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1.  Identification of HNRNPK as Regulator of Hepatitis C Virus Particle Production 
PLoS Pathogens  2015;11(1):e1004573.
Hepatitis C virus (HCV) is a major cause of chronic liver disease affecting around 130 million people worldwide. While great progress has been made to define the principle steps of the viral life cycle, detailed knowledge how HCV interacts with its host cells is still limited. To overcome this limitation we conducted a comprehensive whole-virus RNA interference-based screen and identified 40 host dependency and 16 host restriction factors involved in HCV entry/replication or assembly/release. Of these factors, heterogeneous nuclear ribonucleoprotein K (HNRNPK) was found to suppress HCV particle production without affecting viral RNA replication. This suppression of virus production was specific to HCV, independent from assembly competence and genotype, and not found with the related Dengue virus. By using a knock-down rescue approach we identified the domains within HNRNPK required for suppression of HCV particle production. Importantly, HNRNPK was found to interact specifically with HCV RNA and this interaction was impaired by mutations that also reduced the ability to suppress HCV particle production. Finally, we found that in HCV-infected cells, subcellular distribution of HNRNPK was altered; the protein was recruited to sites in close proximity of lipid droplets and colocalized with core protein as well as HCV plus-strand RNA, which was not the case with HNRNPK variants unable to suppress HCV virion formation. These results suggest that HNRNPK might determine efficiency of HCV particle production by limiting the availability of viral RNA for incorporation into virions. This study adds a new function to HNRNPK that acts as central hub in the replication cycle of multiple other viruses.
Author Summary
As obligate intracellular parasites with limited gene coding capacity viruses exploit host cell machineries for the sake of efficient replication and spread. Thus, identification of these cellular machineries and factors is necessary to understand how a given virus achieves efficient replication and eventually causes host cell damage. Hepatitis C virus (HCV) is an RNA virus replicating in the cytoplasm of hepatocytes. While viral proteins have been studied in great detail, our knowledge about how host cell factors are used by HCV for efficient replication and spread is still scarce. In the present study we conducted a comprehensive RNA-interference-based screen and identified 40 genes that promote the HCV lifecycle and 16 genes that suppress it. Follow-up studies revealed that one of these genes, the heterogeneous nuclear ribonucleoprotein K (HNRNPK), selectively suppresses production of infectious HCV particles. We mapped the domains of HNRNPK required for this suppression and demonstrate that this protein selectively binds to the HCV RNA genome. Based on the correlation between suppression of virus production, HCV RNA binding and recruitment to lipid droplets, we propose that HNRNPK might limit the amount of viral RNA genomes available for incorporation into virus particles. This study provides novel insights into the complexity of reactions that are involved in the formation of HCV virions.
doi:10.1371/journal.ppat.1004573
PMCID: PMC4287573  PMID: 25569684
2.  Hepatitis C: A mouse at the end of the tunnel 
Cell Research  2013;23(12):1343-1344.
Since its discovery in 1989, researchers strive after a small animal model for Hepatitis C virus infection, so far with very limited success. A study recently published in Nature now for the first time reports the recapitulation of the complete life cycle of this virus in inbred mice with a functional adaptive immune system.
doi:10.1038/cr.2013.132
PMCID: PMC3847570  PMID: 24060850
3.  Dengue Virus- and Hepatitis C Virus-Induced Replication and Assembly Compartments: the Enemy Inside—Caught in the Web 
Journal of Virology  2014;88(11):5907-5911.
Dengue virus (DENV) and hepatitis C virus (HCV), members of the family Flaviviridae, are global human health concerns. As positive-strand RNA viruses, they each replicate in the cytoplasm of infected cells and induce distinct membranous replication compartments where most, if not all, steps of the viral life cycle occur. This Gem briefly reviews the most recent insights into the architecture and functional properties of membranous replication and assembly sites induced by DENV and HCV.
doi:10.1128/JVI.03404-13
PMCID: PMC4093888  PMID: 24623440
4.  Aminoterminal Amphipathic α-Helix AH1 of Hepatitis C Virus Nonstructural Protein 4B Possesses a Dual Role in RNA Replication and Virus Production 
PLoS Pathogens  2014;10(11):e1004501.
Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. In concert with other nonstructural proteins, it induces a specific membrane rearrangement, designated as membranous web, which serves as a scaffold for the HCV replicase. The N-terminal part of NS4B comprises a predicted and a structurally resolved amphipathic α-helix, designated as AH1 and AH2, respectively. Here, we report a detailed structure-function analysis of NS4B AH1. Circular dichroism and nuclear magnetic resonance structural analyses revealed that AH1 folds into an amphipathic α-helix extending from NS4B amino acid 4 to 32, with positively charged residues flanking the helix. These residues are conserved among hepaciviruses. Mutagenesis and selection of pseudorevertants revealed an important role of these residues in RNA replication by affecting the biogenesis of double-membrane vesicles making up the membranous web. Moreover, alanine substitution of conserved acidic residues on the hydrophilic side of the helix reduced infectivity without significantly affecting RNA replication, indicating that AH1 is also involved in virus production. Selective membrane permeabilization and immunofluorescence microscopy analyses of a functional replicon harboring an epitope tag between NS4B AH1 and AH2 revealed a dual membrane topology of the N-terminal part of NS4B during HCV RNA replication. Luminal translocation was unaffected by the mutations introduced into AH1, but was abrogated by mutations introduced into AH2. In conclusion, our study reports the three-dimensional structure of AH1 from HCV NS4B, and highlights the importance of positively charged amino acid residues flanking this amphipathic α-helix in membranous web formation and RNA replication. In addition, we demonstrate that AH1 possesses a dual role in RNA replication and virus production, potentially governed by different topologies of the N-terminal part of NS4B.
Author Summary
With an estimated 180 million chronically infected individuals, hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. HCV is a positive-strand RNA virus that builds its replication complex on rearranged intracellular membranes, designated as membranous web. HCV nonstructural protein 4B (NS4B) is a key organizer of HCV membranous web and replication complex formation. Here, we provide a detailed structure-function analysis of an N-terminal amphipathic α-helix of NS4B, named AH1, and demonstrate that it plays key roles in shaping the membranous web as well as in virus production. We also show that the N-terminal part of NS4B adopts a dual membrane topology in a replicative context, possibly reflecting the different roles of this protein in the viral life cycle.
doi:10.1371/journal.ppat.1004501
PMCID: PMC4231108  PMID: 25392992
5.  The lysine methyltransferase SMYD3 interacts with hepatitis C virus NS5A and is a negative regulator of viral particle production 
Virology  2014;462-463(100):34-41.
Hepatitis C virus (HCV) is a considerable global health and economic burden. The HCV nonstructural protein (NS) 5A is essential for the viral life cycle. The ability of NS5A to interact with different host and viral proteins allow it to manipulate cellular pathways and regulate viral processes, including RNA replication and virus particle assembly. As part of a proteomic screen, we identified several NS5A-binding proteins, including the lysine methyltransferase SET and MYND domain containing protein 3 (SMYD3). We confirmed the interaction in the context of viral replication by co-immunoprecipitation and co-localization studies. Mutational analyses revealed that the MYND-domain of SMYD3 and domain III of NS5A are required for the interaction. Overexpression of SMYD3 resulted in decreased intracellular and extracellular virus titers, whilst viral RNA replication remained unchanged, suggesting that SMYD3 negatively affects HCV particle production in a NS5A-dependent manner.
Highlights
•Identification of SMYD3 as interactor of the HCV protein NS5A using a proteomic approach.•Confirmation of SMYD3 as interactor of NS5A in the context of active viral replication.•Identification of SMYD3 as negative regulator of HCV infectious particle assembly.
doi:10.1016/j.virol.2014.05.016
PMCID: PMC4139193  PMID: 25092459
SMYD3; NS5A; HCV; TAP-MS; Virus particle assembly
6.  Membranous Replication Factories Induced by Plus-Strand RNA Viruses 
Viruses  2014;6(7):2826-2857.
In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two “morphotypes” of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER) and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments.
doi:10.3390/v6072826
PMCID: PMC4113795  PMID: 25054883
membrane rearrangements; electron microscopy; electron tomography; ultrastructure; double-membrane vesicles; membranous replication factories; hepatitis C virus; flaviviruses; picornaviruses; coronaviruses
7.  Matrix Conditions and KLF2-Dependent Induction of Heme Oxygenase-1 Modulate Inhibition of HCV Replication by Fluvastatin 
PLoS ONE  2014;9(5):e96533.
Background & Aims
HMG-CoA-reductase-inhibitors (statins) have been shown to interfere with HCV replication in vitro. We investigated the mechanism, requirements and contribution of heme oxygenase-1(HO-1)-induction by statins to interference with HCV replication.
