epidemic in Japan.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/epidemiologia , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Japão/epidemiologia , Estudos de Casos e Controles , Eficácia de Vacinas , RNA Mensageiro , VacinaçãoRESUMO
Alarin is an alternative-splicing form of GALP (galanin-like peptide). It shares only 5 conserved amino acids at the N-terminal region with GALP which is involved in a diverse range of normal brain functions. This study seeks to investigate whether alarin has additional functions due to its differences from GALP. Here, we have shown using a radial diffusion assay that alarin but not GALP inhibited the growth of Escherichia coli (strain ML-35). The conserved N-terminal region, however, remained essential for the antimicrobial activity of alarin as truncated peptides showed reduced killing effect. Moreover, alarin inhibited the growth of E. coli in a similar potency as human cathelicidin LL-37, a well-studied antimicrobial peptide. Electron microscopy further showed that alarin induced bacterial membrane blebbing but unlike LL-37, it did not cause hemolysis of erythrocytes. In addition, alarin is only active against the gram-negative bacteria, E. coli but not the gram-positive bacteria, Staphylococcus aureus. Thus, these data suggest that alarin has potentials as an antimicrobial and should be considered for the development in human therapeutics.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Peptídeo Semelhante a Galanina/análogos & derivados , Peptídeo Semelhante a Galanina/farmacologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos , Catelicidinas/farmacologia , Membrana Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/ultraestrutura , Hemólise , Cavalos/sangue , Humanos , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Gamma-interferon (γ-IFN) significantly inhibits infection by replication-defective viral vectors derived from the human immunodeficiency virus type 1 (HIV-1) or murine leukemia virus (MLV) but the underlying mechanism remains unclear. Previously we reported that knockdown of γ-IFN-inducible lysosomal thiolreductase (GILT) abrogates the antiviral activity of γ-IFN in TE671 cells but not in HeLa cells, suggesting that other γ-IFN-inducible host factors are involved in its antiviral activity in HeLa cells. We identified cellular factors, the expression of which are induced by γ-IFN in HeLa cells, using a microarray, and analyzed the effects of 11 γ-IFN-induced factors on retroviral vector infection. Our results showed that the exogenous expression of FAT10, IFI6, or IDO1 significantly inhibits both HIV-1- and MLV-based vector infections. The antiviral activity of γ-IFN was decreased in HeLa cells, in which the function of IDO1, IFI6, FAT10, and GILT were simultaneously inhibited. IDO1 is an enzyme that metabolizes an essential amino acid, tryptophan. However, IDO1 did not restrict retroviral vector infection in Atg3-silencing HeLa cells, in which autophagy did not occur. This study found that IDO1, IFI6, FAT10, and GILT are involved in the antiviral activity of γ-IFN, and IDO1 inhibits retroviral infection by inducing autophagy.
Assuntos
Infecções por HIV , HIV-1 , Infecções por Retroviridae , Antirretrovirais/farmacologia , Antivirais/farmacologia , Autofagia , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , Células HeLa , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interferon gama/metabolismo , Interferon gama/farmacologia , Vírus da Leucemia Murina , Proteínas Mitocondriais , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Ubiquitinas/farmacologiaRESUMO
We recently identified a CD63-interacting protein to understand the role of CD63 in virion production of the human immunodeficiency virus type 1, and we have found that Rab3a forms a complex with CD63. In this study, we analysed the effect of Rab3a on virion production of the murine leukaemia virus (MLV), which is another member of the retrovirus family. We found that Rab3a silencing induced lysosomal degradation of the MLV Gag protein, and recovery of the Rab3a expression restored the level of the Gag protein through a complex formation of MLV Gag and Rab3a, indicating that Rab3a is required for MLV Gag protein expression. In contrast, CD63 silencing decreased the infectivity of released virions but had no effect on virion production, indicating that CD63 facilitates the infectivity of released MLV particles. Although Rab3a induced CD63 degradation in uninfected cells, the complex of MLV Gag and Rab3a suppressed the Rab3a-mediated CD63 degradation in MLV-infected cells. Finally, we found that the MLV Gag protein interacts with Rab3a to stabilize its own protein and CD63 that facilitates the infectivity of released MLV particles. Considering the involvement of Rab3a in lysosome trafficking to the plasma membrane, it may also induce cell surface transport of the MLV Gag protein.
