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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要甲型肝炎病毒（HAV）是一种属于小核糖核酸病毒科的正链病毒，是急性肝炎的常见原因。肝炎。尽管有疫苗可用，但甲型肝炎发病率的急剧增加导致近年来，美国因严重感染而死亡的人数不断增加。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。一个我们实验室最近进行的全基因组 CRISPR 筛选鉴定出 ZCCHC14（锌指 CCHC 型）含有蛋白质 14) 作为 HAV 复制的重要宿主因子。这令人惊讶，因为 ZCCHC14 不是复制其他小核糖核酸病毒所需的，其已知的活性也与当前的不相符了解 HAV 复制。已知 ZCCHC14 与两个非规范的形成 TRAMP 样复合物聚 (A) RNA 聚合酶 TENT4A 和 TENT4B。该复合物促进乙型肝炎病毒 (HBV) 的复制和人巨细胞病毒 (HCMV)，这两种 DNA 病毒，通过保持多聚 (A) 尾长和稳定性病毒 mRNA。 RG7834 是一种口服二氢喹嗪酮，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 的敲低可强烈抑制 HAV RG7834 对 Huh-7.5 细胞中的 HAV 具有有效的抗病毒活性（IC50=6.2 nM）。我们还表明，RG7834 可阻断甲型肝炎小鼠模型中的 HAV 复制并减少肝损伤使用 Ifnar1-/- 小鼠。然而，与 HBV 不同，RG7834 对 HAV RNA 的聚 (A) 尾长度没有影响，表明它通过独特且新颖的作用机制阻止 HAV 复制。我们假设：（1） ZCCHC14 与 HAV 基因组 RNA 中的特定结构元件结合并招募 TENT4 蛋白以促进病毒 RNA 复制，RG7834 与 TENT4 的结合破坏了其与 ZCCHC14 的关联，病毒 RNA和/或病毒蛋白； (2) RG7834 疗法可以减轻急性甲型肝炎的病程，并且因此，在啮齿动物模型中增强对 HAV 的功能性免疫反应。具体目标 1 将阐明 ZCCHC14-TENT4 在 HAV 生命周期中的作用并研究其潜在机制 RG7834针对HAV的抗病毒活性，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 HAV 的相互作用核糖核酸； 1(c) 鉴定与感染细胞中的 ZCCHC14-TENT4 相互作用的病毒或宿主蛋白。具体的目标2将研究RG7834对小鼠HAV复制和致病性的抗病毒功效，包括：2(a) 口服 RG7834 对肝内病毒复制和肝损伤的功效； 2(b) 是否 RG7834 疗法可诱导针对 HAV 的有益 T 细胞反应。应用程序的总体目标目的是阐明 ZCCHC14-TENT4 复合物在 HAV 复制中的重要作用的机制，并探索二氢喹啉酮类药物（例如 RG7834）用于抗病毒治疗的潜在用途。这项研究将揭示揭示正链 RNA 病毒劫持宿主蛋白以支持其自身功能的新机制复制并可能为甲型肝炎抗病毒治疗打开大门。