Modification Of The Antigenicity & Virulence Of Rotaviruses By Reverse Genetics

抗原性的修饰

• 批准号: 8946326
• 负责人: JOHN PATTON
• 金额: $ 72.44万
• 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/8946326
• 关键词: Accident and Emergency department; Accounting; Affect; Age; Alleles; Amino Acid Sequence; Amino Acids; Animals; Antiviral Agents; Antiviral Response; Astrovirus; BCAR3 gene; Base Sequence; Biology; C-terminal; Cell Line; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Child; Childhood; Clinical; Collection; Colorado tick fever virus; Communities; Data; Developing Countries; Development; Diarrhea; Disease; District of Columbia; Elements; Engineering; Epidemiology; Event; Evolution; Family suidae; Feces; Gastroenteritis; Gastrointestinal Diseases; Gene Proteins; Generations; Genes; Genetic; Genetic Determinism; Genetic Transcription; Genome; Genotype; Goals; Hospitalization; Human; IRF3 gene; Immune response; India; Infant; Infection; Infectious Diseases Research; Interferons; Lead; Life; Link; Location; Luciferases; Maintenance; Mediating; Modification; Morbidity - disease rate; Mutation; N-terminal; Nature; Neonatal; Nonstructural Protein; Nucleotides; Office Visits; Open Reading Frames; Pathogenesis; Pathway interactions; Peptide Sequence Determination; Phylogenetic Analysis; Population; Procedures; Proteins; RNA; RNA Viruses; Recombinant Vaccines; Recombinants; Recovery; Rotavirus; Rotavirus Infections; Rotavirus Vaccines; Rotavirus disease; SH2D3A gene; Sampling; Specificity; Symptoms; System; Trees; Ubiquitin; Vaccination; Vaccines; Variant; Viral; Viral Genes; Viral Pathogenesis; Viral Proteins; Virion; Virulence; Virus; Visit; Work; base; beta-Transducin Repeat-Containing Proteins; cost; cost effective; design; disorder prevention; enteric pathogen; fitness; genetic manipulation; genome sequencing; human disease; improved; mortality; neonate; next generation; next generation sequencing; novel; positional cloning; pressure; prevent; programs; protein function; research study; small hairpin RNA; success; temperature sensitive mutant; ubiquitin-protein ligase; vaccine candidate; vector; virus genetics

The objectives of this project, and related progress in the past year (2013-2014), are reviewed below. (1) DEVELOPMENT OF A REVERSE GENETICS SYSTEM THAT CAN BE USED TO MODIFY RV ANTIGENICITY AND VIRULENCE. Recently, we developed an RV single-segment reverse genetics system that allows replacement of the segment 8 (s8) RNA of the temperature-sensitive mutant virus, tsE, with a recombinant s8 RNA that encodes a fully functional NSP2. Using this system, we were able to engineer recombinant (r)RVs with s8 RNAs that were longer than wildtype, due to the introduction of sequence duplications and heterologous sequences. Subsequently, we have pursued two lines of experiments designed to generate rRVs with s8 RNAs that express foreign protein. (1) T7 transcription vectors for s8 were constructed in which a translational 2A skip element linked to a Gaussia luciferase ORF was inserted into the NSP2 ORF. Reverse genetics experiments to recover rRVs with the modified s8 RNA were attempted using a cell line (MA104-NSP2R) that expresses wildtype NSP2 and an shRNA targeting the tsE s8 RNA. (2) T7 transcription vectors for s8 were constructed in which a ribosomal-readthrough element derived from Colorado Tick Fever Virus (CTFV) segment 9 linked to a HA-ubiquitin ORF was inserted immediately downstream of an intact NSP2 ORF. Reverse genetics experiments to recover rRVs with the modified s8 RNA were attempted using a cell line expressing an shRNA that targeted the tsE s8 RNA. Analysis of virus in cell lysates obtained from reverse genetics experiments suggest that rRVs with the engineered s8 RNAs may be present, but at levels too low to be recoverable. Currently, we are modifying reverse genetics procedures to improve the efficiency of rRV formation and recovery. Generation of rRVs that express foreign proteins may be useful for engineering next generation RV vaccines that provide protection not only against RV disease, but also against diseases caused by other enteric pathogens. (2) ELUCIDATION OF RV MECHANISMS THAT SUBVERT HOST ANTIVIRAL PATHWAYS. The segmented RV genome allows the generation of new virus strains through reassortment. However, the vast majority of human RVs have either of two genotype constellations: G1/3/4/9/12-P8-I1-R1-C1-M1-A1-N1-T1-E1-H1 (genogroup 1) or G2-P4-I2-R2-C2-M2-A2-N2-T2-E2-H2 (genogroup 2). Thus, human RVs appear to be under strong selective pressures that favor the maintenance of certain genotype constellations, despite their opportunity to reassort with large numbers of co-circulating genetically-distinct human and animal RVs. The nature of the barriers that select against the emergence of reassortant strains is not understood, but may reflect the co-evolution of RV RNAs and/or proteins that function optimally when maintained as sets. In addition, adaptation of RVs to select hosts may have given rise to viral proteins that are species specific in their function. These latter proteins likely include the RV nonstructural protein, NSP1, which we have shown is an antagonist of host innate immune responses. NSP1 is a putative viral E3 ubiquitin ligase containing a conserved N-terminal RING domain and a highly variable C-terminal targeting domain. By phylogenetic analysis, we determined that the 18 NSP1 genotypes described to date can be resolved into 3 groups: OSU-like, UK-like, and SA11-4F-like. The OSU-like NSP1 group includes only the A1, A2, and A8 genotypes, and thus nearly all human RVs and select porcine RVs belong to this group. OSU-like NSP1 proteins are characterized by the ability to prevent the function of beta-TrCP, a cellular protein critical to NFkB activation and to the induction of host antiviral responses. As we discovered, a hallmark feature of OSU-like NSP1 proteins is the presence of a phosphodegron-like motif (DSGxS) in the C-terminal targeting domain; this motif mediates the interaction of NSP1 with beta-TrCP. RVs with UK-like and SA11-4F-like NSP1 proteins are almost exclusively animal strains; their NSP1 proteins target interferon regulatory factors (IRF3, IRF5, and/or IRF7), instead of beta-TrCP, and