Protein interactions in the murine cytomegalovirus capsid revealed by cryoEM

Abstract

Cytomegalovirus (CMV) is distinct among members of the Herpesviridae family for having the largest dsDNA genome (230 kb). Packaging of large dsDNA genome is known to give rise to a highly pressurized viral capsid, but molecular interactions conducive to the formation of CMV capsid resistant to pressurization have not been described. Here, we report a cryo electron microscopy (cryoEM) structure of the murine cytomegalovirus (MCMV) capsid at a 9.1 ? resolution and describe the molecular interactions among the ~3000 protein molecules in the CMV capsid at the secondary structure level. Secondary structural elements are resolved to provide landmarks for correlating with results from sequence-based prediction and for structure-based homology modeling. The major capsid protein (MCP) upper domain (MCPud) contains α-helices and β-sheets conserved with those in MCPud of herpes simplex virus type 1 (HSV-1), with the largest differences identified as a “saddle loop” region, located at the tip of MCPud and involved in interaction with the smallest capsid protein (SCP). Interactions among the bacteriophage HK97-like floor domain of MCP, the middle domain of MCP, the hook and clamp domains of the triplex proteins (hoop and clamp domains of TRI-1 and clamp domain of TRI-2) contribute to the formation of a mature capsid. These results offer a framework for understanding how cytomegalovirus uses various secondary structural elements of its capsid proteins to build a robust capsid for packaging its large dsDNA genome inside and for attaching unique functional tegument proteins outside.

Keywords

cytomegalovirus / herpes simplex virus type 1 / cryo electron microscopy / three-dimensional / major capsid protein

