RESUMO
Zoonoses such as ZIKV and SARS-CoV-2 pose a severe risk to global health. There is urgent need for broad antiviral strategies based on host-targets filling gaps between pathogen emergence and availability of therapeutic or preventive strategies. Significant reduction of pathogen titers decreases spread of infections and thereby ensures health systems not being overloaded and public life to continue. Based on previously observed interference with FGFR1/2-signaling dependent impact on interferon stimulated gene (ISG)-expression, we identified Pim kinases as promising druggable cellular target. We therefore focused on analyzing the potential of pan-Pim kinase inhibition to trigger a broad antiviral response. The pan-Pim kinase inhibitor AZD1208 exerted an extraordinarily high antiviral effect against various ZIKV isolates, SARS-CoV-2 and HBV. This was reflected by strong reduction in viral RNA, proteins and released infectious particles. Especially in case of SARS-CoV-2, AZD1208 led to a complete removal of viral traces in cells. Kinome-analysis revealed vast changes in kinase landscape upon AZD1208 treatment, especially for inflammation and the PI3K/Akt-pathway. For ZIKV, a clear correlation between antiviral effect and increase in ISG-expression was observed. Based on a cell culture model with impaired ISG-induction, activation of the PI3K-Akt-mTOR axis, leading to major changes in the endolysosomal equilibrium, was identified as second pillar of the antiviral effect triggered by AZD1208-dependent Pim kinase inhibition, also against HBV. We identified Pim-kinases as cellular target for a broad antiviral activity. The antiviral effect exerted by inhibition of Pim kinases is based on at least two pillars: innate immunity and modulation of the endolysosomal system.
Assuntos
Antivirais , Imunidade Inata , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-pim-1 , SARS-CoV-2 , Transdução de Sinais , Serina-Treonina Quinases TOR , Humanos , Imunidade Inata/efeitos dos fármacos , Antivirais/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Zika virus/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Animais , Vírus da Hepatite B/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Linhagem Celular , COVID-19/imunologia , COVID-19/virologia , Tratamento Farmacológico da COVID-19 , Replicação Viral/efeitos dos fármacos , Compostos de Bifenilo , TiazolidinasRESUMO
BACKGROUND: A peculiar feature of the hepatitis E virus (HEV) is its reliance on the exosomal route for viral release. Genomic replication is mediated via the viral polyprotein pORF1, yet little is known about its subcellular localization. METHODS: Subcellular localization of pORF1 and its subdomains, generated and cloned based on a structural prediciton of the viral replicase, was analyzed via confocal laser scanning microscopy. Exosomes released from cells were isolated via ultracentrifugation and analyzed by isopycnic density gradient centrifugation. This was followed by fluorimetry or Western blot analyses or reverse transcriptase-polymerase chain reaction to analyze separated particles in more detail. RESULTS: We found pORF1 to be accumulating within the endosomal system, most dominantly to multivesicular bodies (MVBs). Expression of the polyprotein's 7 subdomains revealed that the papain-like cysteine-protease (PCP) is the only domain localizing like the full-length protein. A PCP-deficient pORF1 mutant lost its association to MVBs. Strikingly, both pORF1 and PCP can be released via exosomes. Similarly, genomic RNA still is released via exosomes in the absence of pORF2/3. CONCLUSIONS: Taken together, we found that pORF1 localizes to MVBs in a PCP-dependent manner, which is followed by exosomal release. This reveals new aspects of HEV life cycle, because replication and release could be coupled at the endosomal interface. In addition, this may mediate capsid-independent spread or may facilitate the spread of viral infection, because genomes entering the cell during de novo infection readily encounter exosomally transferred pORF1.
Assuntos
Vírus da Hepatite E , Corpos Multivesiculares/metabolismo , Proteínas/metabolismo , Poliproteínas/metabolismo , Peptídeo Hidrolases/metabolismoRESUMO
Worldwide, the hepatitis B and hepatitis C viruses (HBV, HCV) are the most relevant causative viral agents of a chronic hepatitis (inflammation of the liver). At present, more than 250 million people suffer from a chronic HBV infection globally, resulting in 0.8 million deaths per year. A chronic HCV infection accounts for about 70 million cases worldwide, leading to a death toll of about 1 million per year. An approved vaccine is only available against an HBV infection. Both HBV and HCV infections result in a highly increased risk of developing liver fibrosis, cirrhosis, and a hepatocellular carcinoma (HCC).This review aims to describe mechanisms of the HBV- and HCV-associated pathogenesis. The focus is on the interplay between a chronic infection with intracellular signaling transduction, metabolic pathways with an emphasis on lipid metabolism, the establishment of liver fibrosis and cirrhosis during a chronic infection, and the mechanisms of the onset of a virally induced HCC.Despite there being great advances in the characterization of viral life cycles and the development of robust antiviral strategies, significant hurdles persist: gaining a better understanding of the mechanisms that drive virus-associated pathogenesis as well as increasing insights regarding different viral genotypes having impacts on alternate pathogeneses.
Assuntos
Carcinoma Hepatocelular , Hepatite B Crônica , Hepatite B , Hepatite C , Neoplasias Hepáticas , Carcinogênese , Carcinoma Hepatocelular/epidemiologia , Alemanha , Hepatite B Crônica/epidemiologia , Humanos , Neoplasias Hepáticas/epidemiologiaRESUMO
BACKGROUND: The mRNA vaccine BNT162b2 (Comirnaty, BioNTech/Pfizer) and the vaccine candidate CVnCoV (Curevac) each encode a stabilized spike protein of SARS-CoV2 as antigen but differ with respect to the nature of the mRNA (modified versus unmodified nucleotides) and the mRNA amount (30 µg versus 12 µg RNA). This study characterizes antisera elicited by these two vaccines in comparison to convalescent sera. METHODS: Sera from BNT162b2 vaccinated healthcare workers, and sera from participants of a phase I trial vaccinated with 2, 4, 6, 8, or 12 µg CVnCoV and convalescent sera from hospitalized patients were analyzed by ELISA, neutralization tests, surface plasmon resonance (SPR), and peptide arrays. RESULTS: BNT162b2-elicited sera and convalescent sera have a higher titer of spike-RBD-specific antibodies and neutralizing antibodies as compared to the CVnCoV-elicited sera. For all analyzed sera a reduction in binding and neutralizing antibodies was found for the lineage B.1.351 variant of concern. SPR analyses revealed that the CVnCoV-elicited sera have a lower fraction of slow-dissociating antibodies. Accordingly, the CVnCoV sera almost fail to compete with the spike-ACE2 interaction. The significance of common VOC mutations K417N, E484K, or N501Y focused on linear epitopes was analyzed using a peptide array approach. The peptide arrays showed a strong difference between convalescent sera and vaccine-elicited sera. Specifically, the linear epitope at position N501 was affected by the mutation and elucidates the escape of viral variants to antibodies against this linear epitope. CONCLUSION: These data reveal differences in titer, neutralizing capacity, and affinity of the antibodies between BNT162b2- and CVnCoV-elicited sera, which could contribute to the apparent differences in vaccine efficacy.
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacina BNT162 , COVID-19/terapia , Ensaios Clínicos Fase I como Assunto , Epitopos , Humanos , Imunização Passiva , Peptídeos , RNA Mensageiro , RNA Viral , Vacinas Sintéticas , Vacinas de mRNA , Soroterapia para COVID-19RESUMO
Viral hepatitis is characterized as an acute or chronic inflammation of the liver induced by an infection with certain viruses. At present, around 325â¯million humans suffer from the chronic form of the disease worldwide. Each year, about 1.6â¯million people die as a result of viral hepatitis. The causative agents, hepatitis viruses, are subdivided into five groups of pathogens, which are denoted with the letters A to E (HAV to HEV). These differ from each other with respect to phylogeny, transmission, epidemiology, host-specificity, life cycle, structure, and distinct aspects of pathogenesis.The strictly human-pathogenic HAV, a member of the Picornaviridae family, mostly induces acute hepatitis and displays a dominant spread over the Global South. The Hepeviridae-affiliated HEV shows a similar epidemiology, yet spreads further into industrialized countries due to its zoonotic potential. Furthermore, HEV is defined by the capability of inducing chronic hepatitis. This course of disease is also found in a more pronounced manner for the globally prevalent HBV (Hepadnaviridae) and its satellite virus HDV (Kolmioviridae), which further increases their carcinogenic potential. Lastly, a worldwide distribution is similarly described for HCV (Flaviviridae), which displays a high risk of chronifications and therefore a highly increased carcinogenic potential.The aforementioned pathogens differ with respect to their properties and life cycles. Thus, a differentiated look on epidemiology, diagnostic procedures, and disease prevention is required. Despite the presence of therapies, in some cases even a vaccine, there is an urgent need for advances in research on these aspects, especially for poverty-related pathogens.
