Sequence analysis reveals a conserved extension in the capping enzyme of the alphavirus supergroup, and a homologous domain in nodaviruses

Biology Direct

Tero Ahola¹  David G Karlin² 

[1] Department of Food and Environmental Sciences, University of Helsinki, Helsinki, 00014, Finland

[2] The Division of Structural Biology, Henry Wellcome Building, Roosevelt Drive, Oxford OX3 7BN, UK

关键词: Hepatitis E virus;    Sindbis virus;    Chikungunya virus;    Viral replication factory;    Amphipathic alpha-helix;    Protein sequence analysis;    Homology detection;    Nodavirus;    Bromovirus;    Alphavirus;    Capping;    Guanylyltransferase;    Methyltransferase;   

Others  :  1180725
DOI  :  10.1186/s13062-015-0050-0

 received in 2014-12-29, accepted in 2015-03-24,  发布年份 2015

【 摘 要 】

Background

Members of the alphavirus supergroup include human pathogens such as chikungunya virus, hepatitis E virus and rubella virus. They encode a capping enzyme with methyltransferase-guanylyltransferase (MTase-GTase) activity, which is an attractive drug target owing to its unique mechanism. However, its experimental study has proven very difficult.

Results

We examined over 50 genera of viruses by sequence analyses. Earlier studies showed that the MTase-GTase contains a “Core” region conserved in sequence. We show that it is followed by a long extension, which we termed “Iceberg” region, whose secondary structure, but not sequence, is strikingly conserved throughout the alphavirus supergroup. Sequence analyses strongly suggest that the minimal capping domain corresponds to the Core and Iceberg regions combined, which is supported by earlier experimental data. The Iceberg region contains all known membrane association sites that contribute to the assembly of viral replication factories. We predict that it may also contain an overlooked, widely conserved membrane-binding amphipathic helix. Unexpectedly, we detected a sequence homolog of the alphavirus MTase-GTase in taxa related to nodaviruses and to chronic bee paralysis virus. The presence of a capping enzyme in nodaviruses is biologically consistent, since they have capped genomes but replicate in the cytoplasm, where no cellular capping enzyme is present. The putative MTase-GTase domain of nodaviruses also contains membrane-binding sites that may drive the assembly of viral replication factories, revealing an unsuspected parallel with the alphavirus supergroup.

Conclusions

Our work will guide the functional analysis of the alphaviral MTase-GTase and the production of domains for structure determination. The identification of a homologous domain in a simple model system, nodaviruses, which replicate in numerous eukaryotic cell systems (yeast, flies, worms, mammals, and plants), can further help crack the function and structure of the enzyme.

Reviewers

This article was reviewed by Valerian Dolja, Eugene Koonin and Sebastian Maurer-Stroh.

