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There is an urgent need to identify efficient antiviral compounds to combat existing and emerging RNA virus infections, particularly those related to seasonal and pandemic influenza outbreaks. While inhibitors of the influenza viral integral membrane proton channel protein (M2), neuraminidase (NA), and cap-dependent endonuclease are available, circulating influenza viruses acquire resistance over time. Thus, the need for the development of additional anti-influenza drugs with novel mechanisms of action exists. In the present study, a cell-based screening assay and a small molecule library were used to screen for activities that antagonized influenza A non-structural protein 1 (NS1), a highly conserved, multifunctional accessory protein that inhibits the type I interferon response against influenza. Two potential anti-influenza agents, compounds 157 and 164, were identified with anti-NS1 activity, resulting in the reduction of A/PR/8/34(H1N1) influenza A virus replication and the restoration of IFN-ß expression in human lung epithelial A549 cells. A 3D pharmacophore modeling study of the active compounds provided a glimpse of the structural motifs that may contribute to anti-influenza virus activity. This screening approach is amenable to a broader analysis of small molecule compounds to inhibit other viral targets.
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Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Interferón Tipo I , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Interferón Tipo I/metabolismo , Proteínas no Estructurales Virales/metabolismo , Gripe Humana/tratamiento farmacológico , Virus de la Influenza A/genética , Antivirales/farmacología , Antivirales/metabolismo , Replicación ViralRESUMEN
Gene editing may be used to excise the human immunodeficiency virus type 1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection. Here, using cells acutely or latently infected by HIV-1 and treated with long terminal repeat (LTR)-targeting CRISPR/Cas9, we show that the excised HIV-1 provirus persists for a few weeks and may rearrange in circular molecules. Although circular proviral DNA is naturally formed during HIV-1 replication, we observed that gene editing might increase proviral DNA circles with restored LTRs. These extrachromosomal elements were recovered and probed for residual activity through their transfection in uninfected cells. We discovered that they can be transcriptionally active in the presence of Tat and Rev. Although confirming that gene editing is a powerful tool to eradicate HIV-1 infection, this work highlights that, to achieve this goal, the LTRs must be cleaved in several pieces to avoid residual activity and minimize the risk of reintegration in the context of genomic instability, possibly caused by the off-target activity of Cas9. IMPORTANCE The excision of HIV-1 provirus from the host cell genome has proven feasible in vitro and, to some extent, in vivo. Among the different approaches, CRISPR/Cas9 is the most promising tool for gene editing. The present study underlines the remarkable effectiveness of CRISPR/Cas9 in removing the HIV-1 provirus from infected cells and investigates the fate of the excised HIV-1 genome. This study demonstrates that the free provirus may persist in the cell after editing and in appropriate circumstances may reactivate. As an episome, it might be transcriptionally active, especially in the presence of Tat and Rev. The persistence of the HIV-1 episome was strongly decreased by gene editing with multiple targets. Although gene editing has the potential to eradicate HIV-1 infection, this work highlights a potential issue that warrants further investigation.
