RESUMO
The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKß independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.
Assuntos
Terapia Viral Oncolítica , Vírus Oncolíticos , Succinatos , Animais , Humanos , Terapia Viral Oncolítica/métodos , Succinatos/farmacologia , Camundongos , Linhagem Celular Tumoral , Interferon Tipo I/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias do Colo/terapia , Neoplasias do Colo/imunologia , Neoplasias do Colo/tratamento farmacológico , Antivirais/farmacologia , NF-kappa B/metabolismo , Quinase I-kappa B/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Inflamação/tratamento farmacológico , Feminino , Vírus da Estomatite Vesicular Indiana/fisiologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
3-deazaneplanocin A (DzNep) and its 3-brominated analogs inhibit replication of several RNA viruses. This antiviral activity is attributed to inhibition of S-adenosyl homocysteine hydrolase (SAHase) and consequently inhibition of viral methyltransferases, impairing translation of viral transcripts. The L-enantiomers of some derivatives retain antiviral activity despite dramatically reduced inhibition of SAHase in vitro. To better understand the mechanisms by which these compounds exert their antiviral effects, we compared DzNep, its 3-bromo-derivative, CL123, and the related enantiomers, CL4033 and CL4053, for their activities towards the model negative-sense RNA virus vesicular stomatitis virus (VSV). In cell culture, DzNep, CL123 and CL4033 each exhibited 50 percent inhibitory concentrations (IC50s) in the nanomolar range whereas the IC50 for the L-form, CL4053, was 34-85 times higher. When a CL123-resistant mutant (VSVR) was selected, it exhibited cross-resistance to each of the neplanocin analogs, but retained sensitivity to the adenosine analog BCX4430, an RNA chain terminator. Sequencing of VSVR identified a mutation in the C-terminal domain (CTD) of the viral large (L) protein, a domain implicated in regulation of L protein methyltransferase activity. CL123 inhibited VSV viral mRNA 5' cap methylation, impaired viral protein synthesis and decreased association of viral mRNAs with polysomes. Modest impacts on viral transcription were also demonstrated. VSVR exhibited partial resistance in each of these assays but its replication was impaired, relative to the parent VSV, in the absence of the inhibitors. These data suggest that DzNep, CL123 and CL4033 inhibit VSV through impairment of viral mRNA cap methylation and that the L-form, CL4053, based on the cross-resistance of VSVR, may act by a similar mechanism.
Assuntos
Adenosina/análogos & derivados , Antivirais/farmacologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Adenosina/química , Adenosina/farmacologia , Animais , Chlorocebus aethiops , Concentração Inibidora 50 , Metilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Células Vero , Vírus da Estomatite Vesicular Indiana/genéticaRESUMO
Binary ethylenimine (BEI) has been widely used as a virucide to inactivate viruses. For regulatory exclusion of a select agent, the United States Federal Select Agent Program (FSAP) requires an inactivation procedure that renders a select agent non-viable but allows the select agent to retain antigenic characteristics for future use must be validated, and the inactivated agent must be confirmed by a viability testing. In this curve-based validation study, we examined impacts of BEI concentration, treatment temperature, and time on our in-house inactivation procedures of Foot-and-Mouth Disease Virus (FMDV), Vesicular Stomatitis Virus (VSV), and Swine Vesicular Disease Virus (SVDV). The inactivation efficacy was confirmed by virus titration and 3 consecutive blind passages on the monolayers of susceptible cells. A linear correlation between the virus titer reduction and BEI concentration, treatment time, and temperature was established. The results confirmed our in-house BEI inactivation procedure of two doses of 1.5 mM BEI treatment at 37 °C, 1st dose for 24 h, then 2nd dose for 6 more hours for a total of 30 h BEI contact time, can ensure complete inactivation of FMDV, VSV, and SVDV.
