Targeting cellular cathepsins inhibits hepatitis E virus entry.

BACKGROUND AND AIMS: The hepatitis E virus (HEV) is estimated to be responsible for 70,000 deaths annually, yet therapy options remain limited. In the pursuit of effective antiviral therapies, targeting viral entry holds promise and has proven effective for other viruses. However, the precise mechanisms and host factors required during HEV entry remain unclear. Cellular proteases have emerged as host factors required for viral surface protein activation and productive cell entry by many viruses. Hence, we investigated the functional requirement and therapeutic potentials of cellular proteases during HEV infection. APPROACH AND RESULTS: Using our established HEV cell culture model and subgenomic HEV replicons, we found that blocking lysosomal cathepsins (CTS) with small molecule inhibitors, impedes HEV infection without affecting replication. Most importantly, the pan-cathepsin inhibitor K11777 suppressed HEV infections with an EC50 of ~ 0.01 nM. Inhibition by K11777, devoid of notable toxicity in hepatoma cells, was also observed in HepaRG and primary human hepatocytes. Furthermore, through time-of-addition and RNAscope experiments, we confirmed that HEV entry is blocked by inhibition of cathepsins. Cathepsin L (CTSL) knockout cells were less permissive to HEV, suggesting that CTSL is critical for HEV infection. Finally, we observed cleavage of the glycosylated ORF2 protein and virus particles by recombinant CTSL. CONCLUSIONS: In summary, our study highlights the pivotal role of lysosomal cathepsins, especially CTSL, in the HEV entry process. The profound anti-HEV efficacy of the pan-cathepsin inhibitor, K11777, especially with its notable safety profile in primary cells, further underscores its potential as a therapeutic candidate.

Repurposing screen identifies novel candidates for broad-spectrum coronavirus antivirals and druggable host targets.

Libraries composed of licensed drugs represent a vast repertoire of molecules modulating physiological processes in humans, providing unique opportunities for the discovery of host-targeting antivirals. We screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) repurposing library with approximately 12,000 molecules for broad-spectrum coronavirus antivirals and discovered 134 compounds inhibiting an alphacoronavirus and mapping to 58 molecular target categories. Dominant targets included the 5-hydroxytryptamine receptor, the dopamine receptor, and cyclin-dependent kinases. Gene knock-out of the drugs' host targets including cathepsin B and L (CTSB/L; VBY-825), the aryl hydrocarbon receptor (AHR; Phortress), the farnesyl-diphosphate farnesyltransferase 1 (FDFT1; P-3622), and the kelch-like ECH-associated protein 1 (KEAP1; Omaveloxolone), significantly modulated HCoV-229E infection, providing evidence that these compounds inhibited the virus through acting on their respective host targets. Counter-screening of all 134 primary compound candidates with SARS-CoV-2 and validation in primary cells identified Phortress, an AHR activating ligand, P-3622-targeting FDFT1, and Omaveloxolone, which activates the NFE2-like bZIP transcription factor 2 (NFE2L2) by liberating it from its endogenous inhibitor KEAP1, as antiviral candidates for both an Alpha- and a Betacoronavirus. This study provides an overview of HCoV-229E repurposing candidates and reveals novel potentially druggable viral host dependency factors hijacked by diverse coronaviruses.

Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes.

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establish persistence. IMPORTANCE: Hepatitis C virus (HCV) infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms that underly persistence are incompletely defined. We utilized a long-term cell culture-adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.

Spatiotemporal analysis of SARS-CoV-2 infection reveals an expansive wave of monocyte-derived macrophages associated with vascular damage and virus clearance in hamster lungs.

IMPORTANCE: We present the first study of the 3D kinetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the early host response in a large lung volume using a combination of tissue imaging and transcriptomics. This approach allowed us to make a number of important findings: Spatially restricted antiviral response is shown, including the formation of monocytic macrophage clusters and upregulation of the major histocompatibility complex II in infected epithelial cells. The monocyte-derived macrophages are linked to SARS-CoV-2 clearance, and the appearance of these cells is associated with post-infection endothelial damage; thus, we shed light on the role of these cells in infected tissue. An early onset of tissue repair occurring simultaneously with inflammatory and necrotizing processes provides the basis for longer-term alterations in the lungs.

