RESUMO
The pUC-derived plasmid yield from E. coli using polypropylene tubes (PP) was compared among round and conical tubes. The yield from cells grown in a cheaper conical-PP with flat-bottom was 1.5-fold higher (p < 0.001) than other PP. The use of the conical-PP can save research budgets in the current inflationary environment.
Assuntos
Escherichia coli , Plasmídeos , Polipropilenos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Polipropilenos/química , Plasmídeos/genética , Vetores GenéticosRESUMO
Recently, a genome-wide association study using plasma HIV RNA from antiretroviral therapy-naive patients reported that 14 naturally occurring nonsynonymous single-nucleotide polymorphisms (SNPs) in HIV derived from antiretrovirus drug-naive patients were associated with virus load (VL). Those SNPs were detected in reverse transcriptase, RNase H, integrase, envelope, and Nef. However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP and examined their replicative abilities. An HIV variant containing a Met-to-Ile change at codon 50 in integrase [HIV(IN:M50I)] was found as an impaired virus. Despite the mutation being in integrase, the virus release was significantly suppressed (P < 0.001). Transmission electron microscopy analysis revealed that abnormal bud accumulation on the plasma membrane and the released virus particles retained immature forms. Western blot analysis demonstrated a defect in autoprocessing of GagPol and Gag polyproteins' autoprocessing in the HIV(IN:M50I) particles, although Förster resonance energy transfer (FRET) assay displayed that GagPol containing IN:M50I forms a homodimer with a similar efficiency with GagPol (wild type). The impaired maturation and replication were rescued by two other VL-associated SNPs, Ser-to-Asn change at codon 17 of integrase and Asn-to-Ser change at codon 79 of RNase H. These data demonstrate that Gag and GagPol assembly, virus release, and autoprocessing are regulated by not only integrase but also RNase H. IMPORTANCE Nascent HIV-1 is a noninfectious viral particle. Cleaving Gag and GagPol polyproteins in the particle by mature HIV protease (PR), the nascent virus becomes an infectious virus. PR is initially translated as an inactive embedded enzyme in a GagPol polyprotein. The embedded PR in homodimerized GagPol polyproteins catalyzes a proteolytic reaction to release the mature PR. This excision step by self-cleavage is called autoprocessing. Here, during the evaluation of the roles of naturally emerging nonsynonymous SNPs in HIV RNA, we found that autoprocessing is inhibited by Met-to-Ile change at codon 50 in integrase GagPol. Other coexisting SNPs, Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser mutation at codon 79 in RNase H, recovered this defect, suggesting that autoprocessing is regulated by not only integrase but also RNase H in GagPol polyprotein.
Assuntos
Integrase de HIV/metabolismo , HIV-1/fisiologia , Ribonuclease H/metabolismo , Liberação de Vírus/fisiologia , Antirretrovirais/farmacologia , Produtos do Gene gag/genética , Células HEK293 , Infecções por HIV , Integrase de HIV/genética , HIV-1/genética , Humanos , Mutação , Polimorfismo de Nucleotídeo Único , Proteólise , Ribonuclease H/genética , Vírion/metabolismo , Replicação ViralRESUMO
We have previously identified that human Ku70, a nuclear protein, serves as a cytosolic DNA sensor. Upon transfection with DNA or infection with DNA virus, Ku70 translocates from the nucleus into the cytoplasm and then predominately induces interferon lambda1 (IFN-λ1) rather than IFN-alpha or IFN-beta, through a STING-dependent signalling pathway. However, a detailed mechanism for Ku70 cytoplasmic translocation and its correlation with IFN-λ1 induction have not been fully elucidated. Here, we observed that cytoplasmic translocation of Ku70 only occurred in DNA-triggered IFN-λ1-inducible cells. Additionally, infection by Herpes simplex virus type-1 (HSV-1), a DNA virus, induces cytoplasmic translocation of Ku70 and IFN-λ1 induction in a strain-dependent manner: the translocation and IFN-λ1 induction were detected upon infection by HSV-1 McKrae, but not MacIntyre, strain. A kinetic analysis indicated that cytoplasmic translocation of Ku70 was initiated right after DNA transfection and was peaked at 6 hr after DNA stimulation. Furthermore, treatment with leptomycin B, a nuclear export inhibitor, inhibited both Ku70 translocation and IFN-λ1 induction, suggesting that Ku70 translocation is an essential and early event for its cytosolic DNA sensing. We further confirmed that enhancing the acetylation status of the cells promotes Ku70's cytoplasmic accumulation, and therefore increases DNA-mediated IFN-λ1 induction. These findings provide insights into the molecular mechanism by which the versatile sensor detects pathogenic DNA in a localization-dependent manner.
Assuntos
Citoplasma/metabolismo , Herpes Simples/imunologia , Herpesvirus Humano 1/fisiologia , Interferons/metabolismo , Autoantígeno Ku/metabolismo , Acetilação , Antibióticos Antineoplásicos/farmacologia , DNA Viral/genética , DNA Viral/imunologia , Ácidos Graxos Insaturados/farmacologia , Células HEK293 , Humanos , Interferons/genética , Espaço Intracelular/genética , Espaço Intracelular/imunologia , Transporte Proteico , Especificidade da Espécie , Regulação para CimaRESUMO
We previously reported that some small interfering RNA (siRNA) enhances DNA or DNA virus mediated-interferon (IFN)-λ1(a type III IFN) induction through the crosstalk between retinoic acid-inducible gene I (RIG-I) and interferon gamma-inducible protein 16 (IFI16) signalling pathway. Here we provide further evidence of a new role for siRNA. siRNA containing a 5-nucleotide (nt) motif sequence suppresses DNA-mediated not only type III IFNs, but also type I IFNs and inflammatory cytokines. We define that motif siRNA inhibits the induction when the motif is located at the 3' or 5'-terminus of siRNA. Using THP1-Lucia ISG cells with various DNA stimulants, we reveal that motif siRNA inhibits DNA or DNA virus but not RNA virus-mediated signalling. Motif siRNA specifically interrupts IFI16 but not cyclic GMP-AMP synthase (cGAS) binding to DNA and has 2.5-fold higher affinity to IFI16 than that of siRNA without the motif. We further confirm that motif siRNA potently suppresses HSV-1 virus-mediated IFNs and inflammatory cytokines, such as IFNL1, IFNB and TNFA, in human primary immature dendritic cells. Collectively, these findings may shed light on a novel function of siRNA with the unique 5-nt motif as a quencher of innate immunity and facilitate the development of potential therapeutics to regulate innate immune cascades.
Assuntos
Imunidade Inata/imunologia , Proteínas Nucleares/imunologia , Motivos de Nucleotídeos/fisiologia , Fosfoproteínas/imunologia , RNA Interferente Pequeno/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Citocinas/imunologia , DNA/imunologia , Células Dendríticas/imunologia , Células HEK293 , Células HeLa , Herpesvirus Humano 1/imunologia , Humanos , Interferon Tipo I/imunologia , Transdução de Sinais/imunologia , Células THP-1RESUMO
We previously identified Ku70, a subunit of a DNA repair protein complex, as a cytosolic DNA sensor that induces the production of interferon-λ1 (IFN-λ1) by human primary cells and cell lines. IFN-λ1 is a type III IFN and has similar antiviral activity to that of the type I IFNs (IFN-α and IFN-ß). We observed that human embryonic kidney (HEK) 293T cells, which are deficient in the innate immune adaptor protein STING (stimulator of IFN genes), did not produce IFN-λ1 in response to DNA unless they were reconstituted with STING. Conversely, parental HEK 293 cells produced IFN-λ1 after they were exposed to exogenous DNA; however, when STING was knocked out in the HEK 293 cells through the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system, they lost this response. Through confocal microscopy, we demonstrated that endogenous Ku70 was located in the nucleus and then translocated to the cytoplasm upon DNA exposure to form a complex with STING. Additionally, the DNA binding domain of Ku70 was essential for formation of the Ku70-STING complex. Knocking down STING in primary human macrophages inhibited their ability to produce IFN-λ1 in response to transfection with DNA or infection with the DNA virus HSV-2 (herpes simplex virus-2). Together, these data suggest that STING mediates the Ku70-mediated IFN-λ1 innate immune response to exogenous DNA or DNA virus infection.
Assuntos
Interferons/metabolismo , Interleucinas/imunologia , Autoantígeno Ku/metabolismo , Macrófagos/imunologia , Proteínas de Membrana/metabolismo , DNA Viral/imunologia , Células HEK293 , Humanos , Imunidade Inata , Transporte ProteicoRESUMO
Interleukin (IL)-27, a member of the IL-12 cytokine family, plays an important and diverse role in the function of the immune system. We have previously demonstrated that IL-27 is an anti-viral cytokine which inhibits HIV-1, HIV-2, Influenza virus and herpes simplex virus infection, and enhances the potential of reactive oxygen species (ROS) generating activity during differentiation of monocytes to macrophages. In this study, we further investigated the mechanism of the enhanced potential for ROS generation by IL-27. Real time PCR, western blot and knock down assays demonstrate that IL-27 is able to enhance the potential of superoxide production not only during differentiation but also in terminally differentiated-macrophages and immature dendritic cells (iDC) in association with the induction of p47phox, a cytosolic component of the ROS producing enzyme, NADPH oxidase, and the increase in amounts of phosphorylated p47phox upon stimulation. We also demonstrate that IL-27 is able to induce extracellular superoxide dismutase during differentiation of monocytes but not in terminal differentiated macrophages. Since ROS plays an important role in a variety of inflammation, our data demonstrate that IL-27 is a potent regulator of ROS induction and may be a novel therapeutic target.
