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^Min, a variant temperature-sensitive at 33 and 39.5 °C. Compared with WT PV1, P2^Min displayed a vastly reduced specific infectivity (si) (WT, 1 PFU/118 particles vs. P2^Min, 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 (a) by specific recognition between capsid protein(s) and replication proteins (poliovirus), or (b) 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.

中文翻译：

---

大规模编码的脊髓灰质炎病毒基因组的变异，特异性感染性和基因组包装的限制

计算机设计和化学合成产生了脊髓灰质炎病毒1型（PV1）的可行变体，其ORF（6,189个核苷酸）携带多达1,297个“ Max”突变（过量代表同义密码子对）或多达2,104个“ SD”突变（随机混杂的同义词）密码子）。除P2 ^Min（33和39.5°C时对温度敏感的变体）外，“ Min”变体（未充分代表的同义密码子对）不可行。与WT PV1相比，P2 ^Min的比传染性（si）大大降低（WT，1 PFU / 118颗粒比P2 ^Min，1 PFU / 35,000个粒子），将对该表型进行广泛讨论。单倍体PV的Si呈现单一基因型的细胞感染性。我们对PV基因组的序列和结构进行了全面分析，以确定在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，可能在确定包装特异性中起作用的保守同义位点和/或局部二级结构不能幸免于密码子对重新编码。这使得不可能有许多“沿着整个病毒基因组映射的，隐藏的，序列简并的，分散的RNA包装信号” [Patel N等。（2017）Nat Microbiol 2：17098]在脊髓灰质炎病毒包装特异性中起关键作用。考虑到所有现有证据，我们提出了+ ssRNA病毒的两步组装策略：第一步，a）通过衣壳蛋白与复制蛋白（脊髓灰质炎病毒）之间的特异性识别，或（b）通过单个RNA包装信号（PS）与衣壳蛋白的高亲和力相互作用（迄今为止研究最多的+ ssRNA病毒）; 步骤II，基因组/衣壳前体的共缩合，其中发夹结构的阵列在病毒体形成中起作用。