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Sistemas CRISPR-Cas , ADN Circular , VIH-1/genética , Provirus/genética , Secuencias Repetidas Terminales , Proteína 9 Asociada a CRISPR , Edición Génica , Regulación Viral de la Expresión Génica , Terapia Genética , Células HEK293 , Infecciones por VIH/virología , Humanos , ARN Guía de Kinetoplastida/genéticaRESUMEN
The presence of T cell reservoirs in which human immunodeficiency virus (HIV) establishes latency by integrating into the host genome represents a major obstacle to an HIV cure and has prompted the development of strategies aimed at the eradication of HIV from latently infected cells. The "shock-and-kill" strategy is one of the most pursued approaches to the elimination of viral reservoirs. Although several latency-reversing agents (LRAs) have shown promising reactivation activity, they have failed to eliminate the cellular reservoir. In this study, we evaluated a novel immune system-mediated approach to clearing the HIV reservoir, based on a combination of innate immune stimulation and epigenetic reprogramming. The combination of the STING agonist cGAMP (cyclic GMP-AMP) and the FDA-approved histone deacetylase inhibitor resminostat resulted in a significant increase in HIV proviral reactivation and specific apoptosis in HIV-infected cells in vitro Reductions in the proportion of HIV-harboring cells and the total amount of HIV DNA were also observed in CD4+ central memory T (TCM) cells, a primary cell model of latency, where resminostat alone or together with cGAMP induced high levels of selective cell death. Finally, high levels of cell-associated HIV RNA were detected ex vivo in peripheral blood mononuclear cells (PBMCs) and CD4+ T cells from individuals on suppressive antiretroviral therapy (ART). Although synergism was not detected in PBMCs with the combination, viral RNA expression was significantly increased in CD4+ T cells. Collectively, these results represent a promising step toward HIV eradication by demonstrating the potential of innate immune activation and epigenetic modulation for reducing the viral reservoir and inducing specific death of HIV-infected cells.IMPORTANCE One of the challenges associated with HIV-1 infection is that despite antiretroviral therapies that reduce HIV-1 loads to undetectable levels, proviral DNA remains dormant in a subpopulation of T lymphocytes. Numerous strategies to clear residual virus by reactivating latent virus and eliminating the reservoir of HIV-1 (so-called "shock-and-kill" strategies) have been proposed. In the present study, we use a combination of small molecules that activate the cGAS-STING antiviral innate immune response (the di-cyclic nucleotide cGAMP) and epigenetic modulators (histone deacetylase inhibitors) that induce reactivation and HIV-infected T cell killing in cell lines, primary T lymphocytes, and patient samples. These studies represent a novel strategy for HIV eradication by reducing the viral reservoir and inducing specific death of HIV-infected cells.
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Linfocitos T CD4-Positivos/inmunología , Epigénesis Genética , Infecciones por VIH/inmunología , VIH-1/inmunología , Inmunidad Innata/inmunología , Activación Viral/inmunología , Latencia del Virus/inmunología , Regulación Viral de la Expresión Génica , Infecciones por VIH/genética , Infecciones por VIH/virología , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Sulfonamidas/farmacología , Replicación ViralRESUMEN
Current combination antiretroviral therapies (cART) are unable to eradicate HIV-1 from infected individuals because of the establishment of proviral latency in long-lived cellular reservoirs. The shock-and-kill approach aims to reactivate viral replication from the latent state (shock) using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells (kill) by specific therapeutics. The NF-κB RelA/p50 heterodimer has been characterized as an essential component of reactivation of the latent HIV-1 long terminal repeat (LTR). Nevertheless, prolonged NF-κB activation contributes to the development of various autoimmune, inflammatory, and malignant disorders. In the present study, we established a cellular model of HIV-1 latency in J-Lat CD4+ T cells that stably expressed the NF-κB superrepressor IκB-α 2NΔ4 and demonstrate that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivated HIV-1 from latency, even under conditions where NF-κB activation was repressed. Using specific calcineurin phosphatase, p38, and MEK1/MEK2 kinase inhibitors or specific short hairpin RNAs, c-Jun was identified to be an essential factor binding to the LTR enhancer κB sites and mediating the combined synergistic reactivation effect. Furthermore, acetylsalicylic acid (ASA), a potent inhibitor of the NF-κB activator kinase IκB kinase ß (IKK-ß), did not significantly diminish reactivation in a primary CD4+ T central memory (TCM) cell latency model. The present work demonstrates that the shock phase of the shock-and-kill approach to reverse HIV-1 latency may be achieved in the absence of NF-κB, with the potential to avoid unwanted autoimmune- and or inflammation-related side effects associated with latency-reversing strategies.IMPORTANCE The shock-and-kill approach consists of the reactivation of HIV-1 replication from latency using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells. The cellular transcription factor NF-κB is considered a master mediator of HIV-1 escape from latency induced by LRAs. Nevertheless, a systemic activation of NF-κB in HIV-1-infected patients resulting from the combined administration of different LRAs could represent a potential risk, especially in the case of a prolonged treatment. We demonstrate here that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivate HIV-1 from latency, even under conditions where NF-κB activation is repressed. Our study provides a molecular proof of concept for the use of anti-inflammatory drugs, like aspirin, capable of inhibiting NF-κB in patients under combination antiretroviral therapy during the shock-and-kill approach, to avoid potential autoimmune and inflammatory disorders that can be elicited by combinations of LRAs.