【 授权许可】

   
2015 Ahola and Karlin; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

【 参考文献 】

• [1]Goldbach R: Genome similarities between plant and animal RNA viruses. Microbiol Sci 1987, 4(7):197-202.
• [2]Koonin EV, Dolja VV: Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Crit Rev Biochem Mol Biol 1993, 28(5):375-430. doi:10.3109/10409239309078440
• [3]Salonen A, Ahola T, Kaariainen L: Viral RNA replication in association with cellular membranes. Curr Top Microbiol Immunol 2005, 285:139-73.
• [4]Firth AE, Brierley I: Non-canonical translation in RNA viruses. J Gen Virol 2012, 93(Pt 7):1385-409. doi:10.1099/vir. 0.042499-0
• [5]Decroly E, Ferron F, Lescar J, Canard B: Conventional and unconventional mechanisms for capping viral mRNA. Nat Rev Microbiol 2012, 10(1):51-65. doi:10.1038/nrmicro2675
• [6]Ahola T, Kaariainen L: Reaction in alphavirus mRNA capping: formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP. Proc Natl Acad Sci U S A 1995, 92(2):507-11.
• [7]Rozanov MN, Koonin EV, Gorbalenya AE: Conservation of the putative methyltransferase domain: a hallmark of the ‘Sindbis-like’ supergroup of positive-strand RNA viruses. J Gen Virol 1992, 73(Pt 8):2129-34.
• [8]Laakkonen P, Hyvonen M, Peranen J, Kaariainen L: Expression of Semliki Forest virus nsP1-specific methyltransferase in insect cells and in Escherichia coli. J Virol 1994, 68(11):7418-25.
• [9]Mi S, Durbin R, Huang HV, Rice CM, Stollar V: Association of the Sindbis virus RNA methyltransferase activity with the nonstructural protein nsP1. Virology 1989, 170(2):385-91.
• [10]Mi S, Stollar V: Expression of Sindbis virus nsP1 and methyltransferase activity in Escherichia coli. Virology 1991, 184(1):423-7.
• [11]Ahola T, Laakkonen P, Vihinen H, Kaariainen L: Critical residues of Semliki Forest virus RNA capping enzyme involved in methyltransferase and guanylyltransferase-like activities. J Virol 1997, 71(1):392-7.
• [12]Ferron F, Decroly E, Selisko B, Canard B: The viral RNA capping machinery as a target for antiviral drugs. Antiviral Res 2012, 96(1):21-31. doi:10.1016/j.antiviral.2012.07.007
• [13]Magden J, Kaariainen L, Ahola T: Inhibitors of virus replication: recent developments and prospects. Appl Microbiol Biotechnol 2005, 66(6):612-21. doi:10.1007/s00253-004-1783-3
• [14]Huang YL, Hsu YH, Han YT, Meng M: mRNA guanylation catalyzed by the S-adenosylmethionine-dependent guanylyltransferase of bamboo mosaic virus. J Biol Chem 2005, 280(13):13153-62. doi:10.1074/jbc.M412619200
• [15]Magden J, Takeda N, Li T, Auvinen P, Ahola T, Miyamura T, et al.: Virus-specific mRNA capping enzyme encoded by hepatitis E virus. J Virol 2001, 75(14):6249-55. doi:10.1128/JVI. 75.14.6249-6255.2001
• [16]Balistreri G, Caldentey J, Kaariainen L, Ahola T: Enzymatic defects of the nsP2 proteins of Semliki Forest virus temperature-sensitive mutants. J Virol 2007, 81(6):2849-60. doi:10.1128/JVI. 02078-06
• [17]Vasiljeva L, Merits A, Auvinen P, Kaariainen L: Identification of a novel function of the alphavirus capping apparatus. RNA 5′-triphosphatase activity of Nsp2. J Biol Chem 2000, 275(23):17281-7. doi:10.1074/jbc.M910340199
• [18]Li YI, Shih TW, Hsu YH, Han YT, Huang YL, Meng M: The helicase-like domain of plant potexvirus replicase participates in formation of RNA 5′ cap structure by exhibiting RNA 5′-triphosphatase activity. J Virol 2001, 75(24):12114-20. doi:10.1128/JVI. 75.24.12114-12120.2001
• [19]Das PK, Merits A, Lulla A. Functional crosstalk between distant domains of chikungunya virus non-structural protein 2 is decisive for its RNA-modulating activity. J Biol Chem. 2014. doi:10.1074/jbc.M113.503433.
