Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection
Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modifica...
Main Authors: | , , , , , , , , , , , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
MDPI AG
2021-07-01
|
Series: | Viruses |
Subjects: | |
Online Access: | https://www.mdpi.com/1999-4915/13/7/1393 |
id |
doaj-8741c8d60f0b43c88f3ba9fa25d59821 |
---|---|
record_format |
Article |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Thanyaporn Dechtawewat Sittiruk Roytrakul Yodying Yingchutrakul Sawanya Charoenlappanit Bunpote Siridechadilok Thawornchai Limjindaporn Arunothai Mangkang Tanapan Prommool Chunya Puttikhunt Pucharee Songprakhon Kessiri Kongmanas Nuttapong Kaewjew Panisadee Avirutnan Pa-thai Yenchitsomanus Prida Malasit Sansanee Noisakran |
spellingShingle |
Thanyaporn Dechtawewat Sittiruk Roytrakul Yodying Yingchutrakul Sawanya Charoenlappanit Bunpote Siridechadilok Thawornchai Limjindaporn Arunothai Mangkang Tanapan Prommool Chunya Puttikhunt Pucharee Songprakhon Kessiri Kongmanas Nuttapong Kaewjew Panisadee Avirutnan Pa-thai Yenchitsomanus Prida Malasit Sansanee Noisakran Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection Viruses dengue virus NS1 phosphorylation LC-MS/MS virus production |
author_facet |
Thanyaporn Dechtawewat Sittiruk Roytrakul Yodying Yingchutrakul Sawanya Charoenlappanit Bunpote Siridechadilok Thawornchai Limjindaporn Arunothai Mangkang Tanapan Prommool Chunya Puttikhunt Pucharee Songprakhon Kessiri Kongmanas Nuttapong Kaewjew Panisadee Avirutnan Pa-thai Yenchitsomanus Prida Malasit Sansanee Noisakran |
author_sort |
Thanyaporn Dechtawewat |
title |
Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection |
title_short |
Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection |
title_full |
Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection |
title_fullStr |
Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection |
title_full_unstemmed |
Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection |
title_sort |
potential phosphorylation of viral nonstructural protein 1 in dengue virus infection |
publisher |
MDPI AG |
series |
Viruses |
issn |
1999-4915 |
publishDate |
2021-07-01 |
description |
Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design. |
topic |
dengue virus NS1 phosphorylation LC-MS/MS virus production |
url |
https://www.mdpi.com/1999-4915/13/7/1393 |
work_keys_str_mv |
AT thanyaporndechtawewat potentialphosphorylationofviralnonstructuralprotein1indenguevirusinfection AT sittirukroytrakul potentialphosphorylationofviralnonstructuralprotein1indenguevirusinfection AT yodyingyingchutrakul potentialphosphorylationofviralnonstructuralprotein1indenguevirusinfection AT sawanyacharoenlappanit potentialphosphorylationofviralnonstructuralprotein1indenguevirusinfection AT bunpotesiridechadilok potentialphosphorylationofviralnonstructuralprotein1indenguevirusinfection AT thawornchailimjindaporn potentialphosphorylationofviralnonstructuralprotein1indenguevirusinfection AT arunothaimangkang potentialphosphorylationofviralnonstructuralprotein1indenguevirusinfection AT tanapanprommool potentialphosphorylationofviralnonstructuralprotein1indenguevirusinfection AT chunyaputtikhunt potentialphosphorylationofviralnonstructuralprotein1indenguevirusinfection AT puchareesongprakhon potentialphosphorylationofviralnonstructuralprotein1indenguevirusinfection AT kessirikongmanas potentialphosphorylationofviralnonstructuralprotein1indenguevirusinfection AT nuttapongkaewjew potentialphosphorylationofviralnonstructuralprotein1indenguevirusinfection AT panisadeeavirutnan potentialphosphorylationofviralnonstructuralprotein1indenguevirusinfection AT pathaiyenchitsomanus potentialphosphorylationofviralnonstructuralprotein1indenguevirusinfection AT pridamalasit potentialphosphorylationofviralnonstructuralprotein1indenguevirusinfection AT sansaneenoisakran potentialphosphorylationofviralnonstructuralprotein1indenguevirusinfection |
_version_ |
1721285418321707008 |
spelling |
doaj-8741c8d60f0b43c88f3ba9fa25d598212021-07-23T14:11:46ZengMDPI AGViruses1999-49152021-07-01131393139310.3390/v13071393Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus InfectionThanyaporn Dechtawewat0Sittiruk Roytrakul1Yodying Yingchutrakul2Sawanya Charoenlappanit3Bunpote Siridechadilok4Thawornchai Limjindaporn5Arunothai Mangkang6Tanapan Prommool7Chunya Puttikhunt8Pucharee Songprakhon9Kessiri Kongmanas10Nuttapong Kaewjew11Panisadee Avirutnan12Pa-thai Yenchitsomanus13Prida Malasit14Sansanee Noisakran15Division of Molecular Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, ThailandFunctional Proteomics Technology Laboratory, Functional Ingredients and Food Innovation Research Group, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 12120, ThailandFunctional Proteomics Technology Laboratory, Functional Ingredients and Food Innovation Research Group, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 12120, ThailandFunctional Proteomics Technology Laboratory, Functional Ingredients and Food Innovation Research Group, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 12120, ThailandMolecular Biology of Dengue and Flaviviruses Research Team, Medical Molecular Biotechnology Research Group, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 10700, ThailandDepartment of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, ThailandMolecular Biology of Dengue and Flaviviruses Research Team, Medical Molecular Biotechnology Research Group, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 10700, ThailandMolecular Biology of Dengue and Flaviviruses Research Team, Medical Molecular Biotechnology Research Group, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 10700, ThailandMolecular Biology of Dengue and Flaviviruses Research Team, Medical Molecular Biotechnology Research Group, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 10700, ThailandDivision of Molecular Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, ThailandDivision of Dengue Hemorrhagic Fever Research, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, ThailandDivision of Dengue Hemorrhagic Fever Research, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, ThailandDivision of Dengue Hemorrhagic Fever Research, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, ThailandDivision of Molecular Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, ThailandMolecular Biology of Dengue and Flaviviruses Research Team, Medical Molecular Biotechnology Research Group, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 10700, ThailandMolecular Biology of Dengue and Flaviviruses Research Team, Medical Molecular Biotechnology Research Group, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 10700, ThailandDengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design.https://www.mdpi.com/1999-4915/13/7/1393dengue virusNS1phosphorylationLC-MS/MSvirus production |