RESUMEN
Across biological scales, gene-regulatory networks employ autorepression (negative feedback) to maintain homeostasis and minimize failure from aberrant expression. Here, we present a proof of concept that disrupting transcriptional negative feedback dysregulates viral gene expression to therapeutically inhibit replication and confers a high evolutionary barrier to resistance. We find that nucleic-acid decoys mimicking cis-regulatory sites act as "feedback disruptors," break homeostasis, and increase viral transcription factors to cytotoxic levels (termed "open-loop lethality"). Feedback disruptors against herpesviruses reduced viral replication >2-logs without activating innate immunity, showed sub-nM IC50, synergized with standard-of-care antivirals, and inhibited virus replication in mice. In contrast to approved antivirals where resistance rapidly emerged, no feedback-disruptor escape mutants evolved in long-term cultures. For SARS-CoV-2, disruption of a putative feedback circuit also generated open-loop lethality, reducing viral titers by >1-log. These results demonstrate that generating open-loop lethality, via negative-feedback disruption, may yield a class of antimicrobials with a high genetic barrier to resistance.
Asunto(s)
Antivirales , Regulación Viral de la Expresión Génica/efectos de los fármacos , Animales , Antivirales/farmacología , Farmacorresistencia Viral , Redes Reguladoras de Genes/efectos de los fármacos , Ratones , SARS-CoV-2/efectos de los fármacos , Replicación ViralRESUMEN
Rare CD4 T cells that contain HIV under antiretroviral therapy represent an important barrier to HIV cure1-3, but the infeasibility of isolating and characterizing these cells in their natural state has led to uncertainty about whether they possess distinctive attributes that HIV cure-directed therapies might exploit. Here we address this challenge using a microfluidic technology that isolates the transcriptomes of HIV-infected cells based solely on the detection of HIV DNA. HIV-DNA+ memory CD4 T cells in the blood from people receiving antiretroviral therapy showed inhibition of six transcriptomic pathways, including death receptor signalling, necroptosis signalling and antiproliferative Gα12/13 signalling. Moreover, two groups of genes identified by network co-expression analysis were significantly associated with HIV-DNA+ cells. These genes (n = 145) accounted for just 0.81% of the measured transcriptome and included negative regulators of HIV transcription that were higher in HIV-DNA+ cells, positive regulators of HIV transcription that were lower in HIV-DNA+ cells, and other genes involved in RNA processing, negative regulation of mRNA translation, and regulation of cell state and fate. These findings reveal that HIV-infected memory CD4 T cells under antiretroviral therapy are a distinctive population with host gene expression patterns that favour HIV silencing, cell survival and cell proliferation, with important implications for the development of HIV cure strategies.
Asunto(s)
Linfocitos T CD4-Positivos , Regulación Viral de la Expresión Génica , Infecciones por VIH , VIH-1 , Latencia del Virus , Humanos , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , ADN Viral/aislamiento & purificación , Regulación Viral de la Expresión Génica/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/genética , VIH-1/aislamiento & purificación , VIH-1/patogenicidad , Memoria Inmunológica , Microfluídica , Necroptosis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Antirretrovirales/farmacología , Antirretrovirales/uso terapéuticoRESUMEN
Unlike the human genome that comprises mostly noncoding and regulatory sequences, viruses have evolved under the constraints of maintaining a small genome size while expanding the efficiency of their coding and regulatory sequences. As a result, viruses use strategies of transcription and translation in which one or more of the steps in the conventional gene-protein production line are altered. These alternative strategies of viral gene expression (also known as gene recoding) can be uniquely brought about by dedicated viral enzymes or by co-opting host factors (known as host dependencies). Targeting these unique enzymatic activities and host factors exposes vulnerabilities of a virus and provides a paradigm for the design of novel antiviral therapies. In this Review, we describe the types and mechanisms of unconventional gene and protein expression in viruses, and provide a perspective on how future basic mechanistic work could inform translational efforts that are aimed at viral eradication.
Asunto(s)
Antivirales/farmacología , Antivirales/uso terapéutico , Regulación Viral de la Expresión Génica/efectos de los fármacos , Interacciones Microbiota-Huesped/efectos de los fármacos , Interacciones Microbiota-Huesped/genética , Virosis/tratamiento farmacológico , Virosis/virología , Animales , Sistema de Lectura Ribosómico/efectos de los fármacos , Sistema de Lectura Ribosómico/genética , Regulación Viral de la Expresión Génica/genética , Genoma Viral/efectos de los fármacos , Genoma Viral/genética , Humanos , Empalme del ARN/efectos de los fármacos , Empalme del ARN/genéticaRESUMEN
Within the first 15 minutes of infection, herpes simplex virus 1 immediate early proteins repurpose cellular RNA polymerase (Pol II) for viral transcription. An important role of the viral-infected cell protein 27 (ICP27) is to facilitate viral pre-mRNA processing and export viral mRNA to the cytoplasm. Here, we use precision nuclear run-on followed by deep sequencing (PRO-seq) to characterize transcription of a viral ICP27 null mutant. At 1.5 and 3 hours post infection (hpi), we observed increased total levels of Pol II on the mutant viral genome and accumulation of Pol II downstream of poly A sites indicating increased levels of initiation and processivity. By 6 hpi, Pol II accumulation on specific mutant viral genes was higher than that on wild-type virus either at or upstream of poly A signals, depending on the gene. The PRO-seq profile of the ICP27 mutant on late genes at 6 hpi was similar but not identical to that caused by treatment with flavopiridol, a known inhibitor of RNA processivity. This pattern was different from PRO-seq profiles of other α gene mutants and upon inhibition of viral DNA replication with PAA. Together, these results indicate that ICP27 contributes to the repression of aberrant viral transcription at 1.5 and 3 hpi by inhibiting initiation and decreasing RNA processivity. However, ICP27 is needed to enhance processivity on most late genes by 6 hpi in a mechanism distinguishable from its role in viral DNA replication.IMPORTANCEWe developed and validated the use of a processivity index for precision nuclear run-on followed by deep sequencing data. The processivity index calculations confirm infected cell protein 27 (ICP27) induces downstream of transcription termination on certain host genes. The processivity indices and whole gene probe data implicate ICP27 in transient immediate early gene-mediated repression, a process that also requires ICP4, ICP22, and ICP0. The data indicate that ICP27 directly or indirectly regulates RNA polymerase (Pol II) initiation and processivity on specific genes at specific times post infection. These observations support specific and varied roles for ICP27 in regulating Pol II activity on viral genes in addition to its known roles in post transcriptional mRNA processing and export.
Asunto(s)
Genoma Viral , Herpesvirus Humano 1 , Proteínas Inmediatas-Precoces , Mutación , ARN Polimerasa II , Transcripción Viral , Animales , Humanos , Línea Celular , Chlorocebus aethiops , Regulación Viral de la Expresión Génica/efectos de los fármacos , Genes Virales/genética , Genoma Viral/genética , Herpes Simple/virología , Herpes Simple/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces/deficiencia , Proteínas Inmediatas-Precoces/genética , Poli A/genética , Poli A/metabolismo , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Células Vero , Transcripción Viral/efectos de los fármacos , Transcripción Viral/genética , Replicación Viral/genéticaRESUMEN
Bovine alphaherpesvirus 1 (BoHV-1) infection causes respiratory tract disorders and immune suppression and may induce bacterial pneumonia. BoHV-1 establishes lifelong latency in sensory neurons after acute infection. Reactivation from latency consistently occurs following stress or intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two viral transcriptional regulators necessary for productive infection and reactivation from latency. The IEtu1 promoter contains two glucocorticoid receptor (GR) responsive elements (GREs) that are transactivated by activated GR. GC-rich motifs, including consensus binding sites for specificity protein 1 (Sp1), are in the IEtu1 promoter sequences. E2F family members bind a consensus sequence (TTTCCCGC) and certain specificity protein 1 (Sp1) sites. Consequently, we hypothesized that certain E2F family members activate IEtu1 promoter activity. DEX treatment of latently infected calves increased the number of E2F2+ TG neurons. GR and E2F2, but not E2F1, E2F3a, or E2F3b, cooperatively transactivate a 436-bp cis-regulatory module in the IEtu1 promoter that contains both GREs. A luciferase reporter construct containing a 222-bp fragment downstream of the GREs was transactivated by E2F2 unless two adjacent Sp1 binding sites were mutated. Chromatin immunoprecipitation studies revealed that E2F2 occupied IEtu1 promoter sequences when the BoHV-1 genome was transfected into mouse neuroblastoma (Neuro-2A) or monkey kidney (CV-1) cells. In summary, these findings revealed that GR and E2F2 cooperatively transactivate IEtu1 promoter activity, which is predicted to influence the early stages of BoHV-1 reactivation from latency. IMPORTANCE: Bovine alpha-herpesvirus 1 (BoHV-1) acute infection in cattle leads to establishment of latency in sensory neurons in the trigeminal ganglia (TG). A synthetic corticosteroid dexamethasone consistently initiates BoHV-1 reactivation in latently infected calves. The BoHV-1 immediate early transcription unit 1 (IEtu1) promoter regulates expression of infected cell protein 0 (bICP0) and bICP4, two viral transcriptional regulators. Hence, the IEtu1 promoter must be activated for the reactivation to occur. The number of TG neurons expressing E2F2, a transcription factor and cell cycle regulator, increased during early stages of reactivation from latency. The glucocorticoid receptor (GR) and E2F2, but not E2F1, E2F3a, or E2F3b, cooperatively transactivated a 436-bp cis-regulatory module (CRM) in the IEtu1 promoter that contains two GR responsive elements. Chromatin immunoprecipitation studies revealed that E2F2 occupies IEtu1 promoter sequences in cultured cells. GR and E2F2 mediate cooperative transactivation of IEtu1 promoter activity, which is predicted to stimulate viral replication following stressful stimuli.