Assuntos
Aziridinas/farmacologia , Enterovirus Humano B/efeitos dos fármacos , Vírus da Febre Aftosa/efeitos dos fármacos , Febre Aftosa/prevenção & controle , Doenças dos Suínos/prevenção & controle , Estomatite Vesicular/prevenção & controle , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Animais , Contenção de Riscos Biológicos/veterinária , Suínos , Doenças dos Suínos/virologia , Estomatite Vesicular/virologia , Inativação de Vírus/efeitos dos fármacosRESUMO
Interferon (IFN) regulatory factor 3 (IRF3) is the key transcription factor for the induction of IFN and antiviral genes. The absence of antiviral genes in IRF3 deficiency leads to susceptibility to a wide range of viral infections. Previously, we uncovered a function for nontranscriptional IRF3 (nt-IRF3), RLR (RIG-I-like receptor)-induced IRF3-mediated pathway of apoptosis (RIPA), which triggers apoptotic killing of virus-infected cells. Using knock-in mice expressing a transcriptionally inactive, but RIPA-active, IRF3 mutant, we demonstrated the relative contribution of RIPA to host antiviral defense. Given that RIPA is a cellular antiviral pathway, we hypothesized that small molecules that promote RIPA in virus-infected cells would act as antiviral agents. To test this, we conducted a high throughput screen of a library of FDA-approved drugs to identify novel RIPA activators. Our screen identified doxorubicin as a potent RIPA-activating agent. In support of our hypothesis, doxorubicin inhibited the replication of vesicular stomatitis virus, a model rhabdovirus, and its antiviral activity depended on its ability to activate IRF3 in RIPA. Surprisingly, doxorubicin inhibited the transcriptional activity of IRF3. The antiviral activity of doxorubicin was expanded to flavivirus and herpesvirus that also activate IRF3. Mechanistically, doxorubicin promoted RIPA by activating the extracellular signal-regulated kinase (ERK) signaling pathway. Finally, we validated these results using another RIPA-activating compound, pyrvinium pamoate, which showed a similar antiviral effect without affecting the transcriptional activity of IRF3. Therefore, we demonstrate that the RIPA branch of IRF3 can be targeted therapeutically to prevent virus infection.
Assuntos
Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Fator Regulador 3 de Interferon/metabolismo , Transdução de Sinais/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Doxorrubicina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala/métodos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Bibliotecas de Moléculas Pequenas , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacosRESUMO
Interferons (IFNs) induce the expression of interferon-stimulated genes (ISGs), many of which are responsible for the cellular antiviral state in which the replication of numerous viruses is blocked. How the majority of individual ISGs inhibit the replication of particular viruses is unknown. We conducted a loss-of-function screen to identify genes required for the activity of alpha interferon (IFN-α) against vesicular stomatitis virus, Indiana serotype (VSVIND), a prototype negative-strand RNA virus. Our screen revealed that TRIM69, a member of the tripartite motif (TRIM) family of proteins, is a VSVIND inhibitor. TRIM69 potently inhibited VSVIND replication through a previously undescribed transcriptional inhibition mechanism. Specifically, TRIM69 physically associates with the VSVIND phosphoprotein (P), requiring a specific peptide target sequence encoded therein. P is a cofactor for the viral polymerase and is required for viral RNA synthesis, as well as the assembly of replication compartments. By targeting P, TRIM69 inhibits pioneer transcription of the incoming virion-associated minus-strand RNA, thereby preventing the synthesis of viral mRNAs, and consequently impedes all downstream events in the VSVIND replication cycle. Unlike some TRIM proteins, TRIM69 does not inhibit viral replication by inducing degradation of target viral proteins. Rather, higher-order TRIM69 multimerization is required for its antiviral activity, suggesting that TRIM69 functions by sequestration or anatomical disruption of the viral machinery required for VSVIND RNA synthesis.IMPORTANCE Interferons are important antiviral cytokines that work by inducing hundreds of host genes whose products inhibit the replication of many viruses. While the antiviral activity of interferon has long been known, the identities and mechanisms of action of most interferon-induced antiviral proteins remain to be discovered. We identified gene products that are important for the antiviral activity of interferon against vesicular stomatitis virus (VSV), a model virus that whose genome consists of a single RNA molecule with negative-sense polarity. We found that a particular antiviral protein, TRIM69, functions by a previously undescribed molecular mechanism. Specifically, TRIM69 interacts with and inhibits the function of a particular phosphoprotein (P) component of the viral transcription machinery, preventing the synthesis of viral messenger RNAs.
