Identification of Hsc70 As an Influenza Virus Matrix Protein (M1)

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- 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] propagation. Hsp70 has been reported to be involved in Canine distemper 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 nucleus. 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 obtained 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 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 SmartCycler 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 Flex (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- conjugated 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). (C) The purified influenza viral particles were analyzed by M1 after 10% SDS–PAGE. (C) A/PR/8/34-infected cells were subjected 10% SDS–PAGE. Typical bands were excised from the CBB-stained to immunoprecipitation using anti-Hsc70 or rat anti-mouse IgG1 Mab gel and then were subjected to MALDI-TOF MS. The identified as indicated. Immunoprecipitates were analyzed by 10% SDS–PAGE, proteins (P < 0.05) were shown in the figure. and then were visualized with anti-Hsc70 or anti-M1. K. Watanabe et al. / FEBS Letters 580 (2006) 5785–5790 5787

3.2. Interaction of Hsc70 with M1 in infected cells promoter [15] (Fig. 3B). The plasmid encoding M gene Hsc70 is a member of Hsp70 family protein, constitutively (pPolI-WSN-M) is transcribed into a viral genome, and then expressed in the cell (Fig. 2A). The influenza virus infection it is assembled with viral proteins, PA, PB1, PB2 and NP. did not affect the expression level of Hsc70, whereas that of M1 protein is produced from this vRNP complex. Using dele- Hsp70 increased both in HeLa and MDBK cells. Next, the tion mutants of M1, no association between M1D102–201 and interaction between Hsc70 and M1 was confirmed by co- Hsc70 was observed, whereas D76–103, which lacks an RNA immunoprecipitation experiment using anti-M1 (Fig. 2B) or binding domain, still bind to Hsc70 (Fig. 3B). It is also cleared anti-Hsc70 (Fig. 2C). Hsc70 coprecipitates with M1 in HeLa that M1 interacts with Hsc70 in the absence of any other viral cells (Fig. 2B, lane 4), MDBK and MDCK cells (data not proteins (Fig. 3C). These results suggest that the C terminal shown). Using anti-Hsc70 antibody, M1 was co-precipitated half of M1 interacts with Hsc70. with Hsc70 in MDBK, MDCK and HeLa cells (Fig. 2C). 3.4. Localization of Hsc70 in virus infected cells 3.3. C-terminal half of M1 interacts with Hsc70 In uninfected cells, Hsc70 localizes dominantly in the cyto- Fig. 3 shows Hsc70 binding domain of M1. We mimicked plasm at 37 C(Fig. 4a and g). In the early stages (3 h) of virus the late stage of virus infection in the cells by a genome plas- infection, when M1 was not expressed yet (e), an early protein, mid expression system with human RNA-polymerase I (polI) NP, localizes in the nucleus (k). The localization of Hsc70 at

Fig. 3. Determination of Hsc70 binding domain of M1. (A) Diagram of a series of deletion mutants of M1. Hsc70-M1 interaction was confirmed by immunoprecipitation (B) or Far Western blotting (C). (B) Viral RNP-mediated expression of M1 protein. Plasmids were cotransfected into human 293T cells. M1 genome RNA is transcribed under the control of human polI promoter. NP, viral polymerases (PA, PB1 and PB2) and M1 genome RNA forms a viral RNP complex, then M1 protein is synthesized. 293T cells were transfected with pCAGGS-M1 (lane 2), pPolI-WSN-M wild type (lane 3), pPolI-WSN-MD76-103 (lane 4), pPolI-WSN-MD102–201 (lane 5), pPolI-WSN-MD204–252 (lane 6), in the absence (lanes 1 and 2) or the presence of viral polymerases and NP-expressing plasmids (lanes 3–6), respectively. Twenty-four hours after transfection, the cell lysates were subjected to immunoprecipitation using anti-M1 antiserum. (C) Far-western assay. E. coli-expressed crude lysate of His-M1 (lane 1) and its deletion mutants, D1–75 (lane 2), D76–115 (lane 3), D112–252 (lane 4) and D91–252 (lane 5) [4] were separated by 13% SDS–PAGE, transferred onto PVDF membrane, then followed by denaturation with 6 M guanidine HCl in TBST for 10 min. After stepwise renaturating, the membrane was incubated with 1 lg/ml of each purified GST-Hsc70 [23] or GST in TBST at 4 C for 4 h. Specific signals were detected by a Western blotting procedure using anti-Hsc70 antibody. 5788 K. Watanabe et al. / FEBS Letters 580 (2006) 5785–5790

Fig. 4. Localization of Hsc70, M1 and NP in influenza virus infected cells. MDCK cells infected with A/WSN/33 (MOI = 1) were fixed and reacted with anti-M1 (Alexa 546: Red) or anti-NP (Alexa 546: Red) together with anti-Hsc70 (Alexa 488: Green). The fluorescent cells were observed by confocal microscopy (LSM, Carl Zeiss).

