Translational Biology, Medicine, and Health Graduate Program, Virginia Tech, Blacksburg, Virginia, USA.
West Virginia School of Osteopathic Medicine, Lewisburg, West Virginia, USA.
J Virol. 2019 Jan 4;93(2). doi: 10.1128/JVI.01642-18. Print 2019 Jan 15.
Group A rotaviruses (RVAs) are classified according to a nucleotide sequence-based system that assigns a genotype to each of the 11 double-stranded RNA (dsRNA) genome segments. For the segment encoding the VP1 polymerase, 22 genotypes (R1 to R22) are defined with an 83% nucleotide identity cutoff value. For the segment encoding the VP2 core shell protein, which is a functional VP1-binding partner, 20 genotypes (C1 to C20) are defined with an 84% nucleotide identity cutoff value. However, the extent to which the VP1 and VP2 proteins encoded by these genotypes differ in their sequences or interactions has not been described. Here, we sought to (i) delineate the relationships and sites of variation for VP1 and VP2 proteins belonging to the known RVA genotypes and (ii) correlate intergenotypic sequence diversity with functional VP1-VP2 interaction(s) during dsRNA synthesis. Using bioinformatic approaches, we revealed which VP1 and VP2 genotypes encode divergent proteins and identified the positional locations of amino acid changes in the context of known structural domains/subdomains. We then employed an dsRNA synthesis assay to test whether genotype R1, R2, R4, and R7 VP1 polymerases could be enzymatically activated by genotype C1, C2, C4, C5, and C7 VP2 core shell proteins. Genotype combinations that were incompatible informed the rational design and testing of chimeric mutant VP1 and VP2 proteins. The results of this study connect VP1 and VP2 nucleotide-level diversity to protein-level diversity for the first time, and they provide new insights into regions/residues critical for VP1-VP2 interaction(s) during viral genome replication. Group A rotaviruses (RVAs) are widespread in nature, infecting numerous mammalian and avian hosts and causing severe gastroenteritis in human children. RVAs are classified using a system that assigns a genotype to each viral gene according to its nucleotide sequence. To date, 22 genotypes have been described for the gene encoding the viral polymerase (VP1), and 20 genotypes have been described for the gene encoding the core shell protein (VP2). Here, we analyzed if/how the VP1 and VP2 proteins encoded by the known RVA genotypes differ from each other in their sequences. We also used a biochemical approach to test whether the intergenotypic sequence differences influenced how VP1 and VP2 functionally engage each other to mediate RNA synthesis in a test tube. This work is important because it increases our understanding of RVA protein-level diversity and raises new ideas about the VP1-VP2 binding interface(s) that is important for viral replication.
A 组轮状病毒（RVAs）是根据基于核苷酸序列的系统进行分类的，该系统为每个双链 RNA（dsRNA）基因组片段分配一个基因型。对于编码 VP1 聚合酶的片段，使用 83%的核苷酸同一性截断值定义了 22 种基因型（R1 到 R22）。对于编码 VP2 核心壳蛋白的片段，该蛋白是功能性 VP1 结合伴侣，使用 84%的核苷酸同一性截断值定义了 20 种基因型（C1 到 C20）。然而，这些基因型编码的 VP1 和 VP2 蛋白在其序列或相互作用方面的差异程度尚未描述。在这里，我们试图（i）描绘已知 RVA 基因型的 VP1 和 VP2 蛋白的关系和变异位点，以及（ii）在 dsRNA 合成过程中，将基因型间序列多样性与功能性 VP1-VP2 相互作用相关联。使用生物信息学方法，我们揭示了哪些 VP1 和 VP2 基因型编码具有差异的蛋白质，并确定了在已知结构域/亚结构域背景下氨基酸变化的位置。然后，我们采用 dsRNA 合成测定法来测试基因型 R1、R2、R4 和 R7 的 VP1 聚合酶是否可以被基因型 C1、C2、C4、C5 和 C7 的 VP2 核心壳蛋白酶促激活。不兼容的基因型组合为嵌合突变 VP1 和 VP2 蛋白的合理设计和测试提供了信息。这项研究首次将 VP1 和 VP2 的核苷酸水平多样性与蛋白质水平多样性联系起来，并为病毒基因组复制过程中 VP1-VP2 相互作用的关键区域/残基提供了新的见解。A 组轮状病毒（RVAs）在自然界中广泛存在，感染多种哺乳动物和禽类宿主，并导致人类儿童严重的胃肠炎。RVAs 使用根据其核苷酸序列为每个病毒基因分配基因型的系统进行分类。迄今为止，已为编码病毒聚合酶（VP1）的基因描述了 22 种基因型，为编码核心壳蛋白（VP2）的基因描述了 20 种基因型。在这里，我们分析了已知 RVA 基因型编码的 VP1 和 VP2 蛋白在其序列上彼此之间是否存在差异。我们还使用生化方法来测试基因型间序列差异是否影响 VP1 和 VP2 如何以功能性方式相互作用以介导试管中的 RNA 合成。这项工作很重要，因为它增加了我们对 RVA 蛋白质水平多样性的理解，并提出了有关 VP1-VP2 结合界面的新想法，该界面对病毒复制很重要。