Asunto(s)
Ciclo Celular , Factor de Transcripción E2F2 , Regulación Viral de la Expresión Génica , Herpesvirus Bovino 1 , Proteínas Inmediatas-Precoces , Regiones Promotoras Genéticas , Receptores de Glucocorticoides , Activación Transcripcional , Animales , Bovinos , Ratones , Sitios de Unión , Línea Celular , Dexametasona/farmacología , Factor de Transcripción E2F2/metabolismo , Regulación Viral de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica/genética , Infecciones por Herpesviridae/virología , Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/veterinaria , Infecciones por Herpesviridae/genética , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 1/fisiología , Proteínas Inmediatas-Precoces/genética , Neuronas/virología , Receptores de Glucocorticoides/metabolismo , Elementos de Respuesta/genética , Factor de Transcripción Sp1/metabolismo , Transactivadores/metabolismo , Ganglio del Trigémino/citología , Ganglio del Trigémino/virología , Activación Viral , Latencia del VirusRESUMEN
DNA replication of E1-deleted first-generation adenoviruses (AdV) in cultured cancer cells has been reported repeatedly and it was suggested that certain cellular proteins could functionally compensate for E1A, leading to the expression of the early region 2 (E2)-encoded proteins and subsequently virus replication. Referring to this, the observation was named E1A-like activity. In this study, we investigated different cell cycle inhibitors with respect to their ability to increase viral DNA replication of dl70-3, an E1-deleted adenovirus. Our analyses of this issue revealed that in particular inhibition of cyclin-dependent kinases 4/6 (CDK4/6i) increased E1-independent adenovirus E2-expression and viral DNA replication. Detailed analysis of the E2-expression in dl70-3 infected cells by RT-qPCR showed that the increase in E2-expression originated from the E2-early promoter. Mutations of the two E2F-binding sites in the E2-early promoter (pE2early-LucM) caused a significant reduction in E2-early promoter activity in trans-activation assays. Accordingly, mutations of the E2F-binding sites in the E2-early promoter in a virus named dl70-3/E2Fm completely abolished CDK4/6i induced viral DNA replication. Thus, our data show that E2F-binding sites in the E2-early promoter are crucial for E1A independent adenoviral DNA replication of E1-deleted vectors in cancer cells. IMPORTANCE E1-deleted AdV vectors are considered replication deficient and are important tools for the study of virus biology, gene therapy, and large-scale vaccine development. However, deletion of the E1 genes does not completely abolish viral DNA replication in cancer cells. Here, we report, that the two E2F-binding sites in the adenoviral E2-early promoter contribute substantially to the so-called E1A-like activity in tumor cells. With this finding, on the one hand, the safety profile of viral vaccine vectors can be increased and, on the other hand, the oncolytic property for cancer therapy might be improved through targeted manipulation of the host cell.
Asunto(s)
Adenoviridae , Ciclo Celular , Replicación del ADN , Replicación Viral , Adenoviridae/genética , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Sitios de Unión , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Células/efectos de los fármacos , Células/virología , Replicación del ADN/efectos de los fármacos , ADN Viral/metabolismo , Regulación Viral de la Expresión Génica/efectos de los fármacos , Mutación , Regiones Promotoras Genéticas/genética , Inhibidores de Proteínas Quinasas/farmacología , Replicación Viral/fisiología , HumanosRESUMEN
Since its emergence in December 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally and become a major public health burden. Despite its close phylogenetic relationship to SARS-CoV, SARS-CoV-2 exhibits increased human-to-human transmission dynamics, likely due to efficient early replication in the upper respiratory epithelium of infected individuals. Since different temperatures encountered in the human upper and lower respiratory tract (33°C and 37°C, respectively) have been shown to affect the replication kinetics of several respiratory viruses, as well as host innate immune response dynamics, we investigated the impact of temperature on SARS-CoV-2 and SARS-CoV infection using the primary human airway epithelial cell culture model. SARS-CoV-2, in contrast to SARS-CoV, replicated to higher titers when infections were performed at 33°C rather than 37°C. Although both viruses were highly sensitive to type I and type III interferon pretreatment, a detailed time-resolved transcriptome analysis revealed temperature-dependent interferon and pro-inflammatory responses induced by SARS-CoV-2 that were inversely proportional to its replication efficiency at 33°C or 37°C. These data provide crucial insight on pivotal virus-host interaction dynamics and are in line with characteristic clinical features of SARS-CoV-2 and SARS-CoV, as well as their respective transmission efficiencies.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Regulación Viral de la Expresión Génica/genética , SARS-CoV-2/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Animales , Antivirales/farmacología , Células Cultivadas , Chlorocebus aethiops , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/virología , Regulación Viral de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Humanos , Interferones/farmacología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Especificidad de la Especie , Temperatura , Células Vero , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
