Katoh H, Okamoto T, Fukuhara T, Kambara H, Morita E, Mori Y, Kamitani W, Matsuura Y

Katoh H, Okamoto T, Fukuhara T, Kambara H, Morita E, Mori Y, Kamitani W, Matsuura Y. little, if any, effect on viral propagation in cultured cells. Knockdown of Hsp72 caused accumulation of ubiquitinated P protein and delayed P protein degradation. These results show that Hsp72 is recruited to IBs and regulates the degradation of MuV P protein through the ubiquitin-proteasome pathway. IMPORTANCE Formation of cytoplasmic inclusion bodies (IBs) is a common characteristic feature in mononegavirus infections. IBs are considered to be the sites of viral RNA replication and transcription. However, there have been few studies focused on host factors recruited to the IBs and their biological functions. Here, we identified stress-inducible heat shock protein 70 (Hsp72) as the first cellular partner of mumps virus (MuV) phosphoprotein (P Rabbit Polyclonal to CYB5R3 protein), which is an essential component of the IBs and is involved Parimifasor in viral RNA replication/transcription. We found that the Hsp72 mobilized to the IBs promoted degradation of the MuV P protein through the ubiquitin-proteasome pathway. Our data provide new insight into the role played by IBs in mononegavirus infection. INTRODUCTION One of the characteristic features of mononegavirus infection is formation of cytoplasmic inclusion bodies (IBs), which can be observed by light microscopy (1), fluorescence microscopy (2,C6), and electron microscopy (7,C9). It is well known that IBs contain nucleocapsid-like structures, but the detailed compositions and biological functions of IBs remain to be elucidated. In the case of Ebola virus (family within the family (order genetic analyzer (Life Technologies Inc., Rockville, MD). Reagents and antibodies. MG-132 and cycloheximide (CHX) were purchased from Cell Signaling Technology (Danvers, MA) and Sigma (St. Louis, MO), respectively. Lactacystin and epoxomycin were purchased from Peptide Institute Inc. (Osaka, Japan). Anti-N (23D), -P (57A), -M (79D), -F (170C), and -HN (78) mouse monoclonal antibodies (MAbs) and anti-MuV V (T60), -V/P (T61), and -L (L17) rabbit polyclonal antibodies (PAbs) were prepared as described previously (24,C26). Anti-MuV N rabbit PAb was generated with a synthetic peptide derived from the MuV N protein at Sigma. Anti-FLAG (M2) and anti–tubulin mouse MAbs were purchased from Sigma. Anti-Hsp70 (C92F3A-5) and anti-Hsc70 (1F2-H5) mouse MAbs were purchased from Parimifasor StressMarq Bioscience Inc. (Victoria, Canada). Anti-HA mouse MAb (HA11), anti-GRP78 rabbit PAb (ab21685), and anti-ubiquitin rabbit PAb (number 3933) were purchased from Covance (Richmond, Parimifasor CA), Abcam (Cambridge, United Kingdom), and Cell Signaling Technology, respectively. Virus titration. Virus titers were determined by plaque assay in triplicate using Vero cells in 12-well plates. After 1 to 2 2 h of virus adsorption, the cells were cultured in DMEM with 5% FBS and 1% agarose. At 6 days postinoculation, the cells were stained with Neutral Red solution (Sigma), and the plaque counts were determined. Cell extracts, immunoblotting, and immunoprecipitation. For the preparation of cell extracts, cells were washed twice with cold phosphate-buffered saline (PBS) and then lysed in cell lysis buffer (20 mM Tris-HCl, pH 7.5, 135 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail [Complete Mini; Roche, Mannheim, Germany]). For immunoblotting, the cell lysate was boiled in sodium dodecyl sulfate (SDS) sample buffer and subjected to SDS-polyacrylamide gel electrophoresis (PAGE). The proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) and incubated with the appropriate antibodies. Each protein was visualized with SuperSignal West Femto Maximum Sensitivity Substrate (Life Technologies Inc.) and detected by use of an LAS-3000 image analyzer system (Fuji Film, Tokyo, Japan). For immunoprecipitation, Parimifasor the cell lysate was precleaned with protein G-Sepharose (GE Healthcare, Buckinghamshire, United Kingdom). Antibody-protein complexes were purified with protein G beads and washed with cell lysis buffer three times. After boiling in SDS sample buffer, the proteins were separated by SDS-PAGE and processed for immunoblotting. Immunofluorescence microscopy. Vero cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. Then, the cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min, blocked with PBS containing 2% bovine.