Ralf Bartenschlager

MAP-Kinase Regulated Cytosolic Phospholipase A2 Activity Is Essential for Production of Infectious Hepatitis C Virus Particles

Hepatitis C virus (HCV) has infected around 160 million individuals. Current therapies have limited efficacy and are fraught with side effects. To identify cellular HCV dependency factors, possible therapeutic targets, we manipulated signaling cascades with pathway-specific inhibitors. Using this approach we identified the MAPK/ERK regulated, cytosolic, calcium-dependent, group IVA phospholipase A2 (PLA2G4A) as a novel HCV dependency factor. Inhibition of PLA2G4A activity reduced core protein abundance at lipid droplets, core envelopment and secretion of particles. Moreover, released particles displayed aberrant protein composition and were 100-fold less infectious. Exogenous addition of arachidonic acid, the cleavage product of PLA2G4A-catalyzed lipolysis, but not other related poly-unsaturated fatty acids restored infectivity. Strikingly, production of infectious Dengue virus, a relative of HCV, was also dependent on PLA2G4A. These results highlight previously unrecognized parallels in the assembly pathways of these human pathogens, and define PLA2G4A-dependent lipolysis as crucial prerequisite for production of highly infectious viral progeny.

A Plant-Derived Flavonoid Inhibits Entry of All HCV Genotypes Into Human Hepatocytes

BACKGROUND & AIMSnInterferon-based therapies for hepatitis C virus (HCV) infection are limited by side effects and incomplete response rates, particularly among transplant recipients. We screened a library of plant-derived small molecules to identify HCV inhibitors with novel mechanisms.nnnMETHODSnWe isolated phenolic compounds from Marrubium peregrinum L (Lamiaceae). Replication of HCV RNA, virus production, and cell entry were monitored using replicons and infectious HCV. Inhibition of HCV was measured in hepatoma cells and primary human hepatocytes using luciferase reporter gene assays, core enzyme-linked immunosorbent assays, or infectivity titration. We tested the bioavailability of the compound in mice.nnnRESULTSnWe identified a flavonoid, ladanein (BJ486K), with unreported antiviral activity and established its oral bioavailability in mice. Natural and synthetic BJ486K inhibited a post-attachment entry step, but not RNA replication or assembly; its inhibitory concentration 50% was 2.5 μm. BJ486K was effective against all major HCV genotypes, including a variant that is resistant to an entry inhibitor; it prevented infection of primary human hepatocytes. Combined administration of BJ486K and cyclosporine A had a synergistic effect in inhibition of HCV infection.nnnCONCLUSIONSnBJ486K has oral bioavailability and interferes with entry of HCV into cultured human hepatocytes. It synergizes with cyclosporine A to inhibit HCV infection. Its inhibitory effects are independent of HCV genotype, including a variant that is resistant to an entry inhibitor against scavenger receptor class B type I. Flavonoid derivatives therefore might be developed as components of combination therapies because they are potent, broadly active inhibitors of HCV entry that could prevent graft reinfection after liver transplantation.

Apolipoprotein E likely contributes to a maturation step of infectious hepatitis C virus particles and interacts with viral envelope glycoproteins

ABSTRACT The assembly of infectious hepatitis C virus (HCV) particles is tightly linked to components of the very-low-density lipoprotein (VLDL) pathway. We and others have shown that apolipoprotein E (ApoE) plays a major role in production of infectious HCV particles. However, the mechanism by which ApoE contributes to virion assembly/release and how it gets associated with the HCV particle is poorly understood. We found that knockdown of ApoE reduces titers of infectious intra- and extracellular HCV but not of the related dengue virus. ApoE depletion also reduced amounts of extracellular HCV core protein without affecting intracellular core amounts. Moreover, we found that ApoE depletion affected neither formation of nucleocapsids nor their envelopment, suggesting that ApoE acts at a late step of assembly, such as particle maturation and infectivity. Importantly, we demonstrate that ApoE interacts with the HCV envelope glycoproteins, most notably E2. This interaction did not require any other viral proteins and depended on the transmembrane domain of E2 that also was required for recruitment of HCV envelope glycoproteins to detergent-resistant membrane fractions. These results suggest that ApoE plays an important role in HCV particle maturation, presumably by direct interaction with viral envelope glycoproteins. IMPORTANCE The HCV replication cycle is tightly linked to host cell lipid pathways and components. This is best illustrated by the dependency of HCV assembly on lipid droplets and the VLDL component ApoE. Although the role of ApoE for production of infectious HCV particles is well established, it is still poorly understood how ApoE contributes to virion formation and how it gets associated with HCV particles. Here, we provide experimental evidence that ApoE likely is required for an intracellular maturation step of HCV particles. Moreover, we demonstrate that ApoE associates with the viral envelope glycoproteins. This interaction appears to be dispensable for envelopment of virus particles but likely contributes to the quality control of secreted infectious virions. These results shed new light on the exploitation of host cell lipid pathways by HCV and the link of viral particle assembly to the VLDL component ApoE.

Heterologous Src Homology 4 Domains Support Membrane Anchoring and Biological Activity of HIV-1 Nef

Background: HIV-1 Nef is a membrane-associated protein that acts as viral pathogenicity factor. Results: Nef function requires regulated membrane interactions along transport pathways to and from the plasma membrane. Conclusion: Nef function is determined by dynamic anterograde and endocytic transport cycles. Significance: Dynamic transport cycles provide the basis for the multifunctionality of Nef. The HIV-1 pathogenicity factor Nef enhances viral replication by modulation of multiple host cell transport and signaling pathways. Nef associates with membranes via an N-terminal Src homology 4 (SH4) domain, and membrane association is believed to be essential for its biological functions. At which subcellular site(s) Nef exerts its different functions and how kinetics of membrane interactions contribute to its biological activity are unknown. To address how specific characteristics of Nef membrane association affect its biological properties, the SH4 domain of Nef was replaced by heterologous membrane targeting domains. The use of a panel of heterologous SH4 domains resulted in chimeric Nef proteins with distinct steady state subcellular localization, membrane association efficiency, and anterograde transport routes. Irrespective of these modifications, cardinal Nef functions affecting host cell vesicular transport and actin dynamics were fully preserved. In contrast, stable targeting of Nef to the surface of mitochondria, peroxisomes, or the Golgi apparatus, and thus prevention of plasma membrane delivery, caused potent and broad loss of Nef activity. These results support the concept that Nef adopts its active conformation in the membrane-associated state but exclude that membrane-associated Nef simply acts by recruiting soluble factors independently of its local microenvironment. Rather than its steady state subcellular localization or membrane affinity, the ability to undergo dynamic anterograde and internalization cycles appear to determine Nef function. These results reveal that functional membrane interactions of Nef underlie critical spatiotemporal regulation and suggest that delivery to distinct subcellular sites via such transport cycles provides the basis for the multifunctionality of Nef.

Hepatitis C virus core+1/ARF protein decreases hepcidin transcription through an AP1 binding site.

Chronic viral hepatitis C is characterized by iron accumulation in the liver, and hepcidin regulates iron absorption. Hepatitis C virus (HCV) core+1/ARFP is a novel protein produced by a second functional ORF within the core gene. Here, using reporter assays and HCV bicistronic replicons, we show that, similarly to core, core+1/ARFP decreases hepcidin expression in hepatoma cells. The activator protein 1 (AP1) binding site of the human hepcidin promoter, shown here to be relevant to basal promoter activity and to the repression by core, is essential for the downregulation by core+1/ARFP while the previously described C/EBP (CCAAT/enhancer binding protein) and STAT (signal transducer and activator of transcription) sites are not. Consistently, expression of the AP1 components c-jun and c-fos obliterated the repressive effect of core and core+1/ARFP. In conclusion, we provide evidence that core+1/ARFP downregulates AP1-mediated transcription, providing new insights into the biological role of core+1/ARFP, as well as the transcriptional modulation of hepcidin, the main regulator of iron metabolism.

Expression of the Novel Hepatitis C Virus Core+1/ARF Protein in the Context of JFH1-Based Replicons

ABSTRACT Hepatitis C virus contains a second open reading frame within the core gene, designated core+1/ARF. Here we demonstrate for the first time expression of core+1/ARF protein in the context of a bicistronic JFH1-based replicon and report the production of two isoforms, core+1/L (long) and core+1/S (short), with different kinetics.

Maßgeschneiderte Therapie der Virushepatitis der Gegenwart und Zukunft Personalized treatment of viral hepatitis of the present and the future