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VIH-1/efectos de los fármacos , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Antirretrovirales/uso terapéutico , Linfocitos T CD4-Positivos/inmunología , Regulación Viral de la Expresión Génica/genética , Infecciones por VIH/virología , Seropositividad para VIH/inmunología , VIH-1/fisiología , Humanos , Células Jurkat , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/metabolismo , Provirus/efectos de los fármacos , Provirus/fisiología , Receptores Inmunológicos/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Co-infection or superinfection of the host by two or more virus species is a common event, potentially leading to viral interference, viral synergy, or neutral interaction. The simultaneous presence of two or more viruses, even distantly related, within the same cell depends upon viral tropism, i.e., the entry of viruses via receptors present on the same cell type. Subsequently, productive infection depends on the ability of these viruses to replicate efficiently in the same cellular environment. HIV-1 initially targets CCR5-expressing tissue memory CD4+ T cells, and in the absence of early cART initiation, a co-receptor switch may occur, leading to the infection of naïve and memory CXCR4-expressing CD4+ T cells. HIV-1 infection of macrophages at the G1 stage of their cell cycle also occurs in vivo, broadening the possible occurrence of co-infections between HIV-1 and other viruses at the cellular level. Moreover, HIV-1-infected DCs can transfer the virus to CD4+ T cells via trans-infection. This review focuses on the description of reported co-infections within the same cell between HIV-1 and other human pathogenic, non-pathogenic, or low-pathogenic viruses, including HIV-2, HTLV, HSV, HHV-6/-7, GBV-C, Dengue, and Ebola viruses, also discussing the possible reciprocal interactions in terms of virus replication and virus pseudotyping.
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Type I interferon (IFN-I) evasion by Dengue virus (DENV) is key in DENV pathogenesis. The non-structural protein 5 (NS5) antagonizes IFN-I response through the degradation of the signal transducer and activator of transcription 2 (STAT2). We developed a K562 cell-based platform, for high throughput screening of compounds potentially counteracting the NS5-mediated antagonism of IFN-I signaling. Upon a screening with a library of 1220 approved drugs, 3 compounds previously linked to DENV inhibition (Apigenin, Chrysin, and Luteolin) were identified. Luteolin and Apigenin determined a significant inhibition of DENV2 replication in Huh7 cells and the restoration of STAT2 phosphorylation in both cell systems. Apigenin and Luteolin were able to stimulate STAT2 even in the absence of infection. Despite the "promiscuous" and "pan-assay-interfering" nature of Luteolin, Apigenin promotes STAT2 Tyr 689 phosphorylation and activation, highlighting the importance of screening for compounds able to interact with host factors, to counteract viral proteins capable of dampening innate immune responses.
