Mechanism Of Rotavirus Genome Replication And Packaging

轮状病毒基因组复制和包装机制

• 批准号: 8156898
• 负责人: JOHN PATTON
• 金额: $ 50.36万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8156898
• 关键词: Genome; Rotavirus

Rotaviruses (RVs), members of the Reoviridae family, have genomes consisting of eleven segments of double-stranded (ds) RNA. In the infectious RV particle, the genome is contained within a non-enveloped icosahedral capsid composed of three concentric protein layers. The innermost protein layer is a smooth, thin, pseudo T=1 assembly formed from 12 decamers of the core lattice protein VP2. Tethered to the underside of VP2 layer are complexes comprised of the viral RNA-dependent RNA polymerase (RdRP), VP1, and the RNA-capping enzyme, VP3. Together, VP1, VP2, VP3, and the dsRNA genome form the core of the virion. The core proteins function together to transcribe the segmented dsRNA genome, producing eleven capped plus-sense (+)RNAs. The viral RdRP uses the (+)RNAs as templates for the synthesis of the dsRNA genome. Although the RdRP alone can recognize viral (+)RNAs, the polymerase is only active when VP2 is present. The VP2-dependent activity of VP1 provides a means by which genome replication (dsRNA synthesis) can be linked with genome packaging (core assembly). Newly made (+)RNAs pass directly from the RdRP to VP3, an enzyme which introduces m7G caps at the 5'-end of the transcripts through associated guanylyltransferase and methyltransferase activities. Genome replication and core assembly take place in cytoplasmic inclusions bodies, termed viroplasms. Two viral nonstructural proteins, the octamer NSP2 and the dimer NSP5, direct the formation of viroplasms. The interactions of NSP2 and NSP5 with VP1, VP2, and VP3 coordinate genome replication and core assembly. The overriding goal of this project is to characterize the structure and function of the core proteins VP1, VP2, and VP3 and the viroplasm building-blocks NSP2 and NSP5. This includes defining the structural interfaces between the proteins and establishing how these interactions affect and regulate the activities of the proteins. Progress toward this goal is summarized below. (1) RNA interactions with the RV RdRP. The atomic structure of VP1 was determined by X-ray crystallography though a collaboration with Dr. Steve Harrison's group at Harvard, with details described in a publication appearing in 2008. Briefly, the results showed that the RV polymerase is a compact globular protein with three distinct domains: (i) an N-terminal protruding domain, (ii) a polymerase domain comprised of fingers, palm, and thumb subdomains, and (iii) a C-terminal bracelet domain. Together, the N- and C-terminal domains of VP1 sandwich most of the polymerase domain, creating a cage structure with the catalytic region located within a largely hollow center. Four tunnels connect the surface of VP1 to the catalytic center. These tunnels allow for (i) entry of nucleotides, (ii) entry of single-stranded template RNA, (iii) exit of the dsRNA product or the (-)RNA template, and (iv) exit of (+)RNA transcripts. Soaks of VP1 crystals with various RNA oligonucleotides have provided insight into the mechanism by which the RV polymerase binds and recognizes its (+)RNA templates. These analyses have revealed that the RdRP anchors the 3'consensus sequence (3'CS: 5'-UGUGACC-3') of (+)RNAs into the template entry tunnel and catalytic center via stacking interactions and an extensive network of hydrogen bonds. These interactions include specific contacts with the UGUG bases and nonspecific contacts with the sugar-phosphate