Precision medicine is also possible for infectious diseases as shown for the treatment of chronic viral hepatitis, especially if different options are available. In hepatitisxa0B virus (HBV) infection, treatment indication as well as the choice of treatment and the decisions to stop treatment are based on viral markers and alanine aminotransferase (ALT) level. Future therapies for HBV infection aiming for functional cure or even virus elimination may be even more personalized and have to take into account the immune status of axa0given patient. Such treatment modalities might also increase the chance for successful treatment of chronic hepatitis delta where treatment options are still very limited. Some new therapeutic concepts targeting host receptors or host enzymes are promising, but may require individualized approaches. Chronic hepatitisxa0C is axa0good example for precision medicine based on viral and host factors. However, the main reason for individualized direct-acting antiviral (DAA) treatment is to save costs. As DAAs are effective in more than 95% of patients, elimination of HCV seems to be possible at the level of axa0given country or even on axa0global scale. However, owing to high reinfection rates in high-risk groups and limited availability of antiviral therapy in many high endemic countries, it must still be decided whether an HCV vaccine or pre-exposure prophylaxis is required to achieve this goal. Hepatitisxa0E is an emerging topic as this is the most frequent acute hepatitis virus infection. It can result in axa0chronic infection in immunosuppressed individuals. Treatment options are still limited and individualized management is based on tailoring immunosuppressive therapy and therapy with ribavirin. Thus, personalized therapy of hepatitisxa0E virus infection is still limited.ZusammenfassungAuch in der Infektionsmedizin sind maßgeschneiderte Therapien möglich, wie das Beispiel der chronischen Virushepatitis zeigt, insbesondere wenn es bereits verschiedene Therapieoptionen gibt. Bei der Hepatitis-B-Virus(HBV)-Infektion wird zunächst die Therapieindikation anhand von Virusmarkern und Leberwerten des Patienten gestellt. Anschließend wird anhand der Virus- und Patientendaten die Auswahl der Therapieform sowie die Entscheidung über das Therapieende getroffen. Zukünftig wird es vielfältigere Therapieoptionen geben, die vor allem auf die funktionale Kontrolle oder gar Elimination der chronischen HBV-Infektion abzielen und die auch die Therapiechancen bei der Hepatitisxa0Delta erhöhen. Letztere kann bislang nur mit pegyliertem Interferon behandelt werden. Die chronische Hepatitisxa0C ist ein Paradebeispiel für die maßgeschneiderte Therapie anhand von Virus- und Wirtsfaktoren. Die Hauptmotivation ist allerdings, Kosten zu sparen. Aufgrund der hohen Erfolgsraten der direkt antiviral wirkenden Therapie von über 95u2009% ist eine länderweite oder gar globale Elimination der HCV-Infektion prinzipiell möglich. Dennoch muss aufgrund der hohen Reinfektionsraten in Hochrisikogruppen sowie der mangelnden Verfügbarkeit der Medikamente weiterhin die Notwendigkeit einer Impfung oder Präexpositionsprophylaxe ernsthaft diskutiert werden. Bei der Hepatitisxa0E gibt es bislang nicht viele Optionen. Hier ist die Therapieindividualisierung noch stark limitiert.AbstractPrecision medicine is also possible for infectious diseases as shown for the treatment of chronic viral hepatitis, especially if different options are available. In hepatitisxa0B virus (HBV) infection, treatment indication as well as the choice of treatment and the decisions to stop treatment are based on viral markers and alanine aminotransferase (ALT) level. Future therapies for HBV infection aiming for functional cure or even virus elimination may be even more personalized and have to take into account the immune status of axa0given patient. Such treatment modalities might also increase the chance for successful treatment of chronic hepatitis delta where treatment options are still very limited. Some new therapeutic concepts targeting host receptors or host enzymes are promising, but may require individualized approaches. Chronic hepatitisxa0C is axa0good example for precision medicine based on viral and host factors. However, the main reason for individualized direct-acting antiviral (DAA) treatment is to save costs. As DAAs are effective in more than 95% of patients, elimination of HCV seems to be possible at the level of axa0given country or even on axa0global scale. However, owing to high reinfection rates in high-risk groups and limited availability of antiviral therapy in many high endemic countries, it must still be decided whether an HCV vaccine or pre-exposure prophylaxis is required to achieve this goal. Hepatitisxa0E is an emerging topic as this is the most frequent acute hepatitis virus infection. It can result in axa0chronic infection in immunosuppressed individuals. Treatment options are still limited and individualized management is based on tailoring immunosuppressive therapy and therapy with ribavirin. Thus, personalized therapy of hepatitisxa0E virus infection is still limited.

