Interferon lambda restricts herpes simplex virus skin disease by suppressing neutrophil-mediated pathology.

Type III interferons (IFN-λ) are antiviral and immunomodulatory cytokines that have been best characterized in respiratory and gastrointestinal infections, but the effects of IFN-λ against skin infections have not been extensively investigated. We sought to define the skin-specific effects of IFN-λ against the highly prevalent human pathogen, herpes simplex virus (HSV). We infected mice lacking the IFN-λ receptor (Ifnlr1-/-), both the IFN-λ and the IFN-α/ß receptors (Ifnar1-/-Ifnlr1-/-), or IFN-λ cytokines (Ifnl2/3-/-) and found that IFN-λ restricts the severity of HSV-1 and HSV-2 skin lesions without affecting viral loads. We used RNAseq to define IFN-λ- and IFN-ß-induced transcriptional responses in primary mouse keratinocytes. Using conditional knockout mice, we found that IFN-λ signaling in both keratinocytes and neutrophils was necessary to control HSV-1 skin lesion severity and that IFN-λ signaling in keratinocytes suppressed CXCL9-mediated neutrophil recruitment to the skin. Furthermore, depleting neutrophils or blocking CXCL9 protected against severe HSV-1 skin lesions in Ifnlr1-/- mice. Altogether, our results suggest that IFN-λ plays an immunomodulatory role in the skin that restricts neutrophil-mediated pathology during HSV infection and suggests potential applications for IFN-λ in treating viral skin infections.IMPORTANCEType III interferons (IFN-λ) have been shown to have antiviral and immunomodulatory effects at epithelial barriers such as the respiratory and gastrointestinal tracts, but their effects on the skin have not been extensively investigated. We used mice lacking IFN-λ signaling to investigate the skin-specific effects of IFN-λ against the herpes simplex virus (HSV), which targets epithelial tissues to cause cold sores and genital herpes. We found that IFN-λ limited the severity of HSV skin lesions without affecting viral load and that this protective effect required IFN-λ signaling in both keratinocytes and neutrophils. We found that IFN-λ signaling in keratinocytes suppressed neutrophil recruitment to the skin and that depleting neutrophils protected against severe HSV skin lesions in the absence of IFN-λ. Altogether, our results suggest that IFN-λ plays an immunomodulatory role in the skin that restricts neutrophil-mediated pathology during HSV infection and suggests potential applications for IFN-λ in treating viral skin infections.

Mice with FVB-derived sequence on chromosome 17 succumb to disseminated virus infection due to aberrant NK cell and T cell responses.

Zoonotic arenavirus infections can result in viral hemorrhagic disease, characterized by platelet loss, petechia, and multi-organ injury. The mechanisms governing these outcomes are likely impacted by virus strain and infection dose, as well as an individual's genetic background and immune constitution. To better understand the processes leading to severe pathogenesis, we compared two strains of inbred mice, C57BL/6J (B6) and FVB/NJ (FVB), that have diametrically opposed outcomes during disseminated lymphocytic choriomeningitis virus (LCMV) infection. Infection caused minimal pathogenesis in B6 mice, whereas FVB mice developed acute hepatitis and perished due, in part, to aberrant NK cell and T cell responses. Susceptible mice showed an outgrowth of cytolytic CD4+ T cells and loss of Treg cells. B6 congenic mice with the FVB allele at a 25Mb locus on chromosome 17 recapitulated FVB pathogenesis upon infection. A locus containing a limited number of variants in immune-related genes greatly impacts survival during infection.

Interferon lambda restricts herpes simplex virus skin disease by suppressing neutrophil-mediated pathology.

Type III interferons (IFN-λ) are antiviral and immunomodulatory cytokines that have been best characterized in respiratory and gastrointestinal infections, but the effects of IFN-λ against skin infections have not been extensively investigated. We sought to define the skin-specific effects of IFN-λ against the highly prevalent human pathogen herpes simplex virus (HSV). We infected mice lacking the IFN-λ receptor (Ifnlr1-/-), both the IFN-λ and the IFN-αß receptor (Ifnar1-/- Ifnlr1-/-), or IFN-λ cytokines (Ifnl2/3-/-) and found that IFN-λ restricts the severity of HSV-1 and HSV-2 skin lesions, independent of a direct effect on viral load. Using conditional knockout mice, we found that IFN-λ signaling in both keratinocytes and neutrophils was necessary to control HSV-1 skin lesion severity, and that IFN-λ signaling in keratinocytes suppressed CXCL9-mediated neutrophil recruitment to the skin. Furthermore, depleting neutrophils or blocking CXCL9 protected against severe HSV-1 skin lesions in Ifnlr1-/- mice. Altogether, our results suggest that IFN-λ plays an immunomodulatory role in the skin that restricts neutrophil-mediated pathology during HSV infection, and suggest potential applications for IFN-λ in treating viral skin infections.

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.

The ZCCHC14/TENT4 complex is required for hepatitis A virus RNA synthesis.

Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5' untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3' poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3' poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1-/- mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.

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.

T cells protect against hepatitis A virus infection and limit infection-induced liver injury.

BACKGROUND & AIMS: Hepatitis A virus (HAV) is a common cause of enterically transmitted viral hepatitis. In non-immune individuals, infection results in typically transient but occasionally fulminant and fatal inflammatory liver injury. Virus-specific T cell frequencies peak when liver damage is at its zenith, leading to the prevalent notion that T cells exacerbate liver disease, as suspected for other hepatotropic virus infections. However, the overall contribution of T cells to the control of HAV and the pathogenesis of hepatitis A is unclear and has been impeded by a historic lack of small animal models. METHODS: Ifnar1-/- mice are highly permissive for HAV and develop pathogenesis that recapitulates many features of hepatitis A. Using this model, we identified HAV-specific CD8+ and CD4+ T cells by epitope mapping, and then used tetramers and functional assays to quantify T cells in the liver at multiple times after infection. We assessed the relationships between HAV-specific T cell frequency, viral RNA amounts, and liver pathogenesis. RESULTS: A large population of virus-specific T cells accumulated within the livers of Ifnar1-/- mice during the first 1-2 weeks of infection and persisted over time. HAV replication was enhanced and liver disease exacerbated when mice were depleted of T cells. Conversely, immunization with a peptide vaccine increased virus-specific CD8+ T cell frequencies in the liver, reduced viral RNA abundance, and lessened liver injury. CONCLUSION: These data show that T cells protect against HAV-mediated liver injury and can be targeted to improve liver health. LAY SUMMARY: Hepatitis A virus is a leading cause of acute viral hepatitis worldwide. T cells were thought to contribute to liver injury during acute infection. We now show that virus-specific T cells protect against infection and limit liver injury.

Iminosugar Glucosidase Inhibitors Reduce Hepatic Inflammation in Hepatitis A Virus-Infected Ifnar1-/- Mice.

Iminosugar compounds are monosaccharide mimetics with broad but generally weak antiviral activities related to inhibition of enzymes involved in glycobiology. Miglustat (N-butyl-1-deoxynojirimycin), which is approved for the treatment of lipid storage diseases in humans, and UV-4 [N-(9-methoxynonyl)-1-deoxynojirimycin] inhibit the replication of hepatitis A virus (HAV) in cell culture (50% inhibitory concentrations [IC50s] of 32.13 µM and 8.05 µM, respectively) by blocking the synthesis of gangliosides essential for HAV cell entry. We used a murine model of hepatitis A and targeted mass spectrometry to assess the capacity of these compounds to deplete hepatic gangliosides and modify the course of HAV infection in vivo. Miglustat, given by gavage to Ifnar1-/- mice (4,800 mg/kg of body weight/day) depleted hepatic gangliosides by 69 to 75% but caused substantial gastrointestinal toxicity and failed to prevent viral infection. UV-4, similarly administered in high doses (400 mg/kg/day), was well tolerated but depleted hepatic gangliosides by only 20% after 14 days. UV-4 depletion of gangliosides varied by class. Several GM2 species were paradoxically increased, likely due to inhibition of ß-glucosidases that degrade gangliosides. Both compounds enhanced, rather than reduced, virus replication. Nonetheless, both iminosugars had surprising anti-inflammatory effects, blocking the accumulation of inflammatory cells within the liver. UV-4 treatment also resulted in a decrease in serum alanine aminotransferase (ALT) elevations associated with acute hepatitis A. These anti-inflammatory effects may result from iminosugar inhibition of cellular α-glucosidases, leading to impaired maturation of glycan moieties of chemokine and cytokine receptors, and point to the potential importance of paracrine signaling in the pathogenesis of acute hepatitis A. IMPORTANCE Hepatitis A virus (HAV) is a common cause of viral hepatitis. Iminosugar compounds block its replication in cultured cells by inhibiting the synthesis of gangliosides required for HAV cell entry but have not been tested for their ability to prevent or treat hepatitis A in vivo. We show that high doses of the iminosugars miglustat and UV-4 fail to deplete gangliosides sufficiently to block HAV infection in mice lacking a key interferon receptor. These compounds nonetheless have striking anti-inflammatory effects on the HAV-infected liver, reducing the severity of hepatitis despite enhancing chemokine and cytokine expression resulting from hepatocyte-intrinsic antiviral responses. We propose that iminosugar inhibition of cellular α-glucosidases impairs the maturation of glycan moieties of chemokine and cytokine receptors required for effective signaling. These data highlight the potential importance of paracrine signaling pathways in the inflammatory response to HAV and add to our understanding of HAV pathogenesis in mice.

Gangliosides are essential endosomal receptors for quasi-enveloped and naked hepatitis A virus.

The Picornaviridae are a diverse family of positive-strand RNA viruses that includes numerous human and veterinary pathogens1. Among these, hepatitis A virus (HAV), a common cause of acute hepatitis in humans, is unique in that it is hepatotropic and is released from hepatocytes without lysis in small vesicles that resemble exosomes2,3. These quasi-enveloped virions are infectious and are the only form of virus that can be detected in the blood during acute infection2. By contrast, non-enveloped naked virions are shed in faeces and stripped of membranes by bile salts during passage through the bile ducts to the gut4. How these two distinct types of infectious hepatoviruses enter cells to initiate infection is unclear. Here, we describe a genome-wide forward screen that shows that glucosylceramide synthase and other components of the ganglioside synthetic pathway are crucial host factors that are required for cellular entry by hepatoviruses. We show that gangliosides-preferentially disialogangliosides-function as essential endolysosome receptors that are required for infection by both naked and quasi-enveloped virions. In the absence of gangliosides, both virion types are efficiently internalized through endocytosis, but capsids fail to uncoat and accumulate within LAMP1+ endolysosomes. Gangliosides relieve this block, binding to the capsid at low pH and facilitating a late step in entry involving uncoating and delivery of the RNA genome to the cytoplasm. These results reveal an atypical cellular entry pathway for hepatoviruses that is unique among picornaviruses.

Identification of a Locus in Mice that Regulates the Collateral Damage and Lethality of Virus Infection.

Arenaviruses can cause severe hemorrhagic disease in humans, which can progress to organ failure and death. The underlying mechanisms causing lethality and person-to-person variation in outcome remain incompletely explained. Herein, we characterize a mouse model that recapitulates many features of pathogenesis observed in humans with arenavirus-induced hemorrhagic disease, including thrombocytopenia, severe vascular leakage, lung edema, and lethality. The susceptibility of congenic B6.PL mice to lymphocytic choriomeningitis virus (LCMV) infection is associated with increased antiviral T cell responses in B6.PL mice compared with C57BL/6 mice and is T cell dependent. Pathogenesis imparted by the causative locus is inherited in a semi-dominant manner in F1 crosses. The locus includes PL-derived sequence variants in both poorly annotated genes and genes known to contribute to immune responses. This model can be used to further interrogate how limited genetic differences in the host can remarkably alter the disease course of viral infection.

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.

Obesity Expands a Distinct Population of T Cells in Adipose Tissue and Increases Vulnerability to Infection.

Obesity in humans is associated with poorer health outcomes after infections compared with non-obese individuals. Here, we examined the effects of white adipose tissue and obesity on T cell responses to viral infection in mice. We show that lymphocytic choriomeningitis virus (LCMV) grows to high titer in adipose tissue. Virus-specific T cells enter the adipose tissue to resolve infection but then remain as a memory population distinct from memory T cells in lymphoid tissues. Memory T cells in adipose tissue are abundant in lean mice, and diet-induced obesity further increases memory T cell number in adipose tissue and spleen. Upon re-challenge infection, memory T cells rapidly cause severe pathogenesis, leading to increases in lipase levels, calcification of adipose tissue, pancreatitis, and reduced survival in obese mice but not lean mice. Thus, obesity leads to a unique form of viral pathogenesis involving memory T cell-dependent adipocyte destruction and damage to other tissues.

The Innate Immune Sensor NLRC3 Acts as a Rheostat that Fine-Tunes T Cell Responses in Infection and Autoimmunity.

Appropriate immune responses require a fine balance between immune activation and attenuation. NLRC3, a non-inflammasome-forming member of the NLR innate immune receptor family, attenuates inflammation in myeloid cells and proliferation in epithelial cells. T lymphocytes express the highest amounts of Nlrc3 transcript where its physiologic relevance is unknown. We show that NLRC3 attenuated interferon-γ and TNF expression by CD4+ T cells and reduced T helper 1 (Th1) and Th17 cell proliferation. Nlrc3-/- mice exhibited increased and prolonged CD4+ T cell responses to lymphocytic choriomeningitis virus infection and worsened experimental autoimmune encephalomyelitis (EAE). These functions of NLRC3 were executed in a T-cell-intrinsic fashion: NLRC3 reduced K63-linked ubiquitination of TNF-receptor-associated factor 6 (TRAF6) to limit NF-κB activation, lowered phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and diminished glycolysis and oxidative phosphorylation. This study reveals an unappreciated role for NLRC3 in attenuating CD4+ T cell signaling and metabolism.

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.

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.

Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation.

Oxidative tissue injury often accompanies viral infection, yet there is little understanding of how it influences virus replication. We show that multiple hepatitis C virus (HCV) genotypes are exquisitely sensitive to oxidative membrane damage, a property distinguishing them from other pathogenic RNA viruses. Lipid peroxidation, regulated in part through sphingosine kinase-2, severely restricts HCV replication in Huh-7 cells and primary human hepatoblasts. Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals in vitro, suggesting critical regulation of the conformation of the NS3-4A protease and the NS5B polymerase, membrane-bound HCV replicase components. Resistance to lipid peroxidation maps genetically to transmembrane and membrane-proximal residues within these proteins and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain of HCV. Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.

IFN-λ exerts opposing effects on T cell responses depending on the chronicity of the virus infection.

IFN-λ induces an antiviral state in many cell types and may contribute to the overall inflammatory environment after infection. Either of these effects may influence adaptive immune responses, but the role of type 3 IFNs in the development of primary and memory T cell responses to infection has not been evaluated. In this study, we examined T cell responses to acute or persistent lymphocytic choriomeningitis virus infection in IFN-λR1-deficient mice. Following acute infection, we find that IFN-λR1-deficient mice produced normal levels of IFN, robust NK cell responses, but greater than normal CD4+ and CD8+ T cell responses compared with wild type BALB/c mice. There were more T cells that were IL-7R(hi) and, correspondingly, the IFN-λR-deficient mice showed a 2- to 3-fold increase in memory T cell number. The inhibitory effect of IFN-λR expression was independent of direct cytokine signaling into T cells. In contrast with acute infection, the IFN-λR-deficient mice generated markedly diminished T cell responses and had greater weight loss compared with wild type mice when confronted with a highly disseminating variant of lymphocytic choriomeningitis virus. These data indicate that IFN-λR limits T cell responses and memory after transient infection but augments T cell responses during persisting infection. Thus, the immune-regulatory functions for IFN-λR are complex and vary with the overall inflammatory environment.

B cell depletion curtails CD4+ T cell memory and reduces protection against disseminating virus infection.

Dynamic interactions between CD4(+) T cells and B cells are needed for humoral immunity and CD4(+) T cell memory. It is not known whether B cells are needed early on to induce the formation of memory precursor cells or are needed later to sustain memory cells. In this study, primary and memory CD4(+) T cells responses were followed in wild-type mice that were depleted of mature B cells by anti-CD20 before or different times after acute lymphocytic choriomeningitis virus infection. The Ab treatment led to a 1000-fold reduction in B cell number that lasted 6 wk. Primary virus-specific CD4(+) Th1 cells were generated in B cell-depleted mice; however, there was a decrease in the CD4(+)Ly6C(lo)Tbet(+) memory precursor population and a corresponding 4-fold reduction in CD4(+) memory cell number. Memory T cells showed impaired cytokine production when they formed without B cells. B cell depletion had no effect on established memory populations. During disseminating virus infection, B cell depletion led to sustained weight loss and functional exhaustion of CD4(+) and CD8(+) T cells, and prevented mice from resolving the infection. Thus, B cells contribute to the establishment and survival of memory CD4(+) T cells post-acute infection and play an essential role in immune protection against disseminating virus infection.

Differential T cell responses to residual viral antigen prolong CD4+ T cell contraction following the resolution of infection.

The contraction phase of the T cell response is a poorly understood period after the resolution of infection when virus-specific effector cells decline in number and memory cells emerge with increased frequencies. CD8(+) T cells plummet in number and quickly reach stable levels of memory following acute lymphocytic choriomeningitis virus infection in mice. In contrast, virus-specific CD4(+) T cells gradually decrease in number and reach homeostatic levels only after many weeks. In this study, we provide evidence that MHCII-restricted viral Ag persists during the contraction phase following this prototypical acute virus infection. We evaluated whether the residual Ag affected the cell division and number of virus-specific naive and memory CD4(+) T cells and CD8(+) T cells. We found that naive CD4(+) T cells underwent cell division and accumulated in response to residual viral Ag for >2 mo after the eradication of infectious virus. Surprisingly, memory CD4(+) T cells did not undergo cell division in response to the lingering Ag, despite their heightened capacity to recognize Ag and make cytokine. In contrast to CD4(+) T cells, CD8(+) T cells did not undergo cell division in response to the residual Ag. Thus, CD8(+) T cells ceased division within days after the infection was resolved, indicating that CD8(+) T cell responses are tightly linked to endogenous processing of de novo synthesized virus protein. Our data suggest that residual viral Ag delays the contraction of CD4(+) T cell responses by recruiting new populations of CD4(+) T cells.