Upon treatment removal, spontaneous reactivation of latently infected T cells remains a major barrier toward curing HIV. Therapies that reactivate and clear the latent reservoir are only partially effective, while latency-promoting agents (LPAs) used to suppress reactivation and stabilize latency are understudied and lack diversity in their mechanisms of action. Here, we identify additional LPAs using a screen for gene-expression fluctuations (or "noise") that drive cell-fate specification and control HIV reactivation from latency. Single-cell protein dynamics of a minimal HIV gene circuit were monitored with time-lapse fluorescence microscopy. We screened 1,806 drugs, out of which 279 modulate noise magnitude or half autocorrelation time. Next, we tested the strongest noise modulators in a Jurkat T cell latency model and discovered three LPAs that would be overlooked by quantifying their mean expression levels alone. The LPAs reduced reactivation of latency in both Jurkat and primary cell models when challenged by synergistic and potent combinations of HIV activators. The two strongest LPAs, NSC 401005 and NSC 400938, are structurally and functionally related to inhibitors of thioredoxin reductase, a protein involved in maintaining redox balance in host cells. Experiments with multiple functional analogs revealed two additional LPAs, PX12 and tiopronin, and suggest a potential LPA family, within which some are commercially available and Food and Drug Administration-approved. The LPAs presented here may provide new strategies to complement antiretroviral treatments. Screening for gene expression noise holds the potential for drug discovery in other diseases.
Asunto(s)
VIH-1/genética , Latencia del Virus/efectos de los fármacos , Fármacos Anti-VIH/farmacología , Regulación Viral de la Expresión Génica/efectos de los fármacos , VIH-1/fisiología , Humanos , Células JurkatRESUMEN
Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with a liver-specific microRNA called miR-122. miR-122 binds to two sites in the 5' untranslated region of the viral genome and promotes HCV RNA accumulation. This interaction is important for viral RNA accumulation in cell culture, and miR-122 inhibitors have been shown to be effective at reducing viral titers in chronic HCV-infected patients. Herein, we analyzed resistance-associated variants that were isolated in cell culture or from patients who underwent miR-122 inhibitor-based therapy and discovered three distinct resistance mechanisms all based on changes to the structure of the viral RNA. Specifically, resistance-associated variants promoted riboswitch activity, genome stability, or positive-strand viral RNA synthesis, all in the absence of miR-122. Taken together, these findings provide insight into the mechanism(s) of miR-122-mediated viral RNA accumulation and provide mechanisms of antiviral resistance mediated by changes in RNA structure.
Asunto(s)
Antivirales/farmacología , Farmacorresistencia Viral/genética , Hepacivirus/genética , MicroARNs/metabolismo , ARN Viral/metabolismo , Línea Celular Tumoral , Regulación Viral de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica/fisiología , Variación Genética , Hepatitis C Crónica/tratamiento farmacológico , Humanos , MicroARNs/antagonistas & inhibidores , MicroARNs/genéticaRESUMEN
Epstein-Barr virus (EBV) is associated with epithelial and lymphoid malignancies, establishes latent infection in memory B cells, and intermittently produces infectious virions through lytic replication. Released virions play a key role in latent reservoir maintenance and transmission. Lytic EBV transcription differs from cellular transcription in requiring a virus-encoded preinitiation complex that binds to TATT motifs unique to EBV late lytic promoters. Expression of 15 late lytic genes that are important for virion production and infectivity is particularly dependent on the EBV SM protein, a nuclear protein expressed early during lytic reactivation that binds to viral RNAs and enhances RNA stability. We recently discovered that spironolactone blocks EBV virion production by inhibiting EBV SM function. Since spironolactone causes degradation of xeroderma pigmentosum group B-complementing protein (XPB), a component of human transcription factor TFIIH, in both B lymphocytes and epithelial cells, we hypothesized that SM utilizes XPB to specifically activate transcription of SM target promoters. While EBV SM has been thought to act posttranscriptionally, we provide evidence that SM also facilitates EBV gene transcription. We demonstrate that SM binds and recruits XPB to EBV promoters during lytic replication. Depletion of XPB protein, by spironolactone treatment or by siRNA transfection, inhibits SM-dependent late lytic gene transcription but not transcription of other EBV genes or cellular genes. These data indicate that SM acts as a transcriptional activator that has co-opted XPB to specifically target 15 EBV promoters that have uniquely evolved to require XPB for activity, providing an additional mechanism to differentially regulate EBV gene expression.
