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Principal Investigator: You Li
Organization: UNIV OF NORTH CAROLINA CHAPEL HILL
Fiscal Year: 2021
Award: $233,250
Funding agency: National Institute of Allergy and Infectious Diseases
PROJECT SUMMARY/ABSTRACT
Hepatitis A virus (HAV), a plus-strand virus classified in the Picornaviridae, is a common cause of acute
hepatitis. Despite the availability of vaccines, striking increases in the incidence of hepatitis A have led to
increasing numbers of deaths associated with severe infection in the U.S. in recent years. Importantly, no
antiviral therapy exists that is capable of mitigating severe liver injury associated with HAV infection. A
recent genome-wide CRISPR screen carried out in our laboratory identified ZCCHC14 (Zinc finger CCHC-type
containing protein 14) as an essential host factor for HAV replication. This is surprising, as ZCCHC14 is not
required for replication of other picornaviruses, nor are its known activities consistent with current
understanding of HAV replication. ZCCHC14 is known to form a TRAMP-like complex with two non-canonical
poly(A) RNA polymerases TENT4A and TENT4B. This complex facilitates replication of hepatitis B virus (HBV)
and human cytomegalovirus (HCMV), both DNA viruses, by maintaining poly(A) tail length and stability of
viral mRNAs. RG7834, an orally available dihydroquinolizinone, targets TENT4A/B, and has antiviral activity
against HBV in vivo. We have found that knockdown of ZCCHC14 or TENT4A/B strongly inhibits HAV
replication, and that RG7834 has potent antiviral activity against HAV in Huh-7.5 cells (IC50=6.2 nM). We
have also shown that RG7834 blocks HAV replication and reduces liver injury in a murine model of hepatitis A
using Ifnar1-/- mice. However, unlike HBV, RG7834 has no effect on the length of poly(A) tails of HAV RNA,
indicating that it blocks HAV replication via a distinct and novel mechanism of action. We hypothesize: (1)
ZCCHC14 binds to specific structural elements in HAV genomic RNA and recruits TENT4 proteins to promote
viral RNA replication, and that the binding of RG7834 to TENT4 disrupts its association with ZCCHC14, viral
RNA, and/or viral proteins; and (2) that RG7834 therapy can mitigate the course of acute hepatitis A, and
consequently, enhance functional immune responses to HAV in a rodent model. Specific Aim 1 will
elucidate the role of ZCCHC14-TENT4 in the HAV life cycle and investigate the mechanism underlying
antiviral activity of RG7834 against HAV, including: 1(a) determining the step in the replicative cycle
requiring ZCCHC14 and inhibited by RG7834; 1(b) characterizing the interaction of ZCCHC14 with HAV
RNA; and 1(c) identifying viral or host proteins interacting with ZCCHC14-TENT4 in infected cells. Specific
Aim 2 will study the antiviral efficacy of RG7834 on HAV replication and pathogenicity in mice, including: 2(a)
the potency of orally administered RG7834 on intrahepatic viral replication and liver injury; 2(b) whether
RG7834 therapy induces beneficial T-cell responses specific to HAV. The overarching goal of the application
is to elucidate the mechanism underlying the essential role of ZCCHC14-TENT4 complex in HAV replication,
and explore the potential use of dihydroquinolizinones such as RG7834 for antiviral therapy. This study will shed
light on a novel mechanism by which a positive-strand RNA virus hijacks host proteins to support its
replication and potentially open the door to antiviral therapy of hepatitis A.
Terms: <ALT1><Acute Hepatitis><Alanine Aminotransferase><Alanine Transaminase><Alanine-2-Oxoglutarate Aminotransferase><Alpha-Beta-Omega Interferon Receptor-1><Antiviral Agents><Antiviral Drugs><Antiviral Protein Alpha Type><Antiviral Therapy><Antivirals><Apoptosis><Apoptosis Pathway><Binding><Binding Proteins><Bioavailable><Blood Serum><CMV><CRISPR editing screen><CRISPR method><CRISPR methodology><CRISPR screen><CRISPR technique><CRISPR technology><CRISPR-CAS-9><CRISPR-based method><CRISPR-based screen><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 screen><CRISPR/Cas9 technology><Cas nuclease technology><Cell Body><Cell Culture Techniques><Cells><Cessation of life><Chemotactic Cytokines><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Complex><Cytomegalovirus><DNA Viruses><DNA-Dependent RNA Polymerases><DNA-Directed RNA Polymerase><Data><Death><Dose><Drops><Elements><Family Picornaviridae><Gene Expression><Glutamic-Alanine Transaminase><Glutamic-Pyruvate Transaminase><Glutamic-Pyruvic Transaminase><Goals><HBV><HCMV><Hepatitis><Hepatitis A><Hepatitis A Virus><Hepatitis B Virus><Hepatovirus><Homologous Chemotactic Cytokines><Homologous Serum Hepatitis Virus><Host Factor><Host Factor Protein><HuIFN-Alpha-Rec><Human><IFNBR><IFRC><IRES><Immune><Immune response><Immunes><Immunological response><In Vitro><Incidence><Infection><Infection prevention><Infectious Hepatitis Virus><Infiltration><Injury to Liver><Integration Host Factors><Intercrines><Interferon Alpha-Beta Receptor Alpha Chain><Internal Ribosome Entry Segment><Internal Ribosome Entry Site><Knock-out><Knockout><Label><Laboratories><Length><Life Cycle><Life Cycle Stages><Ligand Binding Protein><Ligand Binding Protein Gene><Light><Liver><Mediating><Messenger RNA><Mice><Mice Mammals><Modern Man><Molecular Interaction><Murine><Mus><Oral><Pathogenesis><Pathogenicity><Photoradiation><Picornaviridae><Picornaviruses><Poly(A) Tail><Poly(A)+ RNA><Polyadenylated RNA><Prevent infection><Programmed Cell Death><Protein Binding><Proteins><Proteomics><RNA Polymerases><RNA Stability><RNA Viruses><RNA amplification><RNA chemical synthesis><RNA replication><RNA synthesis><Replication Unit><Replicon><Research><Rhinovirus><Ribosome Entry Site><Rodent Model><Role><SIS cytokines><Salivary Gland Viruses><Serum><Short interfering RNA><Site><Small Interfering RNA><Structure><T cell response><T-Cells><T-Lymphocyte><Time><Translations><Treatment Period><Viral><Viral Antigens><Viral Diseases><Viral Gene Products><Viral Gene Proteins><Viral Genes><Viral Proteins><Viral Shedding><Viral load measurement><Viremia><Virus><Virus Diseases><Virus Replication><Virus Shedding><Zinc Finger Domain><Zinc Finger Motifs><Zinc Fingers><access to vaccination><access to vaccines><anti-viral agents><anti-viral drugs><anti-viral efficacy><anti-viral therapy><anti-virals><antiviral efficacy><base><bound protein><cell culture><chemoattractant cytokine><chemokine><clustered regularly interspaced short palindromic repeats screen><cytokine><cytomegalovirus group><genome scale><genome-wide><genomewide><genomic RNA><hepatic body system><hepatic damage><hepatic injury><hepatic organ system><host response><human pathogen><ifnar1 gene product><immune system response><immunoresponse><in vivo><injury response><intrahepatic><knock-down><knockdown><life course><liver damage><liver injury><mRNA><mouse model><murine model><novel><recruit><response><response to injury><siRNA><social role><thymus derived lymphocyte><treatment days><treatment duration><type I IFN receptor><type I interferon receptor><vaccination access><vaccination availability><vaccine access><vaccine availability><viraemia><viral RNA><viral infection><viral infectious disease treatment><viral multiplication><viral replication><viral sepsis><virus RNA><virus antigen><virus infection><virus load><virus multiplication><virus protein><virus-induced disease><virusemia>