References

[1] Baker, M.L., Jiang, W., Rixon, F.J., and Chiu, W. (2005). Common ancestry of herpesviruses and tailed DNA bacteriophages. J Virol 79, 14967-14970 .10.1128/JVI.79.23.14967-14970.2005
[2] Baldick, C.J., Jr., and Shenk, T. (1996). Proteins associated with purifi ed human cytomegalovirus particles. J Virol 70, 6097-6105 .
[3] Booy, F.P., Newcomb, W.W., Trus, B.L., Brown, J.C., Baker, T.S., and Steven, A.C. (1991). Liquid-crystalline, phage-like packing of encapsidated DNA in herpes simplex virus. Cell 64, 1007-1015 .10.1016/0092-8674(91)90324-R
[4] Borst, E.M., Mathys, S., Wagner, M., Muranyi, W., and Messerle, M. (2001). Genetic evidence of an essential role for cytomegalovirus small capsid protein in viral growth. J Virol 75, 1450-1458 .10.1128/JVI.75.3.1450-1458.2001
[5] Bowman, B.R., Baker, M.L., Rixon, F.J., Chiu, W., and Quiocho, F.A. (2003). Structure of the herpesvirus major capsid protein. EMBO J 22, 757-765 .10.1093/emboj/cdg086
[6] Britt, W.J., and Boppana, S. (2004). Human cytomegalovirus virion proteins. Hum Immunol 65, 395-402 .10.1016/j.humimm.2004.02.008
[7] Buchan, D.W., Ward, S.M., Lobley, A.E., Nugent, T.C., Bryson, K., and Jones, D.T. (2010). Protein annotation and modelling servers at University College London. Nucleic Acids Res 38, W563-568 .10.1093/nar/gkq427
[8] Butcher, S.J., Aitken, J., Mitchell, J., Gowen, B., and Dargan, D.J. (1998). Structure of the human cytomegalovirus B capsid by electron cryomicroscopy and image reconstruction. J Struct Biol 124, 70-76 .10.1006/jsbi.1998.4055
[9] Chee, M., Rudolph, S.A., Plachter, B., Barrel, B., and Jahn, G. (1989). Identification of the major capsid protein gene of human cytomegalovirus. J Virol 63, 1345-1353 .
[10] Chen, D.H., Jiang, H., Lee, M., Liu, F., and Zhou, Z.H. (1999). Threedimensional visualization of tegument/capsid interactions in the intact human cytomegalovirus. Virology 260 , 10-16 .10.1006/viro.1999.9791
[11] Cole, C., Barber, J.D., and Barton, G.J. (2008). The Jpred 3 secondary structure prediction server. Nucleic Acids Res 36, W197-201 .10.1093/nar/gkn238
[12] Conway, J.F., Cockrell, S.K., Copeland, A.M., Newcomb, W.W., Brown, J.C., and Homa, F.L. (2010). Labeling and localization of the herpes simplex virus capsid protein UL25 and its interaction with the two triplexes closest to the penton. J Mol Biol 397, 575-586 .10.1016/j.jmb.2010.01.043
[13] Deng, B., O’Connor, C.M., Kedes, D.H., and Zhou, Z.H. (2008). Cryoelectron tomography of Kaposi’s sarcoma-associated herpesvirus capsids reveals dynamic scaffolding structures essential to capsid assembly and maturation. J Struct Biol 161, 419-427 .10.1016/j.jsb.2007.10.016
[14] Emsley, P., Lohkamp, B., Scott, W.G., and Cowtan, K. (2010). Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66, 486-501 .10.1107/S0907444910007493
[15] Finn, R.D., Mistry, J., Tate, J., Coggill, P., Heger, A., Pollington, J.E., Gavin, O.L., Gunasekaran, P., Ceric, G., Forslund, K., . (2010). The Pfam protein families database. Nucleic Acids Res 38, D211-222 .10.1093/nar/gkp985
[16] Gibson, W., Baxter, M.K., and Clopper, K.S. (1996a). Cytomegalovirus “missing” capsid protein identified as heat-aggregable product of human cytomegalovirus UL46. J Virol 70, 7454-7461 .
[17] Gibson, W., Clopper, K.S., Britt, W.J., and Baxter, M.K. (1996b). Human cytomegalovirus (HCMV) smallest capsid protein identified as product of short open reading frame located between HCMV UL48 and UL49. J Virol 70, 5680-5683 .
[18] Guex, N., and Peitsch, M.C. (1997). SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714-2723 .10.1002/elps.1150181505
[19] Heymann, J.B., Cheng, N., Newcomb, W.W., Trus, B.L., Brown, J.C., and Steven, A.C. (2003). Dynamics of herpes simplex virus capsid maturation visualized by time-lapse cryo-electron microscopy. Nat Struct Biol 10, 334-341 .10.1038/nsb922
[20] Homa, F.L., Huffman, J.B., Toropova, K., Lopez, H.R., Makhov, A.M., and Conway, J.F. (2013). Structure of the pseudorabies virus capsid: comparison with herpes simplex virus type 1 and differential binding of essential minor proteins. J Mol Biol . (In Press).10.1016/j.jmb.2013.06.034
[21] Huang, E., Perkins, E.M., and Desai, P. (2007). Structural features of the scaffold interaction domain at the N terminus of the major capsid protein (VP5) of herpes simplex virus type 1. J Virol 81, 9396-9407 .10.1128/JVI.00986-07
[22] Kelley, L.A., and Sternberg, M.J. (2009). Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4, 363-371 .10.1038/nprot.2009.2
[23] Liang, Y., Ke, E.Y., and Zhou, Z.H. (2002). IMIRS: a high-resolution 3D reconstruction package integrated with a relational image database. J Struct Biol 137, 292-304 .10.1016/S1047-8477(02)00014-X
[24] Liu, F., and Zhou, Z.H. (2007). Comparative virion structures of human herpesviruses. In Human Herpesviruses: Biology, Therapy and Immunoprophylaxis . Arvin, A., Campadelli-Fiume, G., Moore, P., Mocarski, E., Roizman, B., Whitley, R., and Yamanishi, K., eds. (Cambridge, UK, Cambridge University Press), pp . 27-43 .