• [20]Huang YL, Han YT, Chang YT, Hsu YH, Meng M: Critical residues for GTP methylation and formation of the covalent m7GMP-enzyme intermediate in the capping enzyme domain of bamboo mosaic virus. J Virol 2004, 78(3):1271-80.
• [21]Wang HL, O'Rear J, Stollar V: Mutagenesis of the Sindbis virus nsP1 protein: effects on methyltransferase activity and viral infectivity. Virology 1996, 217(2):527-31. doi:10.1006/viro.1996.0147
• [22]Laakkonen P, Ahola T, Kaariainen L: The effects of palmitoylation on membrane association of Semliki forest virus RNA capping enzyme. J Biol Chem 1996, 271(45):28567-71.
• [23]Koonin EV, Gorbalenya AE, Purdy MA, Rozanov MN, Reyes GR, Bradley DW: Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive-strand RNA plant and animal viruses. Proc Natl Acad Sci U S A 1992, 89(17):8259-63.
• [24]Miller DJ, Schwartz MD, Ahlquist P: Flock house virus RNA replicates on outer mitochondrial membranes in Drosophila cells. J Virol 2001, 75(23):11664-76. doi:10.1128/JVI.75.23.11664-11676.2001
• [25]Venter PA, Schneemann A: Recent insights into the biology and biomedical applications of Flock House virus. Cell Mol Life Sci 2008, 65(17):2675-87. doi:10.1007/s00018-008-8037-y
• [26]Dasgupta R, Ghosh A, Dasmahapatra B, Guarino LA, Kaesberg P: Primary and secondary structure of black beetle virus RNA2, the genomic messenger for BBV coat protein precursor. Nucleic Acids Res 1984, 12(18):7215-23.
• [27]Dasmahapatra B, Dasgupta R, Ghosh A, Kaesberg P: Structure of the black beetle virus genome and its functional implications. J Mol Biol 1985, 182(2):183-9.
• [28]Ball LA, Johnson KL: Reverse genetics of nodaviruses. Adv Virus Res 1999, 53:229-44.
• [29]Koonin EV, Wolf YI, Nagasaki K, Dolja VV: The Big Bang of picorna-like virus evolution antedates the radiation of eukaryotic supergroups. Nat Rev Microbiol 2008, 6(12):925-39. doi:10.1038/nrmicro2030
• [30]Johnson KN, Johnson KL, Dasgupta R, Gratsch T, Ball LA: Comparisons among the larger genome segments of six nodaviruses and their encoded RNA replicases. J Gen Virol 2001, 82(Pt 8):1855-66.
• [31]Wang Z, Qiu Y, Liu Y, Qi N, Si J, Xia X, et al.: Characterization of a nodavirus replicase revealed a de novo initiation mechanism of RNA synthesis and terminal nucleotidyltransferase activity. J Biol Chem 2013, 288(43):30785-801. doi:10.1074/jbc.M113.492728
• [32]Dunbrack RL Jr: Sequence comparison and protein structure prediction. Curr Opin Struct Biol 2006, 16(3):374-84. doi:10.1016/j.sbi.2006.05.006
• [33]Kuchibhatla DB, Sherman WA, Chung BY, Cook S, Schneider G, Eisenhaber B, et al.: Powerful sequence similarity search methods and in-depth manual analyses can identify remote homologs in many apparently “orphan” viral proteins. J Virol 2014, 88(1):10-20. doi:10.1128/JVI. 02595-13
• [34]Soding J, Remmert M: Protein sequence comparison and fold recognition: progress and good-practice benchmarking. Curr Opin Struct Biol 2011, 21(3):404-11. doi:10.1016/j.sbi.2011.03.005
• [35]Tomar S, Narwal M, Harms E, Smith JL, Kuhn RJ: Heterologous production, purification and characterization of enzymatically active Sindbis virus nonstructural protein nsP1. Protein Expr Purif 2011, 79(2):277-84. doi:10.1016/j.pep.2011.05.022
• [36]Lin HY, Yu CY, Hsu YH, Meng M: Functional analysis of the conserved histidine residue of Bamboo mosaic virus capping enzyme in the activity for the formation of the covalent enzyme-m7GMP intermediate. FEBS Lett 2012, 586(16):2326-31. doi:10.1016/j.febslet.2012.05.024
