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1.
Cell Mol Gastroenterol Hepatol ; 15(1): 237-259, 2022 Sep 30.
Article in English | MEDLINE | ID: mdl-36184032

ABSTRACT

BACKGROUND & AIMS: Hepatitis B virus (HBV) was identified as an enveloped DNA virus with a diameter of 42 nm. Multivesicular bodies play a central role in HBV egress and exosome biogenesis. In light of this, it was studied whether intact virions wrapped in exosomes are released by HBV-producing cells. METHODS: Robust methods for efficient separation of exosomes from virions were established. Exosomes were subjected to limited detergent treatment for release of viral particles. Electron microscopy of immunogold labeled ultrathin sections of purified exosomes was performed for characterization of exosomal HBV. Exosome formation/release was affected by inhibitors or Crispr/Cas-mediated gene silencing. Infectivity/uptake of exosomal HBV was investigated in susceptible and non-susceptible cells. RESULTS: Exosomes could be isolated from supernatants of HBV-producing cells, which are characterized by the presence of exosomal and HBV markers. These exosomal fractions could be separated from the fractions containing free virions. Limited detergent treatment of exosomes causes stepwise release of intact HBV virions and naked capsids. Inhibition of exosome morphogenesis impairs the release of exosome-wrapped HBV. Electron microscopy confirmed the presence of intact virions in exosomes. Moreover, the presence of large hepatitis B virus surface antigen on the surface of exosomes derived from HBV expressing cells was observed, which conferred exosome-encapsulated HBV initiating infection in susceptible cells in a , large hepatitis B virus surface antigen/Na+-taurocholate co-transporting polypeptide-dependent manner. The uptake of exosomal HBV with low efficiency was also observed in non-permissive cells. CONCLUSION: These data indicate that a fraction of intact HBV virions can be released as exosomes. This reveals a so far not described release pathway for HBV.

2.
Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz ; 65(2): 228-237, 2022 Feb.
Article in German | MEDLINE | ID: mdl-35015106

ABSTRACT

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.


Subject(s)
Carcinoma, Hepatocellular , Hepatitis B, Chronic , Hepatitis B , Hepatitis C , Liver Neoplasms , Carcinogenesis , Carcinoma, Hepatocellular/epidemiology , Germany , Hepatitis B, Chronic/epidemiology , Humans , Liver Neoplasms/epidemiology
3.
J Virol ; 96(4): e0211721, 2022 02 23.
Article in English | MEDLINE | ID: mdl-34935441

ABSTRACT

Zika virus (ZIKV) is a flavivirus that is mainly transmitted by Aedes mosquitos and normally causes mild symptoms. During the outbreak in the Americas in 2015, it was associated with more severe implications, like microcephaly in newborns and the Guillain-Barré syndrome. The lack of specific vaccines and cures strengthens the need for a deeper understanding of the virus life cycle and virus-host interactions. The restriction factor tetherin (THN) is an interferon-inducible cellular protein with broad antiviral properties. It is known to inhibit the release of various enveloped viruses by tethering them to each other and the cell membrane, thereby preventing their further spread. On the other hand, different viruses have developed various escape strategies against THN. Analysis of the cross-talk between ZIKV and THN revealed that, despite a strong induction of THN mRNA expression in ZIKV-infected cells, this is not reflected by an elevated protein level of THN. Contrariwise, the THN protein level is decreased due to a reduced half-life. The increased degradation of THN in ZIKV infected cells involves the endo-lysosomal system but does not depend on the early steps of autophagy. Enrichment of THN by depletion of the ESCRT-0 protein HRS diminishes ZIKV release and spread, which points out the capacity of THN to restrict ZIKV and explains the enhanced THN degradation in infected cells as an effective viral escape strategy. IMPORTANCE Although tetherin expression is strongly induced by ZIKV infection there is a reduction in the amount of tetherin protein. This is due to enhanced lysosomal degradation. However, if the tetherin level is rescued then the release of ZIKV is impaired. This shows that tetherin is a restriction factor for ZIKV, and the induction of an efficient degradation represents a viral escape strategy. To our knowledge, this is the first study that describes and characterizes tetherin as a restriction factor for the ZIKV life cycle.


Subject(s)
Antigens, CD/metabolism , Zika Virus/physiology , Animals , Antigens, CD/genetics , Antiviral Restriction Factors/genetics , Antiviral Restriction Factors/metabolism , Cell Line , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Half-Life , Humans , Lysosomes/drug effects , Lysosomes/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Proteasome Inhibitors/pharmacology , RNA, Messenger/genetics , Virus Release
4.
Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz ; 65(2): 139-148, 2022 Feb.
Article in German | MEDLINE | ID: mdl-34932130

ABSTRACT

Viral hepatitis is characterized as an acute or chronic inflammation of the liver induced by an infection with certain viruses. At present, around 325 million humans suffer from the chronic form of the disease worldwide. Each year, about 1.6 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.


Subject(s)
Hepatitis E virus , Hepatitis, Viral, Human , Viruses , Germany , Hepatitis Viruses , Humans , Prevalence
5.
Front Physiol ; 12: 750544, 2021.
Article in English | MEDLINE | ID: mdl-34858206

ABSTRACT

Cholesterol has gained tremendous attention as an essential lipid in the life cycle of virtually all viruses. These seem to have developed manifold strategies to modulate the cholesterol metabolism to the side of lipid uptake and de novo synthesis. In turn, affecting the cholesterol homeostasis has emerged as novel broad-spectrum antiviral strategy. On the other hand, the innate immune system is similarly regulated by the lipid and stimulated by its derivatives. This certainly requires attention in the design of antiviral strategies aiming to decrease cellular cholesterol, as evidence accumulates that withdrawal of cholesterol hampers innate immunity. Secondly, there are exceptions to the rule of the abovementioned virus-induced metabolic shift toward cholesterol anabolism. It therefore is of interest to dissect underlying regulatory mechanisms, which we aimed for in this minireview. We further collected evidence for intracellular cholesterol concentrations being less important in viral life cycles as compared to the spatial distribution of the lipid. Various routes of cholesterol trafficking were found to be hijacked in viral infections with respect to organelle-endosome contact sites mediating cholesterol shuttling. Thus, re-distribution of cellular cholesterol in the context of viral infections requires more attention in ongoing research. As a final aim, a pan-antiviral treatment could be found just within the transport and re-adjustment of local cholesterol concentrations. Thus, we aimed to emphasize the importance of the regulatory roles the endosomal system fulfils herein and hope to stimulate research in this field.

6.
Cell Microbiol ; 23(12): e13379, 2021 12.
Article in English | MEDLINE | ID: mdl-34272798

ABSTRACT

Although the hepatitis E virus represents an uprising threat to the global community by representing the commonest cause of an acute viral hepatitis worldwide, its life cycle is grossly understudied. Albeit HEV is a non-enveloped virus, its progeny is released as quasi-enveloped virions. Thus, the responsible accessory protein pORF3 gained rising attention in the past years. It mediates viral release via the exosomal route by targeting the viral capsid to the endosomal system, more precisely to multivesicular bodies. As this is followed by quasi-envelopment, pORF3 may in terms represent a substitute to a conventional envelope protein. This feature proofs to be rather unique with respect to other enteric viruses, although the protein's role in the viral life cycle seems to reach far beyond simply maintaining release of progeny viruses. How pORF3 affects viral morphogenesis, how it mediates efficient viral release and how it supports viral spread is summarised in this microreview. With this, we aim to shed light on functions of pORF3 to gain further insights in still enigmatic aspects of the HEV life cycle. TAKE AWAYS: HEV is released as exosome via multivesicular bodies Viral pORF3 mediates release via endosomal complexes required for transport pORF3 modulates various cellular processes in infected cells Elucidation of pORF3-related processes imply novel clinical strategies.