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Virus del Dengue , Apigenina/farmacología , Virus del Dengue/fisiología , Luteolina/farmacología , Transducción de Señal , Factor de Transcripción STAT2/genética , Factor de Transcripción STAT2/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , HumanosRESUMEN
The emergence of the new pathogen SARS-CoV-2 determined a rapid need for monoclonal antibodies (mAbs) to detect the virus in biological fluids as a rapid tool to identify infected individuals to be treated or quarantined. The majority of commercially available antigenic tests for SARS-CoV-2 rely on the detection of N antigen in biologic fluid using anti-N antibodies, and their capacity to specifically identify subjects infected by SARS-CoV-2 is questionable due to several structural analogies among the N proteins of different coronaviruses. In order to produce new specific antibodies, BALB/c mice were immunized three times at 20-day intervals with a recombinant spike (S) protein. The procedure used was highly efficient, and 40 different specific mAbs were isolated, purified and characterized, with 13 ultimately being selected for their specificity and lack of cross reactivity with other human coronaviruses. The specific epitopes recognized by the selected mAbs were identified through a peptide library and/or by recombinant fragments of the S protein. In particular, the selected mAbs recognized different linear epitopes along the S1, excluding the receptor binding domain, and along the S2 subunits of the S protein of SARS-CoV-2 and its major variants of concern. We identified combinations of anti-S mAbs suitable for use in ELISA or rapid diagnostic tests, with the highest sensitivity and specificity coming from proof-of-concept tests using recombinant antigens, SARS-CoV-2 or biological fluids from infected individuals, that represent important additional tools for the diagnosis of COVID-19.
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Crucial steps in high-risk human papillomavirus (HR-HPV)-related carcinogenesis are the integration of HR-HPV into the host genome and loss of viral episomes. The mechanisms that promote cervical neoplastic progression are, however, not clearly understood. During HR-HPV infection, the HPV E5 protein is expressed in precancerous stages but not after viral integration. Given that it has been reported that loss of HPV16 episomes and cervical tumor progression are associated with increased expression of antiviral genes that are inducible by type I interferon (IFN), we asked whether E5, expressed in early phases of cervical carcinogenesis, affects IFN-ß signaling. We show that the HPV type 16 (HPV16) E5 protein expression per se stimulates IFN-ß expression. This stimulation is specifically mediated by the induction of interferon regulatory factor 1 (IRF-1) which, in turn, induces transcriptional activation of IRF-1-targeted interferon-stimulated genes (ISGs) as double-stranded RNA-dependent protein kinase R (PKR) and caspase 8. Our data show a new and unexpected role for HR-HPV E5 protein and indicate that HPV16 E5 may contribute to the mechanisms responsible for cervical carcinogenesis in part via stimulation of IFN-ß and an IFN signature, with IRF-1 playing a pivotal role. HPV16 E5 and IRF-1 may thus serve as potential therapeutic targets in HPV-associated premalignant lesions.
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Interacciones Huésped-Patógeno , Papillomavirus Humano 16/inmunología , Factor 1 Regulador del Interferón/metabolismo , Interferón beta/biosíntesis , Queratinocitos/inmunología , Proteínas Oncogénicas Virales/metabolismo , Línea Celular , Papillomavirus Humano 16/patogenicidad , Humanos , Queratinocitos/virologíaRESUMEN
MOTIVATION: There is an urgent need for new medications to combat influenza pandemics. METHODS: Using the genome analysis of the influenza A virus performed previously, we designed and performed a combinatorial exhaustive systematic methodology for optimal design of universal therapeutic small interfering RNA molecules (siRNAs) targeting all diverse influenza A viral strains. The rationale was to integrate the factors for highly efficient design in a pipeline of analysis performed on possible influenza-targeting siRNAs. This analysis selects specific siRNAs that has the ability to target highly conserved, accessible and biologically significant regions. This would require minimal dosage and side effects. RESULTS AND DISCUSSION: First, >6000 possible siRNAs were designed. Successive filtration followed where a novel method for siRNA scoring filtration layers was implemented. This method excluded siRNAs below the 90% experimental inhibition mapped scores using the intersection of 12 different scoring algorithms. Further filtration of siRNAs is done by eliminating those with off-targets in the human genome and those with undesirable properties and selecting siRNA targeting highly probable single-stranded regions. Finally, the optimal properties of the siRNA were ensured through selection of those targeting 100% conserved, biologically functional short motifs. Validation of a predicted active (sh114) and a predicted inactive (sh113) (that was filtered out in Stage 8) silencer of the NS1 gene showed significant inhibition of the NS1 gene for sh114, with negligible decrease for sh113 which failed target accessibility. This demonstrated the fertility of this methodology. CONTACT: mahef@aucegypt.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Virus de la Influenza A/clasificación , Virus de la Influenza A/genética , Gripe Humana/terapia , ARN Interferente Pequeño/genética , Células HEK293 , Humanos , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/fisiología , Gripe Humana/genética , Programas InformáticosRESUMEN
Among the many activities attributed to the type I interferon (IFN) multigene family, their roles as mediators of the antiviral immune response have emerged as important components of the host response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Viruses likewise have evolved multiple immune evasion strategies to circumvent the host immune response and promote virus propagation and dissemination. Therefore, a thorough characterization of host-virus interactions is essential to understand SARS-CoV-2 pathogenesis. Here, we summarize the virus-mediated evasion of the IFN responses and the viral functions involved, the genetic basis of IFN production in SARS-CoV-2 infection and the progress of clinical trials designed to utilize type I IFN as a potential therapeutic tool.
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Despite the success of highly active antiretroviral therapy (HAART), integrated HIV-1 proviral DNA cannot be eradicated from an infected individual. HAART is not able to eliminate latently infected cells that remain invisible to the immune system. Viral sanctuaries in specific tissues and immune-privileged sites may cause residual viral replication that contributes to HIV-1 persistence. The "Shock or Kick, and Kill" approach uses latency reversing agents (LRAs) in the presence of HAART, followed by cell-killing due to viral cytopathic effects and immune-mediated clearance. Different LRAs may be required for the in vivo reactivation of HIV-1 in different CD4+ T cell reservoirs, leading to the activation of cellular transcription factors acting on the integrated proviral HIV-1 LTR. An important requirement for LRA drugs is the reactivation of viral transcription and replication without causing a generalized immune activation. Toll-like receptors, RIG-I like receptors, and STING agonists have emerged recently as a new class of LRAs that augment selective apoptosis in reactivated T lymphocytes. The challenge is to extend in vitro observations to HIV-1 positive patients. Further studies are also needed to overcome the mechanisms that protect latently infected cells from reactivation and/or elimination by the immune system. The Block and Lock alternative strategy aims at using latency promoting/inducing agents (LPAs/LIAs) to block the ability of latent proviruses to reactivate transcription in order to achieve a long term lock down of potential residual virus replication. The Shock and Kill and the Block and Lock approaches may not be only alternative to each other, but, if combined together (one after the other), or given all at once [namely "Shoc-K(kill) and B(block)-Lock"], they may represent a better approach to a functional cure.
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BACKGROUND: Direct-acting antivirals (DAAs) treatment, although highly efficacious for the treatment of hepatitis C virus (HCV) infection, may not completely reconstitute the HCV-mediated dysregulated immune system, especially in patients co-infected with human immunodeficiency virus (HIV) and HCV. OBJECTIVES: We aimed to evaluate the impact of HCV eradication following DAA therapy on the immune system and liver disease improvement through comparative monitoring of 10 HCV mono-infected and 10 HCV/HIV co-infected patients under combined antiretroviral therapy (cART). Early and late longitudinal phenotypic changes in peripheral blood mononuclear cell (PBMC) subsets, T-cell activation, differentiation and exhaustion, as well as inflammatory biomarkers, indoleamine 2-3 dioxygenase (IDO) activity, and liver stiffness, APRI and FIB-4 scores were assessed. MATERIALS AND METHODS: Samples were obtained at baseline (T0), week 1 (T1), week 2 (T2), week 12 (T3, end of treatment, EOT), and month 9 (T4, end of follow-up, 36 weeks post EOT). RESULTS: All patients achieved a sustained virological response (SVR 12) after DAA treatment. Overall, changes of the T-cell immune phenotypes were greater in HCV/HIV co-infected than in HCV mono-infected, due to an increase in CD4+ and CD8+ T-cell percentages and of CD8+ T-cell activation and memory markers, in particular at the end of follow-up. On the other end, HCV mono-infected showed changes in the activation profile and in the memory CD4+ T-cell compartment. In HCV/HIV co-infected, a decrease in the IDO activity by DAA treatment was observed; conversely, in HCV mono-infected, it resulted unmodified. Regarding inflammatory mediators, viral suppression was associated with a reduction in IP-10 levels, while interferon regulatory factor (IRF)-7, interferon (IFN)-ß, and interferon (IFN)-γ levels were downregulated during therapy and increased post therapy. A decrease in liver stiffness, APRI, and FIB-4 scores was also observed. CONCLUSIONS: Our study suggests that, although patients achieved HCV eradication, the immune activation state in both HCV mono-infected and HCV/HIV co-infected patients remains elevated for a long time after the end of DAA therapy, despite an improvement of liver-specific outcomes, meanwhile highlighting the distinct immunophenotypic and inflammatory biomarker profile between the groups of patients.
