RESUMEN
Virus entry into cells is the initial stage of infection and involves multiple steps, and interfering viral entry represents potential antiviral approaches. Ion channels are pore-forming membrane proteins controlling cellular ion homeostasis and regulating many physiological processes, but their roles during viral infection have rarely been explored. Here, the functional Kv1.3 ion channel was found to be expressed in human hepatic cells and tissues. The Kv1.3 was then revealed to restrict HCV entry via inhibiting endosome acidification-mediated viral membrane fusion. The Kv1.3 was also demonstrated to inhibit DENV and ZIKV with an endosome acidification-dependent entry, but have no effect on SeV with a neutral pH penetration. A Kv1.3 antagonist PAP-1 treatment accelerated animal death in ZIKV-infected Ifnar1-/- mice. Moreover, Kv1.3-deletion was found to promote weight loss and reduce survival rate in ZIKV-infected Kv1.3-/- mice. Altogether, the Kv1.3 ion channel behaves as a host factor restricting viral entry. These findings broaden understanding about ion channel biology.
Asunto(s)
Virus del Dengue/fisiología , Dengue/metabolismo , Hepacivirus/fisiología , Hepatitis C/metabolismo , Canal de Potasio Kv1.3/metabolismo , Infecciones por Respirovirus/metabolismo , Virus Sendai/fisiología , Internalización del Virus , Infección por el Virus Zika/metabolismo , Virus Zika/fisiología , Animales , Chlorocebus aethiops , Dengue/virología , Endosomas/metabolismo , Ficusina/farmacología , Células HEK293 , Hepatitis C/virología , Humanos , Concentración de Iones de Hidrógeno , Canal de Potasio Kv1.3/antagonistas & inhibidores , Canal de Potasio Kv1.3/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Respirovirus/virología , Transfección , Células Vero , Internalización del Virus/efectos de los fármacos , Infección por el Virus Zika/virologíaRESUMEN
Rationale: Many viral infections are known to activate the p38 mitogen-activated protein kinase (MAPK) signaling pathway. However, the role of p38 activation in viral infection and the underlying mechanism remain unclear. The role of virus-hijacked p38 MAPK activation in viral infection was investigated in this study. Methods: The correlation of hepatitis C virus (HCV) infection and p38 activation was studied in patient tissues and primary human hepatocytes (PHHs) by immunohistochemistry and western blotting. Coimmunoprecipitation, GST pulldown and confocal microscopy were used to investigate the interaction of p38α and the HCV core protein. In vitro kinase assays and mass spectrometry were used to analyze the phosphorylation of the HCV core protein. Plaque assays, quantitative real time PCR (qRT-PCR), western blotting, siRNA and CRISPR/Cas9 were used to determine the effect of p38 activation on viral replication. Results: HCV infection was associated with p38 activation in clinical samples. HCV infection increased p38 phosphorylation by triggering the interaction of p38α and TGF-ß activated kinase 1 (MAP3K7) binding protein 1 (TAB1). TAB1-mediated p38α activation facilitated HCV replication, and pharmaceutical inhibition of p38α activation by SB203580 suppressed HCV infection at the viral assembly step. Activated p38α interacted with the N-terminal region of the HCV core protein and subsequently phosphorylated the HCV core protein, which promoted HCV core protein oligomerization, an essential step for viral assembly. As expected, SB203580 or the HCV core protein N-terminal peptide (CN-peptide) disrupted the p38α-HCV core protein interaction, efficiently impaired HCV assembly and impeded normal HCV replication in both cultured cells and primary human hepatocytes. Similarly, severe fever with thrombocytopenia syndrome virus (SFTSV), herpes simplex virus type 1 (HSV-1) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection also activated p38 MAPK. Most importantly, pharmacological blockage of p38 activation by SB203580 effectively inhibited SFTSV, HSV-1 and SARS-CoV-2. Conclusion: Our study shows that virus-hijacked p38 activation is a key event for viral replication and that pharmacological blockage of p38 activation is an antiviral strategy.
