title: Active participation of cellular chaperone Hsp90 in regulating the function of rotavirus nonstructural protein 3 (NSP3).
abstract: Heat shock protein 90 (Hsp90) has been reported to positively regulate rotavirus replication by modulating virus induced PI3K/Akt and NFκB activation. Here, we report the active association of Hsp90 in the folding and stabilization of rotavirus nonstructural protein 3 (NSP3). In pCD-NSP3-transfected cells, treatment with Hsp90 inhibitor (17-N,N-dimethylethylenediamine-geldanamycin (17DMAG)) resulted in the proteasomal degradation of NSP3. Sequence analysis and deletion mutations revealed that the region spanning amino acids 225-258 within the C-terminal eIF4G-binding domain of NSP3 is a putative Hsp90 binding region. Co-immunoprecipitation and mammalian two-hybrid experiments revealed direct interaction of the C-terminal 12-kDa domain of Hsp90 (C90) with residues 225-258 of NSP3. NSP3-Hsp90 interaction is important for the formation of functionally active mature NSP3, because full-length NSP3 in the presence of the Hsp90 inhibitor or NSP3 lacking the amino acid 225-258 region did not show NSP3 dimers following in vitro coupled transcription-translation followed by chase. Disruption of residues 225-258 within NSP3 also resulted in poor RNA binding and eIF4G binding activity. In addition, inhibition of Hsp90 by 17DMAG resulted in reduced nuclear translocation of poly(A)-binding protein and translation of viral proteins. These results highlight the crucial role of Hsp90 chaperone in the regulation of assembly and functionality of a viral protein during the virus replication and propagation in host cells.
authors.affiliation: Division of Virology, National Institute of Cholera and Enteric Diseases, Beliaghata, Kolkata, India.
authors.forename: Dipanjan
authors.initials: D
authors.lastname: Dutta
authors.forename: Shiladitya
authors.initials: S
authors.lastname: Chattopadhyay
authors.forename: Parikshit
authors.initials: P
authors.lastname: Bagchi
authors.forename: Umesh Chandra
authors.initials: UC
authors.lastname: Halder
authors.forename: Satabdi
authors.initials: S
authors.lastname: Nandi
authors.forename: Anupam
authors.initials: A
authors.lastname: Mukherjee
authors.forename: Nobumichi
authors.initials: N
authors.lastname: Kobayashi
authors.forename: Koki
authors.initials: K
authors.lastname: Taniguchi
authors.forename: Mamta
authors.initials: M
authors.lastname: Chawla-Sarkar
mesh_terms: D000818:Animals; D001483:Base Sequence; D016227:Benzoquinones / Q000494:pharmacology; D001665:Binding Sites; D057809:HEK293 Cells; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000882:Haplorhini; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D039102:Poly(A)-Binding Proteins / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000187:drug effects / Q000502:physiology*; D055503:Protein Multimerization / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary; D012401:Rotavirus / Q000502:physiology*; D012400:Rotavirus Infections / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects / Q000502:physiology*
chemical_list: D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; C082740:NSP3 protein, Rotavirus; D039102:Poly(A)-Binding Proteins; D017361:Viral Nonstructural Proteins; C448659:17-(dimethylaminoethylamino)-17-demethoxygeldanamycin; D046988:Proteasome Endopeptidase Complex