Assuntos
Vírus da Hepatite E , Hepatite Viral Humana , Vírus , Alemanha , Vírus de Hepatite , Humanos , PrevalênciaRESUMO
Zika virus (ZIKV) is a flavivirus that is well known for the epidemic in the Americas in 2015 and 2016 in which microcephaly in newborns and other neurological complications were connected to ZIKV infection. Many aspects of the ZIKV viral life cycle, including binding and entry into the host cell, are still enigmatic. Based on the observation that CHO cells lack expression of the epidermal growth factor receptor (EGFR) and are not permissive for various ZIKV strains, the relevance of EGFR for the viral life cycle was analyzed. Infection of A549 cells by ZIKV leads to a rapid internalization of EGFR that colocalizes with the endosomal marker EEA1. Moreover, infection by different ZIKV strains is associated with an activation of EGFR and the subsequent activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling cascade. However, treatment of the cells with methyl-ß-cyclodextrin (MßCD), which on the one hand leads to an activation of EGFR but on the other hand prevents EGFR internalization, impairs ZIKV infection. Specific inhibition of EGFR or of the Ras-Raf-MEK-ERK signal transduction cascade hinders ZIKV infection by inhibition of ZIKV entry. In accordance with this, knockout of EGFR expression impedes ZIKV entry. In the case of an already established infection, inhibition of EGFR or of downstream signaling does not affect viral replication. Taken together, these data demonstrate the relevance of EGFR in the early stages of ZIKV infection and identify EGFR as a target for antiviral strategies. IMPORTANCE These data deepen the knowledge about the ZIKV infection process and demonstrate the relevance of EGFR for ZIKV entry. In light of the fact that a variety of specific and efficient inhibitors of EGFR and of EGFR-dependent signaling have been developed and licensed, repurposing of these substances could be a helpful tool to prevent the spreading of ZIKV infection in an epidemic outbreak.
Assuntos
Internalização do Vírus/efeitos dos fármacos , Zika virus/metabolismo , Células A549 , Animais , Células CHO , Linhagem Celular , Chlorocebus aethiops , Cricetulus , Receptores ErbB/efeitos dos fármacos , Receptores ErbB/metabolismo , Receptores ErbB/fisiologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Estágios do Ciclo de Vida , Transdução de Sinais/efeitos dos fármacos , Células Vero , Replicação Viral/genética , Replicação Viral/fisiologia , Zika virus/patogenicidade , Infecção por Zika virus/virologia , beta-Ciclodextrinas/farmacologiaRESUMO
The N-terminus of the hepatitis B virus (HBV) large surface protein (LHB) differs with respect to genotypes. Compared to the amino terminus of genotype (Gt)D, in GtA, GtB and GtC, an additional identical 11 amino acids (aa) are found, while GtE and GtG share another similar 10 aa. Variants of GtB and GtC affecting this N-terminal part are associated with hepatoma formation. Deletion of these amino-terminal 11 aa in GtA reduces the amount of LHBs and changes subcellular accumulation (GtA-like pattern) to a dispersed distribution (GtD-like pattern). Vice versa, the fusion of the GtA-derived N-terminal 11 aa to GtD causes a GtA-like phenotype. However, insertion of the corresponding GtE-derived 10 aa to GtD has no effect. Deletion of these 11aa decreases filament size while neither the number of released viral genomes nor virion size and infectivity are affected. A negative regulatory element (aa 2-8) and a dominant positive regulatory element (aa 9-11) affecting the amount of LHBs were identified. The fusion of this motif to eGFP revealed that the effect on protein amount and subcellular distribution is not restricted to LHBs. These data identify a novel region in the N-terminus of LHBs affecting the amount and subcellular distribution of LHBs and identify release-promoting and -inhibiting aa residues within this motive.
Assuntos
Genótipo , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/sangue , Morfogênese , Domínios Proteicos/genética , Precursores de Proteínas/genética , Proteínas do Envelope Viral/química , Vírion/crescimento & desenvolvimento , Adulto , Negro ou Afro-Americano/genética , Povo Asiático/genética , Linhagem Celular Tumoral , DNA Viral/sangue , Feminino , Hepatite B Crônica/etnologia , Hepatite B Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas do Envelope Viral/metabolismo , População Branca/genéticaRESUMO
With respect to their genome and their structure, the human hepatitis B virus (HBV) and hepatitis C virus (HCV) are complete different viruses. However, both viruses can cause an acute and chronic infection of the liver that is associated with liver inflammation (hepatitis). For both viruses chronic infection can lead to fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Reactive oxygen species (ROS) play a central role in a variety of chronic inflammatory diseases. In light of this, this review summarizes the impact of both viruses on ROS-generating and ROS-inactivating mechanisms. The focus is on the effect of both viruses on the transcription factor Nrf2 (nuclear factor erythroid 2 (NF-E2)-related factor 2). By binding to its target sequence, the antioxidant response element (ARE), Nrf2 triggers the expression of a variety of cytoprotective genes including ROS-detoxifying enzymes. The review summarizes the literature about the pathways for the modulation of Nrf2 that are deregulated by HBV and HCV and describes the impact of Nrf2 deregulation on the viral life cycle of the respective viruses and the virus-associated pathogenesis.