Assuntos
Interferon-alfa/farmacologia , Proteínas com Motivo Tripartido/antagonistas & inibidores , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vesiculovirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Linhagem Celular , Citocinas/farmacologia , Humanos , Modelos Moleculares , Fosfoproteínas/genética , Conformação Proteica , Domínios Proteicos , RNA Mensageiro/metabolismo , RNA Viral/biossíntese , Proteínas com Motivo Tripartido/química , Ubiquitina-Proteína Ligases/química , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/genética , Vesiculovirus/genética , Proteínas ViraisRESUMO
Vesicular stomatitis virus (VSV) represents a promising platform for developing oncolytic viruses, as well as vaccines against significant human pathogens. To safely control VSV infection in humans, small-molecule drugs that selectively inhibit VSV infection may be needed. Here, using a cell-based high-throughput screening assay followed by an in vitro transcription assay, compounds with a 7-hydroxy-6-methyl-3,4-dihydroquinolin-2(1H)-one structure and an aromatic group at position 4 (named vesiculopolins, VPIs) were identified as VSV RNA polymerase inhibitors. The most effective compound, VPI A, inhibited VSV-induced cytopathic effects and in vitro mRNA synthesis with micromolar to submicromolar 50% inhibitory concentrations. VPI A was found to inhibit terminal de novo initiation rather than elongation for leader RNA synthesis, but not mRNA capping, with the VSV L protein, suggesting that VPI A is targeted to the polymerase domain in the L protein. VPI A inhibited transcription of Chandipura virus, but not of human parainfluenza virus 3, suggesting that it specifically acts on vesiculoviral L proteins. These results suggest that VPIs may serve not only as molecular probes to elucidate the mechanisms of transcription of vesiculoviruses, but also as lead compounds to develop antiviral drugs against vesiculoviruses and other related rhabdoviruses.
Assuntos
Antivirais/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Vesiculovirus/efeitos dos fármacos , Vesiculovirus/genética , Animais , Linhagem Celular , Cricetinae , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , RNA Viral , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/genética , Proteínas Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacosRESUMO
In this study, canine IFNγ was fused by a flexible linker with canine serum albumin to construct the fusion protein IFNγ-CSA for the purpose to design a long-acting canine IFNγ. The fusion protein was successfully expressed in baculovirus-infected Sf9 insect cells and was purified by salting-out and ion exchange chromatography. The IFNγ-CSA fusion possessed potent anti-viral assay against vesicular stomatitis virus in cultured cells. IFNγ-CSA was also stable at 37⯰C up to 72â¯h compared with 8â¯h for IFNγ alone. In vivo pharmacokinetics demonstrated a significantly longer half-life for IFNγ-CSA (15.42â¯h) than for canine reIFNγ (1.51â¯h) in KM mice. These results indicate that IFNγ-CSA expression in the baculovirus system was successful and provide a promising long-acting cytokine for veterinary clinical applications.
Assuntos
Baculoviridae/genética , Interferon gama/genética , Albumina Sérica/genética , Animais , Antivirais/metabolismo , Antivirais/farmacocinética , Baculoviridae/metabolismo , Cães , Feminino , Expressão Gênica , Interferon gama/metabolismo , Interferon gama/farmacocinética , Camundongos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacocinética , Albumina Sérica/metabolismo , Albumina Sérica/farmacocinética , Células Sf9 , Spodoptera , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacosRESUMO
Antiviral immunity has been studied extensively from the perspective of virus-cell interactions, yet the role of virus-virus interactions remains poorly addressed. Here, we demonstrate that viral escape from interferon (IFN)-based innate immunity is a social process in which IFN-stimulating viruses determine the fitness of neighbouring viruses. We propose a general and simple social evolution framework to analyse how natural selection acts on IFN shutdown and validate it in cell cultures and mice infected with vesicular stomatitis virus. Furthermore, we find that IFN shutdown is costly because it reduces short-term viral progeny production, thus fulfilling the definition of an altruistic trait. Hence, in well-mixed populations, the IFN-blocking wild-type virus is susceptible to invasion by IFN-stimulating variants and spatial structure consequently determines whether IFN shutdown can evolve. Our findings reveal that fundamental social evolution rules govern viral innate immunity evasion and virulence and suggest possible antiviral interventions.