Subject(s)
Hepatitis E virus , Hepatitis E , Humans , Viral Proteins , Virion , Virus Release
7.
Cancers (Basel) ; 13(4)2021 Feb 16.
Article in English | MEDLINE | ID: mdl-33669329

ABSTRACT

CXCR4 expression and downstream signaling have been identified as key factors in malignant hematopoiesis. Thus, up to 40% of all patients with Waldenström's macroglobulinemia (WM) carry an activating mutation of CXCR4 that leads to a more aggressive clinical course and inferior outcome upon treatment with the Bruton's tyrosine kinase inhibitor ibrutinib. Nevertheless, little is known about physiological mechanisms counteracting CXCR4 signaling in hematopoietic neoplasms. Recently, the endogenous human peptide EPI-X4 was identified as a natural CXCR4 antagonist that effectively blocks CXCL12-mediated receptor internalization and suppresses the migration and invasion of cancer cells towards a CXCL12 gradient. Here, we demonstrate that EPI-X4 efficiently binds to CXCR4 of WM cells and decreases their migration towards CXCL12. The CXCR4 inhibitory activity of EPI-X4 is accompanied by reduced expression of genes involved in MAPK signaling and energy metabolism. Notably, the anti-WM activity of EPI-X4 could be further augmented by the rational design of EPI-X4 derivatives showing higher binding affinity to CXCR4. In summary, these data demonstrate that a naturally occurring anti-CXCR4 peptide is able to interfere with WM cell behaviour, and that optimized derivatives of EPI-X4 may represent a promising approach in suppressing growth promoting CXCR4 signaling in WM.

8.
Cell Mol Gastroenterol Hepatol ; 12(1): 159-180, 2021.
Article in English | MEDLINE | ID: mdl-33601063

ABSTRACT

BACKGROUND AND AIMS: The Hepatitis E virus hijacks the endosomal system for its release. These structures are highly dependent on cholesterol. Hence, this study investigates the impact of HEV on cholesterol-metabolism, the effect of intracellular cholesterol content on HEV-release and the potential of cholesterol-modulators to serve as antivirals. METHODS: Intracellular cholesterol-content of cells was modulated and impacts on HEV were monitored using qPCR, Western blot, microscopy, virus-titration and density-gradient centrifugation. Blood-lipids and HEV-RNA were routinely quantified in chronically infected patients during follow-up visits. RESULTS: In HEV-infected cells, decreased levels of cholesterol are found. In patients, HEV infection decreases serum-lipid concentrations. Importantly, statin treatment herein increases viral titers. Similarly, reduction of intracellular cholesterol via simvastatin treatment increases viral release in vitro. On the contrary, elevating intracellular cholesterol via LDL or 25-hydroxycholesterol strongly reduces viral release due to enhanced lysosomal degradation of HEV. Drug-induced elevation of intracellular cholesterol via fenofibrate or PSC833 impairs HEV release via the same mechanism. CONCLUSIONS: This study analyses the crosstalk between HEV and intracellular cholesterol. The results highlight the importance of an intact cholesterol homeostasis for HEV-release and thereby identify a potential target for antiviral strategies. Especially fenofibrate is considered a promising novel antiviral against HEV. Beyond this, the study may help clinicians evaluating co-treatments of HEV-infected patients with statins, as this may be counter indicated.


Subject(s)
Antiviral Agents/pharmacology , Cholesterol/metabolism , Cyclosporins/pharmacology , Fenofibrate/pharmacology , Hepatitis E virus/drug effects , Antiviral Agents/chemistry , Cell Survival/drug effects , Cyclosporins/chemistry , Fenofibrate/chemistry , Humans , Microbial Sensitivity Tests , Tumor Cells, Cultured , Virus Replication/drug effects
9.
J Virol ; 2021 Jan 20.
Article in English | MEDLINE | ID: mdl-33472929

