Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, USA.
J Virol. 2012 Sep;86(18):9964-75. doi: 10.1128/JVI.00914-12. Epub 2012 Jul 3.
Polypeptide 2C(ATPase) is one of the most thoroughly studied but least understood proteins in the life cycle of poliovirus. Within the protein, multiple functional domains important for uncoating, host cell membrane alterations, and RNA replication and encapsidation have previously been identified. In this study, charged to alanine-scanning mutagenesis was used to generate conditional-lethal mutations in hitherto-uncharacterized domains of the 2C(ATPase) polypeptide, particularly those involved in morphogenesis. Adjacent or clustered charged amino acids (2 to 4), scattered along the 2C(ATPase) coding sequence, were replaced with alanines. RNA transcripts of mutant poliovirus cDNA clones were transfected into HeLa cells. Subsequently, 10 lethal, 1 severely temperature-sensitive, 2 quasi-infectious, and 3 wild type-like mutants were identified. Using a luciferase-containing reporter virus, we demonstrated RNA replication defects in all lethal and quasi-infectious mutants. Temperature-sensitive mutants were defective in RNA replication only at the restricted temperatures. Furthermore, we characterized a quasi-infectious mutant (K(6)A/K(7)A) that produced a suppressor mutation (G(1)R) and a novel 2B^2C(ATPase) cleavage site (Q^R). Surprisingly, this cleavage site mutation did not interfere with normal processing of the polyprotein. These mutants have led to the identification of several new sites within the 2C(ATPase) polypeptide that are required for RNA replication. In addition, analysis of the suppressor mutants has revealed a new domain near the C terminus of 2C(ATPase) that is involved in encapsidation, possibly achieved through interaction with an amino acid sequence between NTP binding motifs A and B of 2C(ATPase). Most importantly, the identification of suppressor mutations in both 2C(ATPase) and the capsid domains (VP1 and VP3) of poliovirus has confirmed that an interaction between 2C(ATPase) and capsid proteins is involved in viral morphogenesis.
2C(ATPase) 多肤是脊髓灰质炎病毒生命周期中研究得最透彻但了解最少的蛋白质之一。在该蛋白质内，先前已经鉴定出多个对脱壳、宿主细胞膜改变以及 RNA 复制和包装至关重要的功能域。在这项研究中，使用带电荷的丙氨酸扫描诱变生成了 2C(ATPase)多肤中以前未表征的结构域（特别是那些与形态发生有关的结构域）的条件致死突变。沿着 2C(ATPase)编码序列散布的相邻或聚集的带电荷氨基酸（2 到 4 个）被替换为丙氨酸。突变的脊髓灰质炎病毒 cDNA 克隆的 RNA 转录本被转染到 HeLa 细胞中。随后，鉴定出 10 个致死、1 个严重温度敏感、2 个准感染和 3 个野生型样突变体。使用含有荧光素酶的报告病毒，我们证明了所有致死和准感染突变体的 RNA 复制缺陷。温度敏感突变体仅在受限温度下在 RNA 复制中存在缺陷。此外，我们对一个准感染性突变体（K(6)A/K(7)A）进行了表征，该突变体产生了一个抑制突变（G(1)R）和一个新的 2B^2C(ATPase)切割位点（Q^R）。令人惊讶的是，这种切割位点突变不会干扰多蛋白的正常加工。这些突变体导致在 2C(ATPase)多肽内鉴定出几个新的 RNA 复制所必需的位点。此外，对抑制突变体的分析揭示了 2C(ATPase)的 C 末端附近的一个新结构域，该结构域参与了包装，可能通过与 2C(ATPase)的 NTP 结合基序 A 和 B 之间的氨基酸序列相互作用来实现。最重要的是，在 2C(ATPase)和脊髓灰质炎病毒的衣壳蛋白（VP1 和 VP3）中鉴定出抑制突变体，证实了 2C(ATPase)和衣壳蛋白之间的相互作用参与了病毒形态发生。
