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1.
Elife ; 92020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33169665

RESUMO

Since the 1960s, a single class of agent has been licensed targeting virus-encoded ion channels, or 'viroporins', contrasting the success of channel blocking drugs in other areas of medicine. Although resistance arose to these prototypic adamantane inhibitors of the influenza A virus (IAV) M2 proton channel, a growing number of clinically and economically important viruses are now recognised to encode essential viroporins providing potential targets for modern drug discovery. We describe the first rationally designed viroporin inhibitor with a comprehensive structure-activity relationship (SAR). This step-change in understanding not only revealed a second biological function for the p7 viroporin from hepatitis C virus (HCV) during virus entry, but also enabled the synthesis of a labelled tool compound that retained biological activity. Hence, p7 inhibitors (p7i) represent a unique class of HCV antiviral targeting both the spread and establishment of infection, as well as a precedent for future viroporin-targeted drug discovery.


Assuntos
Antivirais/farmacologia , Hepacivirus/metabolismo , Proteínas Virais/antagonistas & inibidores , Animais , Antivirais/química , Biomarcadores , Linhagem Celular , Cães , Descoberta de Drogas , Genótipo , Hepacivirus/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Proteínas Virais/metabolismo
2.
Gut ; 67(3): 562-573, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-27902444

RESUMO

OBJECTIVE: Oncolytic viruses (OVs) represent promising, proinflammatory cancer treatments. Here, we explored whether OV-induced innate immune responses could simultaneously inhibit HCV while suppressing hepatocellular carcinoma (HCC). Furthermore, we extended this exemplar to other models of virus-associated cancer. DESIGN AND RESULTS: Clinical grade oncolytic orthoreovirus (Reo) elicited innate immune activation within primary human liver tissue in the absence of cytotoxicity and independently of viral genome replication. As well as achieving therapy in preclinical models of HCC through the activation of innate degranulating immune cells, Reo-induced cytokine responses efficiently suppressed HCV replication both in vitro and in vivo. Furthermore, Reo-induced innate responses were also effective against models of HBV-associated HCC, as well as an alternative endogenous model of Epstein-Barr virus-associated lymphoma. Interestingly, Reo appeared superior to the majority of OVs in its ability to elicit innate inflammatory responses from primary liver tissue. CONCLUSIONS: We propose that Reo and other select proinflammatory OV may be used in the treatment of multiple cancers associated with oncogenic virus infections, simultaneously reducing both virus-associated oncogenic drive and tumour burden. In the case of HCV-associated HCC (HCV-HCC), Reo should be considered as an alternative agent to supplement and support current HCV-HCC therapies, particularly in those countries where access to new HCV antiviral treatments may be limited.


Assuntos
Carcinoma Hepatocelular/terapia , Hepacivirus/fisiologia , Neoplasias Hepáticas/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/imunologia , Reoviridae/imunologia , Animais , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/terapia , Linfoma de Burkitt/virologia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Hepacivirus/imunologia , Hepatócitos , Herpesvirus Humano 4 , Humanos , Imunidade Inata , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Interferons , Interleucinas/metabolismo , Leucócitos Mononucleares , Fígado/imunologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/virologia , Camundongos , Camundongos SCID , Células T Matadoras Naturais/imunologia , Replicação Viral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
3.
J Gen Virol ; 95(Pt 11): 2427-2441, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25024280

RESUMO

Infectious hepatitis C virus (HCV) particle production in the genotype 2a JFH-1-based cell culture system involves non-structural proteins in addition to canonical virion components. NS2 has been proposed to act as a protein adaptor, co-ordinating the early stages of virion assembly. However, other studies have identified late-acting roles for this protein, making its precise involvement in infectious particle production unclear. Using a robust, bipartite trans-encapsidation system based upon baculovirus expression of HCV structural proteins, we have generated HCV-like particles (HCV-LP) in the absence of NS2 with overt similarity to wild-type virions. HCV-LP could transduce naive cells with trans-encapsidated subgenomic replicon RNAs and shared similar biochemical and biophysical properties with JFH-1 HCV. Both genotype 1b and JFH-1 intracellular HCV-LP were produced in the absence of NS2, whereas restoring NS2 to the JFH-1 system dramatically enhanced secreted infectivity, consistent with a late-acting role. Our system recapitulated authentic HCV particle assembly via trans-complementation of bicistronic, NS2-deleted, chimeric HCV, which is otherwise deficient in particle production. This closely resembled replicon-mediated NS2 trans-complementation, confirming that baculovirus expression of HCV proteins did not unduly affect particle production. Furthermore, this suggests that separation of structural protein expression from replicating HCV RNAs that are destined to be packaged alleviates an early stage requirement for NS2 during particle formation. This highlights our current lack of understanding of how NS2 mediates assembly, yet comparison of full-length and bipartite systems may provide further insight into this process.