Methods
HO-1-induction by fluva-, simva-, rosuva-, atorva- or pravastatin was correlated to HCV replication, using non-infectious replicon systems as well as the infectious cell culture system. The mechanism of HO-1-induction by statins as well as its relevance for interference with HCV replication was investigated using transient or permanent knockdown cell lines. Polyacrylamide(PAA) gels of different density degrees or the Rho-kinase-inhibitor Hydroxyfasudil were used in order to mimic matrix conditions corresponding to normal versus fibrotic liver tissue.
Results
All statins used, except pravastatin, decreased HCV replication and induced HO-1 expression, as well as interferon response in vitro. HO-1-induction was mediated by reduction of Bach1 expression and induction of the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) cofactor Krueppel-like factor 2 (KLF2). Knockdown of KLF2 or HO-1 abrogated effects of statins on HCV replication. HO-1-induction and anti-viral effects of statins were more pronounced under cell culture conditions mimicking advanced stages of liver disease.
Conclusions
Statin-mediated effects on HCV replication seem to require HO-1-induction, which is more pronounced in a microenvironment resembling fibrotic liver tissue. This implicates that certain statins might be especially useful to support HCV therapy of patients at advanced stages of liver disease.
doi:10.1371/journal.pone.0096533
PMCID: PMC4011762  PMID: 24801208
8.  Morphological and Biochemical Characterization of the Membranous Hepatitis C Virus Replication Compartment 
Journal of Virology  2013;87(19):10612-10627.
Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.
doi:10.1128/JVI.01370-13
PMCID: PMC3807400  PMID: 23885072
9.  Analysis of hepatitis C virus resistance to Silibinin in vitro and in vivo points to a novel mechanism involving nonstructural protein 4B 
Hepatology (Baltimore, Md.)  2013;57(3):953-963.
Intravenous silibinin (SIL) is an approved therapeutic that has recently been applied to patients with chronic hepatitis C, successfully clearing hepatitis C virus (HCV) infection in some patients even in monotherapy. Previous studies suggested multiple antiviral mechanisms of SIL, however, the dominant mode of action has not been determined. We first analyzed the impact of SIL on replication of subgenomic replicons from different HCV genotypes in vitro and found a strong inhibition of RNA replication for genotype 1a and genotype 1b. In contrast, RNA replication and infection of genotype 2a were minimally affected by SIL. To identify the viral target of SIL we analyzed resistance to SIL in vitro and in vivo. Selection for drug resistance in cell culture identified a mutation in HCV nonstructural protein (NS) 4B conferring partial resistance to SIL. This was corroborated by sequence analyses of HCV from a liver transplant recipient experiencing viral breakthrough under SIL monotherapy. Again, we identified distinct mutations affecting highly conserved amino acid residues within NS4B, which mediated phenotypic SIL resistance also in vitro. Analyses of chimeric viral genomes suggest that SIL might target an interaction between NS4B and NS3/4A. Ultrastructural studies revealed changes in the morphology of viral membrane alterations upon SIL treatment of a susceptible genotype 1b isolate, but not of a resistant NS4B mutant or genotype 2a, indicating that SIL might interfere with the formation of HCV replication sites.
Conclusion
Mutations conferring partial resistance to SIL treatment in vivo and in cell culture argue for a mechanism involving NS4B. This novel mode of action renders SIL an attractive candidate for combination therapies with other directly acting antiviral drugs, particularly in difficult-to-treat patient cohorts.
doi:10.1002/hep.26260
PMCID: PMC3593759  PMID: 23322644
HCV; NS4B; therapy; antiviral; Legalon-SIL; Silibinin
10.  Dynamic Oscillation of Translation and Stress Granule Formation Mark the Cellular Response to Virus Infection 
Cell host & microbe  2012;12(1):10.1016/j.chom.2012.05.013.
SUMMARY
Virus infection-induced global protein synthesis suppression is linked to assembly of stress granules (SGs), cytosolic aggregates of stalled translation preinitiation complexes. To study long-term stress responses, we developed an imaging approach for extended observation and analysis of SG dynamics during persistent hepatitis C virus (HCV) infection. In combination with type 1 interferon, HCV infection induces highly dynamic assembly/disassembly of cytoplasmic SGs, concomitant with phases of active and stalled translation, delayed cell division, and prolonged cell survival. Double-stranded RNA (dsRNA), independent of viral replication, is sufficient to trigger these oscillations. Translation initiation factor eIF2α phosphorylation by protein kinase R mediates SG formation and translation arrest. This is antagonized by the upregulation of GADD34, the regulatory subunit of protein phosphatase 1 dephosphorylating eIF2α. Stress response oscillation is a general mechanism to prevent long-lasting translation repression and a conserved host cell reaction to multiple RNA viruses, which HCV may exploit to establish persistence.
doi:10.1016/j.chom.2012.05.013
PMCID: PMC3873964  PMID: 22817989
11.  Three-Dimensional Architecture of Tick-Borne Encephalitis Virus Replication Sites and Trafficking of the Replicated RNA 
Journal of Virology  2013;87(11):6469-6481.