Maßgeschneiderte Therapie der Virushepatitis der Gegenwart und Zukunft

Precision medicine is also possible for infectious diseases as shown for the treatment of chronic viral hepatitis, especially if different options are available. In hepatitisxa0B virus (HBV) infection, treatment indication as well as the choice of treatment and the decisions to stop treatment are based on viral markers and alanine aminotransferase (ALT) level. Future therapies for HBV infection aiming for functional cure or even virus elimination may be even more personalized and have to take into account the immune status of axa0given patient. Such treatment modalities might also increase the chance for successful treatment of chronic hepatitis delta where treatment options are still very limited. Some new therapeutic concepts targeting host receptors or host enzymes are promising, but may require individualized approaches. Chronic hepatitisxa0C is axa0good example for precision medicine based on viral and host factors. However, the main reason for individualized direct-acting antiviral (DAA) treatment is to save costs. As DAAs are effective in more than 95% of patients, elimination of HCV seems to be possible at the level of axa0given country or even on axa0global scale. However, owing to high reinfection rates in high-risk groups and limited availability of antiviral therapy in many high endemic countries, it must still be decided whether an HCV vaccine or pre-exposure prophylaxis is required to achieve this goal. Hepatitisxa0E is an emerging topic as this is the most frequent acute hepatitis virus infection. It can result in axa0chronic infection in immunosuppressed individuals. Treatment options are still limited and individualized management is based on tailoring immunosuppressive therapy and therapy with ribavirin. Thus, personalized therapy of hepatitisxa0E virus infection is still limited.ZusammenfassungAuch in der Infektionsmedizin sind maßgeschneiderte Therapien möglich, wie das Beispiel der chronischen Virushepatitis zeigt, insbesondere wenn es bereits verschiedene Therapieoptionen gibt. Bei der Hepatitis-B-Virus(HBV)-Infektion wird zunächst die Therapieindikation anhand von Virusmarkern und Leberwerten des Patienten gestellt. Anschließend wird anhand der Virus- und Patientendaten die Auswahl der Therapieform sowie die Entscheidung über das Therapieende getroffen. Zukünftig wird es vielfältigere Therapieoptionen geben, die vor allem auf die funktionale Kontrolle oder gar Elimination der chronischen HBV-Infektion abzielen und die auch die Therapiechancen bei der Hepatitisxa0Delta erhöhen. Letztere kann bislang nur mit pegyliertem Interferon behandelt werden. Die chronische Hepatitisxa0C ist ein Paradebeispiel für die maßgeschneiderte Therapie anhand von Virus- und Wirtsfaktoren. Die Hauptmotivation ist allerdings, Kosten zu sparen. Aufgrund der hohen Erfolgsraten der direkt antiviral wirkenden Therapie von über 95u2009% ist eine länderweite oder gar globale Elimination der HCV-Infektion prinzipiell möglich. Dennoch muss aufgrund der hohen Reinfektionsraten in Hochrisikogruppen sowie der mangelnden Verfügbarkeit der Medikamente weiterhin die Notwendigkeit einer Impfung oder Präexpositionsprophylaxe ernsthaft diskutiert werden. Bei der Hepatitisxa0E gibt es bislang nicht viele Optionen. Hier ist die Therapieindividualisierung noch stark limitiert.AbstractPrecision medicine is also possible for infectious diseases as shown for the treatment of chronic viral hepatitis, especially if different options are available. In hepatitisxa0B virus (HBV) infection, treatment indication as well as the choice of treatment and the decisions to stop treatment are based on viral markers and alanine aminotransferase (ALT) level. Future therapies for HBV infection aiming for functional cure or even virus elimination may be even more personalized and have to take into account the immune status of axa0given patient. Such treatment modalities might also increase the chance for successful treatment of chronic hepatitis delta where treatment options are still very limited. Some new therapeutic concepts targeting host receptors or host enzymes are promising, but may require individualized approaches. Chronic hepatitisxa0C is axa0good example for precision medicine based on viral and host factors. However, the main reason for individualized direct-acting antiviral (DAA) treatment is to save costs. As DAAs are effective in more than 95% of patients, elimination of HCV seems to be possible at the level of axa0given country or even on axa0global scale. However, owing to high reinfection rates in high-risk groups and limited availability of antiviral therapy in many high endemic countries, it must still be decided whether an HCV vaccine or pre-exposure prophylaxis is required to achieve this goal. Hepatitisxa0E is an emerging topic as this is the most frequent acute hepatitis virus infection. It can result in axa0chronic infection in immunosuppressed individuals. Treatment options are still limited and individualized management is based on tailoring immunosuppressive therapy and therapy with ribavirin. Thus, personalized therapy of hepatitisxa0E virus infection is still limited.