lack phosphodegron-like motifs. These findings indicate that the NSP1 proteins of human/porcine RVs (OSU-like) are functionally distinct from the NSP1 proteins of most animal RVs (UK and SA11-4F-like). Hence, the emergence of human reassortant RVs expressing animal NSP1 proteins as significant causes of human disease seems improbable, given that such reassortants would lack NSP1 activities normally associated with human RVs and that likely influence virulence and viral pathogenesis. (3) ANALYSIS OF THE DIVERSITY AND EVOLUTION OF THE RV GENOME. (a) RVs with G10P11 genotype specificity have been associated with symptomatic and asymptomatic neonatal infections in Vellore, India. To identify possible viral genetic determinants that affect RV pathogenesis, the genome sequences of G10P11 RVs in stool samples of 19 neonates with symptomatic infections and 20 neonates with asymptomatic infections were determined by Sanger and next-generation sequencing. The data showed that all 39 viruses had identical genotype constellations (G10-P11-I2-R2-C2-M2-A1-N1-T1-E2-H3), the same as the previously characterized symptomatic Vellore isolate N155. The data also showed that the RNA and deduced protein sequences of all the Vellore G10P11 viruses were nearly identical; no nucleotide or amino acid differences were found that correlated with symptomatic versus asymptomatic infection. Next-generation sequencing data revealed that some stool samples, both from neonates with symptomatic and asymptomatic infections, also contained one or more positive-strand RNA viruses (Aichi virus, astrovirus, or salivirus/klassevirus) suspected of being potential causes of pediatric gastroenteritis. However, none of the positive-strand RNA viruses could be causally associated with the development of symptoms. These results indicate that the diversity of clinical symptoms in Vellore neonates does not result from genetic differences among G10P11 RVs; instead, other undefined factors appear to influence whether neonates develop gastrointestinal disease symptoms. (b) Genome reassortment allows RV to acquire advantageous genes and adapt in the face of selective pressures. Yet, reassortment may also impose fitness costs if it unlinks genes/proteins that have accumulated compensatory, co-adaptive mutations and operate best when kept together. To better understand human RV evolutionary dynamics, we analyzed the genome sequences of 135 strains (genotype G1/G3/G4-P8-I1-C1-R1-A1-N1-T1-E1-H1) that were collected at a single location in Washington, DC, during the years of 1974-1991. Intra-genotypic phylogenetic trees were constructed for each viral gene using the nucleotide sequences, thereby defining novel allele-level gene constellations (GCs) and illuminating putative reassortment events. The results showed that RVs with distinct GCs co-circulated during the vast majority of the collection years, and that some of these GCs persisted in the community, unchanged by reassortment. To investigate the influence of protein co-adaptation on GC maintenance, we performed a mutual information-based analysis of the concatenated amino acid