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Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.
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Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , Epítopos/inmunología , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/inmunología , Enzima Convertidora de Angiotensina 2/genética , Animales , Sitios de Unión de Anticuerpos/inmunología , Línea Celular Tumoral , Chlorocebus aethiops , Femenino , Glicosilación , Células HEK293 , Humanos , Ratones Endogámicos BALB C , Pruebas de Neutralización , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Células Vero , Tratamiento Farmacológico de COVID-19RESUMEN
Genetic vaccines are safe cost-effective approaches to immunization but DNA immunization is an inefficient process. There is, therefore, a pressing need for adjuvants capable of enhancing the immunogenicity and effectiveness of these vaccines. This is particularly important for diseases for which successful vaccines are still lacking, such as cancer and infectious diseases including HIV-1/AIDS. Here we report an approach to enhance the immunogenicity of DNA vaccines involving the use of transcription factors of the Interferon regulatory factor (IRF) family, specifically IRF-1, IRF-3, and IRF-7 using the tat gene as model antigen. Balb/c mice were immunized by three intramuscular inoculations, using a DNA prime-protein boost protocol, with a DNA encoding tat of HIV-1 and the indicated IRFs and immune responses were compared to those induced by vaccination with tat DNA alone. In vivo administration of plasmid DNA encoding IRF-1, or a mutated version of IRF-1 deleted of the DNA-binding domain, enhanced Tat-specific immune responses and shifted them towards a predominant T helper 1-type immune response with increased IFN-gamma production and cytotoxic T lymphocytes responses. Conversely, the use of IRF-3 or IRF-7 did not affect the tat-induced responses. These findings define IRF-1 and its mutated form as efficacious T helper 1-inducing adjuvants in the context of tat-based vaccination and also providing a new promising candidate for genetic vaccine development.
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Adyuvantes Inmunológicos , Factor 1 Regulador del Interferón/inmunología , Vacunas de ADN/inmunología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Vacunas contra el SIDA/inmunología , Animales , Línea Celular , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Femenino , VIH-1/inmunología , Humanos , Inmunización , Factor 1 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/inmunología , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/inmunología , Ratones , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Transcription of the human immunodeficiency virus (HIV)-1 is controlled by the cooperation of virally encoded and host regulatory proteins. The Tat protein is essential for viral replication, however, expression of Tat after virus entry requires HIV-1 promoter activation. A sequence in the 5' HIV-1 LTR, containing a binding site for transcription factors of the interferon regulatory factors (IRF) family has been suggested to be critical for HIV-1 transcription and replication. Here we show that IRF-1 activates HIV-1 LTR transcription in a dose-dependent fashion and in the absence of Tat. This has biological significance since IRF-1 is produced early upon virus entry, both in cell lines and in primary CD4+ T cells, and before expression of Tat. IRF-1 also cooperates with Tat in amplifying virus gene transcription and replication. This cooperation depends upon a physical interaction that is blocked by overexpression of IRF-8, the natural repressor of IRF-1, and, in turn is released by overexpression of IRF-1. These data suggest a key role of IRF-1 in the early phase of viral replication and/or during viral reactivation from latency, when viral transactivators are absent or present at very low levels, and suggest that the interplay between IRF-1 and IRF-8 may play a key role in virus latency.