Asunto(s)
ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Herpesvirus Humano 4/genética , Interacciones Huésped-Patógeno/genética , Fosfoproteínas/metabolismo , Transactivadores/metabolismo , Línea Celular Tumoral , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Infecciones por Virus de Epstein-Barr/tratamiento farmacológico , Infecciones por Virus de Epstein-Barr/virología , Regulación Viral de la Expresión Génica/efectos de los fármacos , Humanos , Regiones Promotoras Genéticas/genética , Proteolisis/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Espironolactona/farmacología , Espironolactona/uso terapéutico , Activación Transcripcional/efectos de los fármacos , Virión/efectos de los fármacos , Virión/metabolismoRESUMEN
The induction of an interferon-mediated response is the first line of defense against pathogens such as viruses. Yet, the dynamics and extent of interferon alpha (IFNα)-induced antiviral genes vary remarkably and comprise three expression clusters: early, intermediate and late. By mathematical modeling based on time-resolved quantitative data, we identified mRNA stability as well as a negative regulatory loop as key mechanisms endogenously controlling the expression dynamics of IFNα-induced antiviral genes in hepatocytes. Guided by the mathematical model, we uncovered that this regulatory loop is mediated by the transcription factor IRF2 and showed that knock-down of IRF2 results in enhanced expression of early, intermediate and late IFNα-induced antiviral genes. Co-stimulation experiments with different pro-inflammatory cytokines revealed that this amplified expression dynamics of the early, intermediate and late IFNα-induced antiviral genes can also be achieved by co-application of IFNα and interleukin1 beta (IL1ß). Consistently, we found that IL1ß enhances IFNα-mediated repression of viral replication. Conversely, we observed that in IL1ß receptor knock-out mice replication of viruses sensitive to IFNα is increased. Thus, IL1ß is capable to potentiate IFNα-induced antiviral responses and could be exploited to improve antiviral therapies.
Asunto(s)
Regulación Viral de la Expresión Génica/efectos de los fármacos , Factor 2 Regulador del Interferón/metabolismo , Interferón-alfa/farmacología , Coriomeningitis Linfocítica/tratamiento farmacológico , Virus de la Coriomeningitis Linfocítica/efectos de los fármacos , Receptores Tipo I de Interleucina-1/fisiología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/farmacología , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/inmunología , Hepatocitos/virología , Humanos , Factor 2 Regulador del Interferón/genética , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/patología , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/aislamiento & purificación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estabilidad del ARNRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19), one of the most challenging global pandemics of the modern era. Potential treatment strategies against COVID-19 are yet to be devised. It is crucial that antivirals that interfere with the SARS-CoV-2 life cycle be identified and developed. 3-Chymotrypsin-like protease (3CLpro) is an attractive antiviral drug target against SARS-CoV-2, and coronaviruses in general, because of its role in the processing of viral polyproteins. Inhibitors of 3CLpro activity are screened in enzyme assays before further development of the most promising leads. Dimethyl sulfoxide (DMSO) is a common additive used in such assays and enhances the solubility of assay components. However, it may also potentially affect the stability and efficiency of 3CLpro but, to date, this effect had not been analyzed in detail. Here, we investigated the effect of DMSO on 3CLpro-catalyzed reaction. While DMSO (5%-20%) decreased the optimum temperature of catalysis and thermodynamic stability of 3CLpro, it only marginally affected the kinetic stability of the enzyme. Increasing the DMSO concentration up to 20% improved the catalytic efficiency and peptide-binding affinity of 3CLpro. At such high DMSO concentration, the solubility and stability of peptide substrate were improved because of reduced aggregation. In conclusion, we recommend 20% DMSO as the minimum concentration to be used in screens of 3CLpro inhibitors as lead compounds for the development of antiviral drugs against COVID-19.
Asunto(s)
COVID-19/virología , Proteasas 3C de Coronavirus/metabolismo , Dimetilsulfóxido/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica/efectos de los fármacos , SARS-CoV-2/enzimología , Simulación por Computador , Proteasas 3C de Coronavirus/genética , Humanos , Técnicas Analíticas Microfluídicas , Péptidos/metabolismo , Estabilidad ProteicaRESUMEN
HIV-1 persists lifelong in memory cells of the immune system as latent provirus that rebounds upon treatment interruption. Therefore, the latent reservoir is the main target for an HIV cure. Here, we studied the direct link between integration site and transcription using LEDGINs and Barcoded HIV-ensembles (B-HIVE). LEDGINs are antivirals that inhibit the interaction between HIV-1 integrase and the chromatin-tethering factor LEDGF/p75. They were used as a tool to retarget integration, while the effect on HIV expression was measured with B-HIVE. B-HIVE tracks insert-specific HIV expression by tagging a unique barcode in the HIV genome. We confirmed that LEDGINs retarget integration out of gene-dense and actively transcribed regions. The distance to H3K36me3, the marker recognized by LEDGF/p75, clearly increased. LEDGIN treatment reduced viral RNA expression and increased the proportion of silent provirus. Finally, silent proviruses obtained after LEDGIN treatment were located further away from epigenetic marks associated with active transcription. Interestingly, proximity to enhancers stimulated transcription irrespective of LEDGIN treatment, while the distance to H3K36me3 only changed after treatment with LEDGINs. The fact that proximity to these markers are associated with RNA expression support the direct link between provirus integration site and viral expression.
