RESUMEN

S100A8 and S100A9 are members of the Alarmin family; these proteins are abundantly expressed in neutrophils, form a heterodimer complex, and are secreted in plasma on pathogen infection or acute inflammatory diseases. Recently, both proteins were identified as novel biomarkers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and were shown to play key roles in inducing an aggressive inflammatory response by mediating the release of large amounts of pro-inflammatory cytokines, called the "cytokine storm." Although co-infection with SARS-CoV-2 in people living with HIV-1 may result in an immunocompromised status, the role of the S100A8/A9 complex in HIV-1 replication in primary T cells and macrophages is still unclear. Here, we evaluated the roles of the proteins in HIV replication to elucidate their functions. We found that the complex had no impact on virus replication in both cell types; however, the subunits of S100A8 and S100A9 inhibit HIV in macrophages. These findings provide important insights into the regulation of HIV viral loads during SARS-CoV-2 co-infection.

Asunto(s)

COVID-19 , Coinfección , Infecciones por VIH , Biomarcadores/metabolismo , Calgranulina A/metabolismo , Calgranulina B , Infecciones por VIH/metabolismo , Humanos , Macrófagos , SARS-CoV-2 , Replicación Viral

RESUMEN

S100A8 and S100A9 are members of the Alarmin family; these proteins are abundantly expressed in neutrophils and form a heterodimer complex. Recently, both proteins were identified as novel biomarkers of SARS-CoV-2 infection and were shown to play key roles in inducing an aggressive inflammatory response by mediating the release of large amounts of pro-inflammatory cytokines, called the "cytokine storm." Although co-infection with SARS-CoV-2 in people living with HIV-1 may result in an immunocompromised status, the role of the S100A8/A9 complex in HIV-1 replication in primary T cells and macrophages is still unclear. Here, we evaluated the roles of the proteins in HIV replication to elucidate their functions. We found that the complex had no impact on virus replication in both cell types; however, the subunits of S100A8 and S100A9 inhibits HIV in macrophages. These findings provide important insights into the regulation of HIV viral loads in SARS-CoV2 co-infection.

RESUMEN

Cilia on the ventral surface of the hypotrich ciliate Euplotes are clustered into polykinetids or compound ciliary organelles, such as cirri or oral membranelles, used in locomotion and prey capture. A single polykinetid may contain more than 150 individual cilia; these emerge from basal bodies held in a closely spaced array within a scaffold or framework structure that has been referred to as a basal-body "cage". Cage structures were isolated free of cilia and basal bodies; the predominant component of such cages was found on polyacrylamide gels to be a 45-kDa polypeptide. Antisera were raised against this protein band and used for immunolocalizations at the light and electron microscope levels. Indirect immunofluorescence revealed the 45-kDa polypeptide to be localized exclusively to the bases of the ventral polykinetids. Immunogold staining of thin sections of intact cells further localized this reactivity to filaments of a double-layered dense lattice that appears to link adjoining basal bodies into ordered arrays within each polykinetid. Scanning electron microscopy of isolated cages reveals the lower or "basal" cage layer to be a fine lacey meshwork supporting the basal bodies at their proximal ends; adjoining basal bodies are held at their characteristic spacing by filaments of an upper or "medial" cage layer. The isolated cage thus resembles a miniature test-tube rack, able to accommodate varying arrangements of basal-body rows, depending on the particular type of polykinetid. Because of its clear and specific localization to the basal-body cages in Euplotes, we have termed this novel 45-kDa protein "cagein".

Asunto(s)

Euplotes/química , Euplotes/ultraestructura , Orgánulos/química , Orgánulos/ultraestructura , Proteínas Protozoarias/análisis , Electroforesis en Gel de Poliacrilamida , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Peso Molecular

RESUMEN

Cytosolic foreign DNA is detected by pattern recognition receptors and mainly induces type I IFN production. We found that transfection of different types of DNA into various untreated cells induces type III IFN (IFN-λ1) rather than type I IFN, indicating the presence of uncharacterized DNA sensor(s). A pull-down assay using cytosolic proteins identified that Ku70 and Ku80 are the DNA-binding proteins. The knockdown studies and the reporter assay revealed that Ku70 is a novel DNA sensor inducing the IFN-lambda1 activation. The functional analysis of IFNL1 promoter revealed that positive-regulatory domain I and IFN-stimulated response element sites are predominantly involved in the DNA-mediated IFNL1 activation. A pull-down assay using nuclear proteins demonstrated that the IFN-λ1 induction is associated with the activation of IFN regulatory factor-1 and -7. Thus, to our knowledge, we show for the first time that Ku70 mediates type III IFN induction by DNA.