[25] Liu, H., Cheng, L., Zeng, S., Cai, C., Zhou, Z.H., and Yang, Q. (2008). Symmetry-adapted spherical harmonics method for high-resolution 3D single-particle reconstructions. J Struct Biol 161 , 64-73 .10.1016/j.jsb.2007.09.016
[26] Ludtke, S.J., Baldwin, P.R., and Chiu, W. (1999). EMAN: Semi-automated software for high resolution single particle reconstructions. J Struct Biol 128 , 82-97 .10.1006/jsbi.1999.4174
[27] Mettenleiter, T.C. (2002). Herpesvirus assembly and egress. J Virol 76, 1537-1547 .10.1128/JVI.76.4.1537-1547.2002
[28] Mindell, J.A., and Grigorieff, N. (2003). Accurate determination of local defocus and specimen tilt in electron microscopy. J Struct Biol 142, 334-347 .10.1016/S1047-8477(03)00069-8
[29] Mocarski, E.S. (2005). Comparative analysis of herpesvirus-common proteins. In Human Herpesviruses: Biology, Therapy and Immunoprophylaxis . Arvin, A., Campadelli-Fiume, G., Moore, P., Mocarski, E., Roizman, B., Whitley, R., and Yamanishi, K., eds. Cambridge, UK, Cambridge University Press.
[30] Okoye, M.E., Sexton, G.L., Huang, E., McCaffery, J.M., and Desai, P. (2006). Functional analysis of the triplex proteins (VP19C and VP23) of herpes simplex virus type 1. J Virol 80, 929-940 .10.1128/JVI.80.2.929-940.2006
[31] Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., and Ferrin, T.E. (2004). UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25, 1605-1612 .10.1002/jcc.20084
[32] Toropova, K., Huffman, J.B., Homa, F.L., and Conway, J.F. (2011). The herpes simplex virus 1 UL17 protein is the second constituent of the capsid vertex-specific component required for DNA packaging and retention. J Virol 85, 7513-7522 .10.1128/JVI.00837-11
[33] Trus, B.L., Booy, F.P., Newcomb, W.W., Brown, J.C., and Steven, A.C. (1993). Location of VP26 in the herpes simplex virus capsid. Biophys J 64, A64.
[34] Trus, B.L., Gibson, W., Cheng, N., and Steven, A.C. (1999). Capsid structure of simian cytomegalovirus from cryoelectron microscopy: evidence for tegument attachment sites. J Virol 73, 2181-2192 .
[35] Trus, B.L., Heymann, J.B., Nealon, K., Cheng, N., Newcomb, W.W., Brown, J.C., Kedes, D.H., and Steven, A.C. (2001). Capsid structure of Kaposi’s sarcoma-associated herpesvirus, a gammaherpesvirus, compared to those of an alphaherpesvirus, herpes simplex virus type 1, and a betaherpesvirus, cytomegalovirus. J Virol 75, 2879-2890 .10.1128/JVI.75.6.2879-2890.2001
[36] Trus, B.L., Newcomb, W.W., Booy, F.P., Brown, J.C., and Steven, A.C. (1992). Distinct monoclonal antibodies separately label the hexons or the pentons of herpes simples virus capsid. Proc Natl Acad Sci USA 89, 11508-11512 .10.1073/pnas.89.23.11508
[37] Trus, B.L., Newcomb, W.W., Cheng, N., Cardone, G., Marekov, L., Homa, F.L., Brown, J.C., and Steven, A.C. (2007). Allosteric signaling and a nuclear exit strategy: binding of UL25/UL17 heterodimers to DNA-Filled HSV-1 capsids. Mol Cell 26, 479-489 .10.1016/j.molcel.2007.04.010
[38] Wikoff, W.R., Liljas, L., Duda, R.L., Tsuruta, H., Hendrix, R.W., and Johnson, J.E. (2000). Topologically linked protein rings in the bacteriophage HK97 capsid. Science 289, 2129-2133 .10.1126/science.289.5487.2129
[39] Wu, L., Lo, P., Yu, X., Stoops, J.K., Forghani, B., and Zhou, Z.H. (2000). Three-dimensional structure of the human herpesvirus 8 capsid. J Virol 74, 9646-9654 .10.1128/JVI.74.20.9646-9654.2000
[40] Yu, X., Shah, S., Atanasov, I., Lo, P., Liu, F., Britt, W.J., and Zhou, Z.H. (2005a). Three-dimensional localization of the smallest capsid protein in the human cytomegalovirus capsid. J Virol 79, 1327-1332 .10.1128/JVI.79.2.1327-1332.2005
[41] Yu, X., Trang, P., Shah, S., Atanasov, I., Kim, Y.H., Bai, Y., Zhou, Z.H., and Liu, F. (2005b). Dissecting human cytomegalovirus gene function and capsid maturation by ribozyme targeting and electron cryomicroscopy. Proc Natl Acad Sci U S A 102, 7103-7108 .10.1073/pnas.0408826102
[42] Yu, X.K., O’Connor, C.M., Atanasov, I., Damania, B., Kedes, D.H., and Zhou, Z.H. (2003). Three-dimensional structures of the A, B, and C capsids of rhesus monkey rhadinovirus: insights into gammaherpesvirus capsid assembly, maturation, and DNA packaging. J Virol 77, 13182-13193 .10.1128/JVI.77.24.13182-13193.2003
[43] Zhou, Z.H., Chen, D.H., Jakana, J., Rixon, F.J., and Chiu, W. (1999). Visualization of tegument-capsid interactions and DNA in intact herpes simplex virus type 1 virions. J Virol 73, 3210-3218 .
[44] Zhou, Z.H., Dougherty, M., Jakana, J., He, J., Rixon, F.J., and Chiu, W. (2000). Seeing the herpesvirus capsid at 8.5 ?. Science 288, 877-880 .10.1126/science.288.5467.877
[45] Zhou, Z.H., He, J., Jakana, J., Tatman, J.D., Rixon, F.J., and Chiu, W. (1995). Assembly of VP26 in herpes simplex virus-1 inferred from structures of wild-type and recombinant capsids. Nat Struct Biol 2, 1026-1030 .10.1038/nsb1195-1026
[46] Zhou, Z.H., Prasad, B.V., Jakana, J., Rixon, F.J., and Chiu, W. (1994). Protein subunit structures in the herpes simplex virus A-capsid determined from 400 kV spot-scan electron cryomicroscopy. J Mol Biol 242, 456-469 .10.1006/jmbi.1994.1594