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Proteínas de Unión al ADN/metabolismo , Regulación Viral de la Expresión Génica , VIH-1/crecimiento & desarrollo , Fosfoproteínas/metabolismo , Factores de Transcripción/metabolismo , Replicación Viral , Línea Celular , Proteínas de Unión al ADN/genética , Relación Dosis-Respuesta a Droga , Ensayo de Cambio de Movilidad Electroforética , Productos del Gen tat/metabolismo , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , Humanos , Factor 1 Regulador del Interferón , Factor 2 Regulador del Interferón , Factores Reguladores del Interferón , Células Jurkat , Fosfoproteínas/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Elementos de Respuesta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Activación Transcripcional , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Interferon Regulatory Factors (IRFs) are key regulators of immunity, cell survival and apoptosis. IRF transcriptional activity and subcellular localization are tightly regulated by posttranscriptional modifications including phosphorylation. The IκB kinase family member IKK-ε is essential in regulating antiviral innate immunity mediated by IRFs but is now also recognized as an oncoprotein amplified and overexpressed in breast cancer cell lines and patient-derived tumors. In the present study, we report that the tumor suppressor IRF-1 is a specific target of IKK-ε in breast cancer cells. IKK-ε-mediated phosphorylation of IRF-1 dramatically decreases IRF-1 protein stability, accelerating IRF-1 degradation and quenching IRF-1 transcriptional activity. Chemical inhibition of IKK-ε activity, fully restores IRF-1 levels and function and positively correlates with inhibition of cell growth and proliferation of breast cancer cells. By using a breast cancer cell line stably expressing a dominant negative version of IRF-1 we were able to demonstrate that IKK-ε preferentially exerts its oncogenic potential in breast cancer through the regulation of IRF-1 and point to the IKK-ε-mediated phosphorylation of IRF-1 as a therapeutic target to overcome IKK-ε-mediated tumorigenesis.
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Neoplasias de la Mama/patología , Quinasa I-kappa B/metabolismo , Factor 1 Regulador del Interferón/metabolismo , Ubiquitina/metabolismo , Apoptosis , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proliferación Celular , Femenino , Humanos , Quinasa I-kappa B/genética , Factor 1 Regulador del Interferón/genética , Fosforilación , Proteolisis , Transducción de Señal , Células Tumorales Cultivadas , UbiquitinaciónRESUMEN
Several innate cellular antiviral factors exist in mammalian cells that prevent the replication of retroviruses. Among them, the tripartite motif protein (TRIM)5alpha has been shown to block human immunodeficiency virus type 1 (HIV-1) infection in several types of Old World monkey cells. Here we report a novel HIV-1 chronically infected monkey B cell line, F6/HIV-1, characterized by very low levels of TRIM5alpha expression that allows HIV-1 to overcome the restriction. Virus produced by F6/HIV-1 cells fails to infect monkey cells but retains the ability to infect human peripheral blood mononuclear cells (PBMCs) and T cell lines, although with a reduced infectivity compared to the input virus. Ultrastructural analyses revealed the presence of budding virions at the F6/HIV-1 cells plasma membrane characterized by a typical conical core shell. To our knowledge F6/HIV-1 is the first monkey cell line chronically infected by HIV-1 and able to release infectious particles thus representing a useful tool to gain further insights into the molecular mechanisms of HIV-1 pathogenesis.
