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
b
Laboratory of Molecular Biology of Infectious Agents, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan 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
Abstract Influenza virus matrix protein 1 (M1) has been shown to play a crucial role in the virus replication, assembly and budding. We identified heat shock cognate protein 70 (Hsc70) as a M1 binding protein by immunoprecipitation and MALDI-TOF MS. The C terminal domain of M1 interacts with Hsc70. We found that Hsc70 does not correlate with the transport of M1 to the nucleus, however, it does inhibit the nuclear export of M1 and NP, thus resulting in the inhibition of viral production. This is the first demonstration that Hsc70 is directly associated with M1 and therefore is required for viral production. 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Influenza virus; M1; Hsc70; Host factor
1. Introduction The genome of the type A influenza virus in the virion exists as a viral ribonucleoprotein (vRNP) complex with RNAdependent 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), and NP are transported to nucleus where they form vRNPs. The vRNP is surrounded by matrix protein 1 (M1), which has multiple functions in the late stages of infection [1]. Newly synthesized M1 binds to progeny vRNP [2] and suppresses the protease activity of the RdRp [3]. M1 also promotes the termination of viral RNA synthesis. The helix 6 (amino acid residues 91–105) of M1 plays important roles in its nuclear localization [2], in the inhibition of RNA synthesis and RNA binding [4] and in the efficient virus production and virion morphology [5]. M1 also plays an essential role in the export of vRNP from nucleus [6]. However, M1 does not possess
*
Corresponding author. Fax: +81 95 819 2898. E-mail address:
[email protected] (N. Kobayashi). Abbreviations: Hsps, heat shock proteins; vRNP, viral ribonucleoprotein; NP, nucleocapsid protein; M1, matrix protein 1; MALDI-TOF MS, matrix-assisted laser desorption ionization-time of flight mass spectrometry; Hsc70, heat shock cognate protein 70
nuclear export signal (NES), thus mechanism of M1-mediated nuclear export of vRNP is still not well understood. We herein identify heat shock cognate protein 70 (Hsc70), a constitutive form of Hsp70 family protein, as an host factor(s) which bind to M1 in infected cells by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Heat shock proteins (Hsps) were induced by various stimulations, such as heat treatments, chemicals, UV, and viral infection. One of the functions of Hsps is to maintain cellular homeostasis. It has thus been reported that Hsps inhibit the replication of a variety of RNA viruses, such as paramyxoviruses, rhabdoviruses, rotaviruses, retroviruses and 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 knockdown of Hsc70 using SiRNA resulted in a inhibition of vRNP export from the nucleus, thus resulting in a reduction of virus production.
2. Materials and methods 2.1. Viruses, cells, antibodies and SiRNAs Influenza virus A/PR/8/34(H1N1) and A/WSN/33(H1N1) were propagated in 10-day-old embryonated eggs. Purified virions were obtained as described previously [13]. 293T cells were maintained in DMEM. Madin-Darby canine kidney (MDCK), Madin-Darby bovine kidney (MDBK) and HeLa cells were maintained in MEM. The medium was supplemented with 10% FBS. The preparation of anti-M1 and anti-NP were previously described [14]. Anti-Hsc70 (SPA-815) and anti-Hsp70 (SPA-810) were purchased from Stressgen (Victoria BC, Canada). Anti-actin (A-5060) was purchased from Sigma Aldrich. Human Hsc70-specific SiRNA (sc-29349) was purchased from Santa 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 For the construction of pPolI-WSN-MD76–103, pPolI-WSN-M [15] was digested with PstI and HindIII, and then was ligated with linker 5 0 -ACGTTCGA-3 0 . For the construction of pPolI-WSN-MD102-201, pPolI-WSN-M was digested with HindIII and NcoI, and filled with Klenow fragment, and then was self-ligated. For the construction of pPolI-WSN-MD203–252, pPolI-WSN-M was partially digested