Interaction of Hsp40 with influenza virus M2 protein: implications for PKR signaling pathway

Abstract

Influenza virus contains three integral membrane proteins: haemagglutinin, neuraminidase, and matrix protein (M1 and M2). Among them, M2 protein functions as an ion channel, important for virus uncoating in endosomes of virus-infected cells and essential for virus replication. In an effort to explore potential new functions of M2 in the virus life cycle, we used yeast two-hybrid system to search for M2-associated cellular proteins. One of the positive clones was identified as human Hsp40/Hdj1, a DnaJ/Hsp40 family protein. Here, we report that both BM2 (M2 of influenza B virus) and A/M2 (M2 of influenza A virus) interacted with Hsp40 in vitro and in vivo. The region of M2-Hsp40 interaction has been mapped to the CTD1 domain of Hsp40. Hsp40 has been reported to be a regulator of PKR signaling pathway by interacting with p58^IPK that is a cellular inhibitor of PKR. PKR is a crucial component of the host defense response against virus infection. We therefore attempted to understand the relationship among M2, Hsp40 and p58^IPK by further experimentation. The results demonstrated that both A/M2 and BM2 are able to bind to p58^IPKin vitro and in vivo and enhance PKR autophosphorylation probably via forming a stable complex with Hsp40 and P58^IPK, and consequently induce cell death. These results suggest that influenza virus M2 protein is involved in p58^IPK-mediated PKR regulation during influenza virus infection, therefore affecting infected-cell life cycle and virus replication.

Keywords

M2 protein of influenza virus / Hsp40 / P58^IPK / protein interaction / PKR signal pathway

References

[1] Bergmann, M., Garcia-Sastre, A., Carnero, E., Pehamberger, H., Wolff, K., Palese, P., and Muster, T. (2000). Influenza virus NS1 protein counteracts PKR-mediated inhibition of replication. J Virol 74, 6203-6206 .10.1128/JVI.74.13.6203-6206.2000
[2] Briedis, D.J., Lamb, R.A., and Choppin, P.W. (1982). Sequence of RNA segment 7 of the influenza B virus genome: partial amino acid homology between the membrane proteins (M1) of influenza A and B viruses and conservation of a second open reading frame. Virology 116, 581-588 .10.1016/0042-6822(82)90150-7
[3] Cheetham, M.E., and Caplan, A.J. (1998). Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function. Cell Stress Chaperones 3, 28-36 .10.1379/1466-1268(1998)003<0028:SFAEOD>2.3.CO;2
[4] Ciampor, F., Thompson, C.A., Grambas, S., and Hay, A.J. (1992). Regulation of pH by the M2 protein of influenza A viruses. Virus Res 22, 247-258 .10.1016/0168-1702(92)90056-F
[5] Fan, Z., Zhuo, Y., Tan, X., Zhou, Z., Yuan, J., Qiang, B., Yan, J., Peng, X., and Gao, G.F. (2006). SARS-CoV nucleocapsid protein binds to hUbc9, a ubiquitin conjugating enzyme of the sumoylation system. J Med Virol 78, 1365-1373 .10.1002/jmv.20707
[6] Gale, M. Jr, and Katze, M.G. (1998). Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase. Pharmacol Ther 78, 29-46 .10.1016/S0163-7258(97)00165-4
[7] Gale, M. Jr, Tan, S.L., Wambach, M., and Katze, M.G. (1996). Interaction of the interferon-induced PKR protein kinase with inhibitory proteins P58IPK and vaccinia virus K3L is mediated by unique domains: implications for kinase regulation. Mol Cell Biol 16, 4172-4181 .
[8] Goodman, A.G., Fornek, J.L., Medigeshi, G.R., Perrone, L.A., Peng, X., Dyer, M.D., Proll, S.C., Knoblaugh, S.E., Carter, V.S., Korth, M.J., . (2009). P58(IPK): a novel “CIHD” member of the host innate defense response against pathogenic virus infection. PLoS Pathog 5, e1000438.
[9] Hatta, M., Goto, H., and Kawaoka, Y. (2004). Influenza B virus requires BM2 protein for replication. J Virol 78, 5576-5583 .10.1128/JVI.78.11.5576-5583.2004
[10] Horvath, C.M., Williams, M.A., and Lamb, R.A. (1990). Eukaryotic coupled translation of tandem cistrons: identification of the influenza B virus BM2 polypeptide. EMBO J 9, 2639-2647 .
[11] Ichinohe, T., Pang, I.K., and Iwasaki, A. (2010). Influenza virus activates inflammasomes via its intracellular M2 ion channel. Nat Immunol 11, 404-410 .10.1038/ni.1861
[12] Ilyinskii, P.O., Gabai, V.L., Sunyaev, S.R., Thoidis, G., and Shneider, A.M. (2007). Toxicity of influenza A virus matrix protein 2 for mammalian cells is associated with its intrinsic proton-channeling activity. Cell Cycle 6, 2043-2047 .10.4161/cc.6.16.4564
[13] Ilyinskii, P.O., Gambaryan, A.S., Meriin, A.B., Gabai, V., Kartashov, A., Thoidis, G., Shneider, A.M., and Blagosklonny, M. (2008). Inhibition of influenza M2-induced cell death alleviates its negative contribution to vaccination efficiency. PLoS ONE 3, e1417.10.1371/journal.pone.0001417
[14] Katze, M.G., Tomita, J., Black, T., Krug, R.M., Safer, B., and Hovanessian, A. (1988). Influenza virus regulates protein synthesis during infection by repressing autophosphorylation and activity of the cellular 68,000-Mr protein kinase. J Virol 62, 3710-3717 .
[15] Lamb, R.A., Lai, C.J., and Choppin, P.W. (1981). Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: colinear and interrupted mRNAs code for overlapping proteins. Proc Natl Acad Sci U S A 78, 4170-4174 .10.1073/pnas.78.7.4170
[16] Lee, S.B., Green, S.R., Mathews, M.B., and Esteban, M. (1994). Activation of the double-stranded RNA (dsRNA)-activated human protein kinase in vivo in the absence of its dsRNA binding domain. Proc Natl Acad Sci U S A 91, 10551-10555 .10.1073/pnas.91.22.10551
[17] Lee, T.G., and Katze, M.G. (1994). Cellular inhibitors of the interferon-induced, dsRNA-activated protein kinase. Prog Mol Subcell Biol 14, 48-65 .
[18] Liu, D., Liu, X., Yan, J., Liu, W.J., and Gao, G.F. (2009). Interspecies transmission and host restriction of avian H5N1 influenza virus. Sci China C Life Sci 52, 428-438 .10.1007/s11427-009-0062-z
[19] Lu, Y., Wambach, M., Katze, M.G., and Krug, R.M. (1995). Binding of the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the elF-2 translation initiation factor. Virology 214, 222-228 .10.1006/viro.1995.9937
[20] McCown, M.F., and Pekosz, A. (2006). Distinct domains of the influenza a virus M2 protein cytoplasmic tail mediate binding to the M1 protein and facilitate infectious virus production. J Virol 80, 8178-8189 .10.1128/JVI.00627-06
[21] Melville, M.W., Hansen, W.J., Freeman, B.C., Welch, W.J., and Katze, M.G. (1997). The molecular chaperone hsp40 regulates the activity of P58IPK, the cellular inhibitor of PKR. Proc Natl Acad Sci U S A 94, 97-102 .10.1073/pnas.94.1.97
