Manipulation of both virus- and cell-specific factors is required for robust transient replication of a hepatitis C virus genotype 3a sub-genomic replicon.
Manipulation of both virus- and cell-specific factors is required for robust transient replication of a hepatitis C virus genotype 3a sub-genomic replicon.
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DOI：
10.1099/jgv.0.000932
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发表时间：
2017-10
期刊：
影响因子：
--
通讯作者：
Harris M
中科院分区：
文献类型：
--
作者：
Kelly L;Badhan A;Roberts GC;Mbisa JL;Harris M
丙型肝炎病毒（HCV）基因型（GT）3是七种HCV基因型中第二常见的，并且对目前使用的高效直接作用抗病毒药物（DAA）表现出最大的耐药性。先前建立了含有S52 GT3亚基因组复制子（SGR）的稳定细胞系，但该SGR不能稳健地瞬时复制。由于瞬时SGR是DAA开发的关键工具，并且在病毒抗性的研究中，我们试图建立基于S52的瞬时SGR系统。下一代测序用于鉴定在S52 SGR的长期选择期间出现的推定的培养适应性取代。在CpG/UpA-低荧光素酶报告基因的背景下，将这些取代的子集构建回S52 SGR中，其中NS 4A中的单点突变赋予S52最大的复制能力。通过表达副流感病毒5型V蛋白和SEC 14 L2修饰Huh7.5肝癌细胞的先天免疫感应途径导致S52复制的进一步增强。此外，这种瞬时复制的SGR对两种临床相关的NS 5A DAA的敏感性显示出基因型特异性差异。总之，我们报告了NS 4A中的单个取代，加上宿主细胞修饰，使得GT3 S52 SGR能够进行稳定水平的瞬时复制。该系统将在GT3生物学和药物发现的独特方面的基础研究中具有有益的用途。
Hepatitis C virus (HCV) genotype (GT) 3 is the second most prevalent of the seven HCV genotypes and exhibits the greatest resistance to the highly potent, direct-acting antivirals (DAAs) that are currently in use. Previously a stable cell line harbouring the S52 GT3 sub-genomic replicon (SGR) was established, but this SGR was unable to robustly replicate transiently. As transient SGRs are a critical tool in the development of DAAs, and in the study of viral resistance, we sought to establish a transient SGR system based on S52. Next-generation sequencing was used to identify putative culture-adaptive substitutions that had arisen during long-term selection of the S52 SGR. A subset of these substitutions was built back into the S52 SGR in the context of a CpG/UpA-low luciferase reporter, with a single point mutation in NS4A conferring the greatest replication capability upon S52. Modification of the innate immune-sensing pathways of Huh7.5 hepatoma cells by expression of the parainfluenza virus type 5 V protein and SEC14L2 resulted in a further enhancement of S52 replication. Furthermore, this transiently replicating SGR showed genotype-specific differences in sensitivity to two clinically relevant NS5A DAAs. In conclusion, we report that a single substitution in NS4A, coupled with host cell modifications, enabled robust levels of transient replication by the GT3 S52 SGR. This system will have beneficial uses in both basic research into the unique aspects of GT3 biology and drug discovery.
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影响因子：
13.5
作者：
Smith, Donald B.;Bukh, Jens;Kuiken, Carla;Muerhoff, A. Scott;Rice, Charles M.;Stapleton, Jack T.;Simmonds, Peter
通讯作者：
Simmonds, Peter
影响因子：
3.1
作者：
Kylefjord, Helen;Danielsson, Axel;Targett-Adams, Paul
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Targett-Adams, Paul
影响因子：
14.9
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Atkinson, Nicky J.;Witteveldt, Jeroen;Simmonds, Peter
通讯作者：
Simmonds, Peter
影响因子：
29.4
作者：
Li, Yi-Ping;Ramirez, Santseharay;Bukh, Jens
通讯作者：
Bukh, Jens
影响因子：
5.4
作者：
Gottwein, Judith M.;Scheel, Troels K. H.;Bukh, Jens
通讯作者：
Bukh, Jens