Hepatitis C virus cell culture adaptive mutations enhance cell culture propagation by multiple mechanisms but boost antiviral responses in primary human hepatocytes.

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants which underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establishing persistence.

Mouse Liver-Expressed Shiftless Is an Evolutionarily Conserved Antiviral Effector Restricting Human and Murine Hepaciviruses.

Mice are refractory to infection with human-tropic hepatitis C virus (HCV), although distantly related rodent hepaciviruses (RHV) circulate in wild rodents. To investigate whether liver intrinsic host factors can exhibit broad restriction against these distantly related hepaciviruses, we focused on Shiftless (Shfl), an interferon (IFN)-regulated gene (IRG) which restricts HCV in humans. Unusually, and in contrast to selected classical IRGs, human and mouse SHFL orthologues (hSHFL and mSHFL, respectively) were highly expressed in hepatocytes in the absence of viral infection, weakly induced by IFN, and highly conserved at the amino acid level (>95%). Replication of both HCV and RHV subgenomic replicons was suppressed by ectopic expression of mSHFL in human or rodent hepatoma cell lines. Gene editing of endogenous mShfl in mouse liver tumor cells increased HCV replication and virion production. Colocalization of mSHFL protein with viral double-stranded RNA (dsRNA) intermediates was confirmed and could be ablated by mutational disruption of the SHFL zinc finger domain, concomitant with a loss of antiviral activity. In summary, these data point to an evolutionarily conserved function for this gene in humans and rodents: SHFL is an ancient antiviral effector which targets distantly related hepaciviruses via restriction of viral RNA replication. IMPORTANCE Viruses have evolved ways to evade or blunt innate cellular antiviral mechanisms within their cognate host species. However, these adaptations may fail when viruses infect new species and can therefore limit cross-species transmission. This may also prevent development of animal models for human-pathogenic viruses. HCV shows a narrow species tropism likely due to distinct human host factor usage and innate antiviral defenses limiting infection of nonhuman liver cells. Interferon (IFN)-regulated genes (IRGs) partially inhibit HCV infection of human cells by diverse mechanisms. Here, we show that mouse Shiftless (mSHFL), a protein that interferes with HCV replication factories, inhibits HCV replication and infection in human and mouse liver cells. We further report that the zinc finger domain of SHFL is important for viral restriction. These findings implicate mSHFL as a host factor that impairs HCV infection of mice and provide guidance for development of HCV animal models needed for vaccine development.

Vaccine-associated enhanced respiratory pathology in COVID-19 hamsters after TH2-biased immunization.

Vaccine-associated enhanced respiratory disease (VAERD) is a severe complication for some respiratory infections. To investigate the potential for VAERD induction in coronavirus disease 2019 (COVID-19), we evaluate two vaccine leads utilizing a severe hamster infection model: a T helper type 1 (TH1)-biased measles vaccine-derived candidate and a TH2-biased alum-adjuvanted, non-stabilized spike protein. The measles virus (MeV)-derived vaccine protects the animals, but the protein lead induces VAERD, which can be alleviated by dexamethasone treatment. Bulk transcriptomic analysis reveals that our protein vaccine prepares enhanced host gene dysregulation in the lung, exclusively up-regulating mRNAs encoding the eosinophil attractant CCL-11, TH2-driving interleukin (IL)-19, or TH2 cytokines IL-4, IL-5, and IL-13. Single-cell RNA sequencing (scRNA-seq) identifies lung macrophages or lymphoid cells as sources, respectively. Our findings imply that VAERD is caused by the concerted action of hyperstimulated macrophages and TH2 cytokine-secreting lymphoid cells and potentially links VAERD to antibody-dependent enhancement (ADE). In summary, we identify the cytokine drivers and cellular contributors that mediate VAERD after TH2-biased vaccination.

