FEBS Letters 580 (2006) 5785–5790 Identification of Hsc70 as an influenza virus matrix protein (M1) binding factor involved in the virus life cycle Ken Watanabea, Takayuki Fusea, Issay Asanoa, Fujiko Tsukaharab, Yoshiro Marub, Kyosuke Nagatac, Kaio Kitazatoa, Nobuyuki Kobayashia,* a Laboratory of Molecular Biology of Infectious Agents, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan b Department of Pharmacology, Tokyo Women’s Medical University, School of Medicine, 8-1, Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan c Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan Received 22 August 2006; revised 13 September 2006; accepted 15 September 2006 Available online 27 September 2006 Edited by Felix Wieland nuclear export signal (NES), thus mechanism of M1-mediated Abstract Influenza virus matrix protein 1 (M1) has been shown to play a crucial role in the virus replication, assembly and bud- nuclear export of vRNP is still not well understood. ding. We identified heat shock cognate protein 70 (Hsc70) as a We herein identify heat shock cognate protein 70 (Hsc70), a M1 binding protein by immunoprecipitation and MALDI-TOF constitutive form of Hsp70 family protein, as an host factor(s) MS. The C terminal domain of M1 interacts with Hsc70. We which bind to M1 in infected cells by matrix-assisted laser found that Hsc70 does not correlate with the transport of M1 desorption ionization-time of flight mass spectrometry (MAL- to the nucleus, however, it does inhibit the nuclear export of DI-TOF MS). Heat shock proteins (Hsps) were induced by M1 and NP, thus resulting in the inhibition of viral production. various stimulations, such as heat treatments, chemicals, UV, This is the first demonstration that Hsc70 is directly associated and viral infection. One of the functions of Hsps is to maintain with M1 and therefore is required for viral production. cellular homeostasis. It has thus been reported that Hsps inhi- Ó 2006 Federation of European Biochemical Societies. Pub- bit the replication of a variety of RNA viruses, such as para- lished by Elsevier B.V. All rights reserved. myxoviruses, rhabdoviruses, rotaviruses, retroviruses and Keywords: Influenza virus; M1; Hsc70; Host factor influenza virus [7]. On the other hand, many kind of viruses utilize specific Hsps for their replication. Hsc70 is involved in Adenovirus [8], papillomavirus [9], and HTLV-1 [10] propaga- tion. Hsp70 has been reported to be involved in Canine distem- per virus [11] and measles virus [12] propagation. We found a direct interaction and colocalization of Hsc70 and M1. The 1. Introduction knockdown of Hsc70 using SiRNA resulted in a inhibition of vRNP export from the nucleus, thus resulting in a reduction The genome of the type A influenza virus in the virion exists of virus production. as a viral ribonucleoprotein (vRNP) complex with RNA- dependent RNA polymerase (RdRp) and nucleocapsid protein (NP). The transcription of viral RNA takes place in the nu- cleus. The early gene products, RdRp (PB1, PB2, and PA), 2. Materials and methods and NP are transported to nucleus where they form vRNPs. The vRNP is surrounded by matrix protein 1 (M1), which 2.1. Viruses, cells, antibodies and SiRNAs has multiple functions in the late stages of infection [1]. Newly Influenza virus A/PR/8/34(H1N1) and A/WSN/33(H1N1) were synthesized M1 binds to progeny vRNP [2] and suppresses the propagated in 10-day-old embryonated eggs. Purified virions were ob- tained as described previously [13]. 293T cells were maintained in protease activity of the RdRp [3]. M1 also promotes the termi- DMEM. Madin-Darby canine kidney (MDCK), Madin-Darby bovine nation of viral RNA synthesis. The helix 6 (amino acid resi- kidney (MDBK) and HeLa cells were maintained in MEM. The med- dues 91–105) of M1 plays important roles in its nuclear ium was supplemented with 10% FBS. The preparation of anti-M1 and localization [2], in the inhibition of RNA synthesis and RNA anti-NP were previously described [14]. Anti-Hsc70 (SPA-815) and binding [4] and in the efficient virus production and virion anti-Hsp70 (SPA-810) were purchased from Stressgen (Victoria BC, Canada). Anti-actin (A-5060) was purchased from Sigma Aldrich. morphology [5]. M1 also plays an essential role in the export Human Hsc70-specific SiRNA (sc-29349) was purchased from Santa of vRNP from nucleus [6]. However, M1 does not possess Cruz Biotechnology, Inc. Random SiRNA was purchased from iGENE Co. Ltd. (Tsukuba, JAPAN). Transfection of SiRNA into cells was performed according to manufacturer’s instructions. 