Flavivirus replication is accompanied by the rearrangement of cellular membranes that may facilitate viral genome replication and protect viral components from host cell responses. The topological organization of viral replication sites and the fate of replicated viral RNA are not fully understood. We exploited electron microscopy to map the organization of tick-borne encephalitis virus (TBEV) replication compartments in infected cells and in cells transfected with a replicon. Under both conditions, 80-nm vesicles were seen within the lumen of the endoplasmic reticulum (ER) that in infected cells also contained virions. By electron tomography, the vesicles appeared as invaginations of the ER membrane, displaying a pore that could enable release of newly synthesized viral RNA into the cytoplasm. To track the fate of TBEV RNA, we took advantage of our recently developed method of viral RNA fluorescent tagging for live-cell imaging combined with bleaching techniques. TBEV RNA was found outside virus-induced vesicles either associated to ER membranes or free to move within a defined area of juxtaposed ER cisternae. From our results, we propose a biologically relevant model of the possible topological organization of flavivirus replication compartments composed of replication vesicles and a confined extravesicular space where replicated viral RNA is retained. Hence, TBEV modifies the ER membrane architecture to provide a protected environment for viral replication and for the maintenance of newly replicated RNA available for subsequent steps of the virus life cycle.
doi:10.1128/JVI.03456-12
PMCID: PMC3648123  PMID: 23552408
12.  Nuclear Localization of Dengue Virus Nonstructural Protein 5 Does Not Strictly Correlate with Efficient Viral RNA Replication and Inhibition of Type I Interferon Signaling 
Journal of Virology  2013;87(8):4545-4557.
Dengue virus (DENV) is an important human pathogen, especially in the tropical and subtropical parts of the world, causing considerable morbidity and mortality. DENV replication occurs in the cytoplasm; however, a high proportion of nonstructural protein 5 (NS5), containing methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) activities, accumulates in the nuclei of infected cells. The present study investigates the impact of nuclear localization of NS5 on its known functions, including viral RNA replication and subversion of the type I interferon response. By using a mutation analysis approach, we identified the most critical residues within the αβ nuclear localization signal (αβNLS), which are essential for the nuclear accumulation of this protein. Although we observed an overall correlation between reduced nuclear accumulation of NS5 and impaired RNA replication, we identified one mutant with drastically reduced amounts of nuclear NS5 and virtually unaffected RNA replication, arguing that nuclear localization of NS5 does not correlate strictly with DENV replication, at least in cell culture. Because NS5 plays an important role in blocking interferon signaling via STAT-2 (signal transducer and activator of transcription 2) degradation, the abilities of the NLS mutants to block this pathway were investigated. All mutants were able to degrade STAT-2, with accordingly similar type I interferon resistance phenotypes. Since the NLS is contained within the RdRp domain, the MTase and RdRp activities of the mutants were determined by using recombinant full-length NS5. We found that the C-terminal region of the αβNLS is a critical functional element of the RdRp domain required for polymerase activity. These results indicate that efficient DENV RNA replication requires only minimal, if any, nuclear NS5, and they identify the αβNLS as a structural element required for proper RdRp activity.
doi:10.1128/JVI.03083-12
PMCID: PMC3624364  PMID: 23408610
13.  Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines 
Nucleic Acids Research  2013;41(21):e199.
As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.
doi:10.1093/nar/gkt836
PMCID: PMC3834839  PMID: 24049077
14.  Replication Vesicles are Load- and Choke-Points in the Hepatitis C Virus Lifecycle 
PLoS Pathogens  2013;9(8):e1003561.
Hepatitis C virus (HCV) infection develops into chronicity in 80% of all patients, characterized by persistent low-level replication. To understand how the virus establishes its tightly controlled intracellular RNA replication cycle, we developed the first detailed mathematical model of the initial dynamic phase of the intracellular HCV RNA replication. We therefore quantitatively measured viral RNA and protein translation upon synchronous delivery of viral genomes to host cells, and thoroughly validated the model using additional, independent experiments. Model analysis was used to predict the efficacy of different classes of inhibitors and identified sensitive substeps of replication that could be targeted by current and future therapeutics. A protective replication compartment proved to be essential for sustained RNA replication, balancing translation versus replication and thus effectively limiting RNA amplification. The model predicts that host factors involved in the formation of this compartment determine cellular permissiveness to HCV replication. In gene expression profiling, we identified several key processes potentially determining cellular HCV replication efficiency.