The Human Liver-Expressed Lectin CD302 Restricts Hepatitis C Virus Infection.

C-type lectin domain-containing proteins (CTLDcps) shape host responses to pathogens and infectious disease outcomes. Previously, we identified the murine CTLDcp Cd302 as restriction factor, limiting hepatitis C virus (HCV) infection of murine hepatocytes. In this study, we investigated in detail the human orthologue's ability to restrict HCV infection in human liver cells. CD302 overexpression in Huh-7.5 cells potently inhibited infection of diverse HCV chimeras representing seven genotypes. Transcriptional profiling revealed abundant CD302 mRNA expression in human hepatocytes, the natural cellular target of HCV. Knockdown of endogenously expressed CD302 modestly enhanced HCV infection of Huh-7.5 cells and primary human hepatocytes. Functional analysis of naturally occurring CD302 transcript variants and engineered CD302 mutants showed that the C-type lectin-like domain (CTLD) is essential for HCV restriction, whereas the cytoplasmic domain (CPD) is dispensable. Coding single nucleotide polymorphisms occurring in human populations and mapping to different domains of CD302 did not influence the capacity of CD302 to restrict HCV. Assessment of the anti-HCV phenotype at different life cycle stages indicated that CD302 preferentially targets the viral entry step. In contrast to the murine orthologue, overexpression of human CD302 did not modulate downstream expression of nuclear receptor-controlled genes. Ectopic CD302 expression restricted infection of liver tropic hepatitis E virus (HEV), while it did not affect infection rates of two respiratory viruses, including respiratory syncytial virus (RSV) and the alpha coronavirus HVCoV-229E. Together, these findings suggest that CD302 contributes to liver cell-intrinsic defense against HCV and might mediate broader antiviral defenses against additional hepatotropic viruses. IMPORTANCE The liver represents an immunoprivileged organ characterized by enhanced resistance to immune responses. However, the importance of liver cell-endogenous, noncytolytic innate immune responses in pathogen control is not well defined. Although the role of myeloid cell-expressed CTLDcps in host responses to viruses has been characterized in detail, we have little information about their potential functions in the liver and their relevance for immune responses in this organ. Human hepatocytes endogenously express the CTLDcp CD302. Here, we provide evidence that CD302 limits HCV infection of human liver cells, likely by inhibiting a viral cell entry step. We confirm that the dominant liver-expressed transcript variant, as well as naturally occurring coding variants of CD302, maintain the capacity to restrict HCV. We further show that the CTLD of the protein is critical for the anti-HCV activity and that overexpressed CD302 limits HEV infection. Thus, CD302 likely contributes to human liver-intrinsic antiviral defenses.

Intra-host analysis of hepaciviral glycoprotein evolution reveals signatures associated with viral persistence and clearance.

Even 30 years after the discovery of the hepatitis C virus (HCV) in humans there is still no vaccine available. Reasons for this include the high mutation rate of HCV, which allows the virus to escape immune recognition and the absence of an immunocompetent animal model for vaccine development. Phylogenetically distinct hepaciviruses (genus Hepacivirus, family Flaviviridae) have been isolated from diverse species, each with a narrow host range: the equine hepacivirus (EqHV) is the closest known relative of HCV. In this study, we used amplicon-based deep-sequencing to investigate the viral intra-host population composition of the genomic regions encoding the surface glycoproteins E1 and E2. Patterns of E1E2 substitutional evolution were compared in longitudinally sampled EqHV-positive sera of naturally and experimentally infected horses and HCV-positive patients. Intra-host virus diversity was higher in chronically than in acutely infected horses, a pattern which was similar in the HCV-infected patients. However, overall glycoprotein variability was higher in HCV compared to EqHV. Additionally, selection pressure in HCV populations was higher, especially within the N-terminal region of E2, corresponding to the hypervariable region 1 (HVR1) in HCV. An alignment of glycoprotein sequences from diverse hepaciviruses identified the HVR1 as a unique characteristic of HCV: hepaciviruses from non-human species lack this region. Together, these data indicate that EqHV infection of horses could represent a powerful surrogate animal model to gain insights into hepaciviral evolution and HCVs HVR1-mediated immune evasion strategy.

Single-cell analysis of arthritogenic alphavirus-infected human synovial fibroblasts links low abundance of viral RNA to induction of innate immunity and arthralgia-associated gene expression.

Infection by (re-)emerging RNA arboviruses including Chikungunya virus (CHIKV) and Mayaro virus primarily cause acute febrile disease and transient polyarthralgia. However, in a significant subset of infected individuals, debilitating arthralgia persists for weeks over months up to years. The underlying immunopathogenesis of chronification of arthralgia upon primary RNA-viral infection remains unclear. Here, we analysed cell-intrinsic responses to ex vivo arthritogenic alphaviral infection of primary human synovial fibroblasts isolated from knee joints, one the most affected joint types during acute and chronic CHIKV disease. Synovial fibroblasts were susceptible and permissive to alphaviral infection. Base-line and exogenously added type I interferon (IFN) partially and potently restricted infection, respectively. RNA-seq revealed a CHIKV infection-induced transcriptional profile that comprised upregulation of expression of several hundred IFN-stimulated and arthralgia-mediating genes. Single-cell virus-inclusive RNA-seq uncovered a fine-tuned switch from induction to repression of cell-intrinsic immune responses depending on the abundance of viral RNA in an individual cell. Specifically, responses were most pronounced in cells displaying low-to-intermediate amounts of viral RNA and absence of virus-encoded, fluorescent reporter protein expression, arguing for efficient counteraction of innate immunity in cells expressing viral antagonists at sufficient quantities. In summary, cell-intrinsic sensing of viral RNA that potentially persists or replicates at low levels in synovial fibroblasts and other target cell types in vivo may contribute to the chronic arthralgia induced by alphaviral infections. Our findings might advance our understanding of the immunopathophysiology of long-term pathogenesis of RNA-viral infections.

Generation of hiPSC-derived low threshold mechanoreceptors containing axonal termini resembling bulbous sensory nerve endings and expressing Piezo1 and Piezo2.

Somatosensory low threshold mechanoreceptors (LTMRs) sense innocuous mechanical forces, largely through specialized axon termini termed sensory nerve endings, where the mechanotransduction process initiates upon activation of mechanotransducers. In humans, a subset of sensory nerve endings is enlarged, forming bulb-like expansions, termed bulbous nerve endings. There is no in vitro human model to study these neuronal endings. Piezo2 is the main mechanotransducer found in LTMRs. Recent evidence shows that Piezo1, the other mechanotransducer considered absent in dorsal root ganglia (DRG), is expressed at low level in somatosensory neurons. We established a differentiation protocol to generate, from iPSC-derived neuronal precursor cells, human LTMR recapitulating bulbous sensory nerve endings and heterogeneous expression of Piezo1 and Piezo2. The derived neurons express LTMR-specific genes, convert mechanical stimuli into electrical signals and have specialized axon termini that morphologically resemble bulbous nerve endings. Piezo2 is concentrated within these enlarged axon termini. Some derived neurons express low level Piezo1, and a subset co-express both channels. Thus, we generated a unique, iPSCs-derived human model that can be used to investigate the physiology of bulbous sensory nerve endings, and the role of Piezo1 and 2 during mechanosensation.

Strategies to Inhibit Hepatitis B Virus at the Transcript Level.