• [37]Erokhina TN, Vitushkina MV, Zinovkin RA, Lesemann DE, Jelkmann W, Koonin EV, et al.: Ultrastructural localization and epitope mapping of the methyltransferase-like and helicase-like proteins of Beet yellows virus. J Gen Virol 2001, 82(Pt 8):1983-94.
• [38]O'Reilly EK, Wang Z, French R, Kao CC: Interactions between the structural domains of the RNA replication proteins of plant-infecting RNA viruses. J Virol 1998, 72(9):7160-9.
• [39]Pei JM, Kim BH, Tang M, Grishin NV. PROMALS web server for accurate multiple protein sequence alignments. Nucleic Acids Res. 2007;35:W649–W52. doi:10.1093/Nar/Gkm227.
• [40]Hu RH, Lin MC, Hsu YH, Meng M: Mutational effects of the consensus aromatic residues in the mRNA capping domain of Bamboo mosaic virus on GTP methylation and virus accumulation. Virology 2011, 411(1):15-24. doi:10.1016/j.virol.2010.12.022
• [41]Scheidel LM, Stollar V: Mutations that confer resistance to mycophenolic acid and ribavirin on Sindbis virus map to the nonstructural protein nsP1. Virology 1991, 181(2):490-9.
• [42]Rosenblum CI, Scheidel LM, Stollar V: Mutations in the nsP1 coding sequence of Sindbis virus which restrict viral replication in secondary cultures of chick embryo fibroblasts prepared from aged primary cultures. Virology 1994, 198(1):100-8. doi:10.1006/viro.1994.1012
• [43]Li ML, Wang HL, Stollar V: Complementation of and interference with Sindbis virus replication by full-length and deleted forms of the nonstructural protein, nsP1, expressed in stable transfectants of Hela cells. Virology 1997, 227(2):361-9. doi:10.1006/viro.1996.8342
• [44]Li YI, Chen YJ, Hsu YH, Meng M: Characterization of the AdoMet-dependent guanylyltransferase activity that is associated with the N terminus of bamboo mosaic virus replicase. J Virol 2001, 75(2):782-8. doi:10.1128/JVI. 75.2.782-788.2001
• [45]Belov GA, van Kuppeveld FJ. (+)RNA viruses rewire cellular pathways to build replication organelles. Curr Opin Virol. 2012;2(6):740–7. doi:10.1016/j.coviro.2012.09.006.
• [46]Liu L, Westler WM, den Boon JA, Wang X, Diaz A, Steinberg HA, et al.: An amphipathic alpha-helix controls multiple roles of brome mosaic virus protein 1a in RNA replication complex assembly and function. PLoS Pathog 2009, 5(3):e1000351. doi:10.1371/journal.ppat.1000351
• [47]Spuul P, Salonen A, Merits A, Jokitalo E, Kaariainen L, Ahola T: Role of the amphipathic peptide of Semliki forest virus replicase protein nsP1 in membrane association and virus replication. J Virol 2007, 81(2):872-83. doi:10.1128/JVI. 01785-06
• [48]Ahola T, Lampio A, Auvinen P, Kaariainen L: Semliki Forest virus mRNA capping enzyme requires association with anionic membrane phospholipids for activity. EMBO J 1999, 18(11):3164-72. doi:10.1093/emboj/18.11.3164
• [49]Lampio A, Kilpelainen I, Pesonen S, Karhi K, Auvinen P, Somerharju P, et al.: Membrane binding mechanism of an RNA virus-capping enzyme. J Biol Chem 2000, 275(48):37853-9. doi:10.1074/jbc.M004865200
• [50]Cornell RB, Taneva SG: Amphipathic helices as mediators of the membrane interaction of amphitropic proteins, and as modulators of bilayer physical properties. Curr Protein Pept Sci 2006, 7(6):539-52.
• [51]Drin G, Antonny B: Amphipathic helices and membrane curvature. FEBS Lett 2010, 584(9):1840-7. doi:10.1016/j.febslet.2009.10.022
• [52]Ahola T, Kujala P, Tuittila M, Blom T, Laakkonen P, Hinkkanen A, et al.: Effects of palmitoylation of replicase protein nsP1 on alphavirus infection. J Virol 2000, 74(15):6725-33.
