Dechtawewat Thanyaporn, Songprakhon Pucharee, Limjindaporn Thawornchai, Puttikhunt Chunya, Kasinrerk Watchara, Saitornuang Sawanan, Yenchitsomanus Pa-Thai, Noisakran Sansanee
Division of Molecular Medicine, Office of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand.
Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand.
Virol J. 2015 Feb 6;12:14. doi: 10.1186/s12985-014-0219-7.
Host and viral proteins are involved in dengue virus (DENV) replication. Heterogeneous ribonucleoprotein (hnRNP) C1/C2 are abundant host cellular proteins that exhibit RNA binding activity and play important roles in the replication of positive-strand RNA viruses such as poliovirus and hepatitis C virus. hnRNP C1/C2 have previously been shown to interact with vimentin and viral NS1 in DENV-infected cells; however, their functional role in DENV replication is not clearly understood. In the present study, we investigated the role of hnRNP C1/C2 in DENV replication by using an in vitro model of DENV infection in a hepatocyte cell line (Huh7) and siRNA-mediated knockdown of hnRNP C1/C2.
Huh7 cells were transfected with hnRNP C1/C2-specific siRNA or irrelevant siRNA (control) followed by infection with DENV. Mock and DENV-infected knockdown cells were processed for immunoprecipitation using hnRNP C1/C2-specific antibody or their isotype-matched control antibody. The immunoprecipitated samples were subjected to RNA extraction and reverse transcriptase polymerase chain reaction (RT-PCR) for detection of DENV RNA. In addition, the knockdown cells harvested at varying time points after the infection were assessed for cell viability, cell proliferation, percentage of DENV infection, amount of viral RNA, and viral E and NS1 expression. Culture supernatants were subjected to focus forming unit assays to determine titers of infectious DENV. DENV luciferase reporter assay was also set up to determine viral translation.
Immunoprecipitation with the anti-hnRNP C1/C2 antibody and subsequent RT-PCR revealed the presence of DENV RNA in the immunoprecipitated complex containing hnRNP C1/C2 proteins. Transfection with hnRNP C1/C2-specific siRNA resulted in a significant reduction of hnRNP C1/C2 mRNA and protein levels but did not induce cell death during DENV infection. The reduced hnRNP C1/C2 expression decreased the percentage of DENV antigen-positive cells as well as the amount of DENV RNA and the relative levels of DENV E and NS1 proteins; however, it had no direct effect on DENV translation. In addition, a significant reduction of DENV titers was observed in the supernatant from DENV-infected cells following the knockdown of hnRNP C1/C2.
Our findings suggest that hnRNP C1/C2 is involved in DENV replication at the stage of viral RNA synthesis.
宿主蛋白和病毒蛋白参与登革病毒（DENV）的复制。异质性核糖核蛋白（hnRNP）C1/C2是丰富的宿主细胞蛋白，具有RNA结合活性，在脊髓灰质炎病毒和丙型肝炎病毒等正链RNA病毒的复制中发挥重要作用。此前已证明hnRNP C1/C2在DENV感染的细胞中与波形蛋白和病毒NS1相互作用；然而，它们在DENV复制中的功能作用尚不清楚。在本研究中，我们通过在肝细胞系（Huh7）中使用DENV感染的体外模型以及siRNA介导的hnRNP C1/C2敲低，研究了hnRNP C1/C2在DENV复制中的作用。
用hnRNP C1/C2特异性siRNA或无关siRNA（对照）转染Huh7细胞，随后用DENV感染。对模拟感染和DENV感染的敲低细胞，使用hnRNP C1/C2特异性抗体或其同型对照抗体进行免疫沉淀处理。对免疫沉淀样品进行RNA提取和逆转录聚合酶链反应（RT-PCR）以检测DENV RNA。此外，对感染后不同时间点收获的敲低细胞进行细胞活力、细胞增殖、DENV感染百分比、病毒RNA量以及病毒E和NS1表达的评估。对培养上清液进行蚀斑形成单位测定以确定感染性DENV的滴度。还建立了DENV荧光素酶报告基因测定以确定病毒翻译。
用抗hnRNP C1/C2抗体进行免疫沉淀并随后进行RT-PCR，发现在含有hnRNP C1/C2蛋白的免疫沉淀复合物中存在DENV RNA。用hnRNP C1/C2特异性siRNA转染导致hnRNP C1/C2 mRNA和蛋白水平显著降低，但在DENV感染期间未诱导细胞死亡。hnRNP C1/C2表达的降低降低了DENV抗原阳性细胞的百分比以及DENV RNA的量和DENV E和NS1蛋白的相对水平；然而，它对DENV翻译没有直接影响。此外，在敲低hnRNP C1/C2后，在DENV感染细胞的上清液中观察到DENV滴度显著降低。
我们的研究结果表明，hnRNP C1/C2在病毒RNA合成阶段参与DENV复制。