Approximately 240 million people are chronically infected with hepatitis B virus (HBV), despite four decades of effective HBV vaccination. During chronic infection, HBV forms two distinct templates responsible for viral transcription: (1) episomal covalently closed circular (ccc)DNA and (2) host genome-integrated viral templates. Multiple ubiquitous and liver-specific transcription factors are recruited onto these templates and modulate viral gene transcription. This review details the latest developments in antivirals that inhibit HBV gene transcription or destabilize viral transcripts. Notably, nuclear receptor agonists exhibit potent inhibition of viral gene transcription from cccDNA. Small molecule inhibitors repress HBV X protein-mediated transcription from cccDNA, while small interfering RNAs and single-stranded oligonucleotides result in transcript degradation from both cccDNA and integrated templates. These antivirals mediate their effects by reducing viral transcripts abundance, some leading to a loss of surface antigen expression, and they can potentially be added to the arsenal of drugs with demonstrable anti-HBV activity. Thus, these candidates deserve special attention for future repurposing or further development as anti-HBV therapeutics.

Initial HCV infection of adult hepatocytes triggers a temporally structured transcriptional program containing diverse pro- and anti-viral elements.

Transcriptional profiling provides global snapshots of virus-mediated cellular reprogramming, which can simultaneously encompass pro- and antiviral components. To determine early transcriptional signatures associated with HCV infection of authentic target cells, we performed ex vivo infections of adult primary human hepatocytes (PHHs) from seven donors. Longitudinal sampling identified minimal gene dysregulation at six hours post infection (hpi). In contrast, at 72 hpi, massive increases in the breadth and magnitude of HCV-induced gene dysregulation were apparent, affecting gene classes associated with diverse biological processes. Comparison with HCV-induced transcriptional dysregulation in Huh-7.5 cells identified limited overlap between the two systems. Of note, in PHHs, HCV infection initiated broad upregulation of canonical interferon (IFN)-mediated defense programs, limiting viral RNA replication and abrogating virion release. We further find that constitutive expression of IRF1 in PHHs maintains a steady-state antiviral program in the absence of infection, which can additionally reduce HCV RNA translation and replication. We also detected infection-induced downregulation of ∼90 genes encoding components of the EIF2 translation initiation complex and ribosomal subunits in PHHs, consistent with a signature of translational shutoff. As HCV polyprotein translation occurs independently of the EIF2 complex, this process is likely pro-viral: only translation initiation of host transcripts is arrested. The combination of antiviral intrinsic and inducible immunity, balanced against pro-viral programs, including translational arrest, maintains HCV replication at a low-level in PHHs. This may ultimately keep HCV under the radar of extra-hepatocyte immune surveillance while initial infection is established, promoting tolerance, preventing clearance and facilitating progression to chronicity.IMPORTANCEAcute HCV infections are often asymptomatic and therefore frequently undiagnosed. We endeavored to recreate this understudied phase of HCV infection using explanted PHHs and monitored host responses to initial infection. We detected temporally distinct virus-induced perturbations in the transcriptional landscape, which were initially narrow but massively amplified in breadth and magnitude over time. At 72 hpi, we detected dysregulation of diverse gene programs, concurrently promoting both virus clearance and virus persistence. On the one hand, baseline expression of IRF1 combined with infection-induced upregulation of IFN-mediated effector genes suppresses virus propagation. On the other, we detect transcriptional signatures of host translational inhibition, which likely reduces processing of IFN-regulated gene transcripts and facilitates virus survival. Together, our data provide important insights into constitutive and virus-induced transcriptional programs in PHHs, and identifies simultaneous antagonistic dysregulation of pro-and anti-viral programs which may facilitate host tolerance and promote viral persistence.

Liver-expressed Cd302 and Cr1l limit hepatitis C virus cross-species transmission to mice.

Hepatitis C virus (HCV) has no animal reservoir, infecting only humans. To investigate species barrier determinants limiting infection of rodents, murine liver complementary DNA library screening was performed, identifying transmembrane proteins Cd302 and Cr1l as potent restrictors of HCV propagation. Combined ectopic expression in human hepatoma cells impeded HCV uptake and cooperatively mediated transcriptional dysregulation of a noncanonical program of immunity genes. Murine hepatocyte expression of both factors was constitutive and not interferon inducible, while differences in liver expression and the ability to restrict HCV were observed between the murine orthologs and their human counterparts. Genetic ablation of endogenous Cd302 expression in human HCV entry factor transgenic mice increased hepatocyte permissiveness for an adapted HCV strain and dysregulated expression of metabolic process and host defense genes. These findings highlight human-mouse differences in liver-intrinsic antiviral immunity and facilitate the development of next-generation murine models for preclinical testing of HCV vaccine candidates.