ABSTRACT

This study aims to gain deeper insight into HEV-induced innate immunity by characterizing the crosstalk between the virus and the host factor guanylate-binding protein 1 (GBP1). We observe that the amount of GBP1 is elevated upon infection, although number of transcripts is decreased, which is explained by a prolonged protein half-life. Modulation of GBP1 levels via overexpression significantly inhibits the viral life cycle. Use of various GBP-1 mutants revealed that the antiviral effect of GBP-1 on HEV is independent from the GTPase-activity, but depends on the capacity of GBP-1 to form GBP1 homodimers. This connects GBP-1 to the autophagosomal pathway. Indeed, dimerization competent GBP1 targets the viral capsid protein to the lysosomal compartment leading to inactivation of the viral particle. Most importantly, silencing of GBP1 abolishes the antiviral effect of IFNγ on HEV. In IFNγ treated cells the virus is targeted to lysosomal structures and destroyed therein. This process depends in part on GBP1. These observations about the relevance of GBP1 for type II interferon-mediated innate immunity against HEV could be a base for tailoring novel antivirals and improvement of disease management.IMPORTANCE Although HEV represents a worldwide public health problem with 20 million infections and 44.000 death cases per year, there are still no specific antivirals available and many aspects of the viral life cycle are not well understood. Here we identify the guanylate binding protein 1 (GBP1) as a restriction factor affecting life cycle of HEV. Surprisingly, the antiviral effect of GBP1 does not depend on its GTPase function, but on its capacity to homodimerize. We revealed that GBP1 exerts its antiviral activity by targeting HEV to the lysosomal compartment where the virus is inactivated. Most importantly, we observed that the antiviral effect of interferon-γ on HEV strongly depends on GBP1. Our observation that GBP1 impairs HEV and is crucial for the antiviral effect of interferons on HEV extends understanding of host defense-mechanisms. As the interferon-system represents a universal defense-mechanism, our study could help to design novel antivirals targeting.

10.
JCI Insight ; 5(22)2020 11 19.
Article in English | MEDLINE | ID: mdl-33055418

ABSTRACT

Many mutation analyses of the HBV genome have been performed in the search for new prognostic markers. However, the Kozak sequence preceding precore was covered only infrequently in these analyses. In this study, the HBV core promoter/precore region was sequenced in serum samples from European inactive HBV carriers. Quadruple mutation GCAC1809-1812TTCT was found with a high prevalence of 42% in the Kozak sequence preceding precore among all HBV genotypes. GCAC1809-1812TTCT was strongly associated with coexistence of basal core promoter (BCP) double mutation A1762T/G1764A and lower HBV DNA levels. In vitro GCAC1809-1812TTCT lead to drastically diminished synthesis of pregenomic RNA (pgRNA), precore mRNA, core, HBsAg, and HBeAg. Calculation of the pgRNA secondary structure suggests a destabilization of the pgRNA structure by A1762T/G1764A that was compensated by GCAC1809-1812TTCT. In 125 patients with HBV-related cirrhosis, GCAC1809-1812TTCT was not detected. While a strong association of GCAC1809-1812TTCT with inactive carrier status was observed, BCP double mutation was strongly correlated with cirrhosis, but this was only observed in absence of GCAC1809-1812TTCT. In conclusion, our data reveal that GCAC1809-1812TTCT is highly prevalent in inactive carriers and acts as a compensatory mutation for BCP double mutation. GCAC1809-1812TTCT seems to be a biomarker of good prognosis in HBV infection.


Subject(s)
Biomarkers/analysis , DNA, Viral/genetics , Hepatitis B Surface Antigens/genetics , Hepatitis B virus/genetics , Hepatitis B/complications , Liver Cirrhosis/epidemiology , Mutation , Adult , DNA, Viral/analysis , Europe/epidemiology , Female , Genotype , Hepatitis B/genetics , Hepatitis B/virology , Hepatitis B virus/isolation & purification , Heterozygote , Humans , Liver Cirrhosis/diagnosis , Liver Cirrhosis/virology , Male , Middle Aged , Promoter Regions, Genetic
11.
Antiviral Res ; 175: 104706, 2020 03.
Article in English | MEDLINE | ID: mdl-31931103