Assuntos
Produtos do Gene gag , Vírus da Leucemia Murina , Camundongos , Animais , Humanos , Produtos do Gene gag/metabolismo , Vírus da Leucemia Murina/metabolismo , Vírion/metabolismo , Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismoRESUMO
Lamellarin α 20-sulfate is a cell-impenetrable marine alkaloid that can suppress infection that is mediated by the envelope glycoprotein of human immunodeficiency virus type 1. We explored the antiviral action and mechanisms of this alkaloid against emerging enveloped RNA viruses that use endocytosis for infection. The alkaloid inhibited the infection of retroviral vectors that had been pseudotyped with the envelope glycoprotein of Ebola virus and SARS-CoV-2. The antiviral effects of lamellarin were independent of the retrovirus Gag-Pol proteins. Interestingly, although heparin and dextran sulfate suppressed the cell attachment of vector particles, lamellarin did not. In silico structural analyses of the trimeric glycoprotein of the Ebola virus disclosed that the principal lamellarin-binding site is confined to a previously unappreciated cavity near the NPC1-binding site and fusion loop, whereas those for heparin and dextran sulfate were dispersed across the attachment and fusion subunits of the glycoproteins. Notably, lamellarin binding to this cavity was augmented under conditions where the pH was 5.0. These results suggest that the final action of the alkaloid against Ebola virus is specific to events following endocytosis, possibly during conformational glycoprotein changes in the acidic environment of endosomes. Our findings highlight the unique biological and physicochemical features of lamellarin α 20-sulfate and should lead to the further use of broadly reactive antivirals to explore the structural mechanisms of virus replication.
Assuntos
Alcaloides , Tratamento Farmacológico da COVID-19 , Ebolavirus , Doença pelo Vírus Ebola , Alcaloides/farmacologia , Antivirais/química , Sulfato de Dextrana , Ebolavirus/metabolismo , Glicoproteínas , Doença pelo Vírus Ebola/tratamento farmacológico , Heparina/farmacologia , Humanos , SARS-CoV-2 , Internalização do VírusRESUMO
Interferon regulatory factor (IRF)-4 is a member of the IRF transcription factor family, whose expression is primarily restricted to lymphoid and myeloid cells. In T-cells, IRF-4 expression is induced by T-cell receptor (TCR) cross-linking or treatment with phorbol-12-myristate-13-acetate (PMA)/Ionomycin, and IRF-4 is thought to be a critical factor for various functions of T-cells. To elucidate the IRF-4 functions in human adult T-cell leukemia virus type 1 (HTLV-1)-infected T-cells, which constitutively express IRF-4, we isolated IRF-4-binding proteins from T-cells, using a tandem affinity purification (TAP)-mass spectrometry strategy. Fourteen proteins were identified in the IRF-4-binding complex, including endogenous IRF-4 and the nuclear factor-kappaB (NF-κB) family member, c-Rel. The specific association of IRF-4 with c-Rel was confirmed by immunoprecipitation experiments, and IRF-4 was shown to enhance the c-Rel-dependent binding and activation of the interleukin-4 (IL-4) promoter region. We also demonstrated that IL-2 production was also enhanced by exogenously-expressed IRF-4 and c-Rel in the presence of P/I, in T-cells, and that the optimal IL-2 and IL-4 productions in vivo was IRF-4-dependent using IRF-4-/- mice. These data provide molecular evidence to support the clinical observation that elevated expression of c-Rel and IRF-4 is associated with the prognosis in adult T-cell leukemia/lymphoma (ATLL) patients, and present possible targets for future gene therapy.