the early stage did not change significantly (a, b, g and h), how- treated sample. The data obtained at 36 h post infection ever, the localization of Hsc70 changed drastically to the nu- strengthen the results (Fig. 5B). It is also possible that a lack cleus in the late stages (9 h) of viral infection where M1 was of Hsc70 may also affect the infectivity of the virus but not dominantly localized (Fig. 4c). Similar results were observed the production. We therefore examined the number of virus when HeLa cells, and A/PR/8/34(H1N1) viruses were used particles released from the cell by quantifying the RNA gen- (data not shown). ome copy using a real-time PCR analysis (Fig. 5C). Our results suggested that Hsc70 affects the production of the virus but not 3.5. Hsc70 requires for nuclear export of vRNP and virus the infectivity. M1, NP and Hsc70 localized in nucleus at 9 hr production (see Fig. 4) were exported from nucleus and localize both nu- We investigated whether the expression level of Hsc70 affects cleus and cytoplasm at 24 h (Fig. 5D, a, e, h and l) where the the localization of the viral proteins and virus production assembly of the virus occurs. When Hsc70 expression was sup- (Fig. 5). We have introduced SiRNA specific to Hsc70 and suc- pressed by the SiRNA transfection (Fig. 5D, b and i), M1 (f) ceeded in about 90% reduction of Hsc70 in 84 h as shown in and NP (m) were accumulated in nucleus and thus failed to Fig. 5A. Seventy-two hours after an SiRNA transfection, the be exported from the nucleus. These results clearly suggested cells were infected with the virus at a MOI of 0.1 (5000 that Hsc70 is therefore required for the nuclear export of PFU/well). The cells were extensively washed and then were vRNP-M1 complex. incubated further with 500 ll of the medium. About 14000 PFU/ml (without SiRNA) or 20000 PFU/ml (with Ran- 3.6. Roles of Hsps in the late stages of virus infection dom SiRNA) of the virus titer at 12 h post infection was signif- Our previous study and an X-ray structure analysis icantly reduced to 2000 PFU/ml in the Hsc70-specific SiRNA showed amino acid residues 91–105 of M1 is involved in K. Watanabe et al. / FEBS Letters 580 (2006) 5785–5790 5789

