JVI Accepts, published online ahead of print on 31 December 2014 J. Virol. doi:10.1128/JVI.03343-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

J Virol. Ver.3

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Heat shock protein 70 regulates degradation of the mumps virus phosphoprotein via

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the ubiquitin-proteasome pathway

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Hiroshi Katoh1 *, Toru Kubota1, Shunsuke Kita2, Yuichiro Nakatsu1, Natsuko Aoki1,

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Yoshio Mori1, Katsumi Maenaka2, Makoto Takeda1, Minoru Kidokoro1

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1Department

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2Laboratory

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University, Hokkaido, Japan

of Virology III, National Institute of Infectious Diseases, Tokyo, and of Biomolecular Science, Faculty of Pharmaceutical Sciences, Hokkaido

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+To whom correspondence should be addressed:

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Hiroshi Katoh, D.V.M., Ph.D

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Department of Virology III

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National Institute of Infectious Diseases

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4-7-1 Gakuen, Musashimurayama-shi

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Tokyo 208-0011, Japan

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Tel: 81-42-848-7064

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Fax: 81-42-567-5631

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E-mail: [email protected]

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Running title: Hsp72 regulates degradation of MuV P protein

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Key Words: Heat shock protein 70 / Mumps virus / Phosphoprotein / Ubiquitin

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proteasome

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The authors declare no conflict of interest.

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Abstract: 161 words

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Importance: 114 words

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Text: 6,822 words

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Abstract

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Mumps virus (MuV) infection induces formation of cytoplasmic inclusion bodies (IBs).

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Growing evidence indicates that IBs are the site where RNA viruses synthesize their

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viral RNA. However, in the case of MuV infection, little is known about the viral and

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cellular compositions and biological functions of the IBs. In this study, pulldown

35

purification and N-terminal amino acid sequencing revealed that stress inducible heat

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shock protein 70 (Hsp72) was a binding partner of MuV phosphoprotein (P protein),

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which was an essential component of the IBs formation. Immunofluorescence and

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immunoblotting analyses revealed that Hsp72 was colocalized with the P protein in the

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IBs, and its expression was increased during MuV infection. Knockdown of Hsp72

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using siRNAs had little, if any, effect on the viral propagation in cultured cells.

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Knockdown of Hsp72 caused accumulation of ubiquitinated P protein and delayed the

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P protein degradation. These results show that Hsp72 is recruited to IBs and regulates

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degradation of MuV P protein through the ubiquitin-proteasome pathway.

44 45 46

Importance

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Formation of cytoplasmic inclusion bodies (IBs) is a common characteristic feature in

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mononegavirus infections. IBs are considered to be the site of viral RNA replication

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and transcription. However, there have been few studies focused on host factors

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recruited to the IBs and their biological functions. Here, we identified stress inducible

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heat shock protein 70 (Hsp72) as the first cellular partner of mumps virus (MuV)

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phosphoprotein (P), which is an essential component of the IBs and involved in viral

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RNA replication/transcription. We found that the Hsp72 mobilized to the IBs

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promoted degradation of the MuV P protein thorough the ubiquitin-proteasome

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pathway. Our data provide new insight into the role played by IBs in mononegavirus

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infection.

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Introduction

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One of the characteristic features of mononegavirus infection is formation of

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cytoplasmic inclusion bodies (IBs), which can be observed by light microscopy (1),

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fluorescence microscopy (2-6) and electron microscopy (7-9). It is well known that IBs

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contain nucleocapsid-like structures, but the detailed compositions and biological

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functions of IBs remain to be elucidated. In the case of Ebolavirus (family Filoviridae,

66

order Mononegavirales), the IBs have been reported to be the site of viral RNA

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replication (6). Similar findings were also reported for other RNA viruses, including

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rabies virus (RV) (4) and vesicular stomatitis virus (VSV) (5) (both of the family

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Rhabdoviridae, order Mononegavirales). In regard to the significance of IB formation, it is

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currently considered that the IBs concentrate the machinery for viral RNA synthesis. In

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the present study we studied mumps virus (MuV), which is also known to form IBs.

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MuV is the causative agent of mumps, a common childhood illness characterized by

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fever and swelling of the salivary glands (10). It often causes neurological

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complications, including aseptic meningitis, encephalitis, and deafness. MuV belongs

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to the genus Rubulavirus within the family Paramyxoviridae (order Mononegavirales) (11).

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The viral non-segmented negative strand RNA genome encodes eight viral proteins:

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the nucleocapsid (N), V, phospho- (P), matrix (M), haemagglutinin-neuraminidase

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(HN), fusion (F), large (L), and small hydrophobic (SH) proteins. The genome is

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encapsidated by the N protein and forms an active template for RNA replication and

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transcription, a viral ribonucleoprotein (vRNP), with viral polymerases composed of

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the P and L proteins (12). The F and HN proteins are envelope glycoproteins, and the

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M protein is an intra-virion protein that associates with the cytoplasmic tails of the

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envelope glycoproteins and vRNP. The SH protein is also a structural integral

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membrane protein with unknown function. The V protein is a nonstructural protein

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that counteracts the host antiviral responses. The V and P proteins are encoded in the

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same gene using overlapping reading frames. The V protein is translated from the V

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mRNA, a faithful transcript of the V/P gene, whereas the P protein is translated from

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the P mRNA possessing two additional nontemplated guanine residues inserted by an

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RNA editing mechanism. Therefore, the resulting P and V proteins have an identical

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N-terminal region and unique C-terminal regions.

