Publications by authors named "Stanley M Lemon"

178 Publications

Iminosugar glucosidase inhibitors reduce hepatic inflammation in HAV-infected mice.

J Virol 2021 Mar 10. Epub 2021 Mar 10.

Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

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 treatment of lipid storage diseases in humans, and UV-4 (N-(9-methoxynonyl)-1-deoxynojirimycin), inhibit replication of hepatitis A virus (HAV) in cell culture (IC 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 Miglustat, given by gavage to mice (4800 mg/kg/day) depleted hepatic gangliosides by 69-75%, 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 β-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.Hepatitis A virus (HAV) is a common cause of viral hepatitis. Iminosugar compounds block its replication in cultured cells by inhibiting synthesis of gangliosides required for HAV cell entry, but have not been tested for their ability to prevent or treat hepatitis A 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 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.
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http://dx.doi.org/10.1128/JVI.00058-21DOI Listing
March 2021

Interferon regulatory factor 1 (IRF1) and anti-pathogen innate immune responses.

PLoS Pathog 2021 01 21;17(1):e1009220. Epub 2021 Jan 21.

Department of Diseases and Infection, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.

The eponymous member of the interferon regulatory factor (IRF) family, IRF1, was originally identified as a nuclear factor that binds and activates the promoters of type I interferon genes. However, subsequent studies using genetic knockouts or RNAi-mediated depletion of IRF1 provide a much broader view, linking IRF1 to a wide range of functions in protection against invading pathogens. Conserved throughout vertebrate evolution, IRF1 has been shown in recent years to mediate constitutive as well as inducible host defenses against a variety of viruses. Fine-tuning of these ancient IRF1-mediated host defenses, and countering strategies by pathogens to disarm IRF1, play crucial roles in pathogenesis and determining the outcome of infection.
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http://dx.doi.org/10.1371/journal.ppat.1009220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7819612PMC
January 2021

Stimulator of interferon genes (STING) is an essential proviral host factor for human rhinovirus species A and C.

Proc Natl Acad Sci U S A 2020 11 15;117(44):27598-27607. Epub 2020 Oct 15.

Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599;

Human rhinoviruses (RVs) are positive-strand RNA viruses that cause respiratory tract disease in children and adults. Here we show that the innate immune signaling protein STING is required for efficient replication of members of two distinct RV species, RV-A and RV-C. The host factor activity of STING was identified in a genome-wide RNA interference (RNAi) screen and confirmed in primary human small airway epithelial cells. Replication of RV-A serotypes was strictly dependent on STING, whereas RV-B serotypes were notably less dependent. Subgenomic RV-A and RV-C RNA replicons failed to amplify in the absence of STING, revealing it to be required for a step in RNA replication. STING was expressed on phosphatidylinositol 4-phosphate (PI4P)-enriched membranes and was enriched in RV-A16 compared with RV-B14 replication organelles isolated in isopycnic gradients. The host factor activity of STING was species-specific, as murine STING (mSTING) did not rescue RV-A16 replication in STING-deficient cells. This species specificity mapped primarily to the cytoplasmic, ligand-binding domain of STING. Mouse-adaptive mutations in the RV-A16 2C protein allowed for robust replication in cells expressing mSTING, suggesting a role for 2C in recruiting STING to RV-A replication organelles. Palmitoylation of STING was not required for RV-A16 replication, nor was the C-terminal tail of STING that mediates IRF3 signaling. Despite co-opting STING to promote its replication, interferon signaling in response to STING agonists remained intact in RV-A16 infected cells. These data demonstrate a surprising requirement for a key host mediator of innate immunity to DNA viruses in the life cycle of a small pathogenic RNA virus.
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http://dx.doi.org/10.1073/pnas.2014940117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7959528PMC
November 2020

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

Nat Microbiol 2020 09 25;5(9):1069-1078. Epub 2020 May 25.

Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

The Picornaviridae are a diverse family of positive-strand RNA viruses that includes numerous human and veterinary pathogens. 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 exosomes. These quasi-enveloped virions are infectious and are the only form of virus that can be detected in the blood during acute infection. 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 gut. 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.
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http://dx.doi.org/10.1038/s41564-020-0727-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483933PMC
September 2020

TIM1 (HAVCR1): an Essential "Receptor" or an "Accessory Attachment Factor" for Hepatitis A Virus?

J Virol 2019 06 15;93(11). Epub 2019 May 15.

Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

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http://dx.doi.org/10.1128/JVI.01793-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6532101PMC
June 2019

TNRC6 proteins modulate hepatitis C virus replication by spatially regulating the binding of miR-122/Ago2 complexes to viral RNA.

Nucleic Acids Res 2019 07;47(12):6411-6424

Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

The liver-specific microRNA, miR-122, is an essential host factor for replication of the hepatitis C virus (HCV). miR-122 stabilizes the positive-strand HCV RNA genome and promotes its synthesis by binding two sites (S1 and S2) near its 5' end in association with Ago2. Ago2 is essential for both host factor activities, but whether other host proteins are involved is unknown. Using an unbiased quantitative proteomics screen, we identified the TNRC6 protein paralogs, TNRC6B and TNRC6C, as functionally important but redundant components of the miR-122/Ago2 host factor complex. Doubly depleting TNRC6B and TNRC6C proteins reduced HCV replication in human hepatoma cells, dampening miR-122 stimulation of viral RNA synthesis without reducing the stability or translational activity of the viral RNA. TNRC6B/C were required for optimal miR-122 host factor activity only when S1 was able to bind miR-122, and restricted replication when S1 was mutated and only S2 bound by miR-122. TNRC6B/C preferentially associated with S1, and TNRC6B/C depletion enhanced Ago2 association at S2. Collectively, these data suggest a model in which TNRC6B/C regulate the assembly of miR-122/Ago complexes on HCV RNA, preferentially directing miR-122/Ago2 to S1 while restricting its association with S2, thereby fine-tuning the spatial organization of miR-122/Ago2 complexes on the viral genome.
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http://dx.doi.org/10.1093/nar/gkz278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614814PMC
July 2019

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

Nat Microbiol 2019 07 15;4(7):1096-1104. Epub 2019 Apr 15.

UNC Lineberger Comprehensive Cancer Center, Chapel Hill, NC, USA.

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 interferons. RNA viruses have evolved sophisticated strategies to disrupt these signalling pathways and evade elimination by cells, attesting to their importance. 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.
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http://dx.doi.org/10.1038/s41564-019-0425-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588457PMC
July 2019

Hepatovirus 3ABC proteases and evolution of mitochondrial antiviral signaling protein (MAVS).

J Hepatol 2019 07 13;71(1):25-34. Epub 2019 Mar 13.

Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Microbiology & Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address:

Background & Aims: Unlike other hepatitis viruses that have infected primates for millions of years, hepatitis A virus (HAV) likely entered human populations only 10-12 thousand years ago after jumping from a rodent host. The phylogeny of modern hepatoviruses that infect rodents and bats suggest that multiple similar host shifts have occurred in the past. The factors determining such shifts are unknown, but the capacity to overcome innate antiviral responses in a foreign species is likely key.

Methods: We assessed the capacity of diverse hepatovirus 3ABC proteases to cleave mitochondrial antiviral signaling protein (MAVS) and disrupt antiviral signaling in HEK293 and human hepatocyte-derived cell lines. We also applied maximum-likelihood and Bayesian algorithms to identify sites of diversifying selection in MAVS orthologs from 75 chiropteran, rodent and primate species.

Results: 3ABC proteases from bat, but not rodent hepatoviruses efficiently cleaved human MAVS at Glu/Gly, disrupting virus activation of the interferon-β promoter, whereas human HAV 3ABC cleaved at Gln/Val. In contrast, MAVS orthologs from rodents and bats were resistant to cleavage by 3ABC proteases of cognate hepatoviruses and in several cases human HAV. A search for diversifying selection among MAVS orthologs from all 3 orders revealed 90 of ∼540 residues to be under positive selection, including residues in chiropteran MAVS that align with the site of cleavage of human MAVS by bat 3ABC proteases.

Conclusions: 3ABC protease cleavage of MAVS is a conserved attribute of hepatoviruses, acting broadly across different mammalian species and associated with evidence of diversifying selection at cleavage sites in rodent and bat MAVS orthologs. The capacity of hepatoviruses to disrupt MAVS-mediated innate immune responses has shaped evolution of both hepatoviruses and their hosts, and facilitates cross-species transmission of hepatitis A.

Lay Summary: Hepatitis A virus, a common cause of acute hepatitis globally, is likely to have evolved from a virus that jumped from a rodent species to humans within the last 10-12 thousand years. Here we show that distantly related hepatoviruses, that infect bats and rodents today, express proteases that disrupt innate antiviral responses in human cells. This conserved attribute of hepatoviruses may have contributed to that ancient host species shift.
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http://dx.doi.org/10.1016/j.jhep.2019.02.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581616PMC
July 2019

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

Elife 2019 02 25;8. Epub 2019 Feb 25.

Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, United States.

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 β, 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 .
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http://dx.doi.org/10.7554/eLife.43983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422491PMC
February 2019

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

J Virol 2018 12 12;92(23). Epub 2018 Nov 12.

Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

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. YPXL 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 YPXL peptides with ALIX) are required for efficient capsid assembly. However, single Leu-to-Ala substitutions within either VP2 YPXL 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 YPXL 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. 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 YPXL "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 YPXL 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 YPXL motifs and raise questions about the structure of the HAV capsid prior to and following quasi-envelopment.
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http://dx.doi.org/10.1128/JVI.01308-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232465PMC
December 2018

Evolutionary pathways to NS5A inhibitor resistance in genotype 1 hepatitis C virus.

Antiviral Res 2018 10 3;158:45-51. Epub 2018 Aug 3.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Division of Infectious Diseases, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address:

Direct-acting antivirals (DAAs) targeting NS5A are broadly effective against hepatitis C virus (HCV) infections, but sustained virological response rates are generally lower in patients infected with genotype (gt)-1a than gt-1b viruses. The explanation for this remains uncertain. Here, we adopted a highly accurate, ultra-deep primer ID sequencing approach to intensively study serial changes in the NS5A-coding region of HCV in gt-1a- and gt-1b-infected subjects receiving a short course of monotherapy with the NS5A inhibitor, elbasvir. Low or undetectable levels of viremia precluded on-treatment analysis in gt-1b-infected subjects, but variants with the resistance-associated substitution (RAS) Y93H in NS5A dominated rebounding virus populations following cessation of treatment. These variants persisted until the end of the study, two months later. In contrast, while Y93H emerged in multiple lineages and became dominant in subjects with gt-1a virus, these haplotypes rapidly decreased in frequency off therapy. Substitutions at Q30 and L31 emerged in distinctly independent lineages at later time points, ultimately coming to dominate the virus population off therapy. Consistent with this, cell culture studies with gt-1a and gt-1b reporter viruses and replicons demonstrated that Y93H confers a much greater loss of replicative fitness in gt-1a than gt-1b virus, and that L31M/V both compensates for the loss of fitness associated with Q30R (but not Y93H) and also boosts drug resistance. These observations show how differences in the impact of RASs on drug resistance and replicative fitness influence the evolution of gt-1a and gt-1b viruses during monotherapy with an antiviral targeting NS5A.
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http://dx.doi.org/10.1016/j.antiviral.2018.07.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6146077PMC
October 2018

Hepatitis A Virus and Hepatitis E Virus: Emerging and Re-Emerging Enterically Transmitted Hepatitis Viruses.

Cold Spring Harb Perspect Med 2019 06 3;9(6). Epub 2019 Jun 3.

Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital and College of Medicine, The Ohio State University, Columbus, Ohio 43205.

Over the past two decades, progress in understanding human infections with hepatitis A virus (HAV) and hepatitis E virus (HEV) has been eclipsed by the priority of combating persistent hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. During that time, the global burden of liver disease caused by enteric hepatitis viruses has not abated. Because of vaccines, hepatitis A has become increasingly a disease of adults instead of early childhood in many regions of the world, resulting in an age-related shift toward more severe disease. HEV has remained endemic in many developing countries, and in well-developed, economically advanced countries it is now recognized as a cause of chronic, progressive liver disease in individuals with compromised immunity. The goal of this collection of articles is to review recent progress and to shine a bright light on gaps in our understanding of how these viruses replicate, cause disease, interact with the liver and host immune system, and are transmitted, along with prospects for improved control in human populations. Renewed efforts to study and compare HAV and HEV biology in humans and animal models have high potential to enhance our understanding of host-pathogen balance in the liver, and may contribute ultimately to the control of other infectious diseases of the liver.
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http://dx.doi.org/10.1101/cshperspect.a031823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531368PMC
June 2019

Enterically Transmitted Non-A, Non-B Hepatitis and the Discovery of Hepatitis E Virus.

Cold Spring Harb Perspect Med 2019 08 1;9(8). Epub 2019 Aug 1.

Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital and College of Medicine, The Ohio State University, Columbus, Ohio 43205.

The recognition of hepatitis E as a discreet disease entity in the late 1970s followed the development of serological tests for hepatitis A and the discovery that large waterborne outbreaks of hepatitis in India were not caused by hepatitis A virus (HAV). These "enterically transmitted non-A, non-B hepatitis" outbreaks had distinctive epidemiologic features, including the highest attack rates among young adults, little secondary household transmission of infection, and severe disease in pregnant women. The responsible agent, hepatitis E virus (HEV), was identified several years later in extracts of feces from a self-inoculated virologist. Multiple genetically related HEV genotypes are now known to exist, two of which are common in domestic swine herds and the cause of sporadic cases of acute hepatitis in economically well-developed countries. HEV genotypes possess impressive genetic and biologic diversity, and present many unanswered questions concerning their natural host range, potential for zoonotic transmission, and disease pathogenesis.
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http://dx.doi.org/10.1101/cshperspect.a033449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531376PMC
August 2019

Comparative Pathology of Hepatitis A Virus and Hepatitis E Virus Infection.

Cold Spring Harb Perspect Med 2019 04 1;9(4). Epub 2019 Apr 1.

Lineberger Comprehensive Cancer Center, Departments of Medicine and Microbiology & Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7030.

Hepatitis A virus (HAV) and hepatitis E virus (HEV) cause acute, self-limiting hepatic infections that are usually spread by the fecal-oral route in humans. Naturally occurring and experimental infections are possible in a variety of nonhuman primates and, in the case of HEV, a number of other species. Many advances in understanding the pathogenesis of these viruses have come from studies in experimental animals. In general, animals infected with these viruses recapitulate the histologic lesions seen in infected humans, but typically with less severe clinical and histopathological manifestations. This review describes the histopathologic changes associated with HAV and HEV infection in humans and experimental animals.
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http://dx.doi.org/10.1101/cshperspect.a033456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6444697PMC
April 2019

Nonhuman Primate Models of Hepatitis A Virus and Hepatitis E Virus Infections.

Cold Spring Harb Perspect Med 2019 02 1;9(2). Epub 2019 Feb 1.

Departments of Medicine and Microbiology & Immunology, Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7030.

Although phylogenetically unrelated, human hepatitis viruses share an exclusive or near exclusive tropism for replication in differentiated hepatocytes. This narrow tissue tropism may contribute to the restriction of the host ranges of these viruses to relatively few host species, mostly nonhuman primates. Nonhuman primate models thus figure prominently in our current understanding of the replication and pathogenesis of these viruses, including the enterically transmitted hepatitis A virus (HAV) and hepatitis E virus (HEV), and have also played major roles in vaccine development. This review draws comparisons of HAV and HEV infection from studies conducted in nonhuman primates, and describes how such studies have contributed to our current understanding of the biology of these viruses.
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http://dx.doi.org/10.1101/cshperspect.a031815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360867PMC
February 2019

Innate Immunity to Enteric Hepatitis Viruses.

Cold Spring Harb Perspect Med 2019 03 1;9(3). Epub 2019 Mar 1.

Departments of Medicine and Microbiology & Immunology, Lineberger Comprehensive Cancer Center, The University of North Carolina, Chapel Hill, North Carolina 27599.

Although hepatitis A virus (HAV) and hepatitis E virus (HEV) are both positive-strand RNA viruses that replicate in the cytoplasm of hepatocytes, there are important differences in the ways they induce and counteract host innate immune responses. HAV is remarkably stealthy because of its ability to evade and disrupt innate signaling pathways that lead to interferon production. In contrast, HEV does not block interferon production. Instead, it persists in the presence of an interferon response. These differences may provide insight into HEV persistence in immunocompromised patients, an emerging health problem in developed countries.
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http://dx.doi.org/10.1101/cshperspect.a033464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396338PMC
March 2019

Murine Models of Hepatitis A Virus Infection.

Cold Spring Harb Perspect Med 2019 01 2;9(1). Epub 2019 Jan 2.

Department of Microbiology & Immunology, University of North Carolina, Chapel Hill, North Carolina 27599.

Mechanistic analyses of hepatitis A virus (HAV)-induced pathogenesis have long been thwarted by the lack of tractable small animal models that recapitulate disease observed in humans. Several approaches have shown success, including infection of chimeric mice with human liver cells. Other recent studies show that HAV can replicate to high titer in mice lacking expression of the type I interferon (IFN) receptor (IFN-α/β receptor) or mitochondrial antiviral signaling (MAVS) protein. Mice deficient in the IFN receptor show critical features of type A hepatitis in humans when challenged with human HAV, including histological evidence of liver damage, leukocyte infiltration, and the release of liver enzymes into blood. Acute pathogenesis is caused by MAVS-dependent signaling that leads to intrinsic apoptosis of hepatocytes.
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http://dx.doi.org/10.1101/cshperspect.a031674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314078PMC
January 2019

Hepatitis A Virus Genome Organization and Replication Strategy.

Cold Spring Harb Perspect Med 2018 12 3;8(12). Epub 2018 Dec 3.

Departments of Medicine and Microbiology & Immunology, Lineberger Comprehensive Cancer Center, The University of North Carolina, Chapel Hill, North Carolina 27599.

Hepatitis A virus (HAV) is a positive-strand RNA virus classified in the genus of the family It is an ancient virus with a long evolutionary history and multiple features of its capsid structure, genome organization, and replication cycle that distinguish it from other mammalian picornaviruses. HAV proteins are produced by cap-independent translation of a single, long open reading frame under direction of an inefficient, upstream internal ribosome entry site (IRES). Genome replication occurs slowly and is noncytopathic, with transcription likely primed by a uridylated protein primer as in other picornaviruses. Newly produced quasi-enveloped virions (eHAV) are released from cells in a nonlytic fashion in a unique process mediated by interactions of capsid proteins with components of the host cell endosomal sorting complexes required for transport (ESCRT) system.
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http://dx.doi.org/10.1101/cshperspect.a033480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280712PMC
December 2018

Critical challenges and emerging opportunities in hepatitis C virus research in an era of potent antiviral therapy: Considerations for scientists and funding agencies.

Virus Res 2018 03 2;248:53-62. Epub 2018 Mar 2.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

The development and clinical implementation of direct-acting antivirals (DAAs) has revolutionized the treatment of chronic hepatitis C. Infection with any hepatitis C virus (HCV) genotype can now be eliminated in more than 95% of patients with short courses of all-oral, well-tolerated drugs, even in those with advanced liver disease and liver transplant recipients. DAAs have proven so successful that some now consider HCV amenable to eradication, and continued research on the virus of little remaining medical relevance. However, given 400,000 HCV-related deaths annually important challenges remain, including identifying those who are infected, providing access to treatment and reducing its costs. Moreover, HCV infection rarely induces sterilizing immunity, and those who have been cured with DAAs remain at risk for reinfection. Thus, it is very unlikely that global eradication and elimination of the cancer risk associated with HCV infection can be achieved without a vaccine, yet research in that direction receives little attention. Further, over the past two decades HCV research has spearheaded numerous fundamental discoveries in the fields of molecular and cell biology, immunology and microbiology. It will continue to do so, given the unique opportunities afforded by the reagents and knowledge base that have been generated in the development and clinical application of DAAs. Considering these critical challenges and new opportunities, we conclude that funding for HCV research must be sustained.
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http://dx.doi.org/10.1016/j.virusres.2018.02.016DOI Listing
March 2018

An Efficient, Large-Scale Survey of Hepatitis C Viremia in the Democratic Republic of the Congo Using Dried Blood Spots.

Clin Infect Dis 2018 01;66(2):254-260

Abbott Laboratories, Abbott Park, Illinois.

Background: Efficient viral load testing is needed for hepatitis C (HCV) surveillance and diagnosis. HCV viral load testing using dried blood spots (DBSs), made with a single drop of finger-prick whole blood on filter paper, is a promising alternative to traditional serum- or plasma-based approaches.

Methods: We adapted the Abbott Molecular m2000 instrument for high-throughput HCV viremia testing using DBSs with simple specimen processing and applied these methods to estimate the national burden of infection in the Democratic Republic of the Congo (DRC). We tested DBSs collected during the 2013-2014 DRC Demographic and Health Survey, including 1309 adults ≥40 years of age. HCV-positive samples underwent targeted sequencing, genotyping, and phylogenetic analyses.

Results: This high-throughput screening approach reliably identified HCV RNA extracted from DBSs prepared using whole blood, with a 95% limit of detection of 1196 (95% confidence interval [CI], 866-2280) IU/mL for individual 6-mm punches and 494 (95% CI, 372-1228) IU/mL for larger 12-mm punches. Fifteen infections were identified among samples from the DRC Demographic and Health Survey; the weighted country-wide prevalence of HCV viremia was 0.9% (95% CI, 0.3%-1.6%) among adults ≥40 years of age and 0.7% (95% CI, .6%-.8%) among human immunodeficiency virus-infected subjects. All successfully genotyped cases were due to genotype 4 infection.

Conclusions: DBS-based HCV testing represents a useful tool for the diagnosis and surveillance of HCV viremia and can easily be incorporated into specimen referral systems. Among adults ≥40 years of age in the DRC, 100000-200000 may have active infection and be eligible for treatment.
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http://dx.doi.org/10.1093/cid/cix771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850542PMC
January 2018

The Chimpanzee Model of Viral Hepatitis: Advances in Understanding the Immune Response and Treatment of Viral Hepatitis.

ILAR J 2017 12;58(2):172-189

Department of Medicine, Division of Infectious Diseases, University of North Carolina, Chapel Hill, North Carolina.

Chimpanzees (Pan troglodytes) have contributed to diverse fields of biomedical research due to their close genetic relationship to humans and in many instances due to the lack of any other animal model. This review focuses on the contributions of the chimpanzee model to research on hepatitis viruses where chimpanzees represented the only animal model (hepatitis B and C) or the most appropriate animal model (hepatitis A). Research with chimpanzees led to the development of vaccines for HAV and HBV that are used worldwide to protect hundreds of millions from these diseases and, where fully implemented, have provided immunity for entire generations. More recently, chimpanzee research was instrumental in the development of curative therapies for hepatitis C virus infections. Over a span of 40 years, this research would identify the causative agent of NonA,NonB hepatitis, validate the molecular tools for drug discovery, and provide safety and efficacy data on the therapies that now provide a rapid and complete cure of HCV chronic infections. Several cocktails of antivirals are FDA approved that eliminate the virus following 12 weeks of once-per-day oral therapy. This represents the first cure of a chronic viral disease and, once broadly implemented, will dramatically reduce the occurrence of cirrhosis and liver cancer. The recent contributions of chimpanzees to our current understanding of T cell immunity for HCV, development of novel therapeutics for HBV, and the biology of HAV are reviewed. Finally, a perspective is provided on the events leading to the cessation of the use of chimpanzees in research and the future of the chimpanzees previously used to bring about these amazing breakthroughs in human healthcare.
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http://dx.doi.org/10.1093/ilar/ilx028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886334PMC
December 2017

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

Nat Immunol 2017 Dec 2;18(12):1299-1309. Epub 2017 Oct 2.

Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

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.
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http://dx.doi.org/10.1038/ni.3853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5690873PMC
December 2017

Type A viral hepatitis: A summary and update on the molecular virology, epidemiology, pathogenesis and prevention.

J Hepatol 2017 Sep 5. Epub 2017 Sep 5.

Liver Unit, Institute for Gastroenterology and Hepatology, Hadassah-Hebrew University Hospital, P.O.Box 12000, Jerusalem 91120, Israel.

Although epidemic jaundice was well known to physicians of antiquity, it is only in recent years that medical science has begun to unravel the origins of hepatitis A virus (HAV) and the unique pathobiology underlying acute hepatitis A in humans. Improvements in sanitation and the successful development of highly efficacious vaccines have markedly reduced the worldwide prevalence and incidence of this enterically-transmitted infection over the past quarter century, yet the virus persists in vulnerable populations and remains a common cause of food-borne disease outbreaks in economically-advantaged societies. Reductions in the prevalence of HAV have led to increases in the median age at which infection occurs, often resulting in more severe disease in affected persons and paradoxical increases in disease burden in some developing nations. Here, we summarize recent advances in the molecular virology of HAV, an atypical member of the Picornaviridae family, survey what is known of the pathogenesis of hepatitis A in humans and the host-pathogen interactions that typify the infection, and review medical and public health aspects of immunisation and disease prevention.
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http://dx.doi.org/10.1016/j.jhep.2017.08.034DOI Listing
September 2017

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

mBio 2017 09 5;8(5). Epub 2017 Sep 5.

Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

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 and 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 mice. However, intrahepatic HAV RNA and ALT elevations were modestly reduced in mice compared to 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. 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 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.
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http://dx.doi.org/10.1128/mBio.00969-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587907PMC
September 2017

NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains.

PLoS Pathog 2017 Jun 8;13(6):e1006343. Epub 2017 Jun 8.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

Hepatitis C virus (HCV) RNA is synthesized by the replicase complex (RC), a macromolecular assembly composed of viral non-structural proteins and cellular co-factors. Inhibitors of the HCV NS5A protein block formation of new RCs but do not affect RNA synthesis by pre-formed RCs. Without new RC formation, existing RCs turn over and are eventually lost from the cell. We aimed to use NS5A inhibitors to estimate the half-life of the functional RC of HCV. We compared different cell culture-infectious strains of HCV that may be grouped based on their sensitivity to lipid peroxidation: robustly replicating, lipid peroxidation resistant (LPOR) viruses (e.g. JFH-1 or H77D) and more slowly replicating, lipid peroxidation sensitive (LPOS) viruses (e.g. H77S.3 and N.2). In luciferase assays, LPOS HCV strains declined under NS5A inhibitor therapy with much slower kinetics compared to LPOR HCV strains. This difference in rate of decline was not observed for inhibitors of the NS5B RNA-dependent RNA polymerase suggesting that the difference was not simply a consequence of differences in RNA stability. In further analyses, we compared two isoclonal HCV variants: the LPOS H77S.3 and the LPOR H77D that differ only by 12 amino acids. Differences in rate of decline between H77S.3 and H77D following NS5A inhibitor addition were not due to amino acid sequences in NS5A but rather due to a combination of amino acid differences in the non-structural proteins that make up the HCV RC. Mathematical modeling of intracellular HCV RNA dynamics suggested that differences in RC stability (half-lives of 3.5 and 9.9 hours, for H77D and H77S.3, respectively) are responsible for the different kinetics of antiviral suppression between LPOS and LPOR viruses. In nascent RNA capture assays, the rate of RNA synthesis decline following NS5A inhibitor addition was significantly faster for H77D compared to H77S.3 indicating different half-lives of functional RCs.
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http://dx.doi.org/10.1371/journal.ppat.1006343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464671PMC
June 2017

Protein composition of the hepatitis A virus quasi-envelope.

Proc Natl Acad Sci U S A 2017 06 10;114(25):6587-6592. Epub 2017 May 10.

Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7292;

The 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 ( = 105) were significantly enriched for components of the endolysosomal system (>60%, < 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.
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http://dx.doi.org/10.1073/pnas.1619519114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5488923PMC
June 2017

Hepatitis C Virus Indirectly Disrupts DNA Damage-Induced p53 Responses by Activating Protein Kinase R.

mBio 2017 04 25;8(2). Epub 2017 Apr 25.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Many DNA tumor viruses promote cellular transformation by inactivating the critically important tumor suppressor protein p53. In contrast, it is not known whether p53 function is disrupted by hepatitis C virus (HCV), a unique, oncogenic RNA virus that is the leading infectious cause of liver cancer in many regions of the world. Here we show that HCV-permissive, liver-derived HepG2 cells engineered to constitutively express microRNA-122 (HepG2/miR-122 cells) have normal p53-mediated responses to DNA damage and that HCV replication in these cells potently suppresses p53 responses to etoposide, an inducer of DNA damage, or nutlin-3, an inhibitor of p53 degradation pathways. Upregulation of p53-dependent targets is consequently repressed within HCV-infected cells, with potential consequences for cell survival. Despite this, p53 function is not disrupted by overexpression of the complete HCV polyprotein, suggesting that altered p53 function may result from the host response to viral RNA replication intermediates. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated ablation of double-stranded RNA (dsRNA)-activated protein kinase R (PKR) restored p53 responses while boosting HCV replication, showing that p53 inhibition results directly from viral activation of PKR. The hepatocellular abundance of phosphorylated PKR is elevated in HCV-infected chimpanzees, suggesting that PKR activation and consequent p53 inhibition accompany HCV infection These findings reveal a feature of the host response to HCV infection that may contribute to hepatocellular carcinogenesis. Chronic infection with hepatitis C virus (HCV) is the leading cause of liver cancer in most developed nations. However, the mechanisms whereby HCV infection promotes carcinogenesis remain unclear. Here, we demonstrate that HCV infection inhibits the activation of p53 following DNA damage. Contrary to previous reports, HCV protein expression is insufficient to inhibit p53. Rather, p53 inhibition is mediated by cellular protein kinase R (PKR), which is activated by HCV RNA replication and subsequently suppresses global protein synthesis. These results redefine our understanding of how HCV infection influences p53 function. We speculate that persistent disruption of p53-mediated DNA damage responses may contribute to hepatocellular carcinogenesis in chronically infected individuals.
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http://dx.doi.org/10.1128/mBio.00121-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405228PMC
April 2017

Virology: Ins and outs of picornaviruses.

Nature 2017 01 11;541(7637):299-300. Epub 2017 Jan 11.

Departments of Medicine and of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7292, USA, and in the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill.

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http://dx.doi.org/10.1038/nature21116DOI Listing
January 2017

Differential hepatitis C virus RNA target site selection and host factor activities of naturally occurring miR-122 3΄ variants.

Nucleic Acids Res 2017 05;45(8):4743-4755

Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

In addition to suppressing cellular gene expression, certain miRNAs potently facilitate replication of specific positive-strand RNA viruses. miR-122, a pro-viral hepatitis C virus (HCV) host factor, binds and recruits Ago2 to tandem sites (S1 and S2) near the 5΄ end of the HCV genome, stabilizing it and promoting its synthesis. HCV target site selection follows canonical miRNA rules, but how non-templated 3΄ miR-122 modifications impact this unconventional miRNA action is unknown. High-throughput sequencing revealed that a 22 nt miRNA with 3΄G ('22-3΄G') comprised <63% of total miR-122 in human liver, whereas other variants (23-3΄A, 23-3΄U, 21-3΄U) represented 11-17%. All loaded equivalently into Ago2, and when tested individually functioned comparably in suppressing gene expression. In contrast, 23-3΄A and 23-3΄U were more active than 22-3΄G in stabilizing HCV RNA and promoting its replication, whereas 21-3΄U was almost completely inactive. This lack of 21-3΄U HCV host factor activity correlated with reduced recruitment of Ago2 to the HCV S1 site. Additional experiments demonstrated strong preference for guanosine at nt 22 of miR-122. Our findings reveal the importance of non-templated 3΄ miR-122 modifications to its HCV host factor activity, and identify unexpected differences in miRNA requirements for host gene suppression versus RNA virus replication.
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http://dx.doi.org/10.1093/nar/gkw1332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5416874PMC
May 2017

Chronic hepatitis C infection-induced liver fibrogenesis is associated with M2 macrophage activation.

Sci Rep 2016 12 21;6:39520. Epub 2016 Dec 21.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

The immuno-pathogenic mechanisms of chronic hepatitis C virus (HCV) infection remain to be elucidated and pose a major hurdle in treating or preventing chronic HCV-induced advanced liver diseases such as cirrhosis. Macrophages are a major component of the inflammatory milieu in chronic HCV-induced liver disease, and are generally derived from circulating inflammatory monocytes; however very little is known about their role in liver diseases. To investigate the activation and role of macrophages in chronic HCV-induced liver fibrosis, we utilized a recently developed humanized mouse model with autologous human immune and liver cells, human liver and blood samples and cell culture models of monocyte/macrophage and/or hepatic stellate cell activation. We showed that M2 macrophage activation was associated with liver fibrosis during chronic HCV infection in the livers of both humanized mice and patients, and direct-acting antiviral therapy attenuated M2 macrophage activation and associated liver fibrosis. We demonstrated that supernatant from HCV-infected liver cells activated human monocytes/macrophages with M2-like phenotypes. Importantly, HCV-activated monocytes/macrophages promoted hepatic stellate cell activation. These results suggest a critical role for M2 macrophage induction in chronic HCV-associated immune dysregulation and liver fibrosis.
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http://dx.doi.org/10.1038/srep39520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5175173PMC
December 2016