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Role of human heterogeneous nuclear ribonucleoprotein C1/C2 in dengue virus replication
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Metadata
Document Title
Role of human heterogeneous nuclear ribonucleoprotein C1/C2 in dengue virus replication
Author
Dechtawewat T.,Songprakhon P.,Limjindaporn T.,Puttikhunt C.,Kasinrerk W.,Saitornuang S.,Yenchitsomanus P.-T.,Noisakran S.
Name from Authors Collection
Affiliations
Division of Molecular Medicine, Office of Research and Development, Siriraj Hospital, Bangkok, 10700, Thailand; Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand; Department of Anatomy, Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand; Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok, 10700, Thailand; Division of Dengue Hemorrhagic Fever Research Unit, Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand; Division of Clinical Immunology, Department of Medical Technology, Chiang Mai University, Chiang Mai, 50200, Thailand; Biomedical Technology Research Center, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Chiang Mai, 50200, Thailand
Type
Article
Source Title
Virology Journal
ISSN
1743422X
Year
2015
Volume
12
Issue
1
Open Access
All Open Access, Gold, Green
Publisher
BioMed Central Ltd.
DOI
10.1186/s12985-014-0219-7
Abstract
Abstract Background: 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. Methods: 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. Results: 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. Conclusions: Our findings suggest that hnRNP C1/C2 is involved in DENV replication at the stage of viral RNA synthesis. © 2015 Dechtawewat et al.; licensee BioMed Central.
Keyword
Dengue virus | hnRNPC1/C2 | siRNA transfection | Virus replication
Industrial Classification
Knowledge Taxonomy Level 1
Knowledge Taxonomy Level 2
Knowledge Taxonomy Level 3
Funding Sponsor
Chulalongkorn University; National Science and Technology Development Agency; Thailand Research Fund; Korea Foundation for Advanced Studies; National Center for Genetic Engineering and Biotechnology
License
CC BY
Rights
Author
Publication Source
Scopus