Asunto(s)
Regulación Viral de la Expresión Génica , Silenciador del Gen , VIH-1/genética , Provirus/genética , Integración Viral , Línea Celular , Cromatina/metabolismo , Regulación Viral de la Expresión Génica/efectos de los fármacos , Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/metabolismo , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular , ARN Viral/metabolismo , Integración Viral/efectos de los fármacosRESUMEN
Omicron is an emerging SARS-CoV-2 variant, evolved from the Indian delta variant B.1.617.2, which is currently infecting worldwide. The spike glycoprotein, an important molecule in the pathogenesis and transmissions of SARS-CoV-2 variants, especially omicron B.1.1.529, shows 37 mutations distributed over the trimeric protein domains. Notably, fifteen of these mutations reside in the receptor-binding domain of the spike glycoprotein, which may alter transmissibility and infectivity. Additionally, the omicron spike evades neutralization more efficiently than the delta spike. Most of the therapeutic antibodies are ineffective against the omicron variant, and double immunization with BioNTech-Pfizer (BNT162b2) might not adequately protect against severe disease induced by omicron B.1.1.529. So far, no efficient antiviral drugs are available against omicron. The present study identified the promising inhibitors from seaweed's bioactive compounds to inhibit the omicron variant B.1.1.529. We have also compared the seaweed's compounds with the standard drugs ceftriaxone and cefuroxime, which were suggested as beneficial antiviral drugs in COVID-19 treatment. Our molecular docking analysis revealed that caffeic acid hexoside (-6.4 kcal/mol; RMSD = 2.382 Å) and phloretin (-6.3 kcal/mol; RMSD = 0.061 Å) from Sargassum wightii (S. wightii) showed the inhibitory effect against the crucial residues ASN417, SER496, TYR501, and HIS505, which are supported for the inviolable omicron and angiotensin-converting enzyme II (ACE2) receptor interaction. Cholestan-3-ol, 2-methylene-, (3beta, 5 alpha) (CMBA) (-6.0 kcal/mol; RMSD = 3.074 Å) from Corallina officinalis (C. officinalis) manifested the strong inhibitory effect against the omicron RBD mutated residues LEU452 and ALA484, was magnificently observed as the essential residues in Indian delta variant B.1.617.2 previously. The standard drugs (ceftriaxone and cefuroxime) showed no or less inhibitory effect against RBD of omicron B.1.1.529. The present study also emphasized the pharmacological properties of the considered chemical compounds. The results could be used to develop potent seaweed-based antiviral drugs and/or dietary supplements to treat omicron B.1.1529-infected patients.
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Enzima Convertidora de Angiotensina 2/química , Regulación Viral de la Expresión Génica/efectos de los fármacos , Simulación del Acoplamiento Molecular , SARS-CoV-2/metabolismo , Algas Marinas/química , Glicoproteína de la Espiga del Coronavirus/química , Enzima Convertidora de Angiotensina 2/metabolismo , Antivirales/química , Antivirales/farmacología , Humanos , Mutación , Unión Proteica , Conformación Proteica , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Tratamiento Farmacológico de COVID-19RESUMEN
An outbreak, caused by an RNA virus, SARS-CoV-2 named COVID-19 has become pandemic with a magnitude which is daunting to all public health institutions in the absence of specific antiviral treatment. Surface glycoprotein and nucleocapsid phosphoprotein are two important proteins of this virus facilitating its entry into host cell and genome replication. Small interfering RNA (siRNA) is a prospective tool of the RNA interference (RNAi) pathway for the control of human viral infections by suppressing viral gene expression through hybridization and neutralization of target complementary mRNA. So, in this study, the power of RNA interference technology was harnessed to develop siRNA molecules against specific target genes namely, nucleocapsid phosphoprotein gene and surface glycoprotein gene. Conserved sequence from 139 SARS-CoV-2 strains from around the globe was collected to construct 78 siRNA that can inactivate nucleocapsid phosphoprotein and surface glycoprotein genes. Finally, based on GC content, free energy of folding, free energy of binding, melting temperature, efficacy prediction and molecular docking analysis, 8 siRNA molecules were selected which are proposed to exert the best action. These predicted siRNAs should effectively silence the genes of SARS-CoV-2 during siRNA mediated treatment assisting in the response against SARS-CoV-2.
