Mechanism Of Rotavirus Genome Replication And Packaging

轮状病毒基因组复制和包装机制

• 批准号: 8555822
• 负责人: JOHN PATTON
• 金额: $ 55.15万
• 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/8555822
• 关键词: Accident and Emergency department; Active Sites; Acute; Affect; Affinity; Affinity Chromatography; Alanine; Amino Acids; Amylose; Antiviral Agents; Arginine; Aspartic Acid; BCAR3 gene; Binding; Biological Assay; Biology; Bluetongue virus; Capsid; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Characteristics; Child; Collaborations; Complex; Core Assembly; Core Protein; Cryoelectron Microscopy; Cytoplasmic Inclusion; DNA Sequence Rearrangement; DNA-Directed RNA Polymerase; Developing Countries; Development; Diarrhea; Divalent Cations; Double-Stranded RNA; Elements; Engineering; Enzymes; Escherichia coli; Event; Family; Gastroenteritis; Genetic; Genome; Germany; Glycine; Goals; Homologous Gene; Homology Modeling; Hospitalization; Hour; Human; In Vitro; Inclusion Bodies; Individual; Infant; Infection; Lead; Lesion; Life; Life Cycle Stages; Link; Maps; Mediating; Methyltransferase; Modeling; Modification; Mutation; N-terminal; Nucleotides; Outcome; Outpatients; Phenotype; Phosphoproteins; Phosphorylation; Polymerase; Positioning Attribute; Process; Proteins; RNA; RNA Caps; RNA chemical synthesis; RNA-Directed RNA Polymerase; Recombinant Proteins; Regulatory Element; Relative (related person); Reoviridae; Reovirus; Rotavirus; Rotavirus Infections; Rotavirus Vaccines; Signal Transduction; Site; Solubility; Structure; System; TEV protease; Temperature; Transcript; Vaccines; Viral; Viral Nonstructural Proteins; Virion; Virus; Visit; Work; X-Ray Crystallography; base; falls; guanylyltransferase; insight; maltose-binding protein; member; mutant; particle; positional cloning; prevent; programs; protein function; success; tool; viral RNA; virology

Rotaviruses (RVs), members of the Reoviridae family, have genomes consisting of eleven segments of double-stranded (ds) RNA. The genome of the RV virion is contained in 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 the 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 and core assembly. Newly made (+)RNAs pass from the RdRP to VP3, an enzyme which introduces m7G caps to the 5'-end of the transcripts through associated guanylyltransferase and methyltransferase activities. Genome replication and core assembly take place in cytoplasmic inclusions bodies of infected cells; these structures are referred to as viroplasms. Two viral nonstructural proteins, the octamer NSP2 and the phosphoprotein 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 in 2011-2012 is summarized below. (1) RV RNA POLYMERASES RESOLVE INTO TWO PHYLOGENETICALLY DISTINCT CLASSES THAT DIFFER IN THEIR MECHANISM OF TEMPLATE RECOGNITION. Within the Rotavirus genus, eight species (RVA-RVH) have been proposed to exist. The RVA viruses have been the most widely studied since they the primary cause of life-threatening dehydrating diarrhea in infants and young children. In collaboration with Dr. Reimar Johnes group in Germany, we determined the first known sequences for the viral RdRP (VP1)-encoding segments of RVF and RVG viruses and compared these sequences to those of other RV species. Our analysis indicates that the VP1 RNA segments and proteins resolve phylogenetically into two major clades (A and B). RVA, RVC, RVD and RVF species fall within clade A, while RVB, RVG and RVH species fall within clade B. Plus-strand RNAs of clade A viruses, and not clade B viruses, contain a 3'-proximal UGUG cassette that serves as a recognition signal mediating specific interaction with viral polymerases. The polymerase recognition signal in plus-strand RNA of clade B viruses remains to be determined. A VP1 structure for each RV species were predicted using homology modeling. Structural elements involved in interactions with the UGUG cassette were conserved among VP1 of clade A, suggesting a conserved mechanism of viral RNA recognition for these viruses. Based on the distinct characteristics of the polymerases of clade A and B RVs, including poor sequence identity values, the single RV genus should probably be resolved into two genera. Virology (2012) 431:50-57. (2) MUTATIONAL ANALYSIS