Viral protease cleavage of MAVS in genetically modified mice with hepatitis A virus infection.

BACKGROUND & AIMS: Consistent with its relatively narrow host species range, hepatitis A virus (HAV) cannot infect C57BL/6 mice. However, in Mavs-/- mice with genetic deficiency of the innate immune signaling adaptor MAVS, HAV replicates robustly in the absence of disease. The HAV 3ABC protease cleaves MAVS in human cells, thereby disrupting virus-induced IFN responses, but it cannot cleave murine MAVS (mMAVS) due to sequence differences at the site of scission. Here, we sought to elucidate the role of 3ABC MAVS cleavage in determining HAV pathogenesis and host species range. METHODS: Using CRISPR/Cas9 gene editing, we established two independent lineages of C57BL/6 mice with knock-in mutations altering two amino acids in mMAVS ('mMAVS-VS'), rendering it susceptible to 3ABC cleavage without loss of signaling function. We challenged homozygous Mavsvs/vs mice with HAV, and compared infection outcomes with C57BL/6 and genetically deficient Mavs-/- mice. RESULTS: The humanized murine mMAVS-VS protein was cleaved as efficiently as human MAVS when co-expressed with 3ABC in Huh-7 cells. In embyronic fibroblasts from Mavsvs/vs mice, mMAVS-VS was cleaved by ectopically expressed 3ABC, significantly disrupting Sendai virus-induced IFN responses. However, in contrast to Mavs-/- mice with genetic MAVS deficiency, HAV failed to establish infection in Mavsvs/vs mice, even with additional genetic knockout of Trif or Irf1. Nonetheless, when crossed with permissive Ifnar1-/- mice lacking type I IFN receptors, Mavsvs/vsIfnar1-/- mice demonstrated enhanced viral replication coupled with significant reductions in serum alanine aminotransferase, hepatocellular apoptosis, and intrahepatic inflammatory cell infiltrates compared with Ifnar1-/- mice. CONCLUSIONS: MAVS cleavage by 3ABC boosts viral replication and disrupts disease pathogenesis, but it is not by itself sufficient to break the host-species barrier to HAV infection in mice. IMPACT AND IMPLICATIONS: The limited host range of human hepatitis viruses could be explained by species-specific viral strategies that disrupt innate immune responses. Both hepatitis A virus (HAV) and hepatitis C virus express viral proteases that cleave the innate immune adaptor protein MAVS, in human but not mouse cells. However, the impact of this immune evasion strategy has never been assessed in vivo. Here we show that HAV 3ABC protease cleavage of MAVS enhances viral replication and lessens liver inflammation in mice lacking interferon receptors, but that it is insufficient by itself to overcome the cross-species barrier to infection in mice. These results enhance our understanding of how hepatitis viruses interact with the host and their impact on innate immune responses.

Nonlytic Quasi-Enveloped Hepatovirus Release Is Facilitated by pX Protein Interaction with the E3 Ubiquitin Ligase ITCH.

Hepatoviruses are atypical hepatotropic picornaviruses that are released from infected cells without lysis in small membranous vesicles. These exosome-like, quasi-enveloped virions (eHAV) are infectious and the only form of hepatitis A virus (HAV) found circulating in blood during acute infection. eHAV is released through multivesicular endosomes in a process dependent on endosomal sorting complexes required for transport (ESCRT). Capsid protein interactions with the ESCRT-associated Bro1 domain proteins, ALG-2-interacting protein X (ALIX) and His domain-containing protein tyrosine phosphatase (HD-PTP), which are both recruited to the pX domain of 1D (VP1pX), are critical for this process. Previous proteomics studies suggest pX also binds the HECT domain, NEDD4 family E3 ubiquitin ligase, ITCH. Here, we confirm this interaction and show ITCH binds directly to the carboxy-terminal half of pX from both human and bat hepatoviruses independently of ALIX. A small chemical compound (compound 5) designed to disrupt interactions between WW domains of NEDD4 ligases and substrate molecules blocked ITCH binding to pX and demonstrated substantial antiviral activity against HAV. CRISPR deletion or small interfering RNA (siRNA) knockdown of ITCH expression inhibited the release of a self-assembling nanocage protein fused to pX and also impaired the release of eHAV from infected cells. The release could be rescued by overexpression of wild-type ITCH, but not a catalytically inactive ITCH mutant. Despite this, we found no evidence that ITCH ubiquitylates pX or that eHAV release is strongly dependent upon Lys residues in pX. These data indicate ITCH plays an important role in the ESCRT-dependent release of quasi-enveloped hepatovirus, although the substrate molecule targeted for ubiquitylation remains to be determined. IMPORTANCE Mechanisms underlying the cellular release of quasi-enveloped hepatoviruses are only partially understood, yet play a crucial role in the pathogenesis of this common agent of viral hepatitis. Multiple NEDD4 family E3 ubiquitin ligases, including ITCH, have been reported to promote the budding of conventional enveloped viruses but are not known to function in the release of HAV or other picornaviruses from infected cells. Here, we show that the unique C-terminal pX extension of the VP1 capsid protein of HAV interacts directly with ITCH and that ITCH promotes eHAV release in a manner analogous to its role in budding of some conventional enveloped viruses. The catalytic activity of ITCH is required for efficient eHAV release and may potentially function to ubiquitylate the viral capsid or activate ESCRT components.