ABSTRACT

Rocaglates, a class of natural compounds isolated from plants of the genus Aglaia, are potent inhibitors of translation initiation. They are proposed to form stacking interactions with polypurine sequences in the 5'-untranslated region (UTR) of selected mRNAs, thereby clamping the RNA substrate onto eIF4A and causing inhibition of the translation initiation complex. Since virus replication relies on the host translation machinery, it is not surprising that the rocaglate Silvestrol has broad-spectrum antiviral activity. Unfortunately, synthesis of Silvestrol is sophisticated and time-consuming, thus hampering the prospects for further antiviral drug development. Here, we present the less complex structured synthetic rocaglate CR-31-B (-) as a novel compound with potent broad-spectrum antiviral activity in primary cells and in an ex vivo bronchial epithelial cell system. CR-31-B (-) inhibited the replication of corona-, Zika-, Lassa-, Crimean Congo hemorrhagic fever viruses and, to a lesser extent, hepatitis E virus (HEV) at non-cytotoxic low nanomolar concentrations. Since HEV has a polypurine-free 5'-UTR that folds into a stable hairpin structure, we hypothesized that RNA clamping by Silvestrol and its derivatives may also occur in a polypurine-independent but structure-dependent manner. Interestingly, the HEV 5'-UTR conferred sensitivity towards Silvestrol but not to CR-31-B (-). However, if an exposed polypurine stretch was introduced into the HEV 5'-UTR, CR-31-B (-) became an active inhibitor comparable to Silvestrol. Moreover, thermodynamic destabilization of the HEV 5'-UTR led to reduced translational inhibition by Silvestrol, suggesting differences between rocaglates in their mode of action, most probably by engaging Silvestrol's additional dioxane moiety.


Subject(s)
Antiviral Agents/pharmacology , Benzofurans/pharmacology , Triterpenes/pharmacology , Virus Replication/drug effects , Viruses/drug effects , A549 Cells , Animals , Antiviral Agents/chemical synthesis , Benzofurans/chemical synthesis , Bronchi/cytology , Cell Culture Techniques , Cells, Cultured , Epithelial Cells/virology , Eukaryotic Initiation Factor-4A/antagonists & inhibitors , Hepatocytes/virology , Humans , Mice , Viruses/classification
12.
Antiviral Res ; 172: 104644, 2019 12.
Article in English | MEDLINE | ID: mdl-31697958

ABSTRACT

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.


Subject(s)
Flavonoids/pharmacology , Glycosides/pharmacology , Synthetic Drugs/pharmacology , Virus Internalization/drug effects , Zika Virus/drug effects , Animals , Antiviral Agents/pharmacology , Cell Line , Chlorocebus aethiops , Humans , Polynesia , Uganda , Vero Cells , Virus Replication/drug effects , Zika Virus/physiology , Zika Virus Infection/drug therapy
13.
Viruses ; 10(6)2018 06 02.
Article in English | MEDLINE | ID: mdl-29865243

ABSTRACT

Every year, there are about 20 Mio hepatitis E virus (HEV) infections and 60,000 deaths that are associated with HEV worldwide. At the present, there exists no specific therapy for HEV. The natural compound silvestrol has a potent antiviral effect against the (-)-strand RNA-virus Ebola virus, and also against the (+)-strand RNA viruses Corona-, Picorna-, and Zika virus. The inhibitory effect on virus spread is due to an inhibition of the DEAD-box RNA helicase eIF4A, which is required to unwind structured 5'-untranslated regions (UTRs). This leads to an impaired translation of viral RNA. The HEV (+)-strand RNA genome contains a 5'-capped, short 5'-UTR. This study aims to analyze the impact of silvestrol on the HEV life cycle. Persistently infected A549 cells were instrumental. This study identifies silvestrol as a potent inhibitor of the release of HEV infectious viral particles. This goes along with a strongly reduced HEV capsid protein translation, retention of viral RNA inside the cytoplasm, and without major cytotoxic effects. Interestingly, in parallel silvestrol affects the activity of the antiviral major vault protein (MVP) by translocation from the cytoplasm to the perinuclear membrane. These data further characterize the complex antiviral activity of silvestrol and show silvestrol's broad spectrum of function, since HEV is a virus without complex secondary structures in its genome, but it is still affected.


Subject(s)
Antiviral Agents/pharmacology , Hepatitis E virus/drug effects , Triterpenes/pharmacology , Virus Replication/drug effects , A549 Cells , Capsid Proteins/metabolism , Hepatitis E/drug therapy , Humans , RNA, Viral/genetics , Vault Ribonucleoprotein Particles/metabolism , Virus Release/drug effects
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