OF RESIDUES INVOLVED IN NUCLEOTIDE AND DIVALENT CATION STABILIZATION IN THE RV RdRP CATALYTIC POCKET. The RV RdRP, VP1, contains canonical RdRP motifs and a priming loop that is hypothesized to undergo conformational rearrangements during RNA synthesis. In the absence of viral core shell protein VP2, VP1 fails to interact stably with divalent cations or nucleotides and has a retracted priming loop. To identify residues of potential importance to nucleotide and divalent cation stabilization, we aligned VP1 of divergent RVs and the structural homolog reovirus lambda3. VP1 mutants were engineered and characterized for RNA synthetic capacity in vitro. Conserved aspartic acids in RdRP motifs A and C and arginines in motif F that likely stabilize divalent cations and nucleotides were required for efficient RNA synthesis. Mutation of individual priming loop residues diminished or enhanced RNA synthesis efficiency without obviating the need for VP2, which suggests that this structure serves as a dynamic regulatory element that links RdRP activity to particle assembly. Virology (2012) 431:12-20 (3) PREDICTED STRUCTURE AND DOMAIN PURIFICATION OF RV CAPPING ENZYME VP3. VP3 is a critical RV enzyme that is present in virions at low copy number. Several activities involved in viral RNA capping, including guanylyltransferase (GTase) and N7 and 2'-O methyltransferase (MTase), have been ascribed to VP3. The structure of the 835-amino acid VP3 protein, however, remains unknown. Based on homology modeling with the bluetongue virus (BTV)-capping enzyme, VP4, the structure of RV VP3 residues 109-634 was predicted. RV VP3 and BTV VP4 appear to share a similar modular organization, with the 2'-O MTase domain positioned within the N7 MTase domain. In addition, RV VP3 and BTV VP4 have in common several putative MTase active-site residues. The structure of VP3 residues 697-800 was modeled based on homology with members of the 2H phosphoesterase superfamily of enzymes and appears to contain two conserved active-site His-X-Thr/Ser motifs. No homology models were generated with confidence for the N-terminal 108 residues of VP3. Based on predicted structure, we have expressed individual domains of VP3 in E. coli as N-terminal fusions with maltose binding protein. We utilized amylose affinity chromatography to purify fusion constructs representing the predicted N-terminal, 2'-O MTase, combined N7 and 2'-O MTase, and GTase and 2H phosphoesterase domains. The affinity tag was removed by cleavage at an engineered tobacco etch virus protease site. Currently, we are characterizing the solubility and activities of the purified VP3 fragments and working to determine their structures. (4) PROBING NSP5 FUNCTION VIA CHARACTERIZATION OF THE RV TEMPERATURE-SENSITIVE (ts) MUTANT, tsJ. The RV 28kD phosphoprotein NSP5 is an essential building block of viroplasms. NSP5 interacts with NSP2 and VP2 and binds non-specifically to viral RNA. However, the importance of these interactions and the significance of NSP5 phosphorylation in the viral replication cycle are poorly understood. The RV mutant, tsJ, contains a ts lesion that maps to the NSP5-encoding segment. At the non-permissive temperature, the ts lesion causes a 10ex3 reduction in virus titer. The ts lesion is correlated with an alanine to glycine substitution at residue 182 of NSP5. In this study, we analyzed the tsJ phenotype to better understand the function of NSP5. Our results indicate that the level of NSP5 expression in tsJ-infected cells was two-fold higher at non-permissive temperature than at permissive temperature. No similar increase was seen in the expression of NSP2 and VP6. At the non-permissive temperature, viroplasms were initially formed in tsJ-infected cells, but by 9 hours post-infection few inclusions were detected. Additionally, the disappearance of viroplasms was concurrent with mislocalization of NSP2, VP2, and tsJ NSP5. By taking advantage of the reduced replication phenotype of tsJ at the non-permissive temperature, we are currently developing an NSP5-dependent complementation assay. Together, characterization of tsJ and the development of a NSP5 complementation assay will allow for elucidation of NSP5 functions during RV infection.