Nonlytic cellular release of hepatitis A virus requires dual capsid recruitment of the ESCRT-associated Bro1 domain proteins HD-PTP and ALIX.

Although picornaviruses are conventionally considered 'nonenveloped', members of multiple picornaviral genera are released nonlytically from infected cells in extracellular vesicles. The mechanisms underlying this process are poorly understood. Here, we describe interactions of the hepatitis A virus (HAV) capsid with components of host endosomal sorting complexes required for transport (ESCRT) that play an essential role in release. We show release of quasi-enveloped virus (eHAV) in exosome-like vesicles requires a conserved export signal located within the 8 kDa C-terminal VP1 pX extension that functions in a manner analogous to late domains of canonical enveloped viruses. Fusing pX to a self-assembling engineered protein nanocage (EPN-pX) resulted in its ESCRT-dependent release in extracellular vesicles. Mutational analysis identified a 24 amino acid peptide sequence located within the center of pX that was both necessary and sufficient for nanocage release. Deleting a YxxL motif within this sequence ablated eHAV release, resulting in virus accumulating intracellularly. The pX export signal is conserved in non-human hepatoviruses from a wide range of mammalian species, and functional in pX sequences from bat hepatoviruses when fused to the nanocage protein, suggesting these viruses are released as quasi-enveloped virions. Quantitative proteomics identified multiple ESCRT-related proteins associating with EPN-pX, including ALG2-interacting protein X (ALIX), and its paralog, tyrosine-protein phosphatase non-receptor type 23 (HD-PTP), a second Bro1 domain protein linked to sorting of ubiquitylated cargo into multivesicular endosomes. RNAi-mediated depletion of either Bro1 domain protein impeded eHAV release. Super-resolution fluorescence microscopy demonstrated colocalization of viral capsids with endogenous ALIX and HD-PTP. Co-immunoprecipitation assays using biotin-tagged peptides and recombinant proteins revealed pX interacts directly through the export signal with N-terminal Bro1 domains of both HD-PTP and ALIX. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows release requires non-redundant activities of both HD-PTP and ALIX.

IRF3-mediated pathogenicity in a murine model of human hepatitis A.

