Department of Pathology, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan.
Biochem Biophys Res Commun. 2010 Feb 12;392(3):397-402. doi: 10.1016/j.bbrc.2010.01.034. Epub 2010 Jan 13.
We found the 2',5'-oligoadenylate synthetase-like (OASL) gene to be significantly elevated by high virus loads in human liver infected with hepatitis C virus (HCV). Here, we determined whether OASL inhibited HCV replication using an in vitro system. We constructed three expression vectors of OASL to produce isoform a (OASLa), isoform b (OASLb), and the C-terminal ubiquitin-like domain of isoform a (Ub). When Huh7 JFH-1 HCV replicon cells were separately transfected with these three vectors, colony formation of HCV-replicating cells was inhibited by 95%, 94%, and 65%, respectively. Both OASLa and OASLb were also inhibitory for cells as well as the virus because colony formation of OASL-producing cells was reduced to 41% and 8%, respectively. Stable Huh7 clones producing each of the three OASLs were established and assessed for their inhibition of HCV replication using luciferase reporter gene-containing JFH-1 replicon RNA. HCV replication was inhibited by 50-90% in several stable OASL clones. Association analysis in six Ub clones expressing different levels of Ub mRNA showed that the degree of inhibition of HCV replication was significantly associated with the amount of Ub present. In conclusion, OASL possesses two domains with HCV inhibitory activity. The N-terminal OAS-homology domain without OAS activity is inhibitory for cell growth as well as HCV replication, whereas C-terminal Ub is inhibitory only for HCV replication. Therefore, OASLa, a major isoform of this molecule induced in human liver, may mediate anti-HCV activity through two different domains.
我们发现，在感染丙型肝炎病毒（HCV）的人类肝脏中，2'，5'-寡腺苷酸合成酶样（OASL）基因的病毒载量显著升高。在这里，我们使用体外系统来确定 OASL 是否抑制 HCV 复制。我们构建了 OASL 的三个表达载体，以产生同工型 a（OASLa）、同工型 b（OASLb）和同工型 a 的 C 末端泛素样结构域（Ub）。当 Huh7 JFH-1 HCV 复制子细胞分别转染这三个载体时，HCV 复制细胞的集落形成分别被抑制了 95%、94%和 65%。OASLa 和 OASLb 对细胞和病毒都具有抑制作用，因为 OASL 产生细胞的集落形成分别减少到 41%和 8%。建立了稳定产生这三种 OASL 的 Huh7 克隆，并使用含有 JFH-1 复制子 RNA 的荧光素酶报告基因评估它们对 HCV 复制的抑制作用。在几个稳定的 OASL 克隆中，HCV 复制被抑制了 50-90%。在表达不同水平 Ub mRNA 的六个 Ub 克隆中的关联分析表明，抑制 HCV 复制的程度与存在的 Ub 量显著相关。总之，OASL 具有两个具有 HCV 抑制活性的结构域。没有 OAS 活性的 N 端 OAS 同源结构域对细胞生长和 HCV 复制都具有抑制作用，而 C 端 Ub 仅对 HCV 复制具有抑制作用。因此，该分子在人类肝脏中诱导的主要同工型 OASLa 可能通过两个不同的结构域介导抗 HCV 活性。