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Heat shock protein 70 (Hsp70) family proteins are molecular chaperones that

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comprise a set of abundant cellular machines (13). Under normal unstressed conditions,

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Hsp70 proteins play central roles in protein homeostasis, such as assisting in the

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folding or assembly of newly translated proteins, guiding the intracellular trafficking

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of client proteins, disassembling oligomeric protein structures, and facilitating the

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proteolytic degradation of unstable proteins. Under conditions of stress, they prevent

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abnormal protein aggregation and assist in the renaturation or degradation of

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misfolded proteins. Human Hsp70 proteins are comprised of at least eight gene

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products with different amino acid sequences, expression levels and subcellular

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localizations (14). Among the major Hsp70 proteins expressed at high levels in a wide

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range of tissues, stress inducible heat shock protein 70 (Hsp72) and constitutively

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expressed heat shock cognate protein 70 (Hsc70) are present in the cytoplasm and

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nucleus, while glucose-regulated protein (GRP78) and GRP75 are localized in the

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lumen of the ER and the mitochondrial matrix, respectively. During virus infections,

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Hsp70 family proteins are frequently mobilized to the viral replication sites and play

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roles in all steps of the life cycle of many DNA and RNA viruses (4, 15-17). In the case

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of negative-stranded RNA viruses, Hsp70 has been found to have both positive and

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negative regulatory effects on viral propagation. For example, Hsp72 interacts with the

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N protein of measles virus (MV), which is another member of the paramyxovirus

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family, and enhances viral RNA replication (18). Hsp70 is also recruited to the IBs of

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RV and positively regulates RV infection (19). On the other hand, Hsp70 interferes with

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the polymerase activity of influenza virus and negatively regulates viral RNA

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replication (20, 21), highlighting the complexity of the virus-chaperone interaction.

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Our data revealed that MuV-infected cells also recruited Hsp72 to the IBs. In the

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present study, we analyzed the molecular basis and significance of this event in

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MuV-infected cells.

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Materials & Methods

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Cells and virus. Vero (African green monkey kidney), 293T (human kidney) and Huh7

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(human hepatocellular carcinoma) cells were maintained in Dulbecco’s modified

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Eagle’s minimal essential medium (DMEM) (Nacalai Tesque, Kyoto, Japan)

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supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal

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bovine serum (FBS).

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The highly neuropathogenic strain MuV Odate strain was isolated from a patient who

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developed aseptic meningitis (22) and used in this study.

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Plasmids. The cDNA of the P protein was amplified from 293T cells infected with MuV

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Odate

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pCAGPM-N-HA and pCAG-MCS2-FOS for expression in mammalian cells as

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non-tagged, HA-tagged and FLAG-One-STrEP (FOS)-tagged proteins, respectively (23).

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The

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pCAG-P-FOS, respectively. The cDNAs of the N, L and V proteins were also amplified

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and cloned into pCAGGS and/or pCAG-MCS2-FOS, resulting in pCAGGS-N, L and V

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and pCAG-N-FOS, respectively. The cDNAs of human Hsp72, Hsc70, GRP78 and

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ubiquitin were amplified from 293T cells by RT-PCR and cloned into pcDNA3.1-FLAG

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or pCAGPM-N-HA for expression in mammalian cells as a FLAG- or HA-tagged

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protein.

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pcDNA-FLAG-Hsc70, pcDNA-FLAG-GRP78 and pCAGPM-HA-Ub, respectively. A

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series of deletion mutants of the P protein and Hsp72 was generated by PCR-based

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mutagenesis. All plasmids were confirmed by sequencing with an ABI Prism 3130xl

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genetic analyzer (Life Technologies Inc., Rockville, MD).

141

Reagents and antibodies. MG-132 and cycloheximide (CHX) were purchased from

142

Cell Signaling Technology (Danvers, MA) and Sigma (St. Louis, MO), respectively.

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Lactacystin and epoxomicin were purchased from Peptide Institute Inc. (Osaka, Japan).

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Anti-N (23D), P (57A), M (79D), F (170C) and HN (78) mouse monoclonal antibodies

145

(MAbs) and anti-MuV V (T60), V/P (T61) and L (L17) rabbit polyclonal antibodies

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(PAbs) were prepared as described previously (24-26). Anti-MuV N rabbit PAb was

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generated with a synthetic peptide derived from the MuV N protein at Sigma.

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Anti-FLAG (M2) and anti--tubulin mouse MAbs were purchased from Sigma.

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Anti-Hsp70 (C92F3A-5) and anti-Hsc70 (1F2-H5) mouse MAbs were purchased from

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StressMarq Bioscience Inc. (Victoria, Canada). Anti-HA mouse MAb (HA11),

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anti-GRP78 rabbit PAb (ab21685), and anti-ubiquitin rabbit PAb (#3933) were

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purchased from Covance (Richmond, CA), Abcam (Cambridge, United Kingdom), and

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Cell Signaling Technology, respectively.