Asunto(s)
COVID-19/metabolismo , Hepacivirus/metabolismo , Hepatitis C/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células A549 , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , COVID-19/virología , Chlorocebus aethiops , Activación Enzimática , Células HEK293 , Hepatitis C/patología , Hepatitis C/virología , Hepatocitos/metabolismo , Humanos , Imidazoles/farmacología , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Fosforilación , Piridinas/farmacología , Células Vero , Proteínas del Núcleo Viral/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Interferon-inducible transmembrane proteins (IFITM1/2/3) have been reported to suppress the entry of a wide range of viruses. However, their antiviral functional residues and specific mechanisms are still unclear. Here, we firstly resolved the topology of IFITM1 on the plasma membrane where N-terminus points into the cytoplasm and C-terminus resides extracellularly. Further, KRRK basic residues of IFITM1 locating at 62-67 of the conserved intracellular loop (CIL) were found to play a key role in the restriction on the Zika virus (ZIKV) and dengue virus (DENV). Similarly, KRRK basic residues of IFITM2/3 also contributed to suppressing ZIKV replication. Finally, IFITM1 was revealed to be capable of restricting the release of ZIKV particles from endosome to cytosol so as to impede the entry of ZIKV into host cells, which was tightly related with the inhibition of IFITM1 on the acidification of organelles. Overall, our study provided topology, antiviral functional residues and the mechanism of interferon-inducible transmembrane proteins.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Antígenos de Diferenciación/farmacología , Antivirales/metabolismo , Antivirales/farmacología , Virus Zika/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Chlorocebus aethiops , Endosomas/metabolismo , Células HEK293 , Humanos , Interferones/metabolismo , Proteínas de la Membrana/metabolismo , Alineación de Secuencia , Células Vero , Replicación Viral/efectos de los fármacos , Infección por el Virus ZikaRESUMEN
Hepatitis C virus (HCV) infection is a major worldwide health problem which can cause chronic hepatitis, liver fibrosis and hepatocellular carcinoma (HCC). There is still no vaccine to prevent HCV infection. Currently, the clinical treatment of HCV infection mainly relies on the use of direct-acting antivirals (DAAs) which are expensive and have side effects. Here, BmKDfsin3, a scorpion defensin from the venom of Mesobuthus martensii Karsch, is found to dose-dependently inhibit HCV infection at noncytotoxic concentrations and affect viral attachment and post-entry in HCV life cycle. Further experimental results show that BmKDfsin3 not only suppresses p38 mitogen-activated protein kinase (MAPK) activation of HCV-infected Huh7.5.1 cells, but also inhibits p38 activation of Huh7.5.1 cells stimulated by tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) or lipopolysaccharide (LPS). BmKDfsin3 is also revealed to enter into cells. Using an upstream MyD88 dimerization inhibitor ST2345 or kinase IRAK-1/4 inhibitor I, the inhibition of p38 activation represses HCV replication in vitro. Taken together, a scorpion defensin BmKDfsin3 inhibits HCV replication, related to regulated p38 MAPK activation.
RESUMEN
OBJECTIVE: To investigate the expression of kinesin family member 20A (KIF20A) in bladder cancer, the effect of KIF20A on the proliferation and metastasis of bladder cancer cells, and the effect of KIF20A expression on the prognosis of bladder cancer patients. METHODS: Bladder cancer tissue and its adjacent tissues were collected from tumour patients. The mRNA and protein expression levels of KIF20A in the tissue samples were detected by qRT-PCR and western blot. Immunohistochemical (IHC) staining was used to identify the expression and distribution of KIF20A proteins in the tissue samples. The relationship between the KIF20A expression and the clinical pathology of bladder cancer was analysed. The effect of the differential expression of KIF20A on the prognosis of patients with bladder cancer was analysed by the TCGA database. The plasmid was transfected into the bladder cell lines T24 and 5637 to construct two stable cell lines with knocked down KIF20A. The effect of KIF20A expression on the proliferation and invasion of T24 and 5637 bladder cells was explored in vitro using the abovementioned stable cell lines. The effect of the KIF20A expression on the proliferation of bladder cancer cells was evaluated by a mouse xenograft model. RESULTS: The expression of KIF20A was significantly higher in the bladder cancer tissues than in the adjacent control tissues. The expression of KIF20A was significantly associated with the degree of pathological differentiation of bladder cancer. Patients with a higher expression of KIF20A had a higher tumour grade and a more advanced stage. The mean survival of patients with a high KIF20A expression was significantly lower than the mean survival of patients with a low KIF20A expression. The in vitro experiments demonstrated that the knockdown of KIF20A significantly inhibited T24 and 5637 cell proliferation and invasion. The in vivo experiments showed that the knockdown of KIF20A significantly inhibited the proliferation of the bladder tumours. CONCLUSION: KIF20A promotes the proliferation and metastasis of bladder cancer cells. Bladder cancer patients with a high KIF20A expression have a worse tumour differentiation and a poor prognosis. KIF20A may become an independent factor that affects the prognosis of bladder cancer patients and a therapeutic target for bladder cancer.
