Relevance of Rab Proteins for the Life Cycle of Hepatitis C Virus

Although potent direct-acting antiviral drugs for the treatment of chronic hepatitis C virus (HCV) infection are licensed, there are more than 70 million individuals suffering from chronic HCV infection. In light of the limited access to these drugs, high costs, and a lot of undiagnosed cases, it is expected that the number of HCV cases will not decrease worldwide in the next years. Therefore, and due to the paradigmatic character of HCV for deciphering the crosstalk between viral pathogens and the host cell, characterization of HCV life cycle remains a challenge. HCV belongs to the family of Flaviviridae. As an enveloped virus HCV life cycle depends in many steps on intracellular trafficking. Rab GTPases, a large family of small GTPases, play a central role in intracellular trafficking processes controlling fusion, uncoating, vesicle budding, motility by recruiting specific effector proteins. This review describes the relevance of various Rab proteins for the different steps of the HCV life cycle

Charakterisierung des endosomalen Freisetzungswegs von Hepatitis-C-Virionen

Weltweit sind ca. 130–180 Millionen Menschen mit HCV infiziert und jährlich sterben etwa 500.000 Menschen an dessen Folgen. Die neuartigen Therapien versprechen zwar eine sehr hohe Heilungsrate, sind aber aufgrund ihrer enorm hohen Kosten nur in Industrieländern verfügbar. Noch immer gibt es keine prophylaktische Vakzinierung gegen HCV. Deshalb ist es wichtig, den HCV-Lebenszyklus und die Interaktion zwischen Wirtszelle und Virus detailliert zu verstehen, um die Entwicklung von Therapien und Impfungen zu ermöglichen. Außerdem kann ein fundiertes Wissen von HCV translatiert werden und auf neuartige Erreger der Familie der Flaviviridae, wie Denguevirus und Zikavirus, angewendet werden. Während der Zelleintritt und die Replikation von HCV relativ gut charakterisiert sind, bleiben die Assemblierung und Freisetzung der viralen Partikel schlecht verstandene Schritte des HCV-Lebenszyklus. In dieser Arbeit sollte die Rolle des zellulären Proteins α-Taxilin im Lebenszyklus von HCV untersucht werden. In einer späteren Phase der Arbeit wurde der endosomale Freisetzungsweg von HCV untersucht. Dazu wurden HCV Varianten generiert und charakterisiert, die Fluoreszenz-Proteine im NS5A- und E1-Protein enthalten, durch die es möglich ist, den Replikationskomplex und die Viruspartikel zu visualisieren und zu quantifizieren und den viralen Lebenszyklus dadurch besser untersuchen zu können..

Regulation of the Transferrin Receptor Recycling in Hepatitis C Virus-Replicating Cells

After binding of its ligand transferrin, the transferrin receptor (TfR) is internalized via early endosomes. Ligand and receptor can be recycled. α-Taxilin was identified as an essential factor for TfR recycling. Apart from its role for iron uptake, TfR is a coreceptor for hepatitis C virus (HCV) infection. In HCV-replicating cells, the amount of a-taxilin is decreased. This study aims to investigate the effect of decreased α-taxilin levels in HCV-replicating cells on recycling of TfR, its amount on the cell surface, on iron uptake, and the impact of a disturbed TfR recycling on HCV superinfection exclusion. TfR amount and localization were determined by CLSM and surface biotinylation. α-taxilin expression was modulated by CRISPR-Cas9 knockout, siRNA, and stable or transient overexpression. For analysis of HCV superinfection fluorophor-tagged reporter viruses were used. The amount of α-taxilin is decreased in HCV-infected cells. In accordance to this, the protein amount of TfR is significant lower in HCV-positve cells as compared to the control, while TfR expression is not affected. Due to the impaired recycling, internalized TfR is degraded by the endosomal/lysosomal system. The significant lower number of TfR molecules on the cell surface is reflected by reduced transferrin binding/internalization and strong reduction of intracellular iron level. Overexpression of α-taxilin in HCV-replicating cells rescues TfR recycling, augments TfR on the cell surface, and restores transferrin binding. The block of superinfection in HCV-replicating cells could be overcome by overexpression of α-taxilin. Taken together, the diminished level of α-taxilin in HCV-replicating cells prevents recycling of TfR leading to decreased transferrin binding and iron uptake. Disappearance of TfR from the cell surface could be a factor contributing to the exclusion of superinfection by HCV

ZIKV Envelope Domain-Specific Antibodies: Production, Purification and Characterization

Infection with Zika virus (ZIKV) came first to public attention after it was found to be associated with congenital microcephaly during the outbreak in Brazil (2015–2016). Diagnosis of ZIKV suffers from extensive cross-reactivity with other Flaviviruses, which are circulating in many ZIKV epidemic areas. Due to the fatal outcome of ZIKV infection during pregnancy, detailed knowledge about neutralizing and non-neutralizing epitopes is crucial for the development of robust detection systems of protective antibodies. Therefore, additional information about ZIKV immunogenicity and antibody response is required. In this project, we report the production, purification and characterization of six different polyclonal antibodies against ZIKV envelope (E) protein. The produced antibodies bind to isolated ZIKV E protein as well as to the surface of ZIKV particles, interestingly without being potently neutralizing. Surface plasmon resonance measurement showed that these antibodies bind with high affinity to ZIKV E protein. Epitope mapping revealed that the epitopes are distributed among the three ZIKV E domains with seven binding sites. These identified binding sites overlap only partially with the previously described epitopes recognized by neutralizing antibodies, which is in accordance with their lack of potent neutralizing activity. Additionally, these antibodies showed neither cross-reactivity nor potent neutralizing activity against West Nile virus, a related flavivirus. The gained set of data helps to extend our understanding about the distribution of neutralizing and non-/weak-neutralizing epitopes in ZIKV E protein, and provides a rationale for ZIKV vaccine design and development of robust detection assays for neutralizing antibodies