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。 RV病毒体的基因组包含在由三个同心蛋白质层组成的非发达二十面体内衣壳中。最内向的蛋白质层是一个光滑，薄的伪T = 1组件，由核心晶格蛋白的12个decamer形成，VP2。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）与基因组包装和核心组装联系起来。新制造的（+）RNA从RDRP传递到VP3，这是一种通过相关的鸟叶兰氏转移酶和甲基转移酶活性引入转录本的5'末端的酶。 基因组复制和核心组装发生在感染细胞的细胞质包含体中。这些结构称为病毒肿瘤。两种病毒非结构蛋白，即八聚体NSP2和磷酸蛋白NSP5，指导了病毒浮肿的形成。 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2011 - 2012年这一目标的进展。 （1）RV RNA聚合酶分为两个系统发育不同的类别，它们的模板识别机理不同。在轮状病毒属中，已经提出了八个物种（RVA-RVH）。 RVA病毒是研究最广泛的研究，因为它们是婴儿和幼儿威胁生命的脱水腹泻的主要原因。与德国Reimar Johnes Group合作，我们确定了RVF和RVG病毒的病毒RDRP（VP1）编码段的第一个已知序列，并将这些序列与其他RV物种进行了比较。我们的分析表明，VP1 RNA片段和蛋白质将系统发育分解为两个主要进化枝（A和B）。 RVA，RVC，RVD和RVF物种属于进化枝A，而RVB，RVG和RVH物种落在进化枝B中B。促进病毒的加链RNA，而不是进化枝B病毒，含有3'-透明的UGUG盒，可作为识别特定的与病毒polylal Polymerersase的特定相互作用。进化枝B病毒的正链RNA中的聚合酶识别信号尚待确定。使用同源性建模预测每个RV物种的VP1结构。在A的VP1中保守了与ugug盒相互作用的结构元素，这表明这些病毒的病毒RNA识别机制是保守的机制。基于进化枝A和B RV的聚合酶的独特特征，包括较差的序列身份值，单一RV属可能应分解为两个属。病毒学（2012）431：50-57。 （2）对RV RDRP催化口袋中涉及核苷酸和二价阳离子稳定的残基的突变分析。 RV RDRP VP1包含规范的RDRP基序和启动环，该循环被认为在RNA合成过程中可以进行构象重排。在没有病毒核心壳蛋白VP2的情况下，VP1无法与二价阳离子或核苷酸稳定相互作用，并且具有缩回的启动环。为了确定对核苷酸和二价阳离子稳定的潜在重要性的残基，我们将Divergent RVS的VP1和结构同源性异伏病毒lambda3对齐。 VP1突变体经过设计，并在体外具有RNA合成能力的特征。 RDRP基序中的天冬氨酸A和C和精氨酸在基序F中，可能稳定二价阳离子和核苷酸是有效的RNA合成所必需的。单个启动环残基的突变降低或增强了RNA合成效率，而无需避免对VP2的需求，这表明该结构是将RDRP活性与粒子组装联系起来的动态调节元件。病毒学（2012）431：12-20 （3）RV上限酶VP3的预测结构和域纯化。 VP3是一种关键的RV酶，以低拷贝数为单位中存在。涉及病毒RNA封盖涉及的几项活动，包括Guanylyllansferase（GTase）和N7和2'-O甲基转移酶（MTase），已归因于VP3。然而，835-氨基酸VP3蛋白的结构仍然未知。预测，基于使用蓝色病毒（BTV）盖酶，VP4的同源性建模，预测RV VP3残基的结构109-634。 RV VP3和BTV VP4似乎共享一个类似的模块化组织，而2'-O MTase域位于N7 MTase域中。此外，RV VP3和BTV VP4通常具有几种推定的MTase活动点残基。 VP3残基697-800的结构是基于与酶的2H磷酸酯酶超家族成员的同源性建模的，似乎包含两个保守的活性位点His-X-THR/SER基序。没有对VP3的N末端108残基的信心产生的同源模型。基于预测的结构，我们表达了大肠杆菌中VP3的单个结构域作为麦芽糖结合蛋白的N末端融合。我们利用链淀粉亲和力色谱法纯化代表预测的N末端2'-O MTase，N7和2'-O MTase以及GTase以及GTase和2H磷酸酯酶结构域的融合构建体。通过在设计的烟草蚀刻病毒蛋白酶部位切割亲和力标签。当前，我们正在表征纯化的VP3片段的溶解度和活动，并致力于确定其结构。 （4）通过表征RV温度敏感（TS）突变体TSJ来探测NSP5功能。 RV 28KD磷酸蛋白NSP5是Viroplasms的重要组成部分。 NSP5与NSP2和VP2相互作用，并非特异性与病毒RNA结合。 但是，这些相互作用的重要性以及NSP5磷酸化在病毒复制周期中的重要性。 RV突变体TSJ包含映射到NSP5编码段的TS病变。在非抗药性温度下，TS病变导致病毒滴度的10EX3降低。在NSP5的残基182下，TS病变与丙氨酸与甘氨酸取代相关。在这项研究中，我们分析了TSJ表型，以更好地了解NSP5的功能。我们的结果表明，在非允许温度下，TSJ感染细胞中NSP5的表达水平比允许温度高两个。在NSP2和VP6的表达中未看到类似的增加。在非耐药的温度下，最初在TSJ感染的细胞中形成了病毒肿瘤，但在感染后9小时内检测到很少的夹杂物。 此外，病毒肿瘤的消失与NSP2，VP2和TSJ NSP5的错误定位同时存在。 通过利用在非允许温度下TSJ的复制表型减少，我们目前正在开发NSP5依赖性互补测定法。总之，TSJ的表征和NSP5互补测定法的发展将允许在RV感染期间阐明NSP5功能。