Assuntos
Antivirais/imunologia , Evolução Biológica , Evasão da Resposta Imune , Imunidade Inata , Animais , Encéfalo/patologia , Encéfalo/virologia , RNA Polimerases Dirigidas por DNA , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno/imunologia , Interferons/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/patogenicidade , Proteínas ViraisRESUMO
Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV can be sensed by host innate immunity to induce expression of interferons (IFNs) and a number of antiviral effectors. In this study, we found HCV infection induced the expression of neuralized E3 ubiquitin protein ligase 3 (NEURL3), a putative E3 ligase, in a manner that requires the involvement of innate immune sensing but is independent of the IFN action. Furthermore, we showed that NEURL3 inhibited HCV infection while it had little effect on other RNA viruses, including Zika virus (ZIKV), dengue virus (DENV), and vesicular stomatitis virus (VSV). Mechanistic studies demonstrated that NEURL3 inhibited HCV assembly by directly binding HCV envelope glycoprotein E1 to interfere with the E1/E2 heterodimerization, an important prerequisite for virion morphogenesis. Finally, we showed that knockout of NEURL3 significantly enhanced HCV infection. In summary, we identified NEURL3 as a novel inducible antiviral host factor that suppresses HCV assembly. Our results not only shed new insight into how host innate immunity acts against HCV but also revealed a new important biological function for NEURL3.IMPORTANCE The exact biological function of NEURL3, a putative E3 ligase, remains largely unknown. In this study, we found that NEURL3 could be upregulated upon HCV infection in a manner dependent on pattern recognition receptor-mediated innate immune response. NEURL3 inhibits HCV assembly by directly binding viral E1 envelope glycoprotein to disrupt its interaction with E2, an action that requires its Neuralized homology repeat (NHR) domain but not the RING domain. Furthermore, we found that NEURL3 has a pangenotypic anti-HCV activity and interacts with E1 of genotypes 2a, 1b, 3a, and 6a but does not inhibit other closely related RNA viruses, such as ZIKV, DENV, and VSV. To our knowledge, our study is the first report to demonstrate that NEURL3 functions as an antiviral host factor. Our results not only shed new insight into how host innate immunity acts against HCV, but also revealed a new important biological function for NEURL3.
Assuntos
Antivirais/farmacologia , Hepatite C/prevenção & controle , Imunidade Inata/imunologia , Infecções por Vírus de RNA/virologia , Ubiquitina-Proteína Ligases/farmacologia , Proteínas do Envelope Viral/antagonistas & inibidores , Vírus da Dengue/efeitos dos fármacos , Células HEK293 , Hepacivirus/classificação , Hepacivirus/genética , Hepacivirus/imunologia , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Infecções por Vírus de RNA/tratamento farmacológico , Infecções por Vírus de RNA/imunologia , Vírus de RNA/imunologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Montagem de Vírus , Zika virus/efeitos dos fármacosRESUMO
Cellular antiviral programs can efficiently inhibit viral infection. These programs are often initiated through signaling cascades induced by secreted proteins, such as type I interferons, interleukin-6 (IL-6), or tumor necrosis factor alpha (TNF-α). In the present study, we generated an arrayed library of 756 human secreted proteins to perform a secretome screen focused on the discovery of novel modulators of viral entry and/or replication. The individual secreted proteins were tested for the capacity to inhibit infection by two replication-competent recombinant vesicular stomatitis viruses (VSVs) with distinct glycoproteins utilizing different entry pathways. Fibroblast growth factor 16 (FGF16) was identified and confirmed as the most prominent novel inhibitor of both VSVs and therefore of viral replication, not entry. Importantly, an antiviral interferon signature was completely absent in FGF16-treated cells. Nevertheless, the antiviral effect of FGF16 is broad, as it was evident on multiple cell types and also on infection by coxsackievirus. In addition, other members of the FGF family also inhibited viral infection. Thus, our unbiased secretome screen revealed a novel protein family capable of inducing a cellular antiviral state. This previously unappreciated role of the FGF family may have implications for the development of new antivirals and the efficacy of oncolytic virus therapy.IMPORTANCE Viruses infect human cells in order to replicate, while human cells aim to resist infection. Several cellular antiviral programs have therefore evolved to resist infection. Knowledge of these programs is essential for the design of antiviral therapeutics in the future. The induction of antiviral programs is often initiated by secreted proteins, such as interferons. We hypothesized that other secreted proteins may also promote resistance to viral infection. Thus, we tested 756 human secreted proteins for the capacity to inhibit two pseudotypes of vesicular stomatitis virus (VSV). In this secretome screen on viral infection, we identified fibroblast growth factor 16 (FGF16) as a novel antiviral against multiple VSV pseudotypes as well as coxsackievirus. Subsequent testing of other FGF family members revealed that FGF signaling generally inhibits viral infection. This finding may lead to the development of new antivirals and may also be applicable for enhancing oncolytic virus therapy.