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Virus titration. Virus titers were determined by plaque assay in triplicate using Vero

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cells in 12-well plates. After 1 to 2 hr of virus adsorption, the cells were cultured in

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DMEM with 5% FBS and 1% agarose. At 6 days post-inoculation, the cells were stained

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with Neutral Red Solution (Sigma), and the plaque counts were determined.

by

reverse

resulting

The

transcription-PCR

plasmids

resulting

were

(RT-PCR)

designated

plasmids

were

and

cloned

pCAGGS-P,

designated

into

pCAGGS,

pCAGPM-HA-P

and

pcDNA-FLAG-Hsp72,

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Cell extracts, immunoblotting and immunoprecipitation. For the preparation of cell

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extracts, cells were washed twice with cold phosphate-buffered saline (PBS) and then

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lysed in cell lysis buffer (20 mM Tris-HCl, pH 7.5, 135 mM NaCl, 1% Triton-X 100, and

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protease inhibitor cocktail [Complete Mini; Roche, Mannheim, Germany]). For

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immunoblotting, the cell lysate was boiled in sodium dodecyl sulfate (SDS) sample

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buffer and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The

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proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford,

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MA) and incubated with the appropriate antibodies. Each protein was visualized with

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SuperSignal West Femto Maximum Sensitivity Substrate (Life Technologies Inc.) and

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detected by use of an LAS-3000 image analyzer system (Fuji Film, Tokyo, Japan). For

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immunoprecipitation, the cell lysate was pre-cleaned with protein G-sepharose (GE

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Healthcare, Buckinghamshire, United Kingdom). Antibody-protein complexes were

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purified with protein G beads and washed with cell lysis buffer three times. After

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boiling in SDS sample buffer, the proteins were separated by SDS-PAGE and processed

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for immunoblotting.

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Immunofluorescence microscopy. Vero cells were fixed in 4% paraformaldehyde in

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PBS for 15 min at room temperature. Then, the cells were permeabilized with 0.2%

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Triton X-100 in PBS for 10 min, blocked with PBS containing 2% bovine serum albumin

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(BSA) for 30 min at room temperature, and incubated with the appropriate antibodies.

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Nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI). The samples were

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examined under an FV1000D confocal laser-scanning microscope (Olympus, Tokyo,

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Japan).

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Fluorescence in situ hybridization. MuV genomic RNA was detected using the

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QuantiGene ViewRNA ISH cell assay kit (Affymetrix, Santa Clara, CA) and mumps

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virus probe set (Affymetrix) that can hybridize to nucleotides 13,501-14,518 of the MuV

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genome. Vero cells infected with MuV were fixed in 4% paraformaldehyde in PBS for

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30 min at room temperature. Then, cells were permeabilized and hybridized according

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to the manufacturer’s protocol. The samples were examined under an FV1000D

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confocal laser-scanning microscope.

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FOS-tagged purification and N-terminal amino acid sequencing. pCAG-N-FOS,

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pCAG-P-FOS or empty vector was transfected into 293T cells by use of TransIT LT1

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(Mirus, Madison, WI), harvested at 24 hr post-transfection, washed twice with ice-cold

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PBS, suspended in cell lysis buffer, and centrifuged at 14,000 x g for 20 min at 4 ºC. The

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supernatant was pulled down using 50 l of STrEP-Tactin Sepharose (IBA, Gottingen,

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Germany) equilibrated with cell lysis buffer for 2 hr at 4 ºC. The affinity beads were

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washed three times with cell lysis buffer and suspended in 2 x SDS-PAGE sample

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buffer. The proteins were subjected to SDS-PAGE and transferred to membrane,

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followed by Coomassie brilliant blue (CBB) staining using CBB Stain One (Nakalai

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Tesque). Each band was spliced out and subjected to N-terminal amino acid

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sequencing (Procise 491cLC; Applied Biosystems).

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Gene silencing. Commercially available small interfering RNA (siRNA) pool targeting

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Hsp72 (siGENOME SMARTpool, human Hsp72) and control nontargeting siRNA were

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purchased from Dharmacon (Buckinghamshire, United Kingdom) and transfected

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using

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manufacturer’s protocol.

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Quantitative RT-PCR (qRT-PCR). Total RNA was prepared by use of an RNeasy Mini

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Kit (Qiagen), and first-strand cDNA was synthesized using PrimeScript II RTase and

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an oligo(dT) primer (Takara Bio, Shiga, Japan). The amount of each cDNA was

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measured using the Universal ProbeLibrary and the LightCycler 480 system (Roche)

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according to the manufacturer’s instructions. Primers for qRT-PCR were designed by

208

using the Probe Finder software (Roche). The value of each RNA was normalized to

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that of hypoxanthine phosphoribosyltransferase 1 (HPRT1) mRNA.

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TUNEL staining. Vero cells were fixed in 4% paraformaldehyde in PBS for 15 min at

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room temperature. Then, the cells were permeabilized with 0.2% Triton X-100 in PBS

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for

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deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) reaction

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mixture (In situ Apoptosis Detection Kit, Takara Bio) for 90 min at 37ºC. Nuclei were

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stained with DAPI. The samples were examined under a BZ-8000 fluorescence

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microscope (Keyence Co., Osaka, Japan).