Asunto(s)

Antígenos Nucleares/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Interferón Tipo I/metabolismo , Interleucinas/metabolismo , Animales , Antígenos Nucleares/genética , Western Blotting , Línea Celular , Línea Celular Tumoral , Citosol/metabolismo , ADN/genética , Proteínas de Unión al ADN/genética , Femenino , Células HEK293 , Células HeLa , Humanos , Factor 1 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/metabolismo , Interferón Tipo I/genética , Interferones , Interleucinas/genética , Autoantígeno Ku , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Regiones Promotoras Genéticas/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección

RESUMEN

OBJECTIVE: IL-27 is a novel anti-HIV cytokine that inhibits HIV-1 replication in both CD4 T cells and monocyte-derived macrophages (MDM) as IFN-alpha does. To elucidate the mechanism of the antiviral activity, we compared the activity and the gene expression profile of IL-27-treated cells with that of IFN-alpha-treated cells. METHODS: CD4 T cells and monocytes were isolated from peripheral blood mononuclear cells of healthy donors. CD4 T cells were stimulated with phytohemagglutinin, and MDM were induced from monocytes using macrophage-colony stimulating factor. HIV-1 replication was monitored by p24 antigen capture assay. The gene expression profiles were analysed using DNA microarray analysis. The increase in the expression of IFN-inducible genes (IFIG) was confirmed by the Quantigene plex assay. RESULTS: Both cytokines preferentially inhibited HIV-1 replication in MDM compared with CD4 T cells. Quantitative real time polymerase chain reaction, enzyme-linked immunosorbent assay and neutralization assay using anti-IFN indicated that IFN-alpha, IFN-beta and IFN-gamma had no significant impact on IL-27-mediated HIV inhibition. DNA microarray analysis illustrated that IFN-alpha induced 33 and 18 IFIG in MDM and CD4 T cells, respectively. IL-27 induced 28 IFIG in MDM and five IFIG in CD4 T cells. The quantitative assay confirmed that IL-27 activated genes of RNA-dependent kinase, oligoadenylate synthetase, myxovirus protein, and apolipoprotein B messenger RNA-editing enzyme-catalytic polypeptide-like 3G. CONCLUSION: IL-27 differentially regulates the gene expression between CD4 T cells and MDM. IL-27 significantly induces antiviral genes in MDM as does IFN-alpha, suggesting that IL-27 inhibits HIV replication in MDM via mechanism(s) similar to that of IFN-alpha.

Asunto(s)

Regulación Viral de la Expresión Génica , Infecciones por VIH/virología , VIH-1/genética , Interleucinas/genética , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Medios de Cultivo Condicionados/metabolismo , Relación Dosis-Respuesta a Droga , Genes Virales/genética , Interferón-alfa/farmacología , Interleucinas/biosíntesis , Interleucinas/farmacología , Macrófagos/metabolismo , Macrófagos/virología , Análisis de Secuencia por Matrices de Oligonucleótidos , Replicación Viral/inmunología

RESUMEN

The existence of viral latency limits the success of highly active antiretroviral therapy. With the therapeutic intention of reactivating latent virus to induce a cure, in this study we assessed the impact of cell synchronizers on HIV gene activation in latently infected U1 cells and investigated the molecular mechanisms responsible for such effect. Latently infected U1 cells were treated with 10 drugs including hydroxyurea (HU) and HIV-1 replication monitored using a p24 antigen capture assay. We found that HU was able to induce HIV-1 replication by 5-fold. HU has been used in the clinical treatment of HIV-1-infected patients in combination with didanosine; therefore, we investigated the impact of HU on HIV-1 activation in the presence of the proinflammatory cytokines, interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha). IL-6 or TNF-alpha alone induced HIV replication by 18- and approximately 500-fold, respectively. Of interest, in the presence of HU, IL-6-mediated HIV-1 activation was enhanced by >90-fold, whereas TNF-alpha-mediated activation was inhibited by >30%. A reporter gene assay showed that HU and IL-6 synergized to activate HIV promoter activity via the Sp1 binding site. Electrophoretic mobility shift and supershift assays revealed increased binding of the Sp1 and Sp3 transcription factors to this region. Western blot analysis showed that HU and IL-6 co-stimulation resulted in increased levels of Sp1 and Sp3 proteins. In contrast, treatment with HU plus TNF-alpha down-regulated the expression of NF-kappaB. These findings suggest that Sp1/Sp3 is involved in controlling the HU/IL-6-induced reactivation of HIV-1 in latently infected cells.

