Rotavirus Reverse Genetics System to Study Viral Pathogenesis and Receptor Interactions

1篇摘要 2 A组轮状病毒（RVs）是引起幼龄动物急性胃肠炎和死亡的重要原因 3、孩子尽管它们的意义，RVA细胞进入，复制和衰减的机制仍然很差 4项理解表明迫切需要解决这些知识差距并设计通用控制方法。 5我们之前进行了比较序列分析，确定了两个关键的氨基酸取代 6与细胞培养适应和减毒相关：大多数人和猪RVA毒株的VP4中的D385 N， 7仅在猪OSU菌株VP4中的D393H。虽然这些或类似的突变以前被认为是重要的， 8细胞培养适应/减毒，它们的功能尚未得到实验证实。利用我们的猪肠 9肠（PIE）系统，我们已经证明，与宿主聚糖的相互作用的对比模式决定了 10不同RVA菌株的复制效力。OSU复制是独特的，并显着下调， 11去除末端唾液酸，而后者显著增强了新型猪G9P菌株的复制[13]。 12 OSU是一种历史性的显性RVA变异体，其特征在于体内和体外复制稳健，并且与RVA变异体一致。 13与小猪腹泻病相关已有数十年，而G9 P [13]和G9 P [19]是最近在全球范围内发现的 猪和人类中出现的14种RVA变异。后一种菌株可能已经进化到获得一些生物学特性， 15个优点，允许它们在不同主机中有效传播和复制。毒力OSU和G9P [13]菌株具有 在VP4疏水环的aa位置385和393中存在16个独特的aa取代，这可能是VP4疏水环的aa位置385和393中存在16个独特的aa取代的原因。 与宿主聚糖的17种可变相互作用。此外，我们最近的研究表明，宿主（PIE）转录组 18与OSU和G9P感染相关的反应[13]显著不同， 19代谢和先天性免疫信号转导的菌株特异性方式。这些发现表明， RVA对宿主和宿主聚糖的适应是一种关键机制，允许它们的种间（包括 21人畜共患）传播。 22按照之前优化的方案，我们实验室已成功将11个强毒RVA OSU片段克隆到 23 pT7质粒和拯救的感染性病毒。我们现在提议利用这种反向遗传学系统进行深入探索 RVA毒力、细胞附着和复制的分子机制。实现这一目标的具体目标 25项总结如下： 目的1：评估D385N和D393H置换（单独地或与OSU中的其他突变组合）是否与D385N和D393H置换相关。 27 VP4）将赋予后代病毒（ic-attOSU）减毒表型。目的 28 2：评估G9P [13]样突变（S385 N和D393 N）是否将赋予后代G9P [13]样聚糖特异性 29病毒（ic-G9OSU）。目的3：对野生型（ic-virOSU）和突变型（OSU-100）进行比较转录组分析。 30 att和OSU-G9）OSU变体。我们的PIE和Gn猪模型以及新建立的RGS将允许研究 31鉴定的VP4突变如何改变RVA毒力和与宿主聚糖的选择性相互作用。 32

1 Abstract 2 Rotaviruses (RVs) of group A (RVAs) remain an important cause of acute gastroenteritis and mortality in young animals 3 and children. Despite their significance, mechanisms of RVA cell entry, replication and attenuation remain poorly 4 understood indicating a critical need to address these knowledge gaps and design universal control approaches. 5 We have previously conducted comparative sequence analysis that identified two key amino acid substitutions 6 associated with cell culture adaptation and attenuation: D385N in the VP4 of most human and porcine RVA strains and 7 D393H in porcine OSU strain VP4 only. While these or similar mutations were previously suggested to be important for 8 cell culture adaptation/attenuation, their function has not been confirmed experimentally. Using our porcine intestinal 9 enteroid (PIE) system, we have demonstrated that contrasting modes of interactions with the host glycans determine 10 replication efficacy of different RVA strains. OSU replication was uniquely and significantly down-regulated by the 11 removal of terminal sialic acids, while the latter significantly enhanced the replication of a novel porcine G9P[13] strain. 12 OSU is a historic dominant RVA variant characterized by robust in vivo and in vitro replication and consistently 13 associated with diarrheal disease in piglets for several decades, while G9P[13] and G9P[19] are recent, globally 14 emerging RVA variants in swine and humans. It is possible that the latter strains have evolved to acquire some biological 15 advantage allowing for their efficient spread and replication in different hosts. Virulent OSU and G9P[13] strains have 16 unique aa substitutions in the aa positions 385 and 393 of the VP4 hydrophobic loop, which could be a reason for 17 variable interactions with host glycans. Additionally, our recent study demonstrated that the host (PIE) transcriptome 18 response associated with OSU and G9P[13] infections differed drastically altering glycan expression, cholesterol 19 metabolism and innate immune signaling in a strain-specific manner. These findings suggest that evolutionary 20 adaptation of RVAs to the host and host glycans is a critical mechanism allowing for their interspecies (including 21 zoonotic) transmission. 22 Following a previously optimized protocol, our lab has successfully cloned 11 segments of virulent RVA OSU into the 23 pT7 plasmid and rescued infectious virus. We now propose to use this reverse genetics system for in-depth exploration 24 of the molecular mechanisms of RVA virulence, cell attachment and replication. The specific aims to achieve this goal 25 are summarized below: 26 Aim 1: Evaluate if the D385N and D393H substitutions (individually or in combination with other mutations in the OSU 27 VP4) introduced into OSU RGS (ic-virOSU) will confer an attenuated phenotype to the progeny virus (ic-attOSU). Aim 28 2: Evaluate if G9P[13]-like mutations (S385N and D393N) will confer the G9P[13]-like glycan specificity to the progeny 29 virus (ic-G9OSU). Aim 3: To conduct comparative transcriptome analysis of the wild-type (ic-virOSU) and mutant (OSU- 30 att and OSU-G9) OSU variants. Our PIE and Gn pig models as well as the newly established RGS will allow to study 31 how the identified VP4 mutations alter RVA virulence and the selective interactions with host glycans. 32