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Caspase activity and cell viability assays. Caspase 3/7 activity in Vero cells in 96-well

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plates was measured by using a Caspase-Glo 3/7 Assay Kit (Promega, Madison, WI)

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according to the manufacturer’s protocol. Cell viability was measured by using a

220

CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega) and used for

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normalization.

Lipofectamine

10

222 223

Results

min

at

RNAiMAX

room

(Life

Technologies

temperature

and

Inc.)

incubated

according

with

to

the

terminal

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Coexpression of the MuV N and P proteins induces formation of an IB-like structure,

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where these proteins were concentrated. A previously reported analysis using

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electron microscopy suggested that the IBs observed in MuV-infected cells are

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aggregates of nucleocapsids (7). In order to identify viral components of the

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MuV-induced IBs, the intracellular localizations of MuV proteins and genomic RNA

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were analyzed by immunofluorescence microscopy and fluorescence in situ

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hybridization (Fig. 1A and B). The N, V, P, and L proteins were localized mainly to the

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IBs as well as viral genomic RNA, while the M protein was detected not only in the IBs

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but also in the nucleoli and the pericellular region. The F and HN proteins were

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localized mainly in the pericellular region. To further clarify the intracellular

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localizations of the vRNP components, the N, P and L proteins were expressed in cells

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alone or in combination using expression plasmids. Each protein showed a diffuse

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distribution pattern throughout the cytoplasm, when expressed alone (Fig. 1C).

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Co-expression of the N and P proteins but not other combinations led to the formation

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of IB-like structures, where these proteins were concentrated (Fig. 1D).

239 240

MuV P protein associates with Hsp70 family proteins. Data by immunofluorescence

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assay showed that the N, P, V, M, and L proteins were concentrated in the IBs, and

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co-expression of the N and P proteins induced the formation of IB-like structures. As

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an initial step in the search for host factors involved in the formation of IBs, host

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proteins associated with the N and P proteins were analyzed by an FOS affinity tag

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purification method (Fig. 2A). Co-expression of N- and P-FOS proteins led to the

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formation of IB-like structures similar to the untagged N and P proteins, indicating that

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placement of the FOS tag at C-terminus of N and P protein had minimal effects on the

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function of IB-like structure formation (Fig. 2B). The N- and P-FOS proteins were

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expressed in 293T cells and purified together with associated proteins. Several

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polypeptides, including three polypeptides with molecular weights (MWs) of ~72, 73

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and 78 kDa, were co-purified with the P protein, whereas no N protein-associated host

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proteins were detected (Fig. 2C). Analysis by immunoblotting confirmed that the

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purified P-FOS protein was observed at an MW of ~45 kDa, and suggested that several

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other bands were different forms of the P protein, seemingly correspondent to

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phosphorylated and cleaved P-FOS products (Fig. 2D). In order to identify the three

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polypeptides with MWs of ~72, 73 and 78 kDa, individual bands were isolated and

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analyzed by N-terminal amino acid sequencing. The 78 kDa polypeptide was identified

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as GRP78 with a sequence of EEEDKKEDVG (residues 19 to 28), whereas the amino

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acid sequences of the other two polypeptides of 72 and 73 kDa were not identified by

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this assay. It was postulated that they could be Hsp72 and Hsc70, because Hsp70

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family proteins have conserved domains and the MWs of Hsp72 and Hsc70 are 72 kDa

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and 73 kDa, respectively. We investigated this possibility by immunoblotting using

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specific antibodies against Hsp72, Hsc70 and GRP78. As shown in Fig. 2E, Hsp72 and

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Hsc70 as well as GRP78 were clearly detected. These data thus showed that all three

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Hsp70 family proteins were associated with the P protein.

266 267

Hsp72 is up-regulated and recruited to IBs during MuV infection. The interaction

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between the P protein and the three Hsp70 family proteins (Fig. 3A) was analyzed by

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co-immunoprecipitation assay. 293T cells expressing the HA-tagged P protein (HA-P)

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and FLAG-tagged Hsp70 family proteins were used. As shown in Fig. 3B, massive

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amount of FLAG-Hsp72 was co-immunoprecipitated with HA-P, whereas only a small

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amount of FLAG-GRP78 was co-precipitated. Although the binding affinities between

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the P protein and Hsp70 family proteins might be different, all three Hsp70 family

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proteins were indeed capable of associating with the P protein in cell lysates. However,

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it was still unclear whether all three Hsp70 family proteins interact with the P protein

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in living cells. To clarify this point, we investigated the intracellular localizations of

277

Hsp70 family proteins in MuV-infected cells by immunofluorescence microscopy. The

278

results showed that Hsp72 expression was upregulated, and the protein was

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redistributed to the IBs in the MuV-infected cells (Fig. 3C). On the other hand, the

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expression levels and localization of Hsc70 and GRP78 were unchanged (Fig. 3C).

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These results suggested that Hsp72, but not Hsc70 or GRP78, interacts with the P

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protein in MuV-infected cells. Data by immunoblotting assay also demonstrated that

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the expression of Hsp72 was increased by MuV infection (Fig. 3D). To better

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understand the requirements for the up-regulation and localization change of Hsp72,

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the P protein was expressed in cells alone or in combination with the N protein. While

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ectopic expression of the P protein alone did not induce the up-regulation of Hsp72

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and was not co-localized with Hsp72 (Fig. 3E and F), the formation of IB-like structures

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caused by the co-expression of N and P proteins dramatically induced the expression

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of Hsp72 and recruited Hsp72 to the IB-like structures (Fig. 3G). Taken together, the

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up-regulation and recruitment to IBs of Hsp72 occurred with the IB formation during

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MuV infection.