Assuntos
Hepacivirus/fisiologia , Proteínas não Estruturais Virais/fisiologia , Montagem de Vírus/fisiologia , Baculoviridae/genética , Linhagem Celular , Teste de Complementação Genética , Genótipo , Hepacivirus/classificação , Hepacivirus/genética , Humanos , Microscopia Eletrônica de Transmissão , RNA Viral/genética , Replicon , Proteínas não Estruturais Virais/genética , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/fisiologia , Vírion/genética , Vírion/fisiologia , Vírion/ultraestrutura , Montagem de Vírus/genética
4.
J Gen Virol ; 94(Pt 10): 2236-2248, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23907396

RESUMO

Hepatitis C virus (HCV) p7 protein is critical for the efficient production of infectious virions in culture. p7 undergoes genotype-specific protein-protein interactions as well as displaying channel-forming activity, making it unclear whether the phenotypes of deleterious p7 mutations result from the disruption of one or both of these functions. Here, we showed that proton channel activity alone, provided in trans by either influenza virus M2 or genotype 1b HCV p7, was both necessary and sufficient to restore infectious particle production to genotype 2a HCV (JFH-1 isolate) carrying deleterious p7 alanine substitutions within the p7 dibasic loop (R33A, R35A), and the N-terminal trans-membrane region (N15 : C16 : H17/AAA). Both mutations markedly reduced mature p7 abundance, with those in the dibasic loop also significantly reducing levels of mature E2 and NS2. Interestingly, whilst M2 and genotype 1b p7 restored the same level of intracellular infectivity as JFH-1 p7, supplementing with the isogenic protein led to a further increase in secreted infectivity, suggesting a late-acting role for genotype-specific p7 protein interactions. Finally, cells infected by viruses carrying p7 mutations contained non-infectious core-containing particles with densities equivalent to WT HCV, indicating a requirement for p7 proton channel activity in conferring an infectious phenotype to virions.


Assuntos
Proteínas de Transporte/metabolismo , Hepacivirus/metabolismo , Canais Iônicos/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Regulação Viral da Expressão Gênica/fisiologia , Hepacivirus/genética , Hepacivirus/fisiologia , Humanos , Mutação , Prótons , Proteínas Virais/genética , Proteínas Virais/fisiologia , Vírion/genética , Vírion/metabolismo , Vírion/fisiologia , Montagem de Vírus
5.
Hepatology ; 54(1): 79-90, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21520195

RESUMO

UNLABELLED: The hepatitis C virus (HCV) p7 ion channel plays a critical role during infectious virus production and represents an important new therapeutic target. Its activity is blocked by structurally distinct classes of small molecules, with sensitivity varying between isolate p7 sequences. Although this is indicative of specific protein-drug interactions, a lack of high-resolution structural information has precluded the identification of inhibitor binding sites, and their modes of action remain undefined. Furthermore, a lack of clinical efficacy for existing p7 inhibitors has cast doubt over their specific antiviral effects. We identified specific resistance mutations that define the mode of action for two classes of p7 inhibitor: adamantanes and alkylated imino sugars (IS). Adamantane resistance was mediated by an L20F mutation, which has been documented in clinical trials. Molecular modeling revealed that L20 resided within a membrane-exposed binding pocket, where drug binding prevented low pH-mediated channel opening. The peripheral binding pocket was further validated by a panel of adamantane derivatives as well as a bespoke molecule designed to bind the region with high affinity. By contrast, an F25A polymorphism found in genotype 3a HCV conferred IS resistance and confirmed that these compounds intercalate between p7 protomers, preventing channel oligomerization. Neither resistance mutation significantly reduced viral fitness in culture, consistent with a low genetic barrier to resistance occurring in vivo. Furthermore, no cross-resistance was observed for the mutant phenotypes, and the two inhibitor classes showed additive effects against wild-type HCV. CONCLUSION: These observations support the notion that p7 inhibitor combinations could be a useful addition to future HCV-specific therapies.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral/genética , Hepatite C/tratamento farmacológico , Canais Iônicos/antagonistas & inibidores , Mutação/genética , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Amantadina/farmacologia , Sequência de Aminoácidos , Antivirais/uso terapêutico , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Humanos , Imino Açúcares/farmacologia , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Dados de Sequência Molecular , Polimorfismo Genético/genética , Resultado do Tratamento , Proteínas Virais/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
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