backbone of the GACC portions of the 3'CS. Remarkably, these interactions place the 3'-terminal residue of the 3'CS past the site in the catalytic center required to support RNA initiation. In the presence of VP2, it is thought that conformational changes occur that bring the 3'-terminal residue back into proper register to support initiation. To better understand the importance of the interactions between the 3'CS and the RdRP for genome replication, we engineered mutant VP1 proteins and assayed their capacity to synthesize dsRNA in vitro. Our results showed that, individually, mutation of residues that interact specifically with RNA bases did not diminish replication levels. However, simultaneous mutation led to significantly lower levels of dsRNA product, presumably due to impaired recruitment of (+)RNA templates. In contrast, point mutation of nonspecific RNA contact residues in VP1 led to severely diminished replication, likely as a result of improper positioning of templates at the catalytic site. A noteworthy exception was a K419A mutation that enhanced the initiation capacity and product elongation rate of VP1. The specific chemistry of Lys419 and its position at a narrow region of the template entry tunnel appear to contribute to its capacity to moderate replication. Together, our findings suggest that distinct classes of VP1 residues interact with (+)RNA to mediate template recognition and dsRNA synthesis, yet function in concert to promote viral replication at appropriate times and with ideal kinetics. The findings of our analysis have implications for the structure and function of other RdRPs (e.g., HCV, FDHV), as they contain many of the same RNA-contact residues that are used by the RV RdRP to recruit template RNAs into the catalytic center of the enzyme. (2) VP2-dependent activation of the RV RdRP. Most human RV isolates can be classified into one of three groups (A, B, or C). Their segmented genome allows RVs to readily exchange genetic material during co-infections. This reassortment process occurs between viruses belonging to the same group, but not for viruses belonging to different groups. This restriction might reflect the failure of the viral RdRP to recognize and replicate viral RNAs of a different group. To address this question, we have carried out experiments aimed at contrasting the sequences, structures, and functions of RdRPs belonging to RV groups A, B, and C. We found that conserved amino acid residues are located within the hollow center of VP1 near the active site, whereas variable, group-specific residues are mostly surface exposed. By creating a three dimensional homology model of the group C RdRP, based on an atomic structure determined for the group A RdRP, we obtained evidence that these RV RdRPs have nearly identical tertiary folds and share similar mechanisms of recognizing RNA templates. Consistent with their structural analysis, we determined that recombinant group A and C RdRPs are capable of replicating one anothers RNA templates in vitro. However, the activity of both RdRPs is strictly dependent on the presence of their cognate VP2 core lattice protein. That is, the group A RdRP has activity in the presence of group A VP2, but not group C VP2, and vice versa. Thus, the reassortment restriction between rotavirus groups may reflect the inability of their replication proteins to function together in support of genome replication. In the past year, we undertook a study in which we assayed the VP1 and VP2 proteins of various strains of group A and C RVs to determine the limitations of functional