Hepatitis C Virus—From Discovery to Cure: The 2016 Lasker-DeBakey Clinical Medical Research Award

The liver is the largest organ in the human body and is central for metabolism and many other functions. Several viruses specialize in infecting the liver and are called hepatitis viruses. Five such viruses are known, including hepatitis C virus (HCV), which was originally recognized as an agent of posttransfusion non-A, non-B hepatitis. Given that about 6% of patients receiving blood transfusions developed non-A, non-B hepatitis, tremendous efforts were mounted to isolate and molecularly clone this filterable agent, likely a virus. In a landmark paper in 1989, Houghton and his team isolated the first molecular clone of HCV and provided a glimpse of the HCV genome: a positive-strand RNA virus with a genome length of around 9500 nucleotides encoding a long polyprotein that was likely cleaved cotranslationally and posttranslationally into 8 to 10 products.1 Work in many laboratories, subsequently identified 10 HCV proteins generated by the action of host cell and viral proteases, including 2 viral enzymes, the nonstructural proteins (NS) 3-4A serine protease and the NS5B RNA-dependent RNA polymerase, highly attractive HCV drug targets. Subsequent research by Drs Bartenschlager, Rice, and Sofia led to the development of new and effective treatments for HCV.2-7 The bottleneck in drug development was the lack of cell culture systems for HCV, but the availability of molecular HCV clones raised hope because the RNA genome of positive-strand RNA viruses is infectious. Introducing genome RNA or a genome RNA equivalent transcribed from a plasmid into permissive cells can initiate an entire viral life cycle. The genome RNA is recognized by cellular ribosomes, translated to produce the viral proteins, and, in concert with additional factors from the host cell, amplified and used to make infectious virus. However, this approach, which had succeeded for many other viruses, failed for HCV. One reason was a missing piece at the 3′ end of viral genome finally discovered by the laboratories of Kunitada Shimotohno and Charles M. Rice. With the HCV genome now likely complete, making a functional complementary DNA (cDNA) clone should be easy, but how would this be tested without a cell culture system? In 1997, clones reflecting a “consensus” sequence were used to filter out possible lethal mutations present in the patient-derived HCV population or acquired during cDNA cloning in the laboratory. Injection of this synthetic, naked genome RNA into the liver of chimpanzees gave rise to a productive HCV infection and provided the first genetic system for proving that possible HCV-specific drug targets were essential for the virus. With virtually unlimited quantities of HCV genome RNA, validated as infectious in vivo, it might be expected that finding a suitable cell culture system would quickly follow, but that was not the case. The solution came from work in the laboratory of Ralf Bartenschlager that used another HCV consensus genome cloned from the liver of a chronically infected patient. With the aim to isolate rare cells supporting robust HCV replication, “selectable minigenomes,” called replicons, were engineered. These replicons encode the minimal set of viral proteins assumed to be required for autonomous replication and, in addition, a gene conferring resistance against the cytotoxic drug G418. By using drug selection, cells supporting efficient and long-term HCV replication could be isolated. This first robust HCV cell culture model recapitulated all the intracellular steps of the HCV replication cycle and because replication of these HCV RNAs relied on the viral enzymes, most notably the NS3 protease and the NS5B polymerase, the replicon system was suitable for drug development. Subsequent studies conducted in the Rice and Bartenschlager laboratories unveiled the reasons for such high replication efficiency. First, the most HCVpermissive individual cells in a given cell pool had been selected; second, the replicons present in selected cell clones harbored mutations that enhanced HCV RNA replication by orders of magnitude. Insertion of these mutations into the parental replicon allowed direct measurement of HCV replication. Thus, the first widely useful genetic systems for studying HCV biology were created. Subsequent work by Bartenschlager, Rice, and others refined this approach. With the robust HCV replicon whole-cell system available for screening of small molecules, the search for inhibitors of HCV became a major focus of pharmaceutical and biotech companies. This was propelled by the desire to identify direct-acting antivirals with the ultimate goal of replacing the then current standard of care for treating HCV infection, the combination of injectable interferon plus ribavirin, which was limited by severe adverse effects, modest cure rates, and limited genotype coverage. In 2005 efforts in HCV drug discovery were focused on several key viral targets, including the NS5B RNA-dependent RNA polymerase (NS5B RdRp). The VIEWPOINT

Deep Particle Tracker: Automatic Tracking of Particles in Fluorescence Microscopy Images Using Deep Learning

Tracking of particles in fluorescence microscopy image sequences is essential for studying the dynamics of subcellular structures and virus structures. We introduce a novel particle tracking approach using an LSTM-based neural network. Our approach determines assignment probabilities jointly across multiple detections by exploiting both short and long-term temporal dependencies of individual object dynamics. Manually labeled data is not required. We evaluated the performance of our approach using image data of the ISBI Particle Tracking Challenge as well as real fluorescence microscopy image sequences of virus structures. It turned out that the proposed approach outperforms previous methods.