292 293

The N-terminal region of the P protein and the C-terminal region of Hsp72 are

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responsible for their interaction. To determine the interacting regions of Hsp72 and

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the P protein, N-terminally FLAG-tagged Hsp72 (FLAG-Hsp72F), HA-tagged P protein

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(HA-P-Full) and their truncation polypeptides were expressed in cells, and their

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interaction was analyzed by co-immunoprecipitation assays. FLAG-Hsp72N was

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comprised of the N-terminal ATPase domain, while FLAG-Hsp72C was comprised of

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the C-terminal peptide binding domain and variable region (Fig. 4A). HA-P-Full was

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co-precipitated with FLAG-Hsp72F and FLAG-Hsp72C (Fig. 4A), indicating that the

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C-terminal region of Hsp72 interacted with the P protein. HA-P∆N was comprised of

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the oligomerization domain and C-terminal region, while HA-P∆C was comprised of

303

the

304

co-immunoprecipitation assay showed that FLAG-Hsp72F was co-precipitated with

305

HA-P-Full and HA-P∆C (Fig. 4B). The data, taken together, indicated that the

306

N-terminal region of the P protein and the C-terminal region of Hsp72 were

307

responsible for their interaction. A co-immunoprecipitation assay was also performed

308

for the V protein, as the P and V proteins possess the common N-terminal region. The

309

data showed that Hsp72 was also associated with the V protein (Fig. 4C).

N-terminal

region

and

oligomerization

domain

(Fig.

4B).

The

310 311

Hsp72 is nonessential for MuV replication, but suppresses apoptotic cell death of

312

MuV-infected cells. To determine the roles for Hsp72 in MuV infection, the expression

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of Hsp72 was suppressed by Hsp72-specific siRNAs (siHsp72). Transfection of siHsp72

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efficiently knocked down the Hsp72 expression (Fig. 5A). However, the levels of viral

315

RNAs, virus production and IB formation were not affected (Fig. 5B-D), demonstrating

316

that Hsp72 was nonessential for MuV replication. Alternatively, it was noted that ~10%

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of Hsp72-knockdown cells infected with MuV were positive for the TUNEL stain

318

indicating apoptosis induction while only ~2.5% of control cells were TUNEL positive

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(Fig. 6A and B). The induction of apoptosis in Hsp72-knockdown cells was confirmed

320

by the elevated caspase 3/7 activity (Fig. 6C). These data indicated that Hsp72 was

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needed to suppress apoptotic cell death of MuV-infected cells.

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The P and V proteins were ubiquitinated in MuV-infected cells. Since IBs are

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comprised of abundant viral proteins that could lead to deleterious consequences for

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the cells, Hsp72 may play a role in ubiquitin-mediated degradation of the accumulated

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viral proteins. To examine this possibility, HA-Ub-expressing 293T cells were infected

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with MuV, and subjected to immunoprecipitation assays. Polypeptides were

328

immunoprecipitated with the anti-MuV V/P antibody (T61), and detected by

329

immunoblotting using an anti-HA antibody. They showed broad size distributions (Fig.

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7A). The signals of ubiquitinated proteins were increased in the presence of MG132, a

331

proteasomal inhibitor. Since the T61 antibody detects both the P and V proteins, these

332

data suggested that the V protein, the P protein, or both were ubiquitinated in

333

MuV-infected cells. Furthermore, endogenous ubiquitinated P and V proteins were

334

detected in Vero cells infected with MuV (Fig. 7B). Taken together, these results

335

demonstrated that both the P and V proteins were ubiquitinated in MuV-infected cells.

336 337

Hsp72 targets the P protein for degradation through the ubiquitin-proteasome

338

pathway. To investigate the roles of Hsp72 in ubiquitin-mediated degradation of the P

339

and V proteins, the effects of Hsp72 knockdown were analyzed. 293T cells expressing

340

HA-Ub were transfected with either siHsp72 or control siNC. The cells were then

341

infected with MuV, and subjected to immunoprecipitation assays, in which the

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polypeptides were immunoprecipitated with the T61 antibody and detected by

343

immunoblotting using an anti-HA antibody. The signals were clearly higher in the

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Hsp72-knockdown cells than in control cells (Fig. 8A). In fact, the signal levels were as

345

high as those in MG-132-treated cells (Fig. 8A). These data suggested that the

346

ubiquitinated V protein, P protein, or both were accumulated in Hsp72-knockdown

347

cells. Similar experiments were performed using cells expressing the V or P protein

348

individually. Signals for the ubiquitinated P protein were low in control

349

(siNC-transfected) cells, but increased in Hsp72-knockdown (siHsp72-transfected) cells

350

(Fig. 8B). On the other hand, signals for the ubiquitinated V protein were similar

351

between Hsp72-knockdown and control cells (Fig. 8B). These data suggested that

352

Hsp72 was involved in the P protein degradation, but not in the V protein degradation.