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COVID-19/terapia , Química Computacional , Proteínas de la Nucleocápside de Coronavirus/genética , Diseño de Fármacos , Terapia Genética/métodos , Simulación del Acoplamiento Molecular , Interferencia de ARN , ARN Mensajero/antagonistas & inhibidores , ARN Interferente Pequeño/química , ARN Viral/antagonistas & inhibidores , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Proteínas Argonautas/química , Proteínas Argonautas/genética , Composición de Base , COVID-19/virología , Evolución Molecular , Regulación Viral de la Expresión Génica/efectos de los fármacos , Humanos , Pandemias , Fosfoproteínas/genética , Filogenia , Pliegue del ARN , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genética , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , ARN Interferente Pequeño/uso terapéutico , ARN Viral/genética , SARS-CoV-2/efectos de los fármacos , Alineación de Secuencia , Termodinámica , Tratamiento Farmacológico de COVID-19RESUMEN
Non-coding RNAs, particularly lncRNAs and miRNAs, have recently been shown to regulate different steps in viral infections and induction of immune responses against viruses. Expressions of several host and viral lncRNAs have been found to be altered during viral infection. These lncRNAs can exert antiviral function via inhibition of viral infection or stimulation of antiviral immune response. Some other lncRNAs can promote viral replication or suppress antiviral responses. The current review summarizes the interaction between ncRNAs and herpes simplex virus, cytomegalovirus, and Epstein-Barr infections. The data presented in this review helps identify viral-related regulators and proposes novel strategies for the prevention and treatment of viral infection.
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Susceptibilidad a Enfermedades , Interacciones Huésped-Patógeno/genética , ARN no Traducido , Virosis/etiología , Replicación Viral , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Virus ADN/fisiología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno/inmunología , Humanos , Técnicas de Diagnóstico Molecular , Unión Proteica , Especificidad de la Especie , Virosis/diagnóstico , Virosis/tratamiento farmacológico , Virosis/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Antiretroviral therapy effectively controls human immunodeficiency virus (HIV) infection. However, a reservoir of latently infected cells persists under suppressive therapy, constituting a major barrier to an HIV cure. The block-and-lock approach to a functional cure aims at the transcriptional and epigenetic silencing of proviruses, blocking viral reactivation in the absence of therapy, preventing disease progression and transmission, despite the presence of detectable integrated proviruses. This approach has been put forward for exploration based on the activity of didehydro-cortistatin A, an inhibitor of the HIV transcriptional activator Tat. Here we review the mechanisms by which didehydro-cortistatin A inhibition of Tat's feedback loop transcriptional amplification results in epigenetic silencing of the HIV promoter, and we discuss the benefits and limitations of the block-and-lock approach for an HIV cure.
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Fármacos Anti-VIH/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Isoquinolinas/farmacología , Animales , Regulación Viral de la Expresión Génica/efectos de los fármacos , Infecciones por VIH/virología , VIH-1/genética , VIH-1/metabolismo , Humanos , Regiones Promotoras Genéticas , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic virus, has two life cycle modes: the latent and lytic phases. KSHV lytic reactivation is important for both viral propagation and KSHV-induced tumorigenesis. The KSHV replication and transcription activator (RTA) protein is essential for lytic reactivation. Hesperetin, a citrus polyphenolic flavonoid, has antioxidant, anti-inflammatory, hypolipidemic, cardiovascular and anti-tumour effects. However, the effects of hesperetin on KSHV replication and KSHV-induced tumorigenesis have not yet been reported. Here, we report that hesperetin induces apoptotic cell death in BCBL-1 cells in a dose-dependent manner. Hesperetin inhibits KSHV reactivation and reduces the production of progeny virus from KSHV-harbouring cells. We also confirmed that HIF1α promotes the RTA transcriptional activities and lytic cycle-refractory state of KSHV-infected cells. Hesperetin suppresses HIF1α expression to inhibit KSHV lytic reactivation. These results suggest that hesperetin may represent a novel strategy for the treatment of KSHV infection and KSHV-associated lymphomas.