[22] Meurs, E.F., Watanabe, Y., Kadereit, S., Barber, G.N., Katze, M.G., Chong, K., Williams, B.R., and Hovanessian, A.G. (1992). Constitutive expression of human double-stranded RNA-activated p68 kinase in murine cells mediates phosphorylation of eukaryotic initiation factor 2 and partial resistance to encephalomyocarditis virus growth. J Virol 66, 5805-5814 .
[23] Mi, S., Li, Y., Yan J., and GAO, G.F. (2010). Na⁺/K⁺-ATPase β1 subunit interacts with M2 proteins of influenza A and B viruses and affects the virus replication. Sci China C Life Sci 53, 1-8 .
[24] Mohler, P.J., Hoffman, J.A., Davis, J.Q., Abdi, K.M., Kim, C.R., Jones, S.K., Davis, L.H., Roberts, K.F., and Bennett, V. (2004). Isoform specificity among ankyrins. An amphipathic alpha-helix in the divergent regulatory domain of ankyrin-b interacts with the molecular co-chaperone Hdj1/Hsp40. J Biol Chem 279, 25798-25804 .10.1074/jbc.M401296200
[25] Morris, S.J., Price, G.E., Barnett, J.M., Hiscox, S.A., Smith, H., and Sweet, C. (1999). Role of neuraminidase in influenza virus-induced apoptosis. J Gen Virol 80, 137-146 .
[26] Morris, S.J., Smith, H., and Sweet, C. (2002). Exploitation of the Herpes simplex virus translocating protein VP22 to carry influenza virus proteins into cells for studies of apoptosis: direct confirmation that neuraminidase induces apoptosis and indications that other proteins may have a role. Arch Virol 147, 961-979 .10.1007/s00705-001-0779-x
[27] Mould, J.A., Paterson, R.G., Takeda, M., Ohigashi, Y., Venkataraman, P., Lamb, R.A., and Pinto, L.H. (2003). Influenza B virus BM2 protein has ion channel activity that conducts protons across membranes. Dev Cell 5, 175-184 .10.1016/S1534-5807(03)00190-4
[28] Odagiri, T., Hong, J., and Ohara, Y. (1999). The BM2 protein of influenza B virus is synthesized in the late phase of infection and incorporated into virions as a subviral component. J Gen Virol 80, 2573-2581 .
[29] Ohtsuka, K., and Hata, M. (2000). Molecular chaperone function of mammalian Hsp70 and Hsp40—a review. Int J Hyperthermia 16, 231-245 .10.1080/026567300285259
[30] Olsen, C.W., Kehren, J.C., Dybdahl-Sissoko, N.R., and Hinshaw, V.S. (1996). bcl-2 alters influenza virus yield, spread, and hemagglutinin glycosylation. J Virol 70, 663-666 .
[31] Pinto, L.H., Holsinger, L.J., and Lamb, R.A. (1992). Influenza virus M2 protein has ion channel activity. Cell 69, 517-528 .
[32] Pinto, L.H., and Lamb, R.A. (2006). The M2 proton channels of influenza A and B viruses. J Biol Chem 281, 8997-9000 .10.1074/jbc.R500020200
[33] Schultz-Cherry, S., Dybdahl-Sissoko, N., Neumann, G., Kawaoka, Y., and Hinshaw, V.S. (2001). Influenza virus ns1 protein induces apoptosis in cultured cells. J Virol 75, 7875-7881 .10.1128/JVI.75.17.7875-7881.2001
[34] Schultz-Cherry, S., and Hinshaw, V.S. (1996). Influenza virus neuraminidase activates latent transforming growth factor beta. J Virol 70, 8624-8629 .
[35] Shimbo, K., Brassard, D.L., Lamb, R.A., and Pinto, L.H. (1996). Ion selectivity and activation of the M2 ion channel of influenza virus. Biophys J 70, 1335-1346 .10.1016/S0006-3495(96)79690-X
[36] Sohn, S.Y., Kim, J.H., Baek, K.W., Ryu, W.S., and Ahn, B.Y. (2006). Turnover of hepatitis B virus X protein is facilitated by Hdj1, a human Hsp40/DnaJ protein. Biochem Biophys Res Commun 347, 764-768 .10.1016/j.bbrc.2006.06.158
[37] Srivastava, S.P., Kumar, K.U., and Kaufman, R.J. (1998). Phosphorylation of eukaryotic translation initiation factor 2 mediates apoptosis in response to activation of the double-stranded RNA-dependent protein kinase. J Biol Chem 273, 2416-2423 .10.1074/jbc.273.4.2416
[38] Sugrue, R.J., and Hay, A.J. (1991). Structural characteristics of the M2 protein of influenza A viruses: evidence that it forms a tetrameric channel. Virology 180, 617-624 .10.1016/0042-6822(91)90075-M
[39] Takeda, M., Pekosz, A., Shuck, K., Pinto, L.H., and Lamb, R.A. (2002). Influenza a virus M2 ion channel activity is essential for efficient replication in tissue culture. J Virol 76, 1391-1399 .
[40] Tan, S.L., Gale, M.J. Jr, and Katze, M.G. (1998). Double-stranded RNA-independent dimerization of interferon-induced protein kinase PKR and inhibition of dimerization by the cellular P58IPK inhibitor. Mol Cell Biol 18, 2431-2443 .
[41] Wurzer, W.J., Planz, O., Ehrhardt, C., Giner, M., Silberzahn, T., Pleschka, S., and Ludwig, S. (2003). Caspase 3 activation is essential for efficient influenza virus propagation. EMBO J 22, 2717-2728 .10.1093/emboj/cdg279
[42] Yan, J., Zhu, J., Zhong, H., Lu, Q., Huang, C., and Ye, Q. (2003). BRCA1 interacts with FHL2 and enhances FHL2 transactivation function. FEBS Lett 553, 183-189 .10.1016/S0014-5793(03)00978-5
[43] Zamarin, D., García-Sastre, A., Xiao, X., Wang, R., and Palese, P. (2005). Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and VDAC1. PLoS Pathog 1, e4.10.1371/journal.ppat.0010004