with 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 2.3. Immunoprecipitation and MALDI-TOF MS analysis MDCK (1 · 107 cells) were infected with influenza virus (MOI = 1) for 12 h. The cells were lysed with 400 ll of the lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% TritonX-100, 2 mM EDTA, 1 mM DTT and 1 mM PMSF) for 30 min and centrifuged at 15 000 · g for 10 min. In a total volume of 300 ll, the reaction mixture containing 200 ll of the cell lysate, 10 ll of Ab-bound proteinA Sepharose, 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 (Sequencing grade, Promega) was performed as previously described [16]. A MALDI-TOF MS analysis was then performed using Ultra Flex (BRUKER DALTONICS) and Mascot search (Matrix Science Inc.) according to the manufacturer’s instructions. 2.4. Indirect immunofluorecence The cells were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.1% NP-40 in PBS for 20 min, and then were incubated for 1 h with anti-M1 rabbit antiserum or anti-NP rabbit antiserum and anti-Hsc70 rat Mab. After washing with PBS, the cells were incubated for 1 h with the secondary antibodies, Alexa546- conjugated anti-rabbit Ig and Alexa488-conjugated anti-rat Ig. The cells were observed under microscopy. 2.5. Real-time PCR analysis RNA samples from virion particles were extracted by Trizol reagent (Invitrogen) and subjected to reverse transcription using random hexamer and M-MLV RT (Gibco BRL). Real-time PCR reaction was per-
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 successively washed, and then were eluted with 3 M NaCl, and subjected to 10% SDS–PAGE, followed by CBB staining. The arrowhead indicates a specific band detected only in a viral infected sample. (B) Hsc70 spectrum by MALDI-TOF MS. The viral infectionspecific band (panel A, lane 2, arrowhead) was identified as Hsc70 (P < 0.05). (C) The purified influenza viral particles were analyzed by 10% SDS–PAGE. Typical bands were excised from the CBB-stained gel and then were subjected to MALDI-TOF MS. The identified proteins (P < 0.05) were shown in the figure.
K. Watanabe et al. / FEBS Letters 580 (2006) 5785–5790 formed with Cepheid SmartCycler II (Cepheid, Sunnyvale, CA) using SYBR Green I and specific primers, 5 0 -TCTGATCCTCTCGTCATTGCAGCAA-3 0 and 5 0 -AATGACCATCGTCAACATCCACAGC-3 0 , corresponding to nucleotide position between 782 and 984 of A/WSN/33 segment 7 viral genome. The serial dilution of pPolWSN-M plasmid were used to calculate the copy numbers of amplified DNA.
3. Results and discussion 3.1. Identification of M1-binding protein To explore the host factor(s) which interact with M1, the combination of an immunoprecipitation and MALDI-TOF MS was conducted (Fig. 1). The influenza virus infected MDCK cells were lysed and immunoprecipitated with antiM1. The proteins binding to M1 were eluted in a step-wise manner with the increasing NaCl concentration and then were analyzed by SDS–PAGE. Each eluted sample contained several specific bands (data not shown). In 3 M NaCl elute fraction, a specific band (70 kDa) was obtained (Fig. 1A, arrowhead) and identified as Hsc70 (Fig. 1B, P < 0.05). Purified virion were also analyzed by MALDI-TOF MS (Fig. 1C). In addition to viral structural proteins, host proteins including Hsc70 were identified. This finding correlates with those of previous studies in which Hsc70 is incorporated into vesicular stomatitis virus, Newcastle disease virus influenza virus and Rabies virus [17].
Fig. 2. Hsc70- M1 interaction. (A) The cells were infected with the influenza virus A/PR/8/34 (MOI = 1) for 12 h. The cell lysates were subjected to 10% SDS–PAGE, followed by Western blotting as indicated. (B) HeLa cells were infected with A/PR/8/34 (MOI = 1) for 12 h. The cell lysates were immunoprecipitated with anti-M1 or control rabbit normal serum and then were visualized with anti-Hsc70 or antiM1 after 10% SDS–PAGE. (C) A/PR/8/34-infected cells were subjected to immunoprecipitation using anti-Hsc70 or rat anti-mouse IgG1 Mab as indicated. Immunoprecipitates were analyzed by 10% SDS–PAGE, and then were visualized with anti-Hsc70 or anti-M1.