353

Next, the kinetics of the P and V protein degradation was analyzed. Hsp72-knockdown

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and control Vero cells were infected with MuV, and cultured for 24 hr. Then, these cells

355

were incubated for 0 to 12 hr in the presence of CHX. Degradation of the P protein was

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suppressed in Hsp72-knockdown cells (Fig. 8C, DMSO-treated lanes). On the other

357

hand, degradation of the V protein was minimally affected. To further confirm the

358

proteasomal degradation of P protein, we treated the MuV-infected cells with specific

359

proteasome inhibitors, lactacystin and epoxomicin, which have higher degree of

360

specificity than MG-132 (27). Degradation of the P and V proteins was blocked by

361

treatment of proteasome inhibitors (Fig. 8C). Taken together, these data show that

362

Hsp72 binds to both the V and P proteins, but specifically promotes proteasomal

363

degradation of the P protein.

364 365

Discussion

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Many RNA viruses form IBs. However, the precise functions and complete

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compositions of IBs remain to be elucidated. Studies of negative-stranded RNA viruses

368

have reported that the viral genomic RNA and mRNA were present along with the

369

machineries for viral RNA synthesis in IBs (3-6). Therefore, IBs are likely the site of

370

viral RNA replication and transcription (3-6). Also, in the case of MuV infection the

371

vRNP components were concentrated in the IBs (7). This compartmentalization may

372

facilitate virus replication. In addition to viral proteins, host factors involved in innate

373

immune responses are localized to the IBs. This is thought to be a virus strategy to

374

sequester cellular detectors of viral infections (3, 28).

375

Our data demonstrated that Hsp72 was recruited to the IBs and interacted with the P

376

protein during MuV infection. Many viruses use cellular chaperones for their genome

377

replication, protein synthesis and virion assembly (29). In the paramyxovirus family,

378

Hsp72 has been shown to enhance MV RNA replication and transcription through the

379

interaction with the C terminal region of N protein (18). Hsp72 also associates with

380

polymerase complexes of respiratory syncytial virus (RSV) to positively affect viral

381

RNA synthesis (30). Therefore, it was possible that MuV actively used Hsp72 for its

382

replication. However, we considered this unlikely, since knockdown of Hsp72 showed

383

little, if any, effect on MuV propagation in cultured cells. Recent study revealed that

384

MuV P protein forms a unique tetramer structure different from other paramyxovirus

385

(31, 32). Thus, MuV might differ in the requirements for viral RNA replication and

386

propagation from MV and RSV. Further investigation will be required to define the

387

roles of Hsp72 in MuV infection and to explain these differences.

388

Abnormally accumulated proteins disrupt the cellular functions and implicate in

J Virol. Ver.3

389

various cellular disorders. Thus, these abnormal aggregates are required for rapid

390

degradation (33, 34). Recruitment of chaperones is an adaptive cellular response to

391

assist in ubiquitin-dependent degradation. Hsp72 is usually expressed at low basal

392

levels and increases in response to cellular stressors, therefore, the up-regulation and

393

recruitment to IBs of Hsp72 indicates that the formation of abnormal structures

394

consisting of massive viral proteins is deleterious for cell survival. Indeed, our data

395

revealed that Hsp72 bound to the P protein through its C-terminal peptide binding

396

domain and promoted the ubiquitin-proteasomal degradation of P protein, and

397

suppressed apoptotic cell death of MuV-infected cells. Because the accumulation of the

398

ubiquitinated P protein had no effect on viral propagation, Hsp72 might contribute to

399

the degradation of excess amounts of P protein. It is unclear why the P protein, but not

400

N protein, is targeted by Hsp72-mediated degradation. However, these data may

401

suggest a specific role for the P protein degradation in cellular stress responses. One

402

possibility is speculated as follows: the specific degradation of MuV P protein may

403

elicit a high-avidity cytotoxic T lymphocyte (CTL) response, since the degraded

404

paramyxoviral P protein but not other viral proteins has been reported to contribute to

405

effective CTL elicitation via MHC class I presentation (35, 36). Although we did not

406

examine the possibility in this study, Hsp72 might play a crucial role in elicitation of

407

high-avidity CTLs by regulating the ubiquitin-proteasomal degradation of MuV P

408

protein.

409

Hsp70 family chaperone proteins play major roles in protein quality control (PQC)

410

and maintenance of protein homeostasis. They not only support correct folding and

411

re-folding of client proteins but also guide the substrates to protein degradation

412

systems when a native folding state cannot be reached (37, 38). Hsp70 is involved in

413

the ubiquitin-proteasome system, which is the main cellular protein degradation

414

system. Recruitment of carboxyl-terminus of Hsp70 interacting protein (CHIP), which

415

serves as an E3 ubiquitin ligase, is a well-known function of Hsp70 in the

416

ubiquitin-proteasome pathway (39). Hsp70 is also involved in multistep processes such

417

as substrate unfolding, insertion into the proteasomal core and proteasome biogenesis

418

(40, 41). Accumulation of the ubiquitinated P protein in Hsp72-knocked down cells

419

suggests that Hsp72 plays a crucial role in degradation of the P protein after the step of

420

ubiquitination.