Asunto(s)

VIH-1/fisiología , Hidroxiurea/farmacología , Interleucina-6/fisiología , Monocitos/metabolismo , Factor de Transcripción Sp1/fisiología , Factor de Transcripción Sp3/fisiología , Activación Viral/fisiología , Línea Celular Tumoral , Sinergismo Farmacológico , VIH-1/efectos de los fármacos , Humanos , Monocitos/efectos de los fármacos , Monocitos/virología , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Latencia del Virus/fisiología , Replicación Viral/efectos de los fármacos , Replicación Viral/fisiología

RESUMEN

Population-based sequence analysis revealed the presence of a variant of human immunodeficiency virus type 1 (HIV-1) containing an insertion of amino acid Ile in the protease gene at codon 19 (19I) and amino acid substitutions in the protease at codons 21 (E21D) and 22 (A22V) along with multiple mutations associated with drug resistance, M46I/P63L/A71V/I84V/I93L, in a patient who had failed protease inhibitor (PI) therapy. Longitudinal analysis revealed that the P63L/A71V/I93L changes were present prior to PI therapy. Polymorphisms in the Gag sequence were only seen in the p1/p6 cleavage site at the P1' position (Leu to Pro) and the P5' position (Pro to Leu). To characterize the role of these mutations in drug susceptibility and replication capacity, a chimeric HIV-1 strain containing the 19I/E21D/A22V mutations with the M46I/P63L/A71V/I84V/I93L and p1/p6 mutations was constructed. The chimera displayed high-level resistance to multiple PIs, but not to lopinavir, and grew to 30% of that of the wild type. To determine the relative contribution of each mutation to the phenotypic characteristic of the virus, a series of mutants was constructed using site-directed mutagenesis. A high level of resistance was only seen in mutants containing the 19I/A22V and p1/p6 mutations. The E21D mutation enhanced viral replication. These results suggest that the combination of the 19I/E21D/A22V mutations may emerge and lead to high-level resistance to multiple PIs. The combination of the 19I/A22V mutations may be associated with PI resistance; however, the drug resistance may be caused by the presence of a unique set of mutations in the p1/p6 mutations. The E21D mutation contributes to replication fitness rather than drug resistance.

Asunto(s)

Farmacorresistencia Viral , Proteína p24 del Núcleo del VIH/genética , Proteasa del VIH/genética , VIH-1/genética , Mutación , Replicación Viral/genética , Codón , Humanos , Polimorfismo Genético , Inhibidores de Proteasas/uso terapéutico , Insuficiencia del Tratamiento

RESUMEN

Vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) have been implicated as important stimulatory factors for retinal neovascularization. In this study, we used intraocular gene transfer with gutless adenoviral (AGV) vectors to determine the effect of increased intraocular expression of VEGF, IGF-1, or sphingosine kinase (SPK), which produces sphingosine-1-phosphate, another angiogenic factor. Retinal neovascularization did not occur from intravitreous AGV-vectored VEGF, IGF-1, SPK, or combined VEGF and IGF-1, except occasionally adjacent to the retinal penetration site from the injection. However, corneal and iris neovascularization occurred after 2 weeks in all eyes injected with AGV.VEGF, but not those injected with only AGV.IGF-1 or AGV.SPK. These data suggest that the superficial capillary bed of the retina is relatively insensitive to VEGF, IGF-1, or SPK in adult mice, except when combined with retinal trauma. However, AGV-vectored VEGF is sufficient to consistently cause severe corneal and iris neovascularization. This provides a model for anterior segment neovascularization, which unlike previous models is relatively inexpensive and is not plagued by spontaneous regression, and therefore, may be useful for identification of new treatments.

Asunto(s)

Adenoviridae , Ojo/irrigación sanguínea , Neovascularización Retiniana , Transducción Genética , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Adenoviridae/genética , Animales , Cartilla de ADN , Vectores Genéticos , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/genética , Operón Lac , Lisofosfolípidos/metabolismo , Ratones , Neovascularización Fisiológica/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética

RESUMEN

While 51 human adenoviral serotypes have been identified to date, the vast majority of adenoviral vectors designed for gene transfer have been generated in the adenovirus serotype 5 (Ad5) backbone. Viral infections caused by Ad5 are endemic in most human populations and the majority of humans carry preexisting humoral and/or cellular immunity to Ad5 which may severely limit the use of Ad5-based vectors for gene therapy applications. To circumvent this preexisting Ad5 immunity, we have identified Ad35 as an alternative adenoviral serotype to which the majority of humans do not have neutralizing antibodies. Importantly, Ad35 can be grown to high titers with a low particle-to-PFU ratio. As a prerequisite for the development of Ad35 for use as a gene transfer vector, a genome organization map was constructed using the available Ad35 sequence information, and E1a-deficient Ad35 vectors encoding marker genes were generated. Ad35 biodistribution in mice was assessed following intravenous administration and compared with that of Ad5. Extremely low levels of Ad35 were detected in all organs evaluated, including liver, lung, spleen, and bone marrow, while Ad5 displayed high transduction of these organs. Due to the lack of Ad35 liver tropism, minimal hepatotoxicity was observed in mice treated with Ad35. Furthermore, the half-life of Ad35 in mouse blood was found to be two to three times longer than that of Ad5. These data suggest that either mice do not express the Ad35 cell surface receptor or that Ad35 does not efficiently transduce mouse cells in vivo following systemic delivery. Therefore, to begin to elucidate the Ad35 cell entry mechanisms, in vitro competition studies were performed. These data demonstrated that Ad35 cell entry is CAR independent, and may involve protein(s) expressed on most human cells.