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3.2. Interaction of Hsc70 with M1 in infected cells Hsc70 is a member of Hsp70 family protein, constitutively expressed in the cell (Fig. 2A). The influenza virus infection did not affect the expression level of Hsc70, whereas that of Hsp70 increased both in HeLa and MDBK cells. Next, the interaction between Hsc70 and M1 was confirmed by coimmunoprecipitation experiment using anti-M1 (Fig. 2B) or anti-Hsc70 (Fig. 2C). Hsc70 coprecipitates with M1 in HeLa cells (Fig. 2B, lane 4), MDBK and MDCK cells (data not shown). Using anti-Hsc70 antibody, M1 was co-precipitated with Hsc70 in MDBK, MDCK and HeLa cells (Fig. 2C). 3.3. C-terminal half of M1 interacts with Hsc70 Fig. 3 shows Hsc70 binding domain of M1. We mimicked the late stage of virus infection in the cells by a genome plasmid expression system with human RNA-polymerase I (polI)
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promoter [15] (Fig. 3B). The plasmid encoding M gene (pPolI-WSN-M) is transcribed into a viral genome, and then it is assembled with viral proteins, PA, PB1, PB2 and NP. M1 protein is produced from this vRNP complex. Using deletion mutants of M1, no association between M1D102–201 and Hsc70 was observed, whereas D76–103, which lacks an RNA binding domain, still bind to Hsc70 (Fig. 3B). It is also cleared that M1 interacts with Hsc70 in the absence of any other viral proteins (Fig. 3C). These results suggest that the C terminal half of M1 interacts with Hsc70. 3.4. Localization of Hsc70 in virus infected cells In uninfected cells, Hsc70 localizes dominantly in the cytoplasm at 37 C (Fig. 4a and g). In the early stages (3 h) of virus infection, when M1 was not expressed yet (e), an early protein, 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.
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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), however, the localization of Hsc70 changed drastically to the nucleus in the late stages (9 h) of viral infection where M1 was dominantly localized (Fig. 4c). Similar results were observed when HeLa cells, and A/PR/8/34(H1N1) viruses were used (data not shown). 3.5. Hsc70 requires for nuclear export of vRNP and virus production We investigated whether the expression level of Hsc70 affects the localization of the viral proteins and virus production (Fig. 5). We have introduced SiRNA specific to Hsc70 and succeeded in about 90% reduction of Hsc70 in 84 h as shown in Fig. 5A. Seventy-two hours after an SiRNA transfection, the cells were infected with the virus at a MOI of 0.1 (5000 PFU/well). The cells were extensively washed and then were incubated further with 500 ll of the medium. About 14 000 PFU/ml (without SiRNA) or 20 000 PFU/ml (with Random SiRNA) of the virus titer at 12 h post infection was significantly reduced to 2000 PFU/ml in the Hsc70-specific SiRNA
treated sample. The data obtained at 36 h post infection strengthen the results (Fig. 5B). It is also possible that a lack of Hsc70 may also affect the infectivity of the virus but not the production. We therefore examined the number of virus particles released from the cell by quantifying the RNA genome copy using a real-time PCR analysis (Fig. 5C). Our results suggested that Hsc70 affects the production of the virus but not the infectivity. M1, NP and Hsc70 localized in nucleus at 9 hr (see Fig. 4) were exported from nucleus and localize both nucleus and cytoplasm at 24 h (Fig. 5D, a, e, h and l) where the assembly of the virus occurs. When Hsc70 expression was suppressed by the SiRNA transfection (Fig. 5D, b and i), M1 (f) and NP (m) were accumulated in nucleus and thus failed to be exported from the nucleus. These results clearly suggested that Hsc70 is therefore required for the nuclear export of vRNP-M1 complex. 3.6. Roles of Hsps in the late stages of virus infection Our previous study and an X-ray structure analysis showed amino acid residues 91–105 of M1 is involved in
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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 leptomycinB (LMB), an inhibitor of CRM1-mediated nuclear export, inhibits the export of vRNP [19]. However, M1 does not have a nuclear export signal (NES). The mechanism of M1-mediated vRNP export is still unclear. It is possible to speculate the possible existence of a host factor which regulates vRNPs export. In this communication, we clearly demonstrated that Hsc70 directly bind to M1 at C terminal half of M1 (Fig. 3C). Hsc70-specific SiRNA treatment results in reduction of virus production (Fig. 5B and C) by the inhibition
of the nuclear export of M1 and vRNP (Fig. 5D). We recently reported that Hsc70 possess the NES [20], thus suggesting that Hsc70 promotes the nuclear export of M1 and vRNP. We also found M1 to be associated with Hsp90 (data not shown) and PA [3], while Hsp90 stimulates the influenza virus RdRp activity [21]. Hsc70 and Hsp90 may therefore act as a stimulating factor on various steps of the influenza virus life cycle by the association with vRNP. On the other hand, it was reported that Hsp70 induced at 41 C inhibited the export of vRNP in MDCK cells [22]. Further studies are thus
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necessary to explore the detailed mechanisms of Hsps in the export of vRNP. Acknowledgements: We thank Dr. Y. Kawaoka (University of Tokyo) for providing M1, NP, PA, PB1 and PB2 expression vectors. This work was supported in part by a Grant-in-Aid for Ministry of Education, Culture, Sports, Science and Technology, and Scientific Research from Nagasaki University, Japan (K.W.).
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