Insertion position as well as the inserted TRS and gene sequences differentially affect the retention of foreign gene expression by simian hemorrhagic fever virus (SHFV).
Insertion position as well as the inserted TRS and gene sequences differentially affect the retention of foreign gene expression by simian hemorrhagic fever virus (SHFV).
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DOI：
10.1016/j.virol.2018.09.014
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发表时间：
2018-12
期刊：
影响因子：
3.7
通讯作者：
Brinton MA
中科院分区：
文献类型：
--
作者：
Di H;Morantz EK;Sadhwani H;Madden JC Jr;Brinton MA
构建了表达来自额外sg mRNA的外源基因的重组SHFV感染性cDNA克隆。利用ORF 4 ′和ORF 2b之间以及ORF 4和ORF 5之间的两个3′基因组区域位点插入粘液瘤M013基因，该粘液瘤M013基因具有C末端V5标签，随后是三个插入的转录调节序列（TRS）之一，TRS 2 ′、TRS 4 ′或TRS 7。M013插入ORF 4 ′/ORF 2b位点而不是ORF 4/ORF 5位点产生子代病毒，但只有插入TRS 2 ′的重组病毒在第4代中保留了整个M013基因。在ORF 4 ′/ORF 2b位点插入具有插入的TRS 2 ′的自体荧光蛋白基因iLOV产生活的子代病毒，并且iLOV表达维持至第8代。虽然SHFV亚基因组RNA合成的调控是复杂的，但位于两组次要结构蛋白之间的ORF 4 ′/ORF 2b位点能够容忍外源基因插入。
Recombinant SHFV infectious cDNA clones expressing a foreign gene from an additional sg mRNA were constructed. Two 3′ genomic region sites, between ORF4′ and ORF2b and between ORF4 and ORF5, were utilized for insertion of the myxoma M013 gene with a C-terminal V5 tag followed by one of the three inserted transcription regulatory sequences (TRS), TRS2′, TRS4′ or TRS7. M013 insertion at the ORF4′/ORF2b site but not the ORF4/ORF5 site generated progeny virus but only recombinant viruses with an inserted TRS2′ retained the entire M013 gene through passage four. Insertion of an auto-fluorescent protein gene, iLOV, with an inserted TRS2′ at the ORF4′/ORF2b site generated viable progeny virus and iLOV expression was maintained through passage eight. Although regulation of SHFV subgenomic RNA synthesis is complex, the ORF4′/ORF2b site, which is located between the two sets of minor structural proteins, is able to tolerate foreign gene insertion.
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