421

The V protein was also ubiquitinated and associated with Hsp72. However, Hsp72

J Virol. Ver.3

422

was not essential for the V protein degradation. The MuV P protein contains a

423

multimerization domain at the unique C-terminal region, and forms a homo-tetramer

424

(31), while the V protein lacks the multimerization domain and functions as a

425

monomer (42, 43). The rate-limiting step of proteasomal degradation is the unfolding

426

of substrates (44). Therefore, degradation of the P protein may require the support of

427

Hsp72 due to the tetrameric structure.

428

In summary, the formation of IBs is a common strategy of many negative-stranded

429

RNA viruses. The present study demonstrated for the first time the functional

430

interaction between viral and host proteins in the IBs of MuV-infected cells. The host

431

chaperone protein Hsp72 interacted with the MuV P protein in IBs, and targeted the

432

MuV P protein for ubiquitin-mediated degradation. These data should provide

433

insights into the roles of chaperone proteins in virus-infected cells.

434 435

Acknowledgments

436

We thank Nozomi Takeda of the Equipment Management Center (Creative Research

437

Institution, Hokkaido University) for technical support. We also thank all the members

438

of the Department of Virology III, NIID, for their technical advice and critical input.

439

This work was supported by grants from the Ministry of Education, Culture, Sports,

440

Science and Technology (Grant-in Aid for Research Activity Start-up: 25893295) and

441

the Ministry of Health, Labour and Welfare of Japan.

442 443

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574 575

Figure Legends

576

Fig. 1. Intracellular localizations of MuV proteins and genomic RNA. (A) Vero cells

577

infected with MuV were immunostained at 24 hr postinfection (h.p.i.) with mouse

578

anti-N (23D), anti-P (57A), anti-M (79D), anti-F (170C) or anti-HN (78) MAb and rabbit

579

anti-N, anti-V/P (T61), anti-V (T60) or anti-L (L17) PAb, followed by AF488-conjugated

580

anti-mouse IgG and AF594-conjugated anti-rabbit IgG, respectively. Cell nuclei were

581

stained with DAPI (blue). (B) Vero cells infected with MuV were immunostained at 24

582

h.p.i. with rabbit anti-V/P PAb and AF594-conjugated anti-rabbit IgG. Then, the cells

583

were subjected to fluorescence in situ hybridization assay to detect viral genomic RNA.

584

Cell nuclei were stained with DAPI (blue). (C and D) Vero cells transfected with

585

pCAGGS-N, P and/or L alone or in combination were immunostained at 24 hr

586

posttransfection with anti-N or anti-P MAb and anti-V/P or anti-L PAb, followed by

J Virol. Ver.3

587

AF488-conjugated anti-mouse IgG and AF594-conjugated anti-rabbit IgG, respectively.

588

Cell nuclei were stained with DAPI (blue).

589 590

Fig. 2. Identification of cellular proteins associated with the MuV P protein by

591

FOS-tagged purification. (A) Overview of the FOS-tagged purification of cellular

592

proteins associated with the MuV N and P protein. (B) Vero cells transfected with

593

pCAG-N-FOS and/or pCAG–P-FOS were immunostained at 24 hr posttransfection

594

with mouse anti-N MAb and rabbit anti-V/P PAb, followed by AF488-conjugated

595

anti-mouse IgG and AF594-conjugated anti-rabbit IgG. Cell nuclei were stained with

596

DAPI (blue). (C) Purified products were subjected to SDS-PAGE, followed by CBB

597

staining. The empty vector (EV) was used as a negative control. (D) Purification of the

598

P-FOS protein was confirmed by immunoblotting using mouse anti-FLAG MAb (M2)

599

and rabbit anti-V/P PAb. (E) Detection of Hsp70 family proteins in the purified P-FOS

600

complexes was performed by immunoblotting using the appropriate antibodies.

601 602

Fig. 3. Hsp72 was up-regulated and recruited to the IBs during MuV infection. (A)

603

Summary of Hsp70 family protein candidates for binding partners of the P protein. (B)

604

FLAG-Hsp70 proteins were coexpressed with HA-P in 293T cells, immunoprecipitated

605

with anti-HA antibody, and immunoblotted with anti-HA and anti-FLAG antibodies.

606

(C) Vero cells infected with MuV were immunostained at 24 hr postinfection (h.p.i.)

607

with mouse anti-Hsp72 or anti-Hsc70 MAb and rabbit anti-MuV V/P PAb or mouse

608

anti-P MAb and rabbit anti-GRP78 PAb, followed by the appropriated secondary

609

Alexa-conjugated antibodies, respectively. Cell nuclei were stained with DAPI (blue).

610

(D) Vero cells were infected with MuV at an MOI of 1.0. Cell lysates were collected at

611

the indicated times and subjected to immunoblotting with the appropriate antibodies.

612

(E) 293T cells were transfected with pCAGGS-P or empty vector (EV). At 24 hr

613

postransfection, cell lysates were collected and subjected to immunoblotting with the

614

appropriate antibodies. (F and G) Vero cells transfected with pCAGGS-P alone (F) or

615

pCAGGS-N and -P (G) were immunostained at 24 hr posttransfection with mouse

616

anti-Hsp72 MAb and rabbit anti-V/P PAb, followed by AF488-conjugated anti-mouse

617

IgG and AF594-conjugated anti-rabbit IgG. Cell nuclei were stained with DAPI (blue).

618 619

Fig. 4. Determination of the regions responsible for the interaction between the P

J Virol. Ver.3

620

protein and Hsp72. (A) FLAG-Hsp72 and its deletion mutants were coexpressed with

621

HA-P

622

immunoblotted with anti-FLAG and anti-HA antibodies. (B) HA-P and its deletion

623

mutants were coexpressed with FLAG-Hsp72 in 293T cells, immunoprecipitated with

624

anti-HA antibody, and immunoblotted with anti-HA and anti-FLAG antibodies. (C)

625

FLAG-Hsp72 was coexpressed with HA-V in 293T cells, immunoprecipitated with

626

anti-HA antibody, and immunoblotted with anti-HA and anti-FLAG antibodies.

in

293T

cells,

immunoprecipitated

with

anti-FLAG

antibody,

and

627 628

Fig. 5. Effects of Hsp72 knockdown on MuV propagation. (A) At 48 hr posttransfection

629

with either siHsp72 or siNC, Vero cells were inoculated with MuV at an MOI of 1.0.

630

Cell lysates were collected at 24 hr postinfection (h.p.i.) and subjected to

631

immunoblotting with the indicated antibodies. (B) At 48 hr posttransfection with either

632

siHsp72 or siNC, Vero cells were inoculated with MuV at an MOI of 5.0. Total cellular

633

RNA was extracted at 24 h.p.i. and subjected to RT using an oligo(dT) primer. The

634

levels of N mRNA were determined by real-time PCR and calculated as percentages of

635

the control HPRT1 mRNA level. The data are representative of three independent

636

experiments. Error bars indicate the standard deviations of the means. (C) At 48 hr

637

posttransfection with either siHsp72 or siNC, Vero or Huh7 cells were infected with

638

MuV at an MOI of 0.01. The supernatants were collected at 24, 48, 72, and 96 h.p.i., and

639

the infectious titers were determined by plaque assay in Vero cells. The results shown

640

are from three independent experiments, with the error bars representing the standard

641

deviations. (D) At 48 hr posttransfection with either siHsp72 or siNC, Vero cells were

642

inoculated with MuV at an MOI of 1.0. At 24 h.p.i., the cells were fixed, permeabilized

643

and immunostained with mouse anti-Hsp72 MAb and rabbit anti-V/P PAb, followed

644

by AF488-conjugated anti-mouse IgG and AF594-conjugated anti-rabbit IgG. Cell

645

nuclei were stained with DAPI (blue).

646 647

Fig. 6. Increased apoptosis in MuV-infected Hsp72 knockdown cells. (A) At 48 hr

648

posttransfection with either siHsp72 or siNC, Vero cells were infected with MuV at an

649

MOI of 5.0. At 24 hr postinfection (h.p.i.), the cells were fixed, permeabilized and

650

stained with TUNEL stain. (B) TUNEL-positive cells were counted and expressed as

651

percentages of total cells (n > 1,000). Total cells were calculated by DAPI staining. (C)

652

At 48 hr posttransfection with either siHsp72 or siNC, Vero cells were inoculated with

J Virol. Ver.3

653

MuV at an MOI of 5.0. At 24 h.p.i., caspase 3/7 activity was measured, and relative

654

caspase 3/7 activity was calculated by normalization with cell viability.

655 656

Fig. 7. Ubiquitination of MuV P and V proteins. (A) 293T cells were transfected with a

657

plasmid encoding HA-Ub and infected with MuV. At 24 h.p.i., the cells were treated

658

with 10 M of MG-132 for 5 hr, and then the cell lysates were subjected to

659

immunoprecipitation assay with anti-V/P antibody, followed by immunoblotting with

660

the appropriate antibodies. (B) At 24 h.p.i., Vero cells infected with MuV were treated

661

with 10 M of MG-132 for 5 hr, and then the cell lysates were subjected to

662

immunoprecipitation assay with anti-V/P antibody, followed by immunoblotting with

663

the appropriate antibodies.

664 665

Fig. 8. Hsp72 regulates the P protein degradation through the ubiquitin-proteasome

666

system. (A) At 48 hr posttransfection with either siHsp72 or siNC, 293T cells were

667

transfected with a plasmid encoding HA-Ub and infected with MuV. At 24 hr

668

postinfection (h.p.i.), the cells were treated with 10 M of MG-132 for 5 hr, and then the

669

cell lysates were subjected to immunoprecipitation assay with anti-V/P antibody,

670

followed by immunoblotting with anti-HA and anti-V/P antibodies. (B) At 48 hr

671

posttransfection with either siHsp72 or siNC, 293T cells were transfected with

672

plasmids pCAGPM-HA-Ub and pCAGGS-P or -V, respectively. After 24 hr, the cell

673

lysates were subjected to immunoprecipitation assay with anti-V/P antibody, followed

674

by immunoblotting with anti-HA and anti-V/P antibodies. (C) At 48 hr

675

posttransfection with either siHsp72 or siNC, Vero cells were infected with MuV. At 24

676

h.p.i., the cells were treated with MG-132 (10 M), Lactacystin (5 M), Epoxomicin (10

677

M) or DMSO for 2 hr. Then, 50 g/ml of CHX was added, and the cell lysates were

678

collected at the indicated times. The expression levels of the P and V proteins were

679

detected by immunoblotting with anti-V/P antibody.

680