Antiviral inhibition of ZCCHC14-TENT4 complex in hepatitis A virus infection

项目摘要/摘要 甲型肝炎病毒(HAV)是一种正链病毒，归类于短链病毒科，是引起急性肝炎的常见原因 肝炎。尽管有疫苗可用，甲型肝炎发病率的显著增加导致了 近年来，美国与严重感染有关的死亡人数不断增加。重要的是，不 存在能够减轻与甲型肝炎病毒感染相关的严重肝损伤的抗病毒治疗。一个 最近在我们实验室进行的全基因组CRISPR筛查结果表明，ZCCHC14(锌指CCHC-型 包含蛋白质14)作为甲型肝炎病毒复制的基本宿主因子。这令人惊讶，因为ZCCHC14并不是 复制其他小核糖核酸病毒所需的，其已知活性也不符合当前 了解甲型肝炎病毒复制。已知ZCCHC14与两个非正则化合物形成流浪形复合体 聚(A)RNA聚合酶TENT4A和TENT4B。这种复合体促进了乙肝病毒(乙肝病毒)的复制 和人类巨细胞病毒(HCMV)这两种DNA病毒，通过维持Poly(A)的尾长和稳定性 病毒mRNAs。RG7834是一种口服二氢喹诺酮，靶向TENT4A/B，具有抗病毒活性 体内抗乙肝病毒。我们发现ZCCHC14或TENT4A/B基因的敲除对甲型肝炎病毒有很强的抑制作用 RG7834在Huh-7.5细胞中具有较强的抗甲型肝炎病毒活性(IC50=6.2 nM)。我们 RG7834还表明，在甲型肝炎小鼠模型中，RG7834可以阻止甲型肝炎病毒的复制，并减少肝脏损伤 使用Ifnar1-/-小鼠。然而，与乙肝病毒不同，RG7834对甲型肝炎病毒RNA的聚(A)尾巴长度没有影响， 这表明它通过一种独特而新颖的作用机制阻止甲型肝炎病毒的复制。我们假设：(1) ZCCHC14结合甲型肝炎病毒基因组RNA中的特定结构元件并招募TENT4蛋白促进 病毒RNA复制，RG7834与TENT4的结合破坏了其与ZCCHC14病毒的结合 RNA和/或病毒蛋白；以及(2)RG7834疗法可以缓解急性甲型肝炎的病程，以及 因此，在啮齿动物模型中增强对甲型肝炎病毒的功能性免疫反应。具体目标1将 阐明ZCCHC14-TENT4在甲型肝炎病毒生命周期中的作用并探讨其机制 RG7834的抗甲型肝炎病毒活性，包括：1(A)确定复制周期中的步骤 需要ZCCHC14并被RG7834抑制；1(B)表征ZCCHC14与甲型肝炎病毒的相互作用 1(C)鉴定感染细胞中与ZCCHC14-TENT4相互作用的病毒或宿主蛋白。特定的 目的研究RG7834对甲型肝炎病毒复制和小鼠致病性的抗病毒作用，包括：2(A) 口服RG7834对肝内病毒复制和肝损伤的效力；2(B) RG7834治疗可诱导针对甲型肝炎病毒的有益T细胞应答。应用程序的总体目标是 目的是阐明ZCCHC14-TENT4复合体在甲型肝炎病毒复制中的重要作用机制。 并探索RG7834等二氢喹诺酮类药物在抗病毒治疗中的潜在用途。这项研究将使 正链RNA病毒劫持宿主蛋白以支持其功能的新机制 复制并有可能为甲型肝炎的抗病毒治疗打开大门。

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.