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 poten...