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Linfocitos B/metabolismo , Linfocitos B/virología , Proteínas Portadoras/metabolismo , VIH-1/crecimiento & desarrollo , Macaca fascicularis , Sustitución de Aminoácidos/genética , Animales , Factores de Restricción Antivirales , Proteínas Portadoras/genética , Línea Celular , Línea Celular Tumoral , Expresión Génica/genética , Proteína p24 del Núcleo del VIH/metabolismo , VIH-1/genética , VIH-1/ultraestructura , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Leucocitos Mononucleares/virología , Especificidad de la Especie , Linfocitos T/metabolismo , Linfocitos T/virología , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas , Virión/genética , Virión/crecimiento & desarrollo , Virión/ultraestructura , Replicación Viral/fisiología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Human immunodeficiency virus type 1 (HIV-1) gene expression is controlled by a complex interplay between viral and host factors. We have previously shown that interferon-regulatory factor 1 (IRF-1) is stimulated early after HIV-1 infection and regulates promoter transcriptional activity even in the absence of the viral transactivator Tat. In this work we demonstrate that IRF-1 is also required for full NF-kappaB transcriptional activity. We provide evidence that IRF-1 and NF-kappaB form a functional complex at the long terminal repeat (LTR) kappaB sites, which is abolished by specific mutations in the two adjacent kappaB sites in the enhancer region. Silencing IRF-1 with small interfering RNA resulted in impaired NF-kappaB-mediated transcriptional activity and in repressed HIV-1 transcription early in de novo-infected T cells. These data indicate that in early phases of HIV-1 infection or during virus reactivation from latency, when the viral transactivator is absent or present at very low levels, IRF-1 is an additional component of the p50/p65 heterodimer binding the LTR enhancer, absolutely required for efficient HIV-1 replication.
Asunto(s)
Realizador del VIH/genética , Duplicado del Terminal Largo de VIH/genética , VIH-1/fisiología , Factor 1 Regulador del Interferón/metabolismo , FN-kappa B/metabolismo , Sitios de Unión , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Silenciador del Gen , VIH-1/genética , Humanos , Inmunoprecipitación , Factor 1 Regulador del Interferón/antagonistas & inhibidores , Mutación Puntual , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/virologíaRESUMEN
Interferon responsive factor 1 (IRF-1) is a pleiotropic transcription factor, possessing non-redundant biological activities that depend on its interaction with different protein partners and multiple post-translational modifications including phosphorylation. In particular, a 5'-SXXXSXS-3' motif of the protein represents the target of the IκB-related kinases, TANK-binding kinase (TBK)-1 and inhibitor of nuclear factor kappa-B kinase (IKK)-ε. Here, a 3D model of human IRF-1 was determined by using multi-template comparative modeling and molecular dynamics approaches. Models obtained through either phosphorylation or aspartate mutation of residues 215, 219 and 221 were also calculated and compared to the wild type. Calculations indicated that each of these modifications mainly induces a rigidification of the protein structure and only slightly changes in electrostatics and hydrophobicity of IRF-1 surface, resulting in the impairment of the capacity of IRF-1 containing as partate mutations (S221D and S215D/S219D/S221D) to synergize with tumour necrosis factor (TNF)-α stimulation in inducing interferon (IFN) promoter-mediated reporter gene activation. Therefore, these changes are qualitatively correlated to the amount of negative charge located on the 215-221 segments of IRF-1 by phosphorylation or aspartate mutation. Hypotheses on the structural mechanism that governs the phosphorylation-related damping of IRF-1 activity were also drawn. Communicated by Ramaswamy H. Sarma.
Asunto(s)
Factor 1 Regulador del Interferón/química , Factor 1 Regulador del Interferón/genética , Modelos Moleculares , Mutación/genética , Ácido Aspártico/genética , Células HEK293 , Humanos , Factor 1 Regulador del Interferón/metabolismo , Interferón beta/metabolismo , Simulación de Dinámica Molecular , Proteínas Mutantes/química , Fosforilación , Electricidad Estática , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The interferon (IFN) system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24) is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs) and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T) cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE). Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z'- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance.