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

ZCCHC14-TENT4复合物在甲型肝炎病毒感染中的抗病毒抑制作用

• 批准号: 10460644
• 负责人: You Li
• 金额: $ 19.44万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-05 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10460644
• 关键词: Acute Hepatitis; Alanine Transaminase; Antiviral Therapy; Apoptosis; Binding; Binding Proteins; CRISPR screen; CRISPR/Cas technology; Cell Culture Techniques; Cells; Cessation of life; Complex; Cytomegalovirus; DNA Viruses; DNA-Directed RNA Polymerase; Data; Dose; Drops; Elements; Family Picornaviridae; Gene Expression; Goals; Hepatitis; Hepatitis A; Hepatitis A Virus; Hepatitis B Virus; Hepatovirus; Human; Immune; Immune response; In Vitro; Incidence; Infection; Infection prevention; Infiltration; Integration Host Factors; Internal Ribosome Entry Site; Knock-out; Label; Laboratories; Length; Life Cycle Stages; Light; Liver; Mediating; Messenger RNA; Mus; Oral; Pathogenesis; Pathogenicity; Poly(A) Tail; Poly(A)+ RNA; Proteins; Proteomics; RNA Stability; RNA Viruses; RNA amplification; RNA chemical synthesis; RNA replication; Replicon; Research; Rhinovirus; Rodent Model; Role; Serum; Site; Small Interfering RNA; T cell response; T-Lymphocyte; Time; Translations; Viral; Viral Antigens; Viral Genes; Viral Proteins; Viral load measurement; Viremia; Virus; Virus Diseases; Virus Replication; Virus Shedding; Zinc Fingers; anti-viral efficacy; base; chemokine; cytokine; genome-wide; genomic RNA; human pathogen; in vivo; intrahepatic; knock-down; liver injury; mouse model; novel; recruit; response; response to injury; treatment duration; type I interferon receptor; vaccine access; viral RNA

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.

项目总结/摘要 甲型肝炎病毒（HAV）是小核糖核酸病毒科（Picornaviridae）的正链病毒，是急性肝炎的常见原因。 肝炎尽管疫苗的可用性，甲型肝炎发病率的显著增加导致 近年来，美国与严重感染相关的死亡人数不断增加。重要的是没有 存在能够减轻与HAV感染相关的严重肝损伤的抗病毒治疗。一 最近在我们实验室进行的全基因组CRISPR筛选鉴定了ZCCHC 14（锌指CCHC型 含有蛋白质14）作为HAV复制的必需宿主因子。这是令人惊讶的，因为ZCCHC 14不是 其他小核糖核酸病毒复制所需的，也不是其已知的活动与目前的一致。 了解HAV复制。已知ZCCHC 14与两个非典型的 聚（A）RNA聚合酶TENT 4A和TENT 4 B。这种复合物促进B型肝炎病毒（HBV）的复制 和人巨细胞病毒（HCMV），这两种DNA病毒，通过保持聚（A）尾长和稳定性， 病毒mRNA。RG 7834是一种口服二氢喹嗪酮，靶向TENT 4 A/B，具有抗病毒活性 体内抗HBV。我们发现ZCCHC 14或TENT 4 A/B的敲低强烈抑制HAV RG 7834在Huh-7.5细胞中对HAV具有有效的抗病毒活性（IC 50 =6.2 nM）。我们 还显示RG 7834阻断甲型肝炎小鼠模型中HAV复制并减少肝损伤 使用Ifnar 1-/-小鼠。然而，与HBV不同，RG 7834对HAV RNA的poly（A）尾长度没有影响， 表明它通过独特的和新的作用机制阻断HAV复制。我们假设：（1） ZCCHC 14与HAV基因组RNA中的特异性结构元件结合，并招募TENT 4蛋白以促进HAV基因组的表达。 RG 7834与TENT 4的结合破坏了其与ZCCHC 14、病毒RNA复制的结合， RNA和/或病毒蛋白;和（2）RG 7834治疗可缓解急性甲型肝炎的病程，和 从而增强啮齿动物模型中对HAV的功能性免疫应答。具体目标1将 阐明ZCCHC 14-TENT 4在HAV生命周期中的作用，并研究其机制。 RG 7834对HAV的抗病毒活性，包括：1（a）确定复制周期中的步骤 需要ZCCHC 14并被RG 7834抑制; 1（B）表征ZCCHC 14与HAV的相互作用 RNA;和1（c）鉴定感染细胞中与ZCCHC 14-TENT 4相互作用的病毒或宿主蛋白。具体 目的2研究RG 7834对小鼠HAV复制和致病性的影响，包括：2（a） 经口给予RG 7834对肝内病毒复制和肝损伤的效力; 2（B）是否 RG 7834疗法诱导HAV特异性有益T细胞反应。应用程序的首要目标 阐明ZCCHC 14-TENT 4复合物在HAV复制中的重要作用机制， 并探索二氢喹嗪酮类化合物如RG 7834在抗病毒治疗中的潜在用途。这项研究将使 一种新的机制，通过这种机制，正链RNA病毒劫持宿主蛋白质来支持其 复制，并可能打开甲型肝炎抗病毒治疗的大门。