C19orf66 is an interferon-induced inhibitor of HCV replication that restricts formation of the viral replication organelle.

BACKGROUND & AIMS: HCV is a positive-strand RNA virus that primarily infects human hepatocytes. Recent studies have reported that C19orf66 is expressed as an interferon (IFN)-stimulated gene; however, the intrinsic regulation of this gene within the liver as well as its antiviral effects against HCV remain elusive. METHODS: Expression of C19orf66 was quantified in both liver biopsies and primary human hepatocytes, with or without HCV infection. Mechanistic studies of the potent anti-HCV phenotype mediated by C19orf66 were conducted using state-of-the-art virological, biochemical and genetic approaches, as well as correlative light and electron microscopy and transcriptome and proteome analysis. RESULTS: Upregulation of C19orf66 mRNA was observed in both primary human hepatocytes upon HCV infection and in the livers of patients with chronic hepatitis C (CHC). In addition, pegIFNα/ribavirin therapy induced C19orf66 expression in patients with CHC. Transcriptomic profiling and whole cell proteomics of hepatoma cells ectopically expressing C19orf66 revealed no induction of other antiviral genes. Expression of C19orf66 restricted HCV infection, whereas CRIPSPR/Cas9 mediated knockout of C19orf66 attenuated IFN-mediated suppression of HCV replication. Co-immunoprecipitation followed by mass spectrometry identified a stress granule protein-dominated interactome of C19orf66. Studies with subgenomic HCV replicons and an expression system revealed that C19orf66 expression impairs HCV-induced elevation of phosphatidylinositol-4-phosphate, alters the morphology of the viral replication organelle (termed the membranous web) and thereby targets viral RNA replication. CONCLUSION: C19orf66 is an IFN-stimulated gene, which is upregulated in hepatocytes within the first hours post IFN treatment or HCV infection in vivo. The encoded protein possesses specific antiviral activity against HCV and targets the formation of the membranous web. Our study identifies C19orf66 as an IFN-inducible restriction factor with a novel antiviral mechanism that specifically targets HCV replication. LAY SUMMARY: Interferon-stimulated genes are thought to be important to for antiviral immune responses to HCV. Herein, we analysed C19orf66, an interferon-stimulated gene, which appears to inhibit HCV replication. It prevents the HCV-induced elevation of phosphatidylinositol-4-phosphate and alters the morphology of HCV's replication organelle.

Yellow Fever: Integrating Current Knowledge with Technological Innovations to Identify Strategies for Controlling a Re-Emerging Virus.

Yellow fever virus (YFV) represents a re-emerging zoonotic pathogen, transmitted by mosquito vectors to humans from primate reservoirs. Sporadic outbreaks of YFV occur in endemic tropical regions, causing a viral hemorrhagic fever (VHF) associated with high mortality rates. Despite a highly effective vaccine, no antiviral treatments currently exist. Therefore, YFV represents a neglected tropical disease and is chronically understudied, with many aspects of YFV biology incompletely defined including host range, host-virus interactions and correlates of host immunity and pathogenicity. In this article, we review the current state of YFV research, focusing on the viral lifecycle, host responses to infection, species tropism and the success and associated limitations of the YFV-17D vaccine. In addition, we highlight the current lack of available treatments and use publicly available sequence and structural data to assess global patterns of YFV sequence diversity and identify potential drug targets. Finally, we discuss how technological advances, including real-time epidemiological monitoring of outbreaks using next-generation sequencing and CRISPR/Cas9 modification of vector species, could be utilized in future battles against this re-emerging pathogen which continues to cause devastating disease.

Identification of Keratin 23 as a Hepatitis C Virus-Induced Host Factor in the Human Liver.

Keratin proteins form intermediate filaments, which provide structural support for many tissues. Multiple keratin family members are reported to be associated with the progression of liver disease of multiple etiologies. For example, keratin 23 (KRT23) was reported as a stress-inducible protein, whose expression levels correlate with the severity of liver disease. Hepatitis C virus (HCV) is a human pathogen that causes chronic liver diseases including fibrosis, cirrhosis, and hepatocellular carcinoma. However, a link between KRT23 and hepatitis C virus (HCV) infection has not been reported previously. In this study, we investigated KRT23 mRNA levels in datasets from liver biopsies of chronic hepatitis C (CHC) patients and in primary human hepatocytes experimentally infected with HCV, in addition to hepatoma cells. Interestingly, in each of these specimens, we observed an HCV-dependent increase of mRNA levels. Importantly, the KRT23 protein levels in patient plasma decreased upon viral clearance. Ectopic expression of KRT23 enhanced HCV infection; however, CRIPSPR/Cas9-mediated knockout did not show altered replication efficiency. Taken together, our study identifies KRT23 as a novel, virus-induced host-factor for hepatitis C virus.

A central hydrophobic E1 region controls the pH range of hepatitis C virus membrane fusion and susceptibility to fusion inhibitors.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection causes chronic liver disease. Antivirals have been developed and cure infection. However, resistance can emerge and salvage therapies with alternative modes of action could be useful. Several licensed drugs have emerged as HCV entry inhibitors and are thus candidates for drug repurposing. We aimed to dissect their mode of action, identify improved derivatives and determine their viral targets. METHODS: HCV entry inhibition was tested for a panel of structurally related compounds, using chimeric viruses representing diverse genotypes, in addition to viruses containing previously determined resistance mutations. Chemical modeling and synthesis identified improved derivatives, while generation of susceptible and non-susceptible chimeric viruses pinpointed E1 determinants of compound sensitivity. RESULTS: Molecules of the diphenylpiperazine, diphenylpiperidine, phenothiazine, thioxanthene, and cycloheptenepiperidine chemotypes inhibit HCV infection by interfering with membrane fusion. These molecules and a novel p-methoxy-flunarizine derivative with improved efficacy preferentially inhibit genotype 2 viral strains. Viral residues within a central hydrophobic region of E1 (residues 290-312) control susceptibility. At the same time, viral features in this region also govern pH-dependence of viral membrane fusion. CONCLUSIONS: Small molecules from different chemotypes related to flunarizine preferentially inhibit HCV genotype 2 membrane fusion. A hydrophobic region proximal to the putative fusion loop controls sensitivity to these drugs and the pH range of membrane fusion. An algorithm considering viral features in this region predicts viral sensitivity to membrane fusion inhibitors. Resistance to flunarizine correlates with more relaxed pH requirements for fusion. LAY SUMMARY: This study describes diverse compounds that act as HCV membrane fusion inhibitors. It defines viral properties that determine sensitivity to these molecules and thus provides information to identify patients that may benefit from treatment with membrane fusion inhibitors.

Hepatitis C Virus.

Hepatitis C virus (HCV) is an enveloped, RNA virus transmitted through blood-to-blood contact. It infects humans only and primarily targets liver cells. HCV evades innate and adaptive immunity and establishes chronic infections in 70% of cases. If untreated, 20% of patients develop liver cirrhosis, and a fraction of these progress to hepatocellular carcinoma. Annually, 400000 patients die globally due to HCV infection. Direct-acting antivirals (DAAs) are licensed and target three viral proteins: the NS3-4A protease needed for processing the viral polyprotein, the NS5A phosphoprotein that regulates RNA replication and virus assembly, and the viral RNA-dependent RNA polymerase (NS5B) that catalyzes genome replication. Combination therapies cure more than 95% of treated patients. Approximately 71 million people are chronically infected and 1.7 million new infections occur annually. Treatment-induced cure does not protect from viral reinfection. A prophylactic vaccine is under development.