sequences and identified an extensive co-variance network. Unexpectedly, amino acid co-variation was highest between VP4 and VP2, which are structural components of the RV virion that are not thought to directly interact. These results suggest that GCs may be influenced by the selective constraints placed on functionally co-adapted, albeit non-interacting, viral proteins. This work raises important questions about the mutation-reassortment interplay and its impact on human RV evolution.

该项目的目标以及过去一年（2013-2014）相关的进度在下面进行了审查。 （1）可用于修饰RV抗原性和毒力的反向遗传系统的发展。 最近，我们开发了一个RV单段反向遗传学系统，该系统允许用重组S8 RNA替换温度敏感的突变病毒TSE的段8（s8）RNA，该S8 RNA编码功能齐全的NSP2。使用该系统，由于引入序列重复和异源序列，我们能够使用S8 RNA的重组（R）RVS来设计重组（R）RNA。随后，我们进行了两条实验，旨在生成具有表达异物蛋白的S8 RNA的RRV。 （1）构建了S8的T7转录向量，其中将与高斯荧光素酶ORF相关的转换2A跳过元件插入了NSP2 ORF中。尝试使用表达野生型NSP2的细胞系（MA104-NSP2R）和针对TSE S8 RNA的shRNA来尝试使用改良的S8 RNA恢复RRV的反向遗传学实验。 （2）构建了S8的T7转录载体，其中从科罗拉多tick虫发烧病毒（CTFV）段9中衍生出与HA-泛素ORF相关的核糖体阅读元件，立即在完整的NSP2 ORF的下游插入。使用表达针对TSE S8 RNA的shRNA的细胞系，尝试尝试使用改良的S8 RNA回收RRV的反向遗传学实验。从反向遗传学实验获得的细胞裂解物中病毒的分析表明，具有工程化的S8 RNA的RRV可能存在，但水平太低而无法恢复。当前，我们正在修改反向遗传学程序，以提高RRV形成和恢复的效率。表达异物蛋白的RRV产生对于工程下一代RV疫苗可能不仅可以保护RV疾病，而且还针对其他肠道病原体引起的疾病有用。 （2）阐明颠覆宿主抗病毒途径的RV机制。 分段的RV基因组允许通过重新分类产生新的病毒菌株。但是，绝大多数人RV具有两个基因型星座中的任何一个：G1/3/4/9/12-P8-I1-C1-C1-C1-C1-M1-A1-N1-N1-T1-E1-H1（Genogroup 1）或G2-P4-I2-P4-I2-R2-C2-C2-C2-C2-C2-M2-A2-N2-N2-T2-T2-T2-T2-H2（Genogroup 2）。因此，尽管人类RV似乎是有利于维持某些基因型星座的强大选择性压力，尽管他们有机会重新使用大量共同循环的遗传学人类和动物RV。尚不清楚选择反对重新成分菌株出现的障碍的性质，但可能反映了RV RNA和/或蛋白质的共同进化，在保持为集合时可以最佳地发挥作用。此外，RVS对选择宿主的适应可能会引起病毒蛋白，这些病毒蛋白在其功能上是特定的。这些后一种蛋白可能包括RV非结构蛋白NSP1，我们表明的是宿主先天免疫反应的拮抗剂。 NSP1是一种推定的病毒E3泛素连接酶，含有保守的N末端环域和高度可变的C末端靶向结构域。通过系统发育分析，我们确定迄今为止所描述的18种NSP1基因型可以分为3组：类似OSU的，英国和类似SA11-4F的基因型。类似OSU的NSP1组仅包含A1，A2和A8基因型，因此几乎所有人类RVS和Select Porcine RV都属于该组。 OSU样NSP1蛋白的特征在于预防β-TRCP功能的能力，β-TRCP是对NFKB激活至关重要的细胞蛋白和诱导宿主抗病毒反应的功能。正如我们发现的那样，OSU样NSP1蛋白的标志性特征是在C末端靶向域中存在类似磷dy的基序（DSGXS）。该基序介导了NSP1与β-TRCP的相互作用。具有英国和SA11-4F的RVS NSP1蛋白几乎是动物菌株。他们的NSP1蛋白靶向干扰素调节因子（IRF3，IRF5和/或IRF7），而不是β-TRCP，并且缺乏类似磷酸化的基序。这些发现表明，人/猪RV（OSU样）的NSP1蛋白在功能上与大多数动物RV（UK和SA11-4F样）的NSP1蛋白不同。因此，鉴于这种重新分类者缺乏通常与人类RV相关的NSP1活性，并且可能影响毒力和病毒发病机理，因此表达动物NSP1蛋白作为人类疾病的重大原因的人类替代RV的出现似乎是不可能的。 （3）分析RV基因组的多样性和演变。 （A）印度Vellore的具有G10P11基因型特异性的RVS与有症状和无症状的新生儿感染有关。为了鉴定影响RV发病机理的可能的病毒遗传决定因素，通过Sanger和下一代测序确定了19个有症状感染和20种无症状感染的新生儿的粪便样品中G10P11 RV的基因组序列。数据表明，所有39种病毒均具有相同的基因型星座（G10-P11-I2-R2-C2-C2-M2-A1-N1-T1-E2-H3），与先前表征的症状性Vellore分离株N155相同。数据还表明，所有Vellore G10P11病毒的RNA和推导的蛋白质序列几乎相同。没有发现与有症状性和无症状感染相关的核苷酸或氨基酸差异。下一代测序数据表明，从有症状性和无症状感染的新生儿中，一些粪便样本还包含一种或多种阳性链RNA病毒（AICHI病毒，星形病毒或唾液/klassevirus），这些病毒涉嫌可能是儿科胃肠炎的潜在原因。但是，没有任何阳性RNA病毒可能与症状发展有关。这些结果表明，Vellore新生儿的临床症状的多样性不是G10P11 RV之间的遗传差异。取而代之的是，其他不确定的因素似乎会影响新生儿是否患有胃肠道疾病症状。 （b）基因组的重新分类允许RV获取有利的基因并在选择性压力时适应。然而，如果重新分类如果脱离累积补偿性，共同适应突变的基因/蛋白质并在保持在一起时运作良好，则可能会施加健身成本。为了更好地了解人RV进化动力学，我们分析了135个菌株（基因型G1/G3/G4-P8-I1-C1-C1-C1-A1-A1-N1-T1-E1-H1）的基因组序列，这些菌株在1974 - 1991年期间在华盛顿DC的单个位置收集。使用核苷酸序列为每个病毒基因构建了基因型系统发育树，从而定义了新型的等位基因级基因星座（GCS），并启发了推定的重新分层事件。结果表明，具有不同GCS的RVS在绝大多数收集年份中共同循环，并且其中一些GC在社区中持续存在，并没有得到保证。为了研究蛋白质共同适应对GC维持的影响，我们对串联氨基酸序列进行了基于信息的分析，并确定了广泛的共同变异网络。出乎意料的是，VP4和VP2之间的氨基酸共差异最高，它们是RV病毒粒子的结构成分，被认为直接相互作用。这些结果表明，GC可能会受到对功能共同适应的（尽管非相互作用的病毒蛋白）的选择性约束的影响。这项工作提出了有关突变改造相互作用及其对人RV进化的影响的重要问题。