Asunto(s)

Adenoviridae/clasificación , Adenoviridae/fisiología , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Adenoviridae/genética , Adenoviridae/crecimiento & desarrollo , Animales , Línea Celular , Femenino , Ingeniería Genética , Vectores Genéticos/fisiología , Genoma Viral , Humanos , Hígado/virología , Ratones , Ratones Endogámicos C57BL , Serotipificación , Transducción Genética

RESUMEN

Transcriptional regulation that is rapid, reversible, and repeatedly inducible would greatly enhance the safety and efficacy of many gene therapy strategies. We developed a chimeric ligand-inducible regulation system based on the human estrogen receptor. This system has two components, the responsive promoter driving expression of the transgene of interest, and the ligand-inducible chimeric transcription factor. The transcription factor is composed of a novel DNA binding domain and a modified estrogen receptor ligand-binding domain. A point mutation in the ligand-binding domain significantly reduces estrogen binding while allowing binding of the estrogen antagonist, tamoxifen. We used a gutless adenoviral vector system and incorporated both components into two separate vectors. A single gutless vector encoding both system components was also generated. The tamoxifen-mediated induciblity of transgene expression of the gutless vector system was compared in vitro and in vivo with the analogous components incorporated into early generation, E1/E2a/E3-deficient adenoviral vectors. In normal mice, both the gutless vector and early generation systems displayed inducibility in the presence of tamoxifen. Importantly, the gutless vector system was inducible to extremely high levels, at least four times over a 2-month period. In contrast, the early generation vector system was inducible only once. Furthermore, the early generation system displayed significant toxicity, as evidenced by extremely high liver enzyme levels, abnormal liver pathology, and rapid loss of vector DNA from the liver, while the gutless vector system displayed minimal toxicity. These data directly demonstrate the improved in vivo function of the tamoxifen-inducible transcriptional regulation system in the context of the gutless adenoviral vectors.

Asunto(s)

Adenoviridae/genética , Regulación de la Expresión Génica , Vectores Genéticos , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas , Virus Defectuosos/genética , Endostatinas/biosíntesis , Endostatinas/genética , Vectores Genéticos/toxicidad , Células HeLa , Humanos , Ligandos , Hígado/metabolismo , Hígado/patología , Hepatopatías/metabolismo , Hepatopatías/patología , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Transcripción Genética

RESUMEN

Gutless adenoviral vectors are devoid of all viral coding regions and display reduced cytotoxicity, diminished immunogenicity, and an increased coding capacity compared with early generation vectors. Using hemophilia A, a deficiency in clotting factor VIII (FVIII), as a model disease, we generated and evaluated a gutless vector encoding human FVIII. The FVIII gutless vector grew to high titer and was reproducibly scaled-up from vector seed lots. Extensive viral DNA analyses revealed no rearrangements of the vector genome. A quantitative PCR assay demonstrated helper virus contamination levels of <2%, with the best preparation containing 0.3% helper virus. We compared the gutless vector with an E1/E2a/E3-deficient (Av3) early generation vector encoding an identical FVIII expression cassette following intravenous administration to hemophilia A mice. Gutless vector-treated mice displayed 10-fold higher FVIII expression levels that were sustained for at least 9 months. In contrast, mice treated with the Av3 vector displayed FVIII levels below the limit of sensitivity of the assay at 3 months. Assessment of hepatotoxicity by measuring the serum levels of liver enzymes demonstrated that the gutless vector was significantly less toxic than the Av3 vector at time points later than 7 days. At the highest dose used, both vectors caused a transient 10-fold increase in liver enzymes 1 day after vector administration, suggesting that this increase was caused by direct toxicity of the input capsid proteins. These data demonstrate that the gutless vector displayed increased duration and levels of FVIII expression, and was significantly less toxic than an analogous early generation vector.

Asunto(s)

Adenoviridae/genética , Factor VIII/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Hemofilia A/genética , Animales , Secuencia de Bases , Cartilla de ADN , Factor VIII/administración & dosificación , Femenino , Terapia Genética , Hemofilia A/terapia , Humanos , Ratones , Reacción en Cadena de la Polimerasa , Transducción Genética