Assuntos
Fatores de Crescimento de Fibroblastos/farmacologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Replicação Viral/efeitos dos fármacos , Técnicas de Cultura de Células , Linhagem Celular , Meios de Cultivo Condicionados/metabolismo , Biblioteca Gênica , Células HEK293 , Células Hep G2 , Humanos , Biossíntese de Proteínas , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Internalização do VírusRESUMO
Vesicular stomatitis virus (VSV) can cause serious vesicular lesions in pigs, and the matrix (M) protein is its predominant virulence factor. Dendritic cells (DCs) act as the bridge between innate and adaptive immune responses. However, the susceptibility of porcine DCs to VSV infection and the role of M protein in modulating the function of infected DCs are still poorly defined. Thus, this study aimed to determine the ability of virulent wild-type VSV(wtVSV) and two attenuated M protein variants (VSVΔM51 and VSVMT) to induce maturation of porcine monocyte-derived DCs (MoDCs) in vitro. It was found that both wtVSV and the M protein mutant VSVs could productively replicate in porcine MoDCs. Infection with wtVSV resulted in weak proinflammatory cytokine responses and interfered with DC maturation via downregulation of the costimulatory molecule complex CD80/86. Whilst VSVΔM51 could activate porcine MoDCs, VSVMT, a highly attenuated recombinant VSV with triple mutations in the M protein, induced a potent maturation of MoDCs, as evidenced by efficient cytokine induction, and upregulation of CD80/86 and MHC class II. Overall, our findings reveal that porcine MoDCs are differentially activated by VSV, dependent on the presence of a functional M protein. M protein plays a crucial role in modulating porcine DC-VSV interactions. The data further support the potential use of VSVMT as a vaccine vector for pigs.
Assuntos
Células Dendríticas/virologia , Monócitos/virologia , Vírus da Estomatite Vesicular Indiana/genética , Proteínas da Matriz Viral/farmacologia , Animais , Moléculas de Adesão Celular/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Interleucina-1beta/biossíntese , Interleucina-1beta/imunologia , Monócitos/imunologia , Monócitos/fisiologia , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Proteínas Mutantes/farmacologia , Suínos , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/patogenicidade , Proteínas da Matriz Viral/genéticaRESUMO
The methods of the quantitative assay of the antiviral activity of interferons (IFNs) (type I, II or III) are very important during carrying out of the research of them, since they were found. Here a recombinant vesicular stomatitis virus expressing green fluorescent protein (GFP) (VSV/GFP) and MDBK cells were used to develop an antiviral assay (AVA) for IFNs. This method was carried out on a 96-well cell culture plate, and the half reduction of virus replication was quantified by assaying GFP. To quantify GFP, cell lysis buffer was directly added to the wells infected with VSV/GFP to lyse cells, the VSV/GFP was then inactivated, and relative fluorescence unit (RFU) of GFP was measured and used to calculate the antiviral activity. This method needed only one step instead of three steps in the staining method with naphthol blue black, medium with phenol red can be used, and it had good reproducibility. The GFP-containing samples could be stored at 4°C in a wet box for at least 1 week without affecting the assay results. In addition, the results obtained with this method were similar to those obtained with the staining method. In conclusion, a safe, rapid, convenient and low-cost AVA of IFN based on recombinant VSV/GFP was established.
