Modulation of poliovirus replicative fitness in HeLa cells by deoptimization of synonymous codon usage in the capsid region
Modulation of poliovirus replicative fitness in HeLa cells by deoptimization of synonymous codon usage in the capsid region
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DOI：
10.1128/jvi.80.7.3259-3272.2006
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发表时间：
2006-04-01
影响因子：
5.4
通讯作者：
Kew, O
中科院分区：
文献类型：
--
作者：
Burns, CC;Shaw, J;Kew, O
我们用相应的非首选同义密码子替换了Sabin 2型口服脊髓灰质炎病毒疫苗株衣壳区域内9个氨基酸的简并密码子。密码子替换被引入到跨越97%的衣壳区域的四个连续间隔中。在最高度修饰的病毒构建体的衣壳区中，所用密码子的有效数目（N-C）从56.2下降到29.8，CG二核苷酸的数目从97上升到302，并且G+C含量从48.4%增加到56.4%。在HeLa细胞中的复制健身，测量斑块面积和病毒产量在单步生长实验中，下降的置换密码子的数量成比例。空斑面积减少了10倍，病毒产量减少了65倍。也许出乎意料的是，病毒蛋白质的合成和加工似乎在很大程度上不受密码子使用限制的影响。相比之下，感染细胞中病毒RNA的总产率降低了3倍，纯化病毒体的特异性感染性（通过颗粒/PFU比测量）降低了18倍。在HeLa细胞中，密码子替换病毒和未修饰病毒的复制适合度都随着传代次数的增加而增加。经过25次连续传代（类似于50个复制循环）后，大多数密码子替换得以保留，并且修饰病毒的相对适合度仍远低于未修饰病毒的相对适合度。高传代修饰病毒的复制适应性增加与几个CG二核苷酸的消除有关。潜在的应用脊髓灰质炎病毒复制健身的密码子使用的去优化系统调制进行了讨论。
We replaced degenerate codons for nine amino acids within the capsid region of the Sabin type 2 oral poliovirus vaccine strain with corresponding nonpreferred synonymous codons. Codon replacements were introduced into four contiguous intervals spanning 97% of the capsid region. In the capsid region of the most highly modified virus construct, the effective number of codons used (N-C) fell from 56.2 to 29.8, the number of CG dinucleotides rose from 97 to 302, and the G+C content increased from 48.4% to 56.4%. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased in proportion to the number of replacement codons. Plaque areas decreased over an similar to 10-fold range, and virus yields decreased over an similar to 65-fold range. Perhaps unexpectedly, the synthesis and processing of viral proteins appeared to be largely unaltered by the restriction in codon usage. In contrast, total yields of viral RNA in infected cells were reduced similar to 3-fold and specific infectivities of purified virions (measured by particle/PFU ratios) decreased similar to 18-fold in the most highly modified virus. The replicative fitness of both codon replacement viruses and unmodified viruses increased with the passage number in HeLa cells. After 25 serial passages (similar to 50 replication cycles), most codon replacements were retained, and the relative fitness of the modified viruses remained well below that of the unmodified virus. The increased replicative fitness of high-passage modified virus was associated with the elimination of several CG dinucleotides. Potential applications for the systematic modulation of poliovirus replicative fitness by deoptimization of codon usage are discussed.