Fig. 5. Hsc70 requires for export of vRNP and virus production. (A) HeLa cells were transfected with Hsc70 specific or control random SiRNA. The cell lysate was recovered after either 36, 60 or 84 h, and then it was subjected to Western blotting as indicated. Seventy-two (B) or 48 h (C and D) after SiRNA transfection, the cells were infected with A/WSN/33 at a MOI of 0.1 and further incubated. (B) The infectious viral titer in the medium was estimated by a plaque assay. The results represent the means + S.E. The representative data of three independent experiments are indicated. (C) The production of the virus particle was estimated by a real-time PCR analysis. The results represent the means + S.E. The representative data of three independent experiments are indicated. (D) The cells were observed by Axiophot microscopy (LSM, Carl Zeiss). the M1-vRNP interaction [4,18]. We also found that leptomy- of the nuclear export of M1 and vRNP (Fig. 5D). We recently cinB (LMB), an inhibitor of CRM1-mediated nuclear export, reported that Hsc70 possess the NES [20], thus suggesting inhibits the export of vRNP [19]. However, M1 does not have that Hsc70 promotes the nuclear export of M1 and vRNP. a nuclear export signal (NES). The mechanism of M1-medi- We also found M1 to be associated with Hsp90 (data not ated vRNP export is still unclear. It is possible to speculate shown) and PA [3], while Hsp90 stimulates the influenza virus the possible existence of a host factor which regulates vRNPs RdRp activity [21]. Hsc70 and Hsp90 may therefore act as a export. In this communication, we clearly demonstrated that stimulating factor on various steps of the influenza virus life Hsc70 directly bind to M1 at C terminal half of M1 cycle by the association with vRNP. On the other hand, it (Fig. 3C). Hsc70-specific SiRNA treatment results in reduc- was reported that Hsp70 induced at 41 C inhibited the tion of virus production (Fig. 5B and C) by the inhibition export of vRNP in MDCK cells [22]. Further studies are thus 5790 K. Watanabe et al. / FEBS Letters 580 (2006) 5785–5790 necessary to explore the detailed mechanisms of Hsps in the [11] Oglesbee, M.J., Liu, Z., Kenney, H. and Brooks, C.L. (1996) The export of vRNP. highly inducible member of the 70 kDa family of heat shock proteins increases canine distemper virus polymerase activity. J. Gen. Virol. 77 (Pt 9), 2125–2135. Acknowledgements: We thank Dr. Y. Kawaoka (University of Tokyo) [12] Vasconcelos, D.Y., Cai, X.H. and Oglesbee, M.J. (1998) Consti- for providing M1, NP, PA, PB1 and PB2 expression vectors. This work tutive overexpression of the major inducible 70 kDa heat shock was supported in part by a Grant-in-Aid for Ministry of Education, protein mediates large plaque formation by measles virus. J. Gen. Culture, Sports, Science and Technology, and Scientific Research from Virol. 79 (Pt 9), 2239–2247. Nagasaki University, Japan (K.W.). [13] Honda, A., Ueda, K., Nagata, K. and Ishihama, A. (1988) RNA polymerase of influenza virus: role of NP in RNA chain elongation. J. Biochem. (Tokyo) 104, 1021–1026. [14] Takizawa, N., Watanabe, K., Nouno, K., Kobayashi, N. and References Nagata, K. (2006) Association of functional influenza viral proteins and RNAs with nuclear chromatin and sub-chromatin [1] Nayak, D.P., Hui, E.K. and Barman, S. (2004) Assembly and structure. Microbes Infect 8, 823–833. budding of influenza virus. Virus Res. 106, 147–165. [15] Neumann, G., Watanabe, T., Ito, H., Watanabe, S., Goto, H., [2] Ye, Z., Robinson, D. and Wagner, R.R. (1995) Nucleus-targeting Gao, P., Hughes, M., Perez, D.R., Donis, R., Hoffmann, E., domain of the matrix protein (M1) of influenza virus. J. Virol. 69, Hobom, G. and Kawaoka, Y. (1999) Generation of influenza A 1964–1970. viruses entirely from cloned cDNAs. Proc. Natl. Acad. Sci. USA [3] Hara, K., Shiota, M., Kido, H., Watanabe, K., Nagata, K. and 96, 9345–9350. Toyoda, T. (2003) Inhibition of the protease activity of influenza [16] Shevchenko, A., Wilm, M., Vorm, O. and Mann, M. (1996) Mass virus RNA polymerase PA subunit by viral matrix protein. spectrometric sequencing of proteins silver-stained polyacryl- Microbiol. Immunol. 47, 521–526. amide gels. Anal. Chem. 68, 850–858. [4] Watanabe, K., Handa, H., Mizumoto, K. and Nagata, K. (1996) [17] Sagara, J. and Kawai, A. (1992) Identification of heat shock Mechanism for inhibition of influenza virus RNA polymerase protein 70 in the rabies virion. Virology 190, 845–848. activity by matrix protein. J. Virol. 70, 241–247. [18] Sha, B. and Luo, M. (1997) Structure of a bifunctional [5] Burleigh, L.M., Calder, L.J., Skehel, J.J. and Steinhauer, D.A. membrane-RNA binding protein, influenza virus matrix protein (2005) Influenza a viruses with mutations in the m1 helix six M1. Nat. Struct. Biol. 4, 239–244. domain display a wide variety of morphological phenotypes. J. [19] Watanabe, K., Takizawa, N., Katoh, M., Hoshida, K., Kobay- Virol. 79, 1262–1270. ashi, N. and Nagata, K. (2001) Inhibition of nuclear export of [6] Martin, K. and Helenius, A. (1991) Nuclear transport of influenza ribonucleoprotein complexes of influenza virus by leptomycin B. virus ribonucleoproteins: the viral matrix protein (M1) promotes Virus Res. 77, 31–42. export and inhibits import. Cell 67, 117–130. [20] Tsukahara, F. and Maru, Y. (2004) Identification of novel nuclear [7] Santoro, M.G. (1997) Antiviral activity of cyclopentenone pros- export and nuclear localization-related signals in human heat tanoids. Trends Microbiol. 5, 276–281. shock cognate protein 70. J. Biol. Chem. 279, 8867–8872. [8] Saphire, A.C., Guan, T., Schirmer, E.C., Nemerow, G.R. and [21] Momose, F., Naito, T., Yano, K., Sugimoto, S., Morikawa, Y. Gerace, L. (2000) Nuclear import of adenovirus DNA in vitro and Nagata, K. (2002) Identification of Hsp90 as a stimulatory involves the nuclear protein import pathway and hsc70. J. Biol. host factor involved in influenza virus RNA synthesis. J. Biol. Chem. 275, 4298–4304. Chem. 277, 45306–45314. [9] Florin, L., Becker, K.A., Sapp, C., Lambert, C., Sirma, H., [22] Hirayama, E., Atagi, H., Hiraki, A. and Kim, J. (2004) Heat Muller, M., Streeck, R.E. and Sapp, M. (2004) Nuclear translo- shock protein 70 is related to thermal inhibition of nuclear export cation of papillomavirus minor capsid protein L2 requires Hsc70. of the influenza virus ribonucleoprotein complex. J. Virol. 78, J. Virol. 78, 5546–5553. 1263–1270. [10] Sagara, Y., Ishida, C., Inoue, Y., Shiraki, H. and Maeda, Y. [23] Tsukahara, F., Yoshioka, T. and Muraki, T. (2000) Molecular (1998) 71-kilodalton heat shock cognate protein acts as a cellular and functional characterization of HSC54, a novel variant of receptor for syncytium formation induced by human T-cell human heat-shock cognate protein 70. Mol. Pharmacol. 58, 1257– lymphotropic virus type 1. J. Virol. 72, 535–541. 1263.