Assuntos
Antivirais/farmacologia , Bioensaio/métodos , Interferons/farmacologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Animais , Bioensaio/economia , Linhagem Celular , Fluorescência , Proteínas de Fluorescência Verde/genética , Fenolsulfonaftaleína , Reprodutibilidade dos Testes , Replicação Viral/efeitos dos fármacosRESUMO
α1-Antitrypsin (AAT) is widely used to treat patients with congenital AAT deficiency. Cohn Fraction IV (Cohn F IV) is normally discarded during the manufacturing process of albumin but contains approximately 33% of plasma AAT. We established a new process for large-scale purification of AAT from it. liquid chromatography-electrospray ionization-tandem mass spectrometry and high-performance liquid chromatography were applied for qualitative identification and composition analysis, respectively. Stabilizers were optimized for AAT activity protection during lyophilization and dry-heat. Virus inactivation by dry-heat and solvent/detergent (S/D) was validated on a range of viruses. AAT with purity of 95.54%, specific activity of 3,938.5 IU/mg, and yield of 26.79%, was achieved. More than 95% activity was reserved after S/D. More than 96% activity was obtained after lyophilization or dry-heat. After S/D, pseudorabies virus (PRV) and vesicular stomatitis virus (VSV) were inactivated below detectable level within 1 H. Virus titer reductions of more than 5.50 log10 and 5.38 log10 were achieved for PRV and VSV, respectively. Porcine parvovirus and encephalomyocarditis virus were inactivated by 3.17 log10 and 5.88 log10 reduction after dry-heat. The advantages of this process, including suitability for large-scale production, high purity, better utilization of human plasma, viral safety, commercial and inexpensive chromatography medium, may facilitate its further application.
Assuntos
Proteínas Sanguíneas/química , Detergentes/farmacologia , Temperatura Alta , Solventes/farmacologia , Inativação de Vírus/efeitos dos fármacos , alfa 1-Antitripsina/isolamento & purificação , Animais , Linhagem Celular , Detergentes/química , Vírus da Encefalomiocardite/efeitos dos fármacos , Herpesvirus Suídeo 1/efeitos dos fármacos , Parvovirus/efeitos dos fármacos , Solventes/química , Suínos , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , alfa 1-Antitripsina/químicaRESUMO
The role of Ca2+ during dengue virus (DENV) replication is unknown; thus, changes in Ca2+ homeostasis in DENV infected human hepatic HepG2 and Huh-7 cells were analyzed. Infected HepG2 cells, but not Huh-7 cells, showed a significant increase in plasma membrane permeability to Ca2+, while both cell lines showed marked reduced levels of Ca2+ stored in the endoplasmic reticulum. While the expression levels of STIM1 and ORAI1 showed no changes, STIM1 and ORAI1 were shown to co-localized in infected cells, indicating activation of the store-operated Ca2+ entry (SOCE) pathway. Finally, manipulation in the infected cells of the intra and extracellular Ca2+ levels by chelators (BAPTA-AM and EGTA), SOC inhibitor (SKF96365), IP3 Receptor antagonist (2APB) or increase of extracellular [Ca2+], significantly reduced DENV yield, but not vesicular stomatitis virus yield, used as a control. These results show that DENV infection alters cell Ca2+ homeostasis and that such changes favor viral replication.
Assuntos
Quelantes de Cálcio/farmacologia , Cálcio/metabolismo , Vírus da Dengue/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Replicação Viral/efeitos dos fármacos , Animais , Compostos de Boro/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Membrana Celular/virologia , Permeabilidade da Membrana Celular , Chlorocebus aethiops , Vírus da Dengue/fisiologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Retículo Endoplasmático/virologia , Expressão Gênica , Células Hep G2 , Humanos , Imidazóis/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Transporte de Íons , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/antagonistas & inibidores , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Células Vero , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/fisiologia , Replicação Viral/genéticaRESUMO
Asymptomatic infections often proceed undetected, yet can still prime the host to be sensitive to secondary environmental stress. While the mechanisms underlying disease caused by asymptomatic infections are unknown, it is believed that productive pathogen replication is required. We report that the environmental stress of carbon dioxide (CO2) anesthesia converts an asymptomatic rhabdovirus infection in Drosophila to one that is lethal. This lethality results from a pool of infectious virus in glial cells and is regulated by the antiviral RNAi pathway of the host. CO2 sensitivity is caused by the fusogenic activity of the viral glycoprotein, which results in fusion of neurons and glia. Expression of the viral glycoprotein, but not a fusion defective mutant, is sufficient to cause CO2 sensitivity, which can occur even in the absence of productive viral replication. These findings highlight how viral proteins, independent of pathogen replication, may predispose hosts to life-threatening environmental stress.
