Proteomic approach identifies HSP27 as an interacting partner of the hepatitis C virus NS5A protein.
Chronic infection by HCV is closely correlated with liver diseases such as cirrhosis, steatosis, and hepatocellular carcinoma. To understand how long-term interaction between HCV and the host leads to pathogenesis, we identified cellular proteins that interact with NS5A and NS5B using a biochemical approach. Stable cell lines that express flag-NS5A or flag-NS5B under tetracycline induction were generated. The induced flag-tagged proteins were immunoprecipitated (IP'd) and associated proteins separated on 2D gels. Protein spots that specifically co-IP'd with NS5A or NS5B were identified by mass spectrometry. HSP27 was identified as a protein that specifically co-IP'd with NS5A but not with NS5B. The N-terminal regions of NS5A (a.a. 1-181) and HSP27 (a.a. 1-122) were defined to be the domains that interact with each other. HSP27 is generally distributed in the cytoplasm. When heat shocked, HSP27 is concentrated in the ER where NS5A is co-localized.
Choi
Yook-Wah
YW
Collaborative Anti-Viral Research Group, Institute of Molecular and Cell Biology, 30 Medical Drive, 117609 Singapore, Singapore.
Tan
Yee-Joo
YJ
Lim
Seng Gee
SG
Hong
Wanjin
W
Goh
Phuay-Yee
PY
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D015180:Electrophoresis, Gel, Two-Dimensional; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism; D055551:HSP27 Heat-Shock Proteins; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C057725:HSPB1 protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase