Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY, 11794;
Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY, 11794.
Proc Natl Acad Sci U S A. 2017 Oct 10;114(41):E8731-E8740. doi: 10.1073/pnas.1714385114. Epub 2017 Sep 25.
Computer design and chemical synthesis generated viable variants of poliovirus type 1 (PV1), whose ORF (6,189 nucleotides) carried up to 1,297 "Max" mutations (excess of overrepresented synonymous codon pairs) or up to 2,104 "SD" mutations (randomly scrambled synonymous codons). "Min" variants (excess of underrepresented synonymous codon pairs) are nonviable except for P2, a variant temperature-sensitive at 33 and 39.5 °C. Compared with WT PV1, P2 displayed a vastly reduced specific infectivity (si) (WT, 1 PFU/118 particles vs. P2, 1 PFU/35,000 particles), a phenotype that will be discussed broadly. Si of haploid PV presents cellular infectivity of a single genotype. We performed a comprehensive analysis of sequence and structures of the PV genome to determine if evolutionary conserved cis-acting packaging signal(s) were preserved after recoding. We showed that conserved synonymous sites and/or local secondary structures that might play a role in determining packaging specificity do not survive codon pair recoding. This makes it unlikely that numerous "cryptic, sequence-degenerate, dispersed RNA packaging signals mapping along the entire viral genome" [Patel N, et al. (2017) Nat Microbiol 2:17098] play the critical role in poliovirus packaging specificity. Considering all available evidence, we propose a two-step assembly strategy for +ssRNA viruses: step I, acquisition of packaging specificity, either () by specific recognition between capsid protein(s) and replication proteins (poliovirus), or () by the high affinity interaction of a single RNA packaging signal (PS) with capsid protein(s) (most +ssRNA viruses so far studied); step II, cocondensation of genome/capsid precursors in which an array of hairpin structures plays a role in virion formation.
计算机设计和化学合成产生了可行的 1 型脊髓灰质炎病毒 (PV1) 变体，其 ORF（6189 个核苷酸）携带多达 1297 个“Max”突变（过度表达的同义密码子对）或多达 2104 个“SD”突变（随机打乱的同义密码子）。“Min”变体（表达不足的同义密码子对）除了 P2 之外都是不可行的，P2 在 33 和 39.5°C 时是温度敏感的变体。与 WT PV1 相比，P2 的特定感染力（si）大大降低（WT，1 PFU/118 个颗粒对 P2，1 PFU/35000 个颗粒），这是一个将广泛讨论的表型。单倍体 PV 的 si 表现出单个基因型的细胞感染力。我们对 PV 基因组的序列和结构进行了全面分析，以确定在重新编码后是否保留了进化保守的顺式作用包装信号。我们表明，保守的同义位点和/或局部二级结构可能在决定包装特异性方面发挥作用，但在密码子对重新编码后无法存活。这使得许多“隐藏的、序列退化的、分散的 RNA 包装信号映射整个病毒基因组”[Patel N，等人。（2017 年）Nat Microbiol 2:17098]不太可能在脊髓灰质炎病毒包装特异性中发挥关键作用。考虑到所有现有证据，我们提出了一种用于+ssRNA 病毒的两步组装策略：第一步，获得包装特异性，（）通过衣壳蛋白（s）和复制蛋白（脊髓灰质炎病毒）之间的特异性识别，或（）通过单个 RNA 包装信号（PS）与衣壳蛋白（s）的高亲和力相互作用（迄今为止研究的大多数+ssRNA 病毒）；第二步，基因组/衣壳前体的共凝聚，其中发夹结构的阵列在病毒形成中发挥作用。