Assuntos
Regulação da Expressão Gênica , Fatores Reguladores de Interferon/metabolismo , Interleucina-2/genética , Interleucina-4/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-rel/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Humanos , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Proteínas Proto-Oncogênicas c-rel/químicaRESUMO
Lamellarin α and six different types of lamellarin α 20-sulfate analogues were synthesized and their structure-activity relationships were investigated using a single round HIV-1 vector infection assay. All lamellarin sulfates having pentacyclic lamellarin core exhibited anti-HIV-1 activity at a 10 µM concentration range regardless of the number and position of the sulfate group. On the other hand, non-sulfated lamellarin α and ring-opened lamellarin sulfate analogues did not affect HIV-1 vector infection in similar concentrations. The lamellarin sulfates utilized in this study did not exhibit unfavorable cytotoxic effect under the concentrations tested (IC(50)>100 µM). Confocal laser scanning microscopic analysis indicated that hydrophilic lamellarin sulfates were hardly incorporated in the cell. HIV-1 Env-mediated cell-cell fusion was suppressed by lamellarin sulfates. These results suggested that lamellarin sulfates have a novel anti-HIV-1 activity besides the previously reported integrase activity inhibition, possibly at a viral entry step of HIV-1 replication.
Assuntos
Cumarínicos/química , Cumarínicos/farmacologia , Inibidores de Integrase de HIV/química , Inibidores de Integrase de HIV/farmacologia , HIV-1/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Isoquinolinas/química , Isoquinolinas/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Infecções por HIV/tratamento farmacológico , Humanos , Relação Estrutura-AtividadeRESUMO
We have previously reported that gamma-interferon inducible lysosomal thiolreductase (GILT) functions as a host defense factor against retroviruses by digesting disulfide bonds on viral envelope proteins. GILT is widely conserved even in plants and fungi as well as animals. The thiolreductase active site of mammalian GILT is composed of a CXXC amino acid motif, whereas the C-terminal cysteine residue is changed to serine in arthropods including shrimps, crabs, and flies. GILT from Penaeus monodon (PmGILT) also has the CXXS motif instead of the CXXC active site. We demonstrate here that a human GILT mutant (GILT C75S) with the CXXS motif and PmGILT significantly inhibit amphotropic murine leukemia virus vector infection in human cells without alterning its expression level and lysosomal localization, showing that the C-terminal cysteine residue of the active site is not required for the antiviral activity. We have reported that human GILT suppresses HIV-1 particle production by digestion of disulfide bonds on CD63. However, GILT C75S mutant and PmGILT did not digest CD63 disulfide bonds, and had no effect on HIV-1 virion production, suggesting that they do not have thiolreductase activity. Taken together, this study found that antiviral activity, but not thiolreductase activity, is conserved in arthropod GILT proteins. This finding provides a new insight that the common function of GILT is antiviral activity in many animals.
Assuntos
Antivirais/metabolismo , Artrópodes/enzimologia , Artrópodes/virologia , Interferon gama/farmacologia , Oxirredutases/metabolismo , Motivos de Aminoácidos , Animais , Baculoviridae/fisiologia , Células COS , Chlorocebus aethiops , Sequência Conservada , Endossomos/metabolismo , HIV-1/fisiologia , Células HeLa , Humanos , Interferon gama/metabolismo , Vírus da Leucemia Murina/fisiologia , Lisossomos/metabolismo , Oxirredutases/química , Penaeidae/virologia , Especificidade por Substrato , Vírion/fisiologiaRESUMO
BACKGROUND: Fever with jaundice is a common symptom of some infectious diseases. In public health surveillance within the Democratic Republic of the Congo (DRC), yellow fever is the only recognized cause of fever with jaundice. However, only 5% of the surveillance cases are positive for yellow fever and thus indicate the involvement of other pathogens. Leptospira spp. are the causative agents of leptospirosis, a widespread bacterial zoonosis, a known cause of fever with jaundice. This study aimed to determine the seropositivity of anti-Leptospira antibodies among suspected yellow fever cases and map the geographical distribution of possible leptospirosis in the DRC. METHODS: We conducted a retrospective study using 1,300 samples from yellow fever surveillance in the DRC from January 2017 to December 2018. Serum samples were screened for the presence of IgM against Leptospira spp. by a whole cell-based IgM ELISA (Patoc-IgM ELISA) at the Institut National de Recherche Biomedicale in Kinshasa (INRB) according to World Health Organization (WHO) guidance. Exploratory univariable and multivariable logistic regression analyses were undertaken to assess associations between socio-demographic factors and the presence of Leptospira IgM. RESULTS: Of the 1,300 serum samples screened, 88 (7%) showed evidence of IgM against Leptospira spp. Most positive cases (34%) were young adult males in the 20-29-year group. There were statistically significant associations between having Leptospira IgM antibodies, age, sex, and living area. Observed positive cases were mostly located in urban settings, and the majority lived in the province of Kinshasa. There was a statistically significant association between seasonality and IgM Leptospira spp. positivity amongst those living in Kinshasa, where most of the positive cases occurred during the rainy season. CONCLUSIONS: This study showed that leptospirosis is likely an overlooked cause of unexplained cases of fever with jaundice in the DRC and highlights the need to consider leptospirosis in the differential diagnosis of fever with jaundice, particularly in young adult males. Further studies are needed to identify animal reservoirs, associated risk factors, and the burden of human leptospirosis in the DRC.
Assuntos
Febre/diagnóstico , Febre/epidemiologia , Febre/microbiologia , Leptospirose/diagnóstico , Leptospirose/epidemiologia , Adolescente , Adulto , Animais , Anticorpos Antibacterianos/sangue , Criança , Pré-Escolar , República Democrática do Congo/epidemiologia , Humanos , Imunoglobulina M/sangue , Lactente , Recém-Nascido , Icterícia/diagnóstico , Icterícia/epidemiologia , Icterícia/microbiologia , Leptospira/imunologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estudos Retrospectivos , Febre Amarela/diagnóstico , Febre Amarela/epidemiologia , Febre Amarela/microbiologia , Adulto JovemRESUMO
Gene editing using CRISPR/Cas9 is a promising method to cure many human genetic diseases. We have developed an efficient system to deliver Cas9 into the adeno-associated virus integration site 1 (AAVS1) locus, known as a safe harbor, using lentivirus and AAV viral vectors, as a step toward future in vivo transduction. First, we introduced Cas9v1 (derived from Streptococcus pyogenes) at random into the genome using a lentiviral vector. Cas9v1 activity was used when the N-terminal 1.9 kb, and C-terminal 2.3 kb fragments of another Cas9v2 (human codon-optimized) were employed sequentially with specific single-guide RNAs (sgRNAs) and homology donors carried by AAV vectors into the AAVS1 locus. Then, Cas9v1 was removed from the genome by another AAV vector containing sgRNA targeting the long terminal repeat of the lentivirus vector. The reconstituted Cas9v2 in the AAVS1 locus was functional and gene editing was efficient.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Dependovirus/genética , Lentivirus/genética , Streptococcus pyogenes/genética , Transdução Genética , Edição de Genes , Técnicas de Transferência de Genes , Loci Gênicos , Vetores Genéticos/genética , Células HEK293 , Humanos , RNA Guia de Cinetoplastídeos/genética , Transdução Genética/métodosRESUMO
Chikungunya virus (CHIKV) is an enveloped virus that enters host cells and transits within the endosomes before starting its replication cycle, the precise mechanism of which is yet to be elucidated. Endocytosis and endosome acidification inhibitors inhibit infection by CHIKV, murine leukemia virus (MLV), or SARS-coronavirus, indicating that these viral entries into host cells occur through endosomes and require endosome acidification. Although endosomal cathepsin B protease is necessary for MLV, Ebola virus, and SARS-CoV infections, its role in CHIKV infection is unknown. Our results revealed that endocytosis inhibitors attenuated CHIKV-pseudotyped MLV vector infection in 293T cells but not in TE671 cells. In contrast, macropinocytosis inhibitors attenuated CHIKV-pseudotyped MLV vector infection in TE671 cells but not in 293T cells, suggesting that CHIKV host cell entry occurs via endocytosis or macropinocytosis, depending on the cell lines used. Cathepsin B inhibitor and knockdown by an shRNA suppressed CHIKV-pseudotyped MLV vector infection both in 293T and TE671 cells. These results show that cathepsin B facilitates CHIKV infection regardless of the entry pathway.