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Antivirales/farmacología , Infecciones por Herpesviridae/metabolismo , Herpesvirus Humano 8/efectos de los fármacos , Hesperidina/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Activación Viral/efectos de los fármacos , Apoptosis/efectos de los fármacos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/fisiopatología , Infecciones por Herpesviridae/virología , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/fisiología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Although substantial progress has been made in depicting the molecular pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection, the comprehensive mechanism of HIV-1 latency and the most promising therapeutic strategies to effectively reactivate the HIV-1 latent reservoir to achieve a functional cure for AIDS remain to be systematically illuminated. Here, we demonstrated that piwi (P element-induced Wimpy)-like RNA-mediated gene silencing 4 (PIWIL4) played an important role in suppressing HIV-1 transcription and contributed to the latency state in HIV-1-infected cells through its recruitment of various suppressive factors, including heterochromatin protein 1α/ß/γ, SETDB1, and HDAC4. The knockdown of PIWIL4 enhanced HIV-1 transcription and reversed HIV-1 latency in both HIV-1 latently infected Jurkat T cells and primary CD4+ T lymphocytes and resting CD4+ T lymphocytes from HIV-1-infected individuals on suppressive combined antiretroviral therapy (cART). Furthermore, in the absence of PIWIL4, HIV-1 latently infected Jurkat T cells were more sensitive to reactivation with vorinostat (suberoylanilide hydroxamic acid, or SAHA), JQ1, or prostratin. These findings indicated that PIWIL4 promotes HIV-1 latency by imposing repressive marks at the HIV-1 5' long terminal repeat. Thus, the manipulation of PIWIL4 could be a novel strategy for developing promising latency-reversing agents (LRAs).IMPORTANCE HIV-1 latency is systematically modulated by host factors and viral proteins. During this process, the suppression of HIV-1 transcription plays an essential role in promoting HIV-1 latency. In this study, we found that PIWIL4 repressed HIV-1 promoter activity and maintained HIV-1 latency. In particular, we report that PIWIL4 can regulate gene expression through its association with the suppressive activity of HDAC4. Therefore, we have identified a new function for PIWIL4: it is not only a suppressor of endogenous retrotransposons but also plays an important role in inhibiting transcription and leading to latent infection of HIV-1, a well-known exogenous retrovirus. Our results also indicate a novel therapeutic target to reactivate the HIV-1 latent reservoir.
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Proteínas Argonautas/metabolismo , Proteínas Argonautas/farmacología , Epigénesis Genética , Regulación Viral de la Expresión Génica/efectos de los fármacos , VIH-1/fisiología , Latencia del Virus/efectos de los fármacos , Antirretrovirales/uso terapéutico , Proteínas Argonautas/genética , Linfocitos T CD4-Positivos/virología , Células HEK293 , Infecciones por VIH/virología , VIH-1/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Células Jurkat , Proteínas de Unión al ARN , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas Virales/metabolismo , Latencia del Virus/genética , Replicación Viral/efectos de los fármacosRESUMEN
Brain-resident microglia and myeloid cells (perivascular macrophages) are important HIV reservoirs in vivo, especially in the central nervous system (CNS). Despite antiretroviral therapy (ART), low-level persistent HIV replication in these reservoirs remains detectable, which contributes to neuroinflammation and neurological disorders in HIV-infected patients. New approaches complementary to ART to repress residual HIV replication in CNS reservoirs are needed. Our group has recently identified a BRD4-selective small molecule modulator (ZL0580) that induces the epigenetic suppression of HIV. Here, we examined the effects of this compound on HIV in human myeloid cells. We found that ZL0580 induces potent and durable suppression of both induced and basal HIV transcription in microglial cells (HC69) and monocytic cell lines (U1 and OM10.1). Pretreatment of microglia with ZL0580 renders them more refractory to latent HIV reactivation, indicating an epigenetic reprogramming effect of ZL0580 on HIV long terminal repeat (LTR) in microglia. We also demonstrate that ZL0580 induces repressive effect on HIV in human primary monocyte-derived macrophages (MDMs) by promoting HIV suppression during ART treatment. Mechanistically, ZL0580 inhibits Tat transactivation in microglia by disrupting binding of Tat to CDK9, a process key to HIV transcription elongation. High-resolution micrococcal nuclease mapping showed that ZL0580 induces a repressive chromatin structure at the HIV LTR. Taken together, our data suggest that ZL0580 represents a potential approach that could be used in combination with ART to suppress residual HIV replication and/or latent HIV reactivation in CNS reservoirs, thereby reducing HIV-associated neuroinflammation.IMPORTANCE Brain-resident microglia and perivascular macrophages are important HIV reservoirs in the CNS. Persistent viral replication and latent HIV reactivation in the CNS, even under ART, are believed to occur, causing neuroinflammation and neurological disorders in HIV-infected patients. It is critical to identify new approaches that can control residual HIV replication and/or latent HIV reactivation in these reservoirs. We here report that the BRD4-selective small molecule modulator, ZL0580, induces potent and durable suppression of HIV in human microglial and monocytic cell lines. Using an in vitro HIV-infected, ART-treated MDM model, we show that ZL0580 also induces suppressive effect on HIV in human primary macrophages. The significance of our research is that it suggests a potential new approach that has utility in combination with ART to suppress residual HIV replication and/or HIV reactivation in CNS reservoirs, thereby reducing neuroinflammation and neurological disorders in HIV-infected individuals.