Inhibition of Zika Virus Replication by Silvestrol

The Zika virus (ZIKV) outbreak in 2016 in South America with specific pathogenic outcomes highlighted the need for new antiviral substances with broad-spectrum activities to react quickly to unexpected outbreaks of emerging viral pathogens. Very recently, the natural compound silvestrol isolated from the plant Aglaia foveolata was found to have very potent antiviral effects against the (−)-strand RNA-virus Ebola virus as well as against Corona- and Picornaviruses with a (+)-strand RNA-genome. This antiviral activity is based on the impaired translation of viral RNA by the inhibition of the DEAD-box RNA helicase eukaryotic initiation factor-4A (eIF4A) which is required to unwind structured 5´-untranslated regions (5′-UTRs) of several proto-oncogenes and thereby facilitate their translation. Zika virus is a flavivirus with a positive-stranded RNA-genome harboring a 5′-capped UTR with distinct secondary structure elements. Therefore, we investigated the effects of silvestrol on ZIKV replication in A549 cells and primary human hepatocytes. Two different ZIKV strains were used. In both infected A549 cells and primary human hepatocytes, silvestrol has the potential to exert a significant inhibition of ZIKV replication for both analyzed strains, even though the ancestor strain from Uganda is less sensitive to silvestrol. Our data might contribute to identify host factors involved in the control of ZIKV infection and help to develop antiviral concepts that can be used to treat a variety of viral infections without the risk of resistances because a host protein is targeted

A synthetic derivative of houttuynoid B prevents cell entry of Zika virus

Zika virus (ZIKV) is a re-emerging virus belonging to the family of Flaviviridae, which contains several human pathogens. A great deal of attention came through the association of ZIKV infection with an increasing number of microcephaly cases in newborns during the 2016 outbreak in Brazil. Currently, no anti-viral drug or vaccine is available. Houttuynoids are a group of structurally related flavonoid glycosides that can be isolated from Houttuynia cordata belonging to the family of Sauraceae. Moreover, H. cordata was described to have an antiviral effect on herpes simplex virus type 1 (HSV-1), human immunodeficiency virus type 1 (HIV-1) and influenza A virus (Hayashi et al., 1995). In light of this, this study aimed to investigate a potential antiviral effect of the synthetic houttuynoids TK1023 and TK1024 (i.e. houttuynoid B) on two ZIKV isolates (Uganda and French Polynesia). A significant decrease in the amount of intra- and extracellular viral genomes as well as infectious viral particles was observed after treatment with the tetra-O-acetylated houttuynoid TK1023 independent from the analyzed virus isolate. In contrast, TK1024 (houttuynoid B) had no effect on ZIKV. Treatment with TK1023 significantly decreases the number of infected cells 24 h and 48 h after infection, as compared to the control. Analysis of the mode of action revealed that TK1023 neither affects the viral genome replication nor the production of viral proteins nor morphogenesis or release. Binding and entry assays showed that TK1023 interferes with the entry of the virus in the cell. Thereby, the spread of ZIKV infection is impaired as the infection of the individual cell is inhibited. These data indicate that for both analyzed virus isolates the spread of ZIKV infection can be impaired by the synthetic houttuynoid TK1023 due to an inhibition of the viral entry

Impact of Zika Virus Infection on Human Neural Stem Cell MicroRNA Signatures

Zika virus (ZIKV) is a mosquito-borne virus, which can cause brain abnormalities in newborns, including microcephaly. MicroRNAs (miRNAs) are small non-coding RNAs, which post- transcriptionally regulate gene expression. They are involved in various processes including neurological development and host responses to viral infection, but their potential role in ZIKV pathogenesis remains poorly understood. MiRNAs can be incorporated into extracellular vesicles (EVs) and mediate cell-to-cell communication. While it is well known that in viral infections EVs carrying miRNAs can play a crucial role in disease pathogenesis, ZIKV effects on EV-delivered miRNAs and their contribution to ZIKV pathogenesis have not been elucidated. In the present study, we profiled intracellular and EV-derived miRNAs by next generation sequencing and analyzed the host mRNA transcriptome of neural stem cells during infection with ZIKV Uganda and French Polynesia strains. We identified numerous miRNAs, including miR-4792, which were dysregulated at the intracellular level and had altered levels in EVs during ZIKV infection. Integrated analyses of differentially expressed genes and miRNAs showed that ZIKV infection had an impact on processes associated with neurodevelopment and oxidative stress. Our results provide insights into the roles of intracellular and EV-associated host miRNAs in ZIKV pathogenesis