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

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

展开

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.

项目摘要/摘要肝炎病毒（HAV），一种在picornaviridae中分类的链链病毒，是急性的常见原因肝炎。尽管有疫苗的可用性，但乙型肝炎的发生率的惊人增加导致近年来，与美国严重感染有关的死亡人数增加。重要的是，不存在能够减轻与HAV感染相关的严重肝损伤的抗病疗法。一个在我们的实验室确定的ZCCHC14（锌指CCHC型）中进行的最新全基因组CRISPR屏幕包含蛋白质14）作为HAV复制的基本宿主因素。这是令人惊讶的，因为ZCCHC14不是复制其他picornavirus的必需品，其已知活动也不与当前了解HAV复制。已知ZCCHC14形成一个流浪汉，具有两个非典型的复合物聚（A）RNA聚合酶Tent4a和Tent4b。这种复合物促进了丙型肝炎病毒（HBV）的复制和人类巨细胞病毒（HCMV），两种DNA病毒，都通过维持poly（a）的尾巴长度和稳定性病毒mRNA。 RG7834是一种口服二氢喹啉酮，靶向Tent4a/b，具有抗病毒活性反对体内HBV。我们发现ZCCHC14或TENT4A/B的敲低强烈抑制HAV 复制，RG7834在HUH-7.5细胞中对HAV具有有效的抗病毒活性（IC50 = 6.2 nm）。我们还表明，RG7834阻止了HAV复制并减少肝炎鼠模型中的肝损伤使用IFNAR1 - / - 小鼠。但是，与HBV不同，RG7834对HAV RNA的聚（A）尾巴的长度没有影响，表明它通过独特而新颖的作用机理阻止了HAV复制。我们假设：（1） ZCCHC14与HAV基因组RNA中的特定结构元素结合，并募集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 RNA和/或病毒蛋白；（2）RG7834治疗可以减轻急性肝炎的进程，并因此，在啮齿动物模型中增强对HAV的功能免疫反应。具体目标1将阐明ZCCHC14-TENT4在HAV生命周期中的作用并研究基本机制 RG7834对HAV的抗病毒活性，包括：1（a）确定复制循环中的步骤需要ZCCHC14并被RG7834抑制； 1（b）表征ZCCHC14与HAV的相互作用 RNA； 1（c）识别感染细胞中与ZCCHC14-TENT4相互作用的病毒或宿主蛋白。具体的 AIM 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）口服的RG7834在肝内病毒复制和肝损伤方面的效力； 2（b）是否 RG7834治疗诱导对HAV的有益T细胞反应。应用程序的总体目标是为了阐明ZCCHC14-TENT4复合物在HAV复制中的基本作用的基础机制，并探索二氢喹啉酮（例如RG7834）的潜在用途。这项研究将脱落对一种新型机制的照明，阳性RNA病毒劫持了宿主蛋白以支持其复制并有可能打开肝炎抗病毒治疗的大门。