Assuntos
Dióxido de Carbono/farmacologia , Meio Ambiente , Neurônios/virologia , Estresse Fisiológico , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/patogenicidade , Viroses , Animais , Animais Geneticamente Modificados , Anopheles/imunologia , Anopheles/virologia , Antivirais/farmacologia , Comportamento Animal , Drosophila melanogaster/imunologia , Drosophila melanogaster/virologia , Glicoproteínas/metabolismo , Concentração de Íons de Hidrogênio , Imunidade Inata , Neuroglia/imunologia , Neuroglia/virologia , Neurônios/imunologia , Interferência de RNA , Sindbis virus/imunologia , Sindbis virus/patogenicidade , Ensaio de Placa Viral , Proteínas Virais/metabolismo , Viroses/imunologia , Viroses/virologia , Replicação Viral/efeitos dos fármacosRESUMO
This paper describes computer-aided design of new anti-viral agents against Vaccinia virus (VACV) potentially acting as nucleic acid intercalators. Earlier obtained experimental data for DNA intercalation affinities and activities against Vesicular stomatitis virus (VSV) have been used to build, respectively, pharmacophore and QSAR models. These models were used for virtual screening of a database of 245 molecules generated around typical scaffolds of known DNA intercalators. This resulted in 12 hits which then were synthesized and tested for antiviral activity against VaV together with 43 compounds earlier studied against VSV. Two compounds displaying high antiviral activity against VaV and low cytotoxicity were selected for further antiviral activity investigations.
Assuntos
Antivirais/farmacologia , DNA/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Antivirais/síntese química , Antivirais/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Quantitativa Estrutura-AtividadeRESUMO
Accumulating evidence shows that innate immune responses are associated with extracellular nucleotides, particularly ATP. In this article, we demonstrate extensive protection of ATP/P2X7 signaling in a host against viral infection. Interestingly, we observed a significant increase in ATP as a danger signal in vesicular stomatitis virus (VSV)-infected cell supernatant and VSV-infected mice in an exocytosis- and pannexin channel-dependent manner. Furthermore, extracellular ATP reduces the replication of VSV, Newcastle disease virus, murine leukemia virus, and HSV in vivo and in vitro through the P2X7 receptor. Meanwhile, ATP significantly increases IFN-ß expression in a concentration- and time-dependent manner. Mechanistically, ATP facilitates IFN-ß secretion through P38/JNK/ATF-2 signaling pathways, which are crucial in promoting antiviral immunity. Taken together, these results demonstrate the protective role of extracellular ATP and P2X7 in viral infection and suggest a potential therapeutic role for ATP/P2X7 in viral diseases.
Assuntos
Trifosfato de Adenosina/metabolismo , Interferon beta/biossíntese , Receptores Purinérgicos P2X7/metabolismo , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Imunidade Inata , Interferon beta/genética , Interferon beta/imunologia , Vírus da Leucemia Murina/efeitos dos fármacos , Vírus da Leucemia Murina/imunologia , Medições Luminescentes , Camundongos , Vírus da Doença de Newcastle/efeitos dos fármacos , Vírus da Doença de Newcastle/imunologia , Células RAW 264.7 , Receptores Purinérgicos P2X7/imunologia , Transdução de Sinais , Simplexvirus/efeitos dos fármacos , Simplexvirus/imunologia , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/imunologia , Replicação Viral/efeitos dos fármacosRESUMO
Oncolytic viruses (OVs) offer a promising therapeutic approach to treat multiple types of cancer. In this study, we show that the manipulation of the antioxidant network via transcription factor Nrf2 augments vesicular stomatitis virus Δ51 (VSVΔ51) replication and sensitizes cancer cells to viral oncolysis. Activation of Nrf2 signaling by the antioxidant compound sulforaphane (SFN) leads to enhanced VSVΔ51 spread in OV-resistant cancer cells and improves the therapeutic outcome in different murine syngeneic and xenograft tumor models. Chemoresistant A549 lung cancer cells that display constitutive dominant hyperactivation of Nrf2 signaling are particularly vulnerable to VSVΔ51 oncolysis. Mechanistically, enhanced Nrf2 signaling stimulated viral replication in cancer cells and disrupted the type I IFN response via increased autophagy. This study reveals a previously unappreciated role for Nrf2 in the regulation of autophagy and the innate antiviral response that complements the therapeutic potential of VSV-directed oncolysis against multiple types of OV-resistant or chemoresistant cancer.