Assuntos
Células Dendríticas/efeitos dos fármacos , Interleucinas/farmacologia , Macrófagos/efeitos dos fármacos , NADPH Oxidases/genética , Superóxidos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/imunologia , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-4/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/imunologia , NADPH Oxidases/imunologia , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase/imunologia , Superóxidos/imunologiaRESUMO
In addition to silencing specific genes, small interfering RNA (siRNA) transfection is also associated with the non-specific induction of inflammatory cytokines and type I interferon. Those so-called "off-target" effects have considerable implications for the interpretation of in vitro studies and clinical application of siRNA. The present study attempted to develop a better understanding of the mechanism involved in these off target effects. Synthesized siRNA significantly enhances DNA-mediated interferon lambda-1 response (IFN-λ1/IL-29), a newly characterized antiviral interferon in non-immune or primary immune cells. This enhancement was most pronounced by double-stranded siRNA with at least a 2-nucleotide overhang at one 3' terminus in a dose-dependent manner, while the presence of DNA was indispensable. A pull-down assay using biotinylated siRNA- or DNA-conjugated beads indicated that retinoic acid-inducible gene I (RIG-I) and interferon gamma-inducible protein 16 (IFI16) were involved in the sensing of siRNA and DNA, respectively. Co-immunoprecipitation analysis further revealed that RIG-I and IFI16 formed a complex via siRNA, and the dissociation of IFI16 from this complex in the presence of DNA activated the downstream STING-TBK1-IRF3 (stimulator of interferon genes - tank-binding kinase 1 - interferon regulatory factor 3) pathway, shedding light on a new physiological signalling pathway to activate innate immunity. Collectively, these findings may provide rational information for siRNA-induced innate immunity, with important implications for developing siRNA-based reagents to control human diseases.
Assuntos
RNA Helicases DEAD-box/metabolismo , DNA/metabolismo , Interleucinas/biossíntese , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Linhagem Celular , Células Cultivadas , Proteína DEAD-box 58 , Células HeLa , Herpesvirus Humano 1/fisiologia , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferons , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/química , Receptores ImunológicosRESUMO
We have previously reported that a subunit protein vaccine based on the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein and a recombinant adeno-associated virus (rAAV)-based RBD (RBD-rAAV) vaccine could induce highly potent neutralizing Ab responses in immunized animals. In this study, systemic, mucosal, and cellular immune responses and long-term protective immunity induced by RBD-rAAV were further characterized in a BALB/c mouse model, with comparison of the i.m. and intranasal (i.n.) routes of administration. Our results demonstrated that: 1) the i.n. vaccination induced a systemic humoral immune response of comparable strength and shorter duration than the i.m. vaccination, but the local humoral immune response was much stronger; 2) the i.n. vaccination elicited stronger systemic and local specific cytotoxic T cell responses than the i.m. vaccination, as evidenced by higher prevalence of IL-2 and/or IFN-gamma-producing CD3+/CD8+ T cells in both lungs and spleen; 3) the i.n. vaccination induced similar protection as the i.m. vaccination against SARS-CoV challenge in mice; 4) higher titers of mucosal IgA and serum-neutralizing Ab were associated with lower viral load and less pulmonary pathological damage, while no Ab-mediated disease enhancement effect was observed; and 5) the vaccination could provide long-term protection against SARS-CoV infection. Taken together, our findings suggest that RBD-rAAV can be further developed into a vaccine candidate for prevention of SARS and that i.n. vaccination may be the preferred route of administration due to its ability to induce SARS-CoV-specific systemic and mucosal immune responses and its better safety profile.
Assuntos
Glicoproteínas de Membrana/administração & dosagem , Glicoproteínas de Membrana/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Administração Intranasal , Animais , Formação de Anticorpos , Células 3T3 BALB , Dependovirus/genética , Humanos , Imunidade nas Mucosas , Imunoglobulina A/imunologia , Pulmão/imunologia , Glicoproteínas de Membrana/genética , Camundongos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Glicoproteína da Espícula de Coronavírus , Baço/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinação , Proteínas do Envelope Viral/genética , Vacinas Virais/genética , Replicação Viral/efeitos dos fármacosRESUMO
A competitive indicator displacement assay has been successfully developed for the ratiometric determination of sulfhydryl-containing amino acids and peptides using heterobimetallic donor-acceptor complexes as chemodosimetric ensembles. Chromotropic cis-[ML2(CN)2](M = FeII, RuII, OsII; L = diimine) are used as signaling indicators and PtII(DMSO)Cl2 acceptor moiety is used as the receptor for the sulfhydryl-containing analytes. A series of three heterobimetallic donor-acceptor complexes: cis-FeII(bpy)2[CN-PtII(DMSO)Cl2]2 (1), cis-Ru(II)(bpy)2[CN-PtII(DMSO)Cl2]2 (2) and cis-Os(II)(bpy)2[CN-PtII(DMSO)Cl2]2 (3) are synthesized and characterized by X-ray crystallography. All the three ensembles are able to produce specific colorimetric/fluorimetric responses to sulfhydryl-containing amino acids (cysteine, homocysteine and methionine) as well as the sulfhydryl-containing small peptide glutathione. The mechanism of the competitive displacement assay is evaluated by examining the thermodynamics of formation of the donor-acceptor linkage and adducts between the acceptor metal and the sulfhydryl-containing analytes as well as by systematic variation of the donor and acceptor metals in the chemodosimetric ensembles.