compatibility for in vitro dsRNA synthesis. By engineering chimeric group A/C VP2 proteins capable of activating non-cognate VP1, we delineated core shell subdomains important for turning on polymerase function. Our results demonstrate that the amino termini of VP2, which interact to form an internal protruding hub underneath each fivefold axis (vis--vis, a VP2 decamer) of the viral core, play an important but non-specific role in VP1 activation. The VP2 residues that correlate with polymerase-activation specificity are located on the inner face of the principal (scaffold) domain, within the apical and/or central subdomains. These results indicate that function of the RV polymerase requires multiple interactions with the VP2 decamer, including with regions of both the hub and scaffold domain.

轮状病毒（RVS）是依伏迪科家族的成员，其基因组由11个段的双链（DS）RNA组成。在传染性的RV颗粒中，基因组包含在由三个同心蛋白质层组成的非发育二十面体内衣壳中。最内向的蛋白质层是一个光滑，薄的伪T = 1组件，由核心晶格蛋白VP2的12个decamer形成。束缚在VP2层的底面的是由病毒RNA依赖性RNA聚合酶（RDRP），VP1和RNA限制酶VP3组成的复合物。 VP1，VP2，VP3和DSRNA基因组一起构成了病毒体的核心。核心蛋白共同发挥作用以转录分段的DSRNA基因组，产生11个上限的加sense（+）RNA。病毒RDRP使用（+）RNA作为DSRNA基因组合成的模板。尽管单独的RDRP可以识别病毒（+）RNA，但仅在存在VP2时，聚合酶才有活性。 VP1的VP2依赖性活性提供了一种方法，可以将基因组复制（DSRNA合成）与基因组包装（核心组装）联系起来。新制造的（+）RNA直接从RDRP传递到VP3，这是一种酶，该酶通过相关的Guanylyllyllansferase和甲基转移酶活性在转录本的5'末端引入M7G盖。基因组复制和核心组装发生在细胞质内包含体中，称为病毒肿瘤。两个病毒非结构蛋白，即八聚体NSP2和二聚体NSP5，指导ViroPlasms的形成。 NSP2和NSP5与VP1，VP2和VP3坐标基因组复制和核心组件的相互作用。 该项目的重大目标是表征核心蛋白VP1，VP2和VP3的结构和功能以及VirOplast building-Bluide-Bluide-blude-bluide nsp2和nsp5。这包括定义蛋白质之间的结构接口并确定这些相互作用如何影响和调节蛋白质的活性。下面总结了朝着这个目标的进展。 （1）与RV RDRP的RNA相互作用。 The atomic structure of VP1 was determined by X-ray crystallography though a collaboration with Dr. Steve Harrison's group at Harvard, with details described in a publication appearing in 2008. Briefly, the results showed that the RV polymerase is a compact globular protein with three distinct domains: (i) an N-terminal protruding domain, (ii) a polymerase domain comprised of fingers, palm, and thumb subdomains, and （iii）C末端手镯域。 VP1三明治的N-和C末端结构域大多数聚合酶结构域，形成了一个笼子结构，催化区域位于大部分空心中心。四个隧道将VP1的表面连接到催化中心。这些隧道允许（i）进入核苷酸，（ii）进入单链模板RNA，（iii）DSRNA产物或（ - ）RNA模板的出口，以及（iv）RNA转录本的出口。具有各种RNA寡核苷酸的VP1晶体的浸泡提供了有关RV聚合酶结合并识别其（+）RNA模板的机制的洞察力。这些分析表明，RDRP通过堆叠相互作用和广泛的氢键网络将（+）RNA的3'Consensus序列（3'CS：5'-GugugAcc-3'）固定在模板进入隧道和催化中心。这些相互作用包括与乌干达基地的特定接触以及与3'C的GACC部分的糖磷酸骨架的非特异性接触。值得注意的是，这些相互作用将3'C的3'-末端残基置于支撑RNA启动所需的催化中心中的位置。在存在VP2的情况下，人们认为发生构象变化，将3'-末端残基重新回到适当的登记册中以支持启动。 为了更好地理解3'CS与RDRP对基因组复制之间相互作用的重要性，我们设计了突变体VP1蛋白，并分析了它们在体外合成DSRNA的能力。我们的结果表明，与RNA碱基专门相互作用的残基的突变不会降低复制水平。然而，同时突变导致DSRNA产物的水平显着降低，这可能是由于（+）RNA模板的募集受损。相反，VP1中非特异性RNA接触残基的点突变导致复制严重减少，这可能是由于模板在催化位点的定位不当而导致的。值得注意的例外是K419A突变，增强了VP1的启动能力和产品伸长率。 LYS419的特定化学及其在模板进入隧道狭窄区域的位置似乎有助于其中度复制的能力。总之，我们的发现表明，不同类别的VP1残基与（+）RNA相互作用以介导模板识别和DSRNA合成，但在适当的时间和理想动力学的情况下协同促进病毒复制。我们的分析发现对其他RDRP（例如HCV，FDHV）的结构和功能具有影响，因为它们包含RV RDRP使用的许多相同的RNA接触残基来募集酶的催化中心。 （2）RV RDRP的VP2依赖性激活。大多数人RV分离株可以分为三组之一（A，B或C）。它们的分段基因组允许RV在共同感染期间容易交换遗传物质。这种保证过程发生在属于同一组的病毒之间，而不是针对属于不同组的病毒。该限制可能反映了病毒RDRP无法识别和复制不同群体的病毒RNA的失败。为了解决这个问题，我们进行了旨在对比属于RV组A，B和C的RDRP的序列，结构和功能的实验。我们发现，保守的氨基酸残基位于活性位点附近的VP1的空心中心内，而群体可变，组特定的残基大部分表面暴露。通过基于确定A组RDRP的原子结构，创建C组CRDR的三维同源模型，我们获得了证据，证明这些RV RDRP几乎具有相同的第三次折叠，并具有识别RNA模板的相似机制。与它们的结构分析一致，我们确定重组A组和C RDRP能够在体外复制一个Anothers RNA模板。但是，两个RDRP的活性严格取决于其同源VP2核心晶格蛋白的存在。也就是说，A组RDRP在A组VP2的存在下具有活性，而不是C组VP2，反之亦然。因此，轮状病毒组之间的重新分类限制可能反映其复制蛋白无法共同起作用以支持基因组复制。 在过去的一年中，我们进行了一项研究，其中我们测定了A和C组的各种菌株的VP1和VP2蛋白，以确定体外DSRNA合成的功能兼容性的局限性。 通过工程嵌合A/C VP2蛋白能够激活非认知VP1，我们描绘了对打开聚合酶功能很重要的核心壳子域。我们的结果表明，VP2相互作用的氨基末端在病毒核的每个五倍轴（Vis-vis，VP2 decamer）下面形成内部突出中心，在VP1激活中起重要但非特异性的作用。与聚合酶激活特异性相关的VP2残基位于基本和/或中央子域内主（支架）结构域的内表面。这些结果表明，RV聚合酶的功能需要与VP2 Decamer多次相互作用，包括与集线器和支架域的区域。