Assuntos
Autofagia , Fator 2 Relacionado a NF-E2/metabolismo , Vírus Oncolíticos/fisiologia , Transdução de Sinais , Estomatite Vesicular/metabolismo , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Animais , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Autofagia/efeitos dos fármacos , Linhagem Celular , Terapia Combinada , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Isotiocianatos/farmacologia , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Neoplasias/metabolismo , Neoplasias/mortalidade , Neoplasias/patologia , Neoplasias/terapia , Terapia Viral Oncolítica , Deleção de Sequência , Transdução de Sinais/efeitos dos fármacos , Sulfóxidos , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Proteínas da Matriz Viral/genética , Replicação Viral/efeitos dos fármacosRESUMO
The transcription and replication machinery of negative-stranded RNA viruses presents a possible target for interference in the viral life cycle. We demonstrate the validity of this concept through the use of cytosolically expressed single-domain antibody fragments (VHHs) that protect cells from a lytic infection with vesicular stomatitis virus (VSV) by targeting the viral nucleoprotein N. We define the binding sites for two such VHHs, 1004 and 1307, by X-ray crystallography to better understand their inhibitory properties. We found that VHH 1307 competes with the polymerase cofactor P for binding and thus inhibits replication and mRNA transcription, while binding of VHH 1004 likely only affects genome replication. The functional relevance of these epitopes is confirmed by the isolation of escape mutants able to replicate in the presence of the inhibitory VHHs. The escape mutations allow identification of the binding site of a third VHH that presumably competes with P for binding at another site than 1307. Collectively, these binding sites uncover different features on the N protein surface that may be suitable for antiviral intervention.
Assuntos
Anticorpos Antivirais/metabolismo , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/imunologia , Anticorpos de Domínio Único/metabolismo , Vírus da Estomatite Vesicular Indiana/fisiologia , Replicação Viral , Células A549 , Animais , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Sítios de Ligação , Cristalografia por Raios X , Replicação do DNA , Humanos , Mutação , Proteínas do Nucleocapsídeo/metabolismo , RNA Viral , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Transcrição Gênica , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologiaRESUMO
Virus resistance to antiviral therapies is an increasing concern that makes the development of broad-spectrum antiviral drugs urgent. Targeting of the viral envelope, a component shared by a large number of viruses, emerges as a promising strategy to overcome this problem. Natural and synthetic porphyrins are good candidates for antiviral development due to their relative hydrophobicity and pro-oxidant character. In the present work, we characterized the antiviral activities of protoprophyrin IX (PPIX), Zn-protoporphyrin IX (ZnPPIX), and mesoporphyrin IX (MPIX) against vesicular stomatitis virus (VSV) and evaluated the mechanisms involved in this activity. Treatment of VSV with PPIX, ZnPPIX, and MPIX promoted dose-dependent virus inactivation, which was potentiated by porphyrin photoactivation. All three porphyrins inserted into lipid vesicles and disturbed the viral membrane organization. In addition, the porphyrins also affected viral proteins, inducing VSV glycoprotein cross-linking, which was enhanced by porphyrin photoactivation. Virus incubation with sodium azide and α-tocopherol partially protected VSV from inactivation by porphyrins, suggesting that singlet oxygen (1O2) was the main reactive oxygen species produced by photoactivation of these molecules. Furthermore, 1O2 was detected by 9,10-dimethylanthracene oxidation in photoactivated porphyrin samples, reinforcing this hypothesis. These results reveal the potential therapeutic application of PPIX, ZnPPIX, and MPIX as good models for broad antiviral drug design.