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.
Highlights
From the ‡Division of Virology, National Institute of Cholera and Enteric Diseases, P-33, Calcutta Improvement Trust Road, Scheme XM, Beliaghata, Kolkata 700010, India, the §Department of Hygiene, Sapporo Medical University, S-1 W-17, Chuo-ku, Sapporo 060-8556, Japan, and the ¶Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake 470-1100, Aichi, Japan
The cells were washed with cold PBS and incubated with monoclonal poly(A)-binding protein (PABP) and polyclonal RV-NSP3 antibodies at room temperature for 2 h followed by Rhodamine Red X-conjugated anti-mouse and fluorescein isothiocyanate-conjugated anti-rabbit antibodies (The Jackson Laboratory, West Grove, PA) for 1 h
Inhibition of Heat shock protein 90 (Hsp90) Results in Reduced Expression of Transiently Transfected NSP3 and Negatively Affects Nuclear Localization of PABP in Virus-infected Cells—To analyze whether Hsp90 directly affects the expression of any rotaviral protein, plasmids encoding the full-length virus structural gene (VP6) and nonstructural genes (NSP1, NSP2, NSP3, and NSP5) were transfected in 293T cells either untreated or treated with Hsp90 inhibitor (17DMAG)
Summary
Reagents—17-N,N-Dimethylethylenediamine-geldanamycin (17DMAG) was purchased from Invivogen (San Diego, CA). Plasmid Construction, Transfection, and Protein Purification— Full-length NSP1, NSP2, NSP3, NSP5, and VP6 from rotavirus SA11-H96 were amplified from extracted RNA by RT-PCR with the respective primers (Table 1) and cloned into the pCDNA6 (Invitrogen) mammalian expression vector under the control of the CMV promoter (The GenBankTM accession numbers for the cloned cDNAs are JF791801–JF791803 and JF791805–JF791806, respectively). Three gel-purified PCR fragments that included the internal chimeric cDNA encoding the NSP3-(225–258) region of each of the three virus strains along with two adjacent SA11 fragments were mixed in equimolar amounts to perform an overlap extension PCR; this produced recombinant chimeric NSP3 products that were cloned in-frame into the pCDNA-6 vector. His6-tagged full-length NSP3 and ⌬225–258 deletion and point mutants were purified under native conditions using the QIAexpressionistTM protein purification kit (Qiagen) following the manufacturer’s instructions. For the gel retardation assay, purified proteins (100 nM) were incubated with biotinylated NSP5 RNA probe (5 nM) in 25 l of RNA binding buffer (10 mM HEPES, pH 7.9, 40 mM KCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), and 20% glycerol) for 20 min
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