HAV-infected Ifnar1-/- mice recapitulate many of the cardinal features of hepatitis A in humans, including serum alanine aminotransferase (ALT) elevation, hepatocellular apoptosis, and liver inflammation. Previous studies implicate MAVS-IRF3 signaling in pathogenesis, but leave unresolved the role of IRF3-mediated transcription versus the non-transcriptional, pro-apoptotic activity of ubiquitylated IRF3. Here, we compare the intrahepatic transcriptomes of infected versus naïve Mavs-/- and Ifnar1-/- mice using high-throughput sequencing, and identify IRF3-mediated transcriptional responses associated with hepatocyte apoptosis and liver inflammation. Infection was transcriptionally silent in Mavs-/- mice, in which HAV replicates robustly within the liver without inducing inflammation or hepatocellular apoptosis. By contrast, infection resulted in the upregulation of hundreds of genes in Ifnar1-/- mice that develop acute hepatitis closely modeling human disease. Upregulated genes included pattern recognition receptors, interferons, chemokines, cytokines and other interferon-stimulated genes. Compared with Ifnar1-/- mice, HAV-induced inflammation was markedly attenuated and there were few apoptotic hepatocytes in livers of infected Irf3S1/S1Ifnar1-/- mice in which IRF3 is transcriptionally-inactive due to alanine substitutions at Ser-388 and Ser-390. Although transcriptome profiling revealed remarkably similar sets of genes induced in Irf3S1/S1Ifnar1-/- and Ifnar1-/- mice, a subset of genes was differentially expressed in relation to the severity of the liver injury. Prominent among these were both type 1 and type III interferons and interferon-responsive genes associated previously with apoptosis, including multiple members of the ISG12 and 2'-5' oligoadenylate synthetase families. Ifnl3 and Ifnl2 transcript abundance correlated strongly with disease severity, but mice with dual type 1 and type III interferon receptor deficiency remained fully susceptible to liver injury. Collectively, our data show that IRF3-mediated transcription is required for HAV-induced liver injury in mice and identify key IRF3-responsive genes associated with pathogenicity, providing a clear distinction from the transcription-independent role of IRF3 in liver injury following binge exposure to alcohol.

A herpesvirus transactivator and cellular POU proteins extensively regulate DNA binding of the host Notch signaling protein RBP-Jκ to the virus genome.

Reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) from latency requires the viral transactivator Rta to contact the host protein Jκ recombination signal-binding protein (RBP-Jκ or CSL). RBP-Jκ normally binds DNA sequence-specifically to determine the transcriptional targets of the Notch-signaling pathway, yet Notch alone cannot reactivate KSHV. We previously showed that Rta stimulates RBP-Jκ DNA binding to the viral genome. On a model viral promoter, this function requires Rta to bind to multiple copies of an Rta DNA motif (called "CANT" or Rta-c) proximal to an RBP-Jκ motif. Here, high-resolution ChIP/deep sequencing from infected primary effusion lymphoma cells revealed that RBP-Jκ binds nearly exclusively to different sets of viral genome sites during latency and reactivation. RBP-Jκ bound DNA frequently, but not exclusively, proximal to Rta bound to single, but not multiple, Rta-c motifs. To discover additional regulators of RBP-Jκ DNA binding, we used bioinformatics to identify cellular DNA-binding protein motifs adjacent to either latent or reactivation-specific RBP-Jκ-binding sites. Many of these cellular factors, including POU class homeobox (POU) proteins, have known Notch or herpesvirus phenotypes. Among a set of Rta- and RBP-Jκ-bound promoters, Rta transactivated only those that also contained POU motifs in conserved positions. On some promoters, POU factors appeared to inhibit RBP-Jκ DNA binding unless Rta bound to a proximal Rta-c motif. Moreover, POU2F1/Oct-1 expression was induced during KSHV reactivation, and POU2F1 knockdown diminished infectious virus production. Our results suggest that Rta and POU proteins broadly regulate DNA binding of RBP-Jκ during KSHV reactivation.

Basal expression of interferon regulatory factor 1 drives intrinsic hepatocyte resistance to multiple RNA viruses.

Current models of cell-intrinsic immunity to RNA viruses centre on virus-triggered inducible antiviral responses initiated by RIG-I-like receptors or Toll-like receptors that sense pathogen-associated molecular patterns, and signal downstream through interferon regulatory factors (IRFs), transcription factors that induce synthesis of type I and type III interferons1. RNA viruses have evolved sophisticated strategies to disrupt these signalling pathways and evade elimination by cells, attesting to their importance2. Less attention has been paid to how IRFs maintain basal levels of protection against viruses. Here, we depleted antiviral factors linked to RIG-I-like receptor and Toll-like receptor signalling to map critical host pathways restricting positive-strand RNA virus replication in immortalized hepatocytes and identified an unexpected role for IRF1. We show that constitutively expressed IRF1 acts independently of mitochondrial antiviral signalling (MAVS) protein, IRF3 and signal transducer and activator of transcription 1 (STAT1)-dependent signalling to provide intrinsic antiviral protection in actinomycin D-treated cells. IRF1 localizes to the nucleus, where it maintains the basal transcription of a suite of antiviral genes that protect against multiple pathogenic RNA viruses, including hepatitis A and C viruses, dengue virus and Zika virus. Our findings reveal an unappreciated layer of hepatocyte-intrinsic immunity to these positive-strand RNA viruses and identify previously unrecognized antiviral effector genes.

Cellular entry and uncoating of naked and quasi-enveloped human hepatoviruses.

Many 'non-enveloped' viruses, including hepatitis A virus (HAV), are released non-lytically from infected cells as infectious, quasi-enveloped virions cloaked in host membranes. Quasi-enveloped HAV (eHAV) mediates stealthy cell-to-cell spread within the liver, whereas stable naked virions shed in feces are optimized for environmental transmission. eHAV lacks virus-encoded surface proteins, and how it enters cells is unknown. We show both virion types enter by clathrin- and dynamin-dependent endocytosis, facilitated by integrin ß1, and traffic through early and late endosomes. Uncoating of naked virions occurs in late endosomes, whereas eHAV undergoes ALIX-dependent trafficking to lysosomes where the quasi-envelope is enzymatically degraded and uncoating ensues coincident with breaching of endolysosomal membranes. Neither virion requires PLA2G16, a phospholipase essential for entry of other picornaviruses. Thus naked and quasi-enveloped virions enter via similar endocytic pathways, but uncoat in different compartments and release their genomes to the cytosol in a manner mechanistically distinct from other Picornaviridae.

Redundant Late Domain Functions of Tandem VP2 YPX3L Motifs in Nonlytic Cellular Egress of Quasi-enveloped Hepatitis A Virus.

The quasi-envelopment of hepatitis A virus (HAV) capsids in exosome-like virions (eHAV) is an important but incompletely understood aspect of the hepatovirus life cycle. This process is driven by recruitment of newly assembled capsids to endosomal vesicles into which they bud to form multivesicular bodies with intraluminal vesicles that are later released at the plasma membrane as eHAV. The endosomal sorting complexes required for transport (ESCRT) are key to this process, as is the ESCRT-III-associated protein, ALIX, which also contributes to membrane budding of conventional enveloped viruses. YPX1or3L late domains in the structural proteins of these viruses mediate interactions with ALIX, and two such domains exist in the HAV VP2 capsid protein. Mutational studies of these domains are confounded by the fact that the Tyr residues (important for interactions of YPX1or3L peptides with ALIX) are required for efficient capsid assembly. However, single Leu-to-Ala substitutions within either VP2 YPX3L motif (L1-A and L2-A mutants) were well tolerated, albeit associated with significantly reduced eHAV release. In contrast, simultaneous substitutions in both motifs (L1,2-A) eliminated virus release but did not inhibit assembly of infectious intracellular particles. Immunoprecipitation experiments suggested that the loss of eHAV release was associated with a loss of ALIX recruitment. Collectively, these data indicate that HAV YPX3L motifs function as redundant late domains during quasi-envelopment and viral release. Since these motifs present little solvent-accessible area in the crystal structure of the naked extracellular capsid, the capsid structure may be substantially different during quasi-envelopment.IMPORTANCE Nonlytic release of hepatitis A virus (HAV) as exosome-like quasi-enveloped virions is a unique but incompletely understood aspect of the hepatovirus life cycle. Several lines of evidence indicate that the host protein ALIX is essential for this process. Tandem YPX3L "late domains" in the VP2 capsid protein could be sites of interaction with ALIX, but they are not accessible on the surface of an X-ray model of the extracellular capsid, raising doubts about this putative late domain function. Here, we describe YPX3L domain mutants that assemble capsids normally but fail to bind ALIX and be secreted as quasi-enveloped eHAV. Our data support late domain function for the VP2 YPX3L motifs and raise questions about the structure of the HAV capsid prior to and following quasi-envelopment.

NLRX1 promotes immediate IRF1-directed antiviral responses by limiting dsRNA-activated translational inhibition mediated by PKR.

NLRX1 is unique among the nucleotide-binding-domain and leucine-rich-repeat (NLR) proteins in its mitochondrial localization and ability to negatively regulate antiviral innate immunity dependent on the adaptors MAVS and STING. However, some studies have suggested a positive regulatory role for NLRX1 in inducing antiviral responses. We found that NLRX1 exerted opposing regulatory effects on viral activation of the transcription factors IRF1 and IRF3, which might potentially explain such contradictory results. Whereas NLRX1 suppressed MAVS-mediated activation of IRF3, it conversely facilitated virus-induced increases in IRF1 expression and thereby enhanced control of viral infection. NLRX1 had a minimal effect on the transcription of IRF1 mediated by the transcription factor NF-kB and regulated the abundance of IRF1 post-transcriptionally by preventing translational shutdown mediated by the double-stranded RNA (dsRNA)-activated kinase PKR and thereby allowed virus-induced increases in the abundance of IRF1 protein.

TIM1 (HAVCR1) Is Not Essential for Cellular Entry of Either Quasi-enveloped or Naked Hepatitis A Virions.

Receptor molecules play key roles in the cellular entry of picornaviruses, and TIM1 (HAVCR1) is widely accepted to be the receptor for hepatitis A virus (HAV), an unusual, hepatotropic human picornavirus. However, its identification as the hepatovirus receptor predated the discovery that hepatoviruses undergo nonlytic release from infected cells as membrane-cloaked, quasi-enveloped HAV (eHAV) virions that enter cells via a pathway distinct from naked, nonenveloped virions. We thus revisited the role of TIM1 in hepatovirus entry, examining both adherence and infection/replication in cells with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-engineered TIM1 knockout. Cell culture-derived, gradient-purified eHAV bound Huh-7.5 human hepatoma cells less efficiently than naked HAV at 4°C, but eliminating TIM1 expression caused no difference in adherence of either form of HAV, nor any impact on infection and replication in these cells. In contrast, TIM1-deficient Vero cells showed a modest reduction in quasi-enveloped eHAV (but not naked HAV) attachment and replication. Thus, TIM1 facilitates quasi-enveloped eHAV entry in Vero cells, most likely by binding phosphatidylserine (PtdSer) residues on the eHAV membrane. Both Tim1-/-Ifnar1-/- and Tim4-/-Ifnar1-/- double-knockout mice were susceptible to infection upon intravenous challenge with infected liver homogenate, with fecal HAV shedding and serum alanine aminotransferase (ALT) elevations similar to those in Ifnar1-/- mice. However, intrahepatic HAV RNA and ALT elevations were modestly reduced in Tim1-/-Ifnar1-/- mice compared to Ifnar1-/- mice challenged with a lower titer of gradient-purified HAV or eHAV. We conclude that TIM1 is not an essential hepatovirus entry factor, although its PtdSer-binding activity may contribute to the spread of quasi-enveloped virus and liver injury in mice.IMPORTANCE T cell immunoglobulin and mucin-containing domain protein 1 (TIM1) was reported more than 2 decades ago to be an essential cellular receptor for hepatitis A virus (HAV), a picornavirus in the Hepatovirus genus, resulting in its designation as "hepatitis A virus cellular receptor 1" (HAVCR1) by the Human Genome Organization Gene Nomenclature Committee. However, recent studies have shown that HAV exists in nature as both naked, nonenveloped (HAV) virions and membrane-cloaked, quasi-enveloped infectious virus (eHAV), prompting us to revisit the role of TIM1 in viral entry. We show here that TIM1 (HAVCR1) is not an essential cellular receptor for HAV entry into cultured cells or required for viral replication and pathogenesis in permissive strains of mice, although it may facilitate early stages of infection by binding phosphatidylserine on the eHAV surface. This work thus corrects the published record and sets the stage for future efforts to identify specific hepatovirus entry factors.

Protein composition of the hepatitis A virus quasi-envelope.

The Picornaviridae are a diverse family of RNA viruses including many pathogens of medical and veterinary importance. Classically considered "nonenveloped," recent studies show that some picornaviruses, notably hepatitis A virus (HAV; genus Hepatovirus) and some members of the Enterovirus genus, are released from cells nonlytically in membranous vesicles. To better understand the biogenesis of quasi-enveloped HAV (eHAV) virions, we conducted a quantitative proteomics analysis of eHAV purified from cell-culture supernatant fluids by isopycnic ultracentrifugation. Amino acid-coded mass tagging (AACT) with stable isotopes followed by tandem mass spectrometry sequencing and AACT quantitation of peptides provided unambiguous identification of proteins associated with eHAV versus unrelated extracellular vesicles with similar buoyant density. Multiple peptides were identified from HAV capsid proteins (53.7% coverage), but none from nonstructural proteins, indicating capsids are packaged as cargo into eHAV vesicles via a highly specific sorting process. Other eHAV-associated proteins (n = 105) were significantly enriched for components of the endolysosomal system (>60%, P < 0.001) and included many common exosome-associated proteins such as the tetraspanin CD9 and dipeptidyl peptidase 4 (DPP4) along with multiple endosomal sorting complex required for transport III (ESCRT-III)-associated proteins. Immunoprecipitation confirmed that DPP4 is displayed on the surface of eHAV produced in cell culture or present in sera from humans with acute hepatitis A. No LC3-related peptides were identified by mass spectrometry. RNAi depletion studies confirmed that ESCRT-III proteins, particularly CHMP2A, function in eHAV biogenesis. In addition to identifying surface markers of eHAV vesicles, the results support an exosome-like mechanism of eHAV egress involving endosomal budding of HAV capsids into multivesicular bodies.

The PHES battery does not detect all cirrhotic patients with early neurological deficits, which are different in different patients.

BACKGROUND AND AIMS: The psychometric hepatic encephalopathy score (PHES) is the "gold standard" for minimal hepatic encephalopathy (MHE) diagnosis. Some reports suggest that some cirrhotic patients "without" MHE according to PHES show neurological deficits and other reports that neurological alterations are not homogeneous in all cirrhotic patients. This work aimed to assess whether: 1) a relevant proportion of cirrhotic patients show neurological deficits not detected by PHES; 2) cirrhotic patients with mild neurological deficits are a homogeneous population or may be classified in sub-groups according to specific deficits. METHODS: Cirrhotic patients "without" (n = 56) or "with" MHE (n = 41) according to PHES and controls (n = 52) performed psychometric tests assessing attention, concentration, mental processing speed, working memory and bimanual and visuomotor coordination. Heterogeneity of neurological alterations was analysed using Hierarchical Clustering Analysis. RESULTS: PHES classified as "with" MHE 42% of patients. Around 40% of patients "without" MHE according to PHES fail two psychometric tests. Oral SDMT, d2, bimanual and visuo-motor coordination tests are failed by 54, 51, 51 and 43% of patients, respectively. The earliest neurological alterations are different for different patients. Hierarchical clustering analysis shows that patients "without" MHE according to PHES may be classified in clusters according to the tests failed. In some patients coordination impairment appear before cognitive impairment while in others concentration and attention deficits appear before. CONCLUSIONS: PHES is not sensitive enough to detect early neurological alterations in a relevant proportion of cirrhotic patients. Oral SDMT, d2 and bimanual and visuo-motor coordination tests are more sensitive. The earliest neurological alterations are different in different cirrhotic patients. These data also have relevant clinical implications. Patients classified as "without MHE" by PHES belonging to clusters 3 and 4 in our study have a high risk of suffering clinical complications, including overt HE and must be diagnosed and clinically followed.

Altered postural control and stability in cirrhotic patients with minimal hepatic encephalopathy correlate with cognitive deficits.

BACKGROUND & AIMS: Cognitive dysfunction in cirrhotic patients with minimal hepatic encephalopathy (MHE) is associated with falls. Alterations in postural control and stability could contribute to increase falls risk in these patients. We aimed to assess whether postural control and direction-specific limits of stability are altered in cirrhotic patients with MHE compared to patients without MHE and controls. We also assessed if alterations in postural control correlate with neurological impairment and/or blood biomarkers. METHODS: Posturography analysis, attention Stroop test and bimanual and visuo-motor coordination tests were performed in 18 controls, 19 patients with cirrhosis without MHE and 17 with MHE, diagnosed by PHES. Posturography was assessed by NedSVE® /IBV system under four sensory conditions. Limits of stability and rhythmic weight-shifting tests were also performed. Blood ammonia and serum interleukins were also measured. Falls were assessed after 12-24 months follow-up. RESULTS: MHE patients show impaired balance, mainly on unstable surface with eyes open, with longer reaction and confinement times and lower success in Limits of Stability test compared to patients without MHE. Performance in attention and motor coordination tests correlated with most posturography parameters alterations. Logistic regression analysis shows that posturography parameters and bimanual coordination test are good predictors of falls. CONCLUSION: Balance patterns and limits of stability in MHE patients are impaired compared to patients without MHE and controls. This seems to contribute to a higher falls risk. Attention and motor coordination deficits could contribute to balance impairment in patients with MHE.

MAVS-dependent host species range and pathogenicity of human hepatitis A virus.

Hepatotropic viruses are important causes of human disease, but the intrahepatic immune response to hepatitis viruses is poorly understood because of a lack of tractable small- animal models. We describe a murine model of hepatitis A virus (HAV) infection that recapitulates critical features of type A hepatitis in humans. We demonstrate that the capacity of HAV to evade MAVS-mediated type I interferon responses defines its host species range. HAV-induced liver injury was associated with interferon-independent intrinsic hepatocellular apoptosis and hepatic inflammation that unexpectedly resulted from MAVS and IRF3/7 signaling. This murine model thus reveals a previously undefined link between innate immune responses to virus infection and acute liver injury, providing a new paradigm for viral pathogenesis in the liver.

Is cognitive impairment in cirrhotic patients due to increased peroxynitrite and oxidative stress?

Cirrhotic patients may suffer minimal hepatic encephalopathy (MHE), with mild cognitive impairment. 3-Nitro-tyrosine levels are a good biomarker for diagnosis of the cognitive impairment and MHE in cirrhotic patients. This suggests that oxidative stress could be involved in the induction of cognitive and motor alterations in MHE. We have observed that patients with MHE show increased oxidative stress in blood compared with cirrhotic patients without MHE, with increased lipid peroxidation, DNA oxidation, protein carbonylation, 3-nitrotyrosine, oxidized glutathione (GSSG)/reduced glutathione (GSH) ratio, and GSH levels. The activities of antioxidant enzymes are enhanced in erythrocytes and mononuclear cells from patients with and without MHE compared with control subjects. Only glutathione peroxidase activity was increased in MHE patients compared with patients without MHE. Oxidative stress markers in blood, especially GSSG/GSH ratio, GSH, malondialdehyde, and 3-nitrotyrosine, correlate with deficits in attention and motor coordination. The increase in antioxidant activities in patients would be an adaptive mechanism to cope with enhanced oxidative stress, although it is not effective enough to normalize it. Our observations lead to the hypothesis that oxidative stress and increased peroxynitrite formation would mediate the synergistic effects of hyperammonemia and inflammation on cognitive and motor impairment in MHE.

Debut de hepatitis crónica autoinmune como prúrigo nodular / Autoimmune chronic hepatitis presenting as prurigo nodularis

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Patients with minimal hepatic encephalopathy show impaired mismatch negativity correlating with reduced performance in attention tests.

UNLABELLED: Attention deficit is an early event in the cognitive impairment of patients with minimal hepatic encephalopathy (MHE). The underlying mechanisms remain unclear. Mismatch negativity (MMN) is an auditory event-related potential that reflects an attentional trigger. Patients with schizophrenia show impaired attention and cognitive function, which are reflected in altered MMN. We hypothesized that patients with MHE, similarly to those with schizophrenia, should show MMN alterations related with attention deficits. The aims of this work were to assess whether (1) MMN is altered in cirrhotic patients with MHE, compared to those without MHE, (2) MMN changes in parallel with performance in attention tests and/or MHE in a longitudinal study, and (3) MMN predicts performance in attention tests and/or in the Psychometric Hepatic Encephalopathy Score (PHES). We performed MMN analysis as well as attention and coordination tests in 34 control subjects and in 37 patients with liver cirrhosis without MHE and 23 with MHE. Patients with MHE show reduced performance in selective and sustained attention tests and in visuomotor and bimanual coordination tests. The MMN wave area was reduced in patients with MHE, but not in those without MHE. In the longitudinal study, MMN area improved in parallel with performance in attention tests and PHES in 4 patients and worsened in parallel in another 4. Logistic regression analyses showed that MMN area predicts performance in attention tests and in PHES, but not in other tests or critical flicker frequency. Receiver operating characteristic curve analyses showed that MMN area predicts attention deficits in the number connection tests A and B, Stroop tasks, and MHE, with sensitivities of 75%-90% and specificities of 76%-83%. CONCLUSION: MMN area is useful to diagnose attention deficits and MHE in patients with liver cirrhosis.

Kaposi's sarcoma-associated herpesvirus Rta tetramers make high-affinity interactions with repetitive DNA elements in the Mta promoter to stimulate DNA binding of RBP-Jk/CSL.

Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 [HHV-8]) is the etiologic agent of Kaposi's sarcoma (KS) and lymphoproliferative diseases. We previously demonstrated that the KSHV lytic switch protein Rta stimulates DNA binding of the cellular RBP-Jk/CSL protein, the nuclear component of the Notch pathway, on Rta target promoters. In the current study, we define the promoter requirements for formation of transcriptionally productive Rta/RBP-Jk/DNA complexes. We show that highly pure Rta footprints 7 copies of a previously undescribed repetitive element in the promoter of the essential KSHV Mta gene. We have termed this element the "CANT repeat." CANT repeats are found on both strands of DNA and have a consensus sequence of ANTGTAACANT(A/T)(A/T)T. We demonstrate that Rta tetramers make high-affinity interactions (i.e., nM) with 64 bp of the Mta promoter but not single CANT units. The number of CANT repeats, their presence in palindromes, and their positions relative to the RBP-Jk binding site determine the optimal target for Rta stimulation of RBP-Jk DNA binding and formation of ternary Rta/RBP-Jk/DNA complexes. DNA binding and tetramerization mutants of Rta fail to stimulate RBP-Jk DNA binding. Our chromatin immunoprecipitation assays show that RBP-Jk DNA binding is broadly, but selectively, stimulated across the entire KSHV genome during reactivation. We propose a model in which tetramerization of Rta allows it to straddle RBP-Jk and contact repeat units on both sides of RBP-Jk. Our study integrates high-affinity Rta DNA binding with the requirement for a cellular transcription factor in Rta transactivation.

3-nitro-tyrosine as a peripheral biomarker of minimal hepatic encephalopathy in patients with liver cirrhosis.

OBJECTIVES: Between 30 and 50% of the cirrhotic patients who do not show symptoms of clinical hepatic encephalopathy (HE) present minimal hepatic encephalopathy (MHE), with mild cognitive impairment. MHE impairs the quality of life, increases the risk of suffering accidents, predicts the appearance of clinical HE, and is associated with shortened lifespan. Early detection of MHE would be very useful. The "gold standard" for MHE diagnosis is the psychometric hepatic encephalopathy score (PHES). However, it is time consuming and needs adjusting for age and educational level. It would be very useful to have some blood biomarker reflecting the presence of MHE in cirrhotic patients. The aim of this work was to identify serum molecules useful as biomarkers for MHE. METHODS: We measured in 63 controls, 43 cirrhotic patients without MHE, and 44 patients with MHE, from Hospital Clinico de Valencia, serum levels of different amino acids, cyclic guanosine monophosphate (cGMP), nitrites+nitrates, and 3-nitrotyrosine. We analyzed for each parameter its diagnostic accuracy as an indicator of MHE, as assessed using the PHES. RESULTS: These studies supported that 3-nitro-tyrosine is a good marker for MHE. To validate its utility as a biomarker for MHE, we analyzed in a second cohort of 44 cirrhotic patients without MHE and 18 patients with MHE, from Hospital Arnau de Vilanova, serum levels of 3-nitro-tyrosine, methionine, and citrulline. Citrulline (173±17%), methionine (173±16%), and 3-nitro-tyrosine (857±92%) were increased in sera from patients with MHE when compared with those without MHE. The receiver operating characteristic (ROC) curve analysis of 3-nitro-tyrosine for the diagnosis of MHE in the first cohort showed an area under the curve (AUC) value of 0.96 (95% confidence interval 0.93-0.99). At the cutoff of 14 nM, the specificity was 93%, sensitivity 89%, and positive and negative predictive values were both 91%. When the same cutoff was applied to the second cohort, the specificity was 83% and sensitivity was 94%. The positive and negative predictive values were 70 and 97%, respectively. CONCLUSIONS: This pilot study, to be validated in a larger cohort, shows that determination of 3-nitro-tyrosine in serum, which is easy and not time consuming, is useful to identify patients with MHE, with good sensitivity, specificity, and positive and negative predictive values.