Asunto(s)
Biomarcadores de Tumor/genética , Cinesinas/genética , Neoplasias de la Vejiga Urinaria/metabolismo , Anciano , Biomarcadores de Tumor/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Cinesinas/metabolismo , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Viral infections still threaten human health all over the world, and many people die from viral diseases every year. However, there are no effective vaccines or drugs for preventing or managing most viral diseases. Thus, the discovery and development of broad-spectrum antiviral agents remain urgent. Here, we expressed and purified a venom peptide, Ev37, from the scorpion Euscorpiops validus in a prokaryotic system. We found that rEv37 can inhibit dengue virus type 2 (DENV-2), hepatitis C virus (HCV), Zika virus (ZIKV), and herpes simplex virus type 1 (HSV-1) infections in a dose-dependent manner at noncytotoxic concentrations, but that it has no effect on Sendai virus (SeV) and adenovirus (AdV) infections in vitro Furthermore, rEv37 alkalized acidic organelles to prevent low pH-dependent fusion of the viral membrane-endosomal membrane, which mainly blocks the release of the viral genome from the endosome to the cytoplasm and then restricts viral late entry. Taken together, our results indicate that the scorpion venom peptide Ev37 is a broad-spectrum antiviral agent with a specific molecular mechanism against viruses undergoing low pH-dependent fusion activation during entry into host cells. We conclude that Ev37 is a potential candidate for development as an antiviral drug.
Asunto(s)
Citoplasma/metabolismo , Virus del Dengue/fisiología , Endosomas/metabolismo , Venenos de Escorpión/farmacología , Escorpiones/química , Internalización del Virus/efectos de los fármacos , Adenoviridae/fisiología , Animales , Chlorocebus aethiops , Citoplasma/virología , Endosomas/virología , Células HEK293 , Humanos , Fusión de Membrana/efectos de los fármacos , Venenos de Escorpión/química , Virus Sendai/fisiología , Células VeroRESUMEN
Dengue virus (DENV) and Zika virus (ZIKV) have spread throughout many countries in the developing world and infect millions of people every year, causing severe harm to human health and the economy. Unfortunately, there are few effective vaccines and therapies available against these viruses. Therefore, the discovery of new antiviral agents is critical. Herein, a scorpion venom peptide (Smp76) characterized from Scorpio maurus palmatus was successfully expressed and purified in Escherichia coli BL21(DE3). The recombinant Smp76 (rSmp76) was found to effectively inhibit DENV and ZIKV infections in a dose-dependent manner in both cultured cell lines and primary mouse macrophages. Interestingly, rSmp76 did not inactivate the viral particles directly but suppressed the established viral infection, similar to the effect of interferon (IFN)-ß. Mechanistically, rSmp76 was revealed to upregulate the expression of IFN-ß by activating interferon regulatory transcription factor 3 (IRF3) phosphorylation, enhancing the type-I IFN response and inhibiting viral infection. This mechanism is significantly different from traditional virucidal antimicrobial peptides (AMPs). Overall, the scorpion venom peptide Smp76 is a potential new antiviral agent with a unique mechanism involving type-I IFN responses, demonstrating that natural AMPs can enhance immunity by functioning as immunomodulators.