Suppression of the CpG dinucleotide is widespread in RNA viruses infecting vertebrates and plants, and in the genomes of retroviruses and small mammalian DNA viruses. The functional basis for CpG suppression in the latter was investigated through the construction of mutants of the parvovirus, minute virus of mice (MVM) with increased CpG or TpA dinucleotides in the VP gene. CpG-high mutants displayed extraordinary attenuation in A9 cells compared to wild-type MVM (>six logs), while TpA elevation showed no replication effect. Attenuation was independent of Toll-like receptor 9 and STING-mediated DNA recognition pathways and unrelated to effects on translation efficiency. While translation from codon-optimized VP RNA was enhanced in a cell-free assay, MVM containing this sequence was highly attenuated. Further mutational analysis indicated that this arose through its increased numbers of CpG dinucleotides (7→70) and separately from its increased G+C content (42.3→57.4 %), which independently attenuated replication. CpG-high viruses showed impaired NS mRNA expression by qPCR and reduced NS and particularly VP protein expression detected by immunofluorescence and replication in A549 cells, effects reversed in zinc antiviral protein (ZAP) knockout cells, even though nuclear relocalization of VP remained defective. The demonstrated functional basis for CpG suppression in MVM and potentially other small DNA viruses and the observed intolerance of CpGs in coding sequences, even after codon optimization, has implications for the use of small DNA virus vectors in gene therapy and immunization.

中文翻译：

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使用小型DNA病毒模型研究CpG二核苷酸诱导的病毒复制衰减的机制。

在感染脊椎动物和植物的RNA病毒以及逆转录病毒和小型哺乳动物DNA病毒的基因组中，CpG二核苷酸的抑制普遍存在。通过构建细小病毒突变体，小鼠细小病毒（MVM）的突变体，在VP基因中增加CpG或TpA二核苷酸，研究了后者抑制CpG的功能基础。与野生型MVM相比，高CpG高突变体在A9细胞中显示出非凡的衰减（> 6个对数），而TpA升高则无复制作用。衰减独立于Toll样受体9和STING介导的DNA识别途径，与翻译效率的影响无关。尽管在无细胞分析中增强了密码子优化的VP RNA的翻译，但包含该序列的MVM却被高度减毒。进一步的突变分析表明，这是由于其CpG二核苷酸的数量增加（7→70）和其G + C含量增加（42.3→57.4％）而引起的，后者独立地减弱了复制。高CpG高病毒通过qPCR显示NS mRNA表达受损，并通过免疫荧光和复制在A549细胞中检测到NS降低，特别是VP蛋白表达降低，即使VP的核重新定位仍存在缺陷，但在锌抗病毒蛋白（ZAP）敲除细胞中这种作用却相反。甚至在密码子优化之后，MVM和潜在的其他小DNA病毒中CpG抑制的已证明功能基础，以及在编码序列中观察到的CpG不耐受，甚至对在基因治疗和免疫中使用小DNA病毒载体都具有影响。