2.2. Construction of plasmids *Corresponding author. Fax: +81 95 819 2898. For the construction of pPolI-WSN-MD76–103, pPolI-WSN-M [15] E-mail address: [email protected] (N. Kobayashi). was digested with PstI and HindIII, and then was ligated with linker 50-ACGTTCGA-30. For the construction of pPolI-WSN-MD102-201, Abbreviations: Hsps, heat shock proteins; vRNP, viral ribonucleopro- pPolI-WSN-M was digested with HindIII and NcoI, and filled with tein; NP, nucleocapsid protein; M1, matrix protein 1; MALDI-TOF Klenow fragment, and then was self-ligated. For the construction of MS, matrix-assisted laser desorption ionization-time of flight mass pPolI-WSN-MD203–252, pPolI-WSN-M was partially digested with spectrometry; Hsc70, heat shock cognate protein 70 NcoI, filled with Klenow fragment, and then was self-ligated. 0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.09.040 5786 K. Watanabe et al. / FEBS Letters 580 (2006) 5785–5790 2.3. Immunoprecipitation and MALDI-TOF MS analysis formed with Cepheid SmartCyclerÒ II (Cepheid, Sunnyvale, CA) MDCK (1 · 107 cells) were infected with influenza virus (MOI = 1) using SYBR Green I and specific primers, 50-TCTGATCCTCTCGT- for 12 h. The cells were lysed with 400 ll of the lysis buffer (50 mM CATTGCAGCAA-30 and 50-AATGACCATCGTCAACATCCA- Tris, pH 7.5, 150 mM NaCl, 0.5% TritonX-100, 2 mM EDTA, CAGC-30, corresponding to nucleotide position between 782 and 984 1 mM DTT and 1 mM PMSF) for 30 min and centrifuged at of A/WSN/33 segment 7 viral genome. The serial dilution of pPol- 15000 · g for 10 min. In a total volume of 300 ll, the reaction mixture WSN-M plasmid were used to calculate the copy numbers of amplified containing 200 ll of the cell lysate, 10 ll of Ab-bound proteinA Se- DNA. pharose, and the lysis buffer containing 0.1% BSA was incubated at 4 °C for overnight. After successive washings, Ab-bound proteins were eluted in a step-wise manner with the wash buffer containing 0.5, 1, 2 2.5 and 3 M NaCl, respectively. The in-gel digestion with trypsin 3. Results and discussion (Sequencing grade, Promega) was performed as previously described [16]. A MALDI-TOF MS analysis was then performed using Ultra 3.1. Identification of M1-binding protein Flexä (BRUKER DALTONICS) and Mascot search (Matrix Science To explore the host factor(s) which interact with M1, the Inc.) according to the manufacturer’s instructions. combination of an immunoprecipitation and MALDI-TOF MS was conducted (Fig. 1). The influenza virus infected 2.4. Indirect immunofluorecence The cells were fixed with 4% paraformaldehyde in PBS for 10 min MDCK cells were lysed and immunoprecipitated with anti- and permeabilized with 0.1% NP-40 in PBS for 20 min, and then were M1. The proteins binding to M1 were eluted in a step-wise incubated for 1 h with anti-M1 rabbit antiserum or anti-NP rabbit manner with the increasing NaCl concentration and then were antiserum and anti-Hsc70 rat Mab. After washing with PBS, the cells analyzed by SDS–PAGE. Each eluted sample contained sev- were incubated for 1 h with the secondary antibodies, Alexa546- con- jugated anti-rabbit Ig and Alexa488-conjugated anti-rat Ig. The cells eral specific bands (data not shown). In 3 M NaCl elute frac- were observed under microscopy. tion, a specific band (70 kDa) was obtained (Fig. 1A, arrowhead) and identified as Hsc70 (Fig. 1B, P < 0.05). Puri- 2.5. Real-time PCR analysis fied virion were also analyzed by MALDI-TOF MS RNA samples from virion particles were extracted by Trizol reagent (Fig. 1C). In addition to viral structural proteins, host proteins (Invitrogen) and subjected to reverse transcription using random hex- including Hsc70 were identified. This finding correlates with amer and M-MLV RT (Gibco BRL). Real-time PCR reaction was per- those of previous studies in which Hsc70 is incorporated into vesicular stomatitis virus, Newcastle disease virus influenza virus and Rabies virus [17]. Fig. 1. Identification of Hsc70 as a M1 binding protein. (A) MDCK cells, infected with influenza virus (A/PR/8/34) were lysed and immunoprecipitated using anti-M1. The immunoprecipitates were Fig. 2. Hsc70- M1 interaction. (A) The cells were infected with the successively washed, and then were eluted with 3 M NaCl, and influenza virus A/PR/8/34 (MOI = 1) for 12 h. The cell lysates were subjected to 10% SDS–PAGE, followed by CBB staining. The subjected to 10% SDS–PAGE, followed by Western blotting as arrowhead indicates a specific band detected only in a viral infected indicated. (B) HeLa cells were infected with A/PR/8/34 (MOI = 1) for sample. (B) Hsc70 spectrum by MALDI-TOF MS. The viral infection- 12 h. The cell lysates were immunoprecipitated with anti-M1 or control specific band (panel A, lane 2, arrowhead) was identified as Hsc70 rabbit normal serum and then were visualized with anti-Hsc70 or anti- (P < 0.05).