Assuntos
Catepsina B/metabolismo , Febre de Chikungunya/patologia , Vírus Chikungunya/fisiologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Catepsina B/antagonistas & inibidores , Linhagem Celular Tumoral , Endocitose/fisiologia , Endossomos/virologia , Células HEK293 , Células HeLa , Humanos , Vírus da Leucemia Murina/fisiologia , Pinocitose/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Human immunodeficiency virus type 1 (HIV-1)-based viral vector is widely used as a biomaterial to transfer a gene of interest into target cells in many biological study fields including gene therapy. Vesicular stomatitis virus glycoprotein (VSV-G)-containing HIV-1 vector much more efficiently transduces various mammalian cells than other viral envelope proteins-containing vectors. Understanding the mechanism would contribute to development of a novel method of efficient HIV-1 vector production. HIV-1 vector is generally constructed by transient transfection of human 293T or African green monkey COS7 cells. It was found in this study that HIV-1 Gag protein is constitutively digested in lysosomes of African green monkey cells. Surprisingly, VSV-G elevated HIV-1 Gag protein levels, suggesting that VSV-G protects Gag protein from the lysosomal degradation. Unphosphorylated ezrin, but not phosphorylated ezrin, was detected in COS7 cells, and ezrin silencing elevated Gag protein levels in the presence of VSV-G. Expression of unphosphorylated ezrin reduced Gag protein amounts. These results indicate that unphosphorylated ezrin proteins inhibit the VSV-G-mediated stabilization of HIV-1 Gag protein. Trafficking of HIV-1 Gag-associated intracellular vesicles may be controlled by ezrin. Finally, this study found that ezrin silencing yields higher amount of VSV-G-pseudotyped HIV-1 vector.
RESUMO
Sarcopenia is characterized by a progressive skeletal muscle disorder that involves the loss of muscle mass and low muscle strength, which contributes to increased adverse outcomes. Few studies have investigated the association between chronic infection and sarcopenia. This study aimed to examine the association between human T-cell lymphotropic virus type-1 (HTLV-1) and sarcopenia. We conducted a cross-sectional study and enrolled 2,811 participants aged ≥ 40 years from a prospective cohort study in Japanese community dwellers during 2017-2019. Sarcopenia was defined as low appendicular skeletal muscle mass and low handgrip strength. The association between HTLV-1 seropositivity and sarcopenia was assessed using multivariable logistic regression. Odds ratio (OR) and 95% confidence interval (CI) of sarcopenia were analysed using HTLV-1 seropositivity. We adjusted for age, sex, body mass index, physical activity, systolic blood pressure, glycated haemoglobin, low-density lipoprotein cholesterol, and smoking and drinking status. Of 2,811 participants, 484 (17.2%) HTLV-1 infected participants were detected. HTLV-1 infection was significantly associated with sarcopenia (adjusted OR 1.46, 95% CI 1.03-2.07, P = 0.034). HTLV-1 was associated with sarcopenia among community-dwelling adults. Active surveillance and early detection of asymptomatic HTLV-1 infection might be beneficial to reinforce countermeasures to inhibit the progress of HTLV infection-associated sarcopenia.