Author Summary
Hepatitis C is a severe disease and a prime cause for liver transplantation. Up to 3% of the world's population are chronically infected with its causative agent, the Hepatitis C virus (HCV). This capacity to establish long (decades) lasting persistent infection sets HCV apart from other plus-strand RNA viruses typically causing acute, self-limiting infections. A prerequisite for its capacity to persist is HCV's complex and tightly regulated intracellular replication strategy. In this study, we therefore wanted to develop a comprehensive understanding of the molecular processes governing HCV RNA replication in order to pinpoint the most vulnerable substeps in the viral life cycle. For that purpose, we used a combination of biological experiments and mathematical modeling. Using the model to study HCV's replication strategy, we recognized diverse but crucial roles for the membraneous replication compartment of HCV in regulating RNA amplification. We further predict the existence of an essential limiting host factor (or function) required for establishing active RNA replication and thereby determining cellular permissiveness for HCV. Our model also proved valuable to understand and predict the effects of pharmacological inhibitors of HCV and might be a solid basis for the development of similar models for other plus-strand RNA viruses.
doi:10.1371/journal.ppat.1003561
PMCID: PMC3749965  PMID: 23990783
15.  The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication 
PLoS Pathogens  2013;9(7):e1003440.
Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.
Author Summary
Viruses are obligate intracellular parasites that rely on cellular functions for efficient replication. As most biological processes are sustained by protein-protein interactions, the identification of interactions between viral and host proteins can provide a global overview about the cellular functions engaged during viral replication. Influenza viruses express 13 viral proteins, including NS1 and NS2, which are translated from an alternatively spliced RNA derived from the same genome segment. We present here a comprehensive overview of possible interactions of cellular proteins with NS1 and NS2 from 9 viral strains. Seventy nine cellular proteins were identified to interact with NS1, NS2 or both NS1 and NS2. These interacting host cell proteins are potentially involved in many steps of the virus life cycle and 7 can directly control the viral replication. Most of the cellular targets are shared by the majority of the virus strains, especially the double-stranded RNA binding domain protein family that is strikingly targeted by NS1. One of its members, ADAR1, is essential for influenza virus replication. ADAR1 colocalizes with NS1 in nuclear structures and its editing activity is enhanced by NS1 expressed on its own and during virus infection. A similar phenomenon is observed for dengue virus whose NS3 protein also interacts with ADAR1, suggesting a parallel virus-host co-evolution.
doi:10.1371/journal.ppat.1003440
PMCID: PMC3701712  PMID: 23853584
16.  A Novel Inhibitor of Dengue Virus Replication That Targets the Capsid Protein 
Dengue viruses (DENV) infect 50 to 100 million people worldwide per year, of which 500,000 develop severe life-threatening disease. This mosquito-borne illness is endemic in most tropical and subtropical countries and has spread significantly over the last decade. While there are several promising vaccine candidates in clinical trials, there are currently no approved vaccines or therapeutics available for treatment of dengue infection. Here, we describe a novel small-molecule compound, ST-148, that is a potent inhibitor of all four serotypes of DENV in vitro. ST-148 significantly reduced viremia and viral load in vital organs and tended to lower cytokine levels in the plasma in a nonlethal model of DENV infection in AG129 mice. Compound resistance mapped to the DENV capsid (C) gene, and a direct interaction of ST-148 with C protein is suggested by alterations of the intrinsic fluorescence of the protein in the presence of compound. Thus, ST-148 appears to interact with the DENV C protein and inhibits a distinct step(s) of the viral replication cycle.
doi:10.1128/AAC.01429-12
PMCID: PMC3535982  PMID: 23070172
17.  Evidence for Novel Hepaciviruses in Rodents 
PLoS Pathogens  2013;9(6):e1003438.
Hepatitis C virus (HCV) is among the most relevant causes of liver cirrhosis and hepatocellular carcinoma. Research is complicated by a lack of accessible small animal models. The systematic investigation of viruses of small mammals could guide efforts to establish such models, while providing insight into viral evolutionary biology. We have assembled the so-far largest collection of small-mammal samples from around the world, qualified to be screened for bloodborne viruses, including sera and organs from 4,770 rodents (41 species); and sera from 2,939 bats (51 species). Three highly divergent rodent hepacivirus clades were detected in 27 (1.8%) of 1,465 European bank voles (Myodes glareolus) and 10 (1.9%) of 518 South African four-striped mice (Rhabdomys pumilio). Bats showed anti-HCV immunoblot reactivities but no virus detection, although the genetic relatedness suggested by the serologic results should have enabled RNA detection using the broadly reactive PCR assays developed for this study. 210 horses and 858 cats and dogs were tested, yielding further horse-associated hepaciviruses but none in dogs or cats. The rodent viruses were equidistant to HCV, exceeding by far the diversity of HCV and the canine/equine hepaciviruses taken together. Five full genomes were sequenced, representing all viral lineages. Salient genome features and distance criteria supported classification of all viruses as hepaciviruses. Quantitative RT-PCR, RNA in-situ hybridisation, and histopathology suggested hepatic tropism with liver inflammation resembling hepatitis C. Recombinant serology for two distinct hepacivirus lineages in 97 bank voles identified seroprevalence rates of 8.3 and 12.4%, respectively. Antibodies in bank vole sera neither cross-reacted with HCV, nor the heterologous bank vole hepacivirus. Co-occurrence of RNA and antibodies was found in 3 of 57 PCR-positive bank vole sera (5.3%). Our data enable new hypotheses regarding HCV evolution and encourage efforts to develop rodent surrogate models for HCV.
Author Summary
The hepatitis C virus (HCV) is one of the most relevant causes of liver disease and cancer in humans. The lack of a small animal models represents an important hurdle on our way to understanding, treating, and preventing hepatitis C. The investigation of small mammals could identify virus infections similar to hepatitis C in animals that can be kept in laboratories, such as rodents, and can also yield insights into the evolution of those ancestral virus lineages out of which HCV developed. Here, we investigated a worldwide sample of 4,770 rodents, 2,939 bats, 210 horses and 858 cats and dogs for HCV-related viruses. New viruses were discovered in European bank voles (Myodes glareolus) and South African four-striped mice (Rhabdomys pumilio). The disease in bank voles was studied in more detail, suggesting that infection of the liver occurs with similar symptoms to those caused by HCV in humans. These rodents might thus enable the development of new laboratory models of hepatitis C. Moreover, the phylogenetic history of those viruses provides fascinating new ideas regarding the evolution of HCV ancestors.
doi:10.1371/journal.ppat.1003438
PMCID: PMC3688547  PMID: 23818848
18.  Architecture and biogenesis of plus-strand RNA virus replication factories 
World Journal of Virology  2013;2(2):32-48.
Plus-strand RNA virus replication occurs in tight association with cytoplasmic host cell membranes. Both, viral and cellular factors cooperatively generate distinct organelle-like structures, designated viral replication factories. This compartmentalization allows coordination of the different steps of the viral replication cycle, highly efficient genome replication and protection of the viral RNA from cellular defense mechanisms. Electron tomography studies conducted during the last couple of years revealed the three dimensional structure of numerous plus-strand RNA virus replication compartments and highlight morphological analogies between different virus families. Based on the morphology of virus-induced membrane rearrangements, we propose two separate subclasses: the invaginated vesicle/spherule type and the double membrane vesicle type. This review discusses common themes and distinct differences in the architecture of plus-strand RNA virus-induced membrane alterations and summarizes recent progress that has been made in understanding the complex interplay between viral and co-opted cellular factors in biogenesis and maintenance of plus-strand RNA virus replication factories.
doi:10.5501/wjv.v2.i2.32
PMCID: PMC3785047  PMID: 24175228
Viral replication factory; Viral replication complex; Plus-strand RNA virus; Membrane remodeling; Virus-host interaction; Alphavirus; Enterovirus; Coronavirus; Flavivirus; Hepatitis C virus
19.  High-throughput RNA interference screens integrative analysis: Towards a comprehensive understanding of the virus-host interplay 
World Journal of Virology  2013;2(2):18-31.
Viruses are extremely heterogeneous entities; the size and the nature of their genetic information, as well as the strategies employed to amplify and propagate their genomes, are highly variable. However, as obligatory intracellular parasites, replication of all viruses relies on the host cell. Having co-evolved with their host for several million years, viruses have developed very sophisticated strategies to hijack cellular factors that promote virus uptake, replication, and spread. Identification of host cell factors (HCFs) required for these processes is a major challenge for researchers, but it enables the identification of new, highly selective targets for anti viral therapeutics. To this end, the establishment of platforms enabling genome-wide high-throughput RNA interference (HT-RNAi) screens has led to the identification of several key factors involved in the viral life cycle. A number of genome-wide HT-RNAi screens have been performed for major human pathogens. These studies enable first inter-viral comparisons related to HCF requirements. Although several cellular functions appear to be uniformly required for the life cycle of most viruses tested (such as the proteasome and the Golgi-mediated secretory pathways), some factors, like the lipid kinase Phosphatidylinositol 4-kinase IIIα in the case of hepatitis C virus, are selectively required for individual viruses. However, despite the amount of data available, we are still far away from a comprehensive understanding of the interplay between viruses and host factors. Major limitations towards this goal are the low sensitivity and specificity of such screens, resulting in limited overlap between different screens performed with the same virus. This review focuses on how statistical and bioinformatic analysis methods applied to HT-RNAi screens can help overcoming these issues thus increasing the reliability and impact of such studies.
doi:10.5501/wjv.v2.i2.18
PMCID: PMC3785050  PMID: 24175227
RNA interference; High-throughput; Cell population; Dependency factors; Bioinformatics; Human immunodeficiency virus; Hepatitis C virus; Dengue virus; Viral infection; Virus-host interactions
20.  The Lipid Kinase Phosphatidylinositol-4 Kinase III Alpha Regulates the Phosphorylation Status of Hepatitis C Virus NS5A 
PLoS Pathogens  2013;9(5):e1003359.
The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an essential host factor of hepatitis C virus (HCV) replication. PI4KIIIα catalyzes the synthesis of phosphatidylinositol 4-phosphate (PI4P) accumulating in HCV replicating cells due to enzyme activation resulting from its interaction with nonstructural protein 5A (NS5A). This study describes the interaction between PI4KIIIα and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in PI4KIIIα interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIIIα functional interaction site (PFIS) encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIIIα binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIIIα. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58), indicating that PI4KIIIα affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIIIα or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIIIα increased relative abundance of basally phosphorylated NS5A (p56). PI4KIIIα therefore regulates the phosphorylation status of NS5A and viral RNA replication by favoring p56 or repressing p58 synthesis. Replication deficiencies of PFIS mutants in NS5A could not be rescued by increasing PI4P levels, but by supplying functional NS5A, supporting an essential role of PI4KIIIα in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIIIα activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIIIα in the morphogenesis of viral replication sites and its regulation by facilitating p56 synthesis.
Author Summary
Hepatitis C virus (HCV) infections affect about 170 million people worldwide and often result in severe chronic liver disease. HCV is a positive-strand RNA virus inducing massive rearrangements of intracellular membranes to generate the sites of genome replication, designated the membranous web. The complex biogenesis of the membranous web is still poorly understood, but requires the concerted action of several viral nonstructural proteins and cellular factors. Recently, we and others identified the lipid kinase phosphatidylinositol-4 kinase III alpha (PI4KIIIα), catalyzing the synthesis of phosphatidylinositol 4-phosphate (PI4P), as an essential host factor involved in the formation of the membranous web. In this study, we characterized the virus-host interaction in greater detail using a genetic approach. We identified a highly conserved region in the viral phosphoprotein NS5A crucial for the interaction with PI4KIIIα. Surprisingly, we found that PI4KIIIα, despite being a lipid kinase, appeared to regulate the phosphorylation status of NS5A, thus contributing to viral replication. Our results furthermore suggest that the morphology of the membranous web is regulated by NS5A phosphorylation, providing novel insights into the complex regulation of viral RNA replication.
doi:10.1371/journal.ppat.1003359
PMCID: PMC3649985  PMID: 23675303
21.  Reconstitution of the Entire Hepatitis C Virus Life Cycle in Nonhepatic Cells 
Journal of Virology  2012;86(21):11919-11925.
Hepatitis C virus (HCV) is a human hepatotropic virus, but the relevant host factors restricting HCV infection to hepatocytes are only partially understood. We demonstrate that exogenous expression of defined host factors reconstituted the entire HCV life cycle in human nonhepatic 293T cells. This study shows robust HCV entry, RNA replication, and production of infectious virus in human nonhepatic cells and highlights key host factors required for liver tropism of HCV.
doi:10.1128/JVI.01066-12
PMCID: PMC3486316  PMID: 22896615
22.  Divergent Roles of Autophagy in Virus Infection 
Cells  2013;2(1):83-104.
Viruses have played an important role in human evolution and have evolved diverse strategies to co-exist with their hosts. As obligate intracellular pathogens, viruses exploit and manipulate different host cell processes, including cellular trafficking, metabolism and immunity-related functions, for their own survival. In this article, we review evidence for how autophagy, a highly conserved cellular degradative pathway, serves either as an antiviral defense mechanism or, alternatively, as a pro-viral process during virus infection. Furthermore, we highlight recent reports concerning the role of selective autophagy in virus infection and how viruses manipulate autophagy to evade lysosomal capture and degradation.
doi:10.3390/cells2010083
PMCID: PMC3972664  PMID: 24709646
selectively autophagy; virophagy; antiviral and proviral autophagy
23.  Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Hepatitis C Virus Replication 
PLoS Pathogens  2012;8(12):e1003056.
All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.
Author Summary
All positive-strand RNA viruses replicate in the cytoplasm in distinct membranous compartments acting as ‘replication factories’. Membranes building up these factories are recruited from different sources and serve as platforms for the assembly of multi-subunit protein complexes (the ‘replicase’) that catalyze the amplification of the viral RNA genome. In this study we found that hepatitis C virus (HCV), a major causative agent of chronic liver disease, induces profound remodeling of primarily endoplasmic reticulum-derived membranes. Surprisingly, the 3D architecture of these membrane rearrangements is similar to those induced by the unrelated picorna- and coronaviruses, but in striking contrast to the closely related flaviviruses. Early in infection HCV induces double membrane vesicles (DMVs) that emerge as protrusions of the ER; later on, HCV induces in addition multi-membrane vesicles that are probably the result of a cellular stress reaction and that are reminiscent to an autophagic response. These profound membrane rearrangements are induced by the concerted action of HCV-encoded nonstructural proteins of which NS5A is the only one capable to induce DMVs. These results provide important insights into the 3D architecture of the membrane alterations induced by HCV and reveal unexpected similarities between HCV and the very distantly related picorna- and coronaviruses.
doi:10.1371/journal.ppat.1003056
PMCID: PMC3516559  PMID: 23236278
24.  Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment 
Cell host & microbe  2011;9(1):32-45.
SUMMARY
Hepatitis C virus (HCV) is a major causative agent of chronic liver disease in humans. To gain insight into host factor requirements for HCV replication we performed a siRNA screen of the human kinome and identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIα) as required for HCV replication. Consistent with elevated levels of the PI4KIIIα product phosphatidylinositol-4-phosphate (PI4P) detected in HCV infected cultured hepatocytes and liver tissue from chronic hepatitis C patients, the enzymatic activity of PI4KIIIα was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIα and stimulate its kinase activity. The absence of PI4KIIIα activity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. Our analysis suggests that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex.
doi:10.1016/j.chom.2010.12.002
PMCID: PMC3433060  PMID: 21238945
25.  MAP-Kinase Regulated Cytosolic Phospholipase A2 Activity Is Essential for Production of Infectious Hepatitis C Virus Particles 
PLoS Pathogens  2012;8(7):e1002829.
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.
Author Summary
The human genome encodes more than 30 phospholipase A2s. These enzymes cleave fatty acids at the C2 atom of phosphoglycerides and thus modulate membrane properties. Among all PLA2s only PLA2G4A, which is recruited to perinuclear membranes by Ca2+ and activated by extracellular stimuli via the mitogen activated protein kinase pathway, specifically cleaves lipids with arachidonic acid. Metabolism of arachidonic acid yields prostaglandins and leukotriens, important lipid mediators of inflammation. We show that inhibition of PLA2G4A produces aberrant HCV particles and that infectivity is rescued by addition of arachidonic acid. Our results suggest that a specific lipid (arachidonic acid) is essential for production of highly infectious HCV progeny, likely by creating a membrane environment conducive for efficient incorporation of crucial host and viral factors into the lipid envelope of nascent particles. Strikingly, PLA2G4A is also essential for production of highly infectious Dengue Virus (DENV) particles but not for vesicular stomatitis virus (VSV). These observations argue that HCV and DENV which unlike VSV produce particles at intracellular membranes usurp a common host factor (PLA2G4A) for assembly of highly infectious progeny. These findings open new perspectives for antiviral intervention and highlight thus far unrecognized parallels in the assembly pathway of HCV and DENV.
doi:10.1371/journal.ppat.1002829
PMCID: PMC3406102  PMID: 22911431

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