• [53]den Boon JA, Chen J, Ahlquist P: Identification of sequences in Brome mosaic virus replicase protein 1a that mediate association with endoplasmic reticulum membranes. J Virol 2001, 75(24):12370-81. doi:10.1128/JVI. 75.24.12370-12381.2001
• [54]Sapay N, Guermeur Y, Deleage G: Prediction of amphipathic in-plane membrane anchors in monotopic proteins using a SVM classifier. BMC Bioinformatics 2006, 7:255. doi:10.1186/1471-2105-7-255 BioMed Central Full Text
• [55]Gautier R, Douguet D, Antonny B, Drin G: HELIQUEST: a web server to screen sequences with specific alpha-helical properties. Bioinformatics 2008, 24(18):2101-2. doi:10.1093/bioinformatics/btn392
• [56]Salanki K, Gellert A, Naray-Szabo G, Balazs E: Modeling-based characterization of the elicitor function of amino acid 461 of Cucumber mosaic virus 1a protein in the hypersensitive response. Virology 2007, 358(1):109-18. doi:10.1016/j.virol.2006.08.014
• [57]Yokoi T, Yamashita S, Hibi T: The nucleotide sequence and genome organization of Sclerophthora macrospora virus A. Virology 2003, 311(2):394-9.
• [58]Heller-Dohmen M, Gopfert JC, Pfannstiel J, Spring O: The nucleotide sequence and genome organization of Plasmopara halstedii virus. Virol J 2011, 8:123. doi:10.1186/1743-422X-8-123 BioMed Central Full Text
• [59]Olivier V, Blanchard P, Chaouch S, Lallemand P, Schurr F, Celle O, et al.: Molecular characterisation and phylogenetic analysis of Chronic bee paralysis virus, a honey bee virus. Virus Res 2008, 132(1–2):59-68. doi:10.1016/j.virusres.2007.10.014
• [60]Cook S, Chung BY, Bass D, Moureau G, Tang S, McAlister E, et al.: Novel virus discovery and genome reconstruction from field RNA samples reveals highly divergent viruses in dipteran hosts. PLoS One 2013, 8(11):e80720. doi:10.1371/journal.pone.0080720
• [61]Runckel C, Flenniken ML, Engel JC, Ruby JG, Ganem D, Andino R, et al.: Temporal analysis of the honey bee microbiome reveals four novel viruses and seasonal prevalence of known viruses, Nosema, and Crithidia. PLoS One 2011, 6(6):e20656. doi:10.1371/journal.pone.0020656
• [62]Felix MA, Ashe A, Piffaretti J, Wu G, Nuez I, Belicard T, et al.: Natural and experimental infection of Caenorhabditis nematodes by novel viruses related to nodaviruses. Plos Biol 2011, 9(1):e1000586. doi:10.1371/journal.pbio.1000586
• [63]Franz CJ, Zhao G, Felix MA, Wang D: Complete genome sequence of Le Blanc virus, a third Caenorhabditis nematode-infecting virus. J Virol 2012, 86(21):11940. doi:10.1128/JVI. 02025-12
• [64]Hildebrand A, Remmert M, Biegert A, Soding J: Fast and accurate automatic structure prediction with HHpred. Proteins 2009, 77(Suppl 9):128-32. doi:10.1002/prot.22499
• [65]Nishikiori M, Meshi T, Ishikawa M: Guanylylation-competent replication proteins of Tomato mosaic virus are disulfide-linked. Virology 2012, 434(1):118-28. doi:10.1016/j.virol.2012.09.011
• [66]Vlot AC, Menard A, Bol JF: Role of the alfalfa mosaic virus methyltransferase-like domain in negative-strand RNA synthesis. J Virol 2002, 76(22):11321-8.
• [67]Yi G, Kao C. cis- and trans-acting functions of brome mosaic virus protein 1a in genomic RNA1 replication. J Virol. 2008;82(6):3045–53. doi:10.1128/JVI.02390-07.
• [68]Dye BT, Miller DJ, Ahlquist P: In vivo self-interaction of nodavirus RNA replicase protein a revealed by fluorescence resonance energy transfer. J Virol 2005, 79(14):8909-19. doi:10.1128/JVI. 79.14.8909-8919.2005
• [69]Guo YX, Chan SW, Kwang J: Membrane association of greasy grouper nervous necrosis virus protein A and characterization of its mitochondrial localization targeting signal. J Virol 2004, 78(12):6498-508. doi:10.1128/JVI. 78.12.6498-6508.2004
• [70]Mezeth KB, Nylund S, Henriksen H, Patel S, Nerland AH, Szilvay AM: RNA-dependent RNA polymerase from Atlantic halibut nodavirus contains two signals for localization to the mitochondria. Virus Res 2007, 130(1–2):43-52. doi:10.1016/j.virusres.2007.05.014
• [71]Miller DJ, Ahlquist P: Flock house virus RNA polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion. J Virol 2002, 76(19):9856-67.
• [72]Qiu Y, Wang Z, Liu Y, Qi N, Miao M, Si J, et al.: Membrane association of Wuhan nodavirus protein A is required for its ability to accumulate genomic RNA1 template. Virology 2013, 439(2):140-51. doi:10.1016/j.virol.2013.02.010
• [73]Gant VU Jr, Moreno S, Varela-Ramirez A, Johnson KL: Two membrane-associated regions within the Nodamura virus RNA-dependent RNA polymerase are critical for both mitochondrial localization and RNA replication. J Virol 2014, 88(11):5912-26. doi:10.1128/JVI. 03032-13
• [74]Qiu Y, Wang Z, Liu Y, Han Y, Miao M, Qi N, et al.: The self-interaction of a nodavirus replicase is enhanced by mitochondrial membrane lipids. PLoS One 2014, 9(2):e89628. doi:10.1371/journal.pone.0089628
• [75]Reichert E, Clase A, Bacetty A, Larsen J: Alphavirus antiviral drug development: scientific gap analysis and prospective research areas. Biosecur Bioterror 2009, 7(4):413-27. doi:10.1089/bsp.2009.0032
• [76]Fogg MJ, Alzari P, Bahar M, Bertini I, Betton JM, Burmeister WP, et al.: Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens. Acta Crystallogr D Biol Crystallogr 2006, 62(Pt 10):1196-207. doi:10.1107/S0907444906030915
• [77]Koonin EV: The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses. J Gen Virol 1991, 72(Pt 9):2197-206.
• [78]Bhardwaj G, Ko KD, Hong Y, Zhang Z, Ho NL, Chintapalli SV, et al.: PHYRN: a robust method for phylogenetic analysis of highly divergent sequences. PLoS One 2012, 7(4):e34261. doi:10.1371/journal.pone.0034261
• [79]Monttinen HA, Ravantti JJ, Stuart DI, Poranen MM. Automated Structural Comparisons Clarify the Phylogeny of the Right-Hand-Shaped Polymerases. Mol Biol Evol. 2014. doi:msu219.
• [80]Cerny J, Cerna Bolfikova B, Valdes JJ, Grubhoffer L, Ruzek D: Evolution of tertiary structure of viral RNA dependent polymerases. PLoS One 2014, 9(5):e96070. doi:10.1371/journal.pone.0096070
• [81]Diaz A, Gallei A, Ahlquist P: Bromovirus RNA replication compartment formation requires concerted action of 1a's self-interacting RNA capping and helicase domains. J Virol 2012, 86(2):821-34. doi:10.1128/JVI. 05684-11
• [82]Kopek BG, Perkins G, Miller DJ, Ellisman MH, Ahlquist P: Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle. PLoS Biol 2007, 5(9):e220. doi:10.1371/journal.pbio.0050220
• [83]Schwartz M, Chen J, Janda M, Sullivan M, den Boon J, Ahlquist P: A positive-strand RNA virus replication complex parallels form and function of retrovirus capsids. Mol Cell 2002, 9(3):505-14.
• [84]Kallio K, Hellstrom K, Balistreri G, Spuul P, Jokitalo E, Ahola T. Template RNA length determines the size of replication complex spherules for Semliki forest virus. J Virol. 2013. doi:10.1128/JVI.00660-13.
• [85]Soding J, Biegert A, Lupas AN. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 2005;33(Web Server issue):W244–8. doi:10.1093/nar/gki408.
• [86]Abrescia NG, Bamford DH, Grimes JM, Stuart DI: Structure unifies the viral universe. Annu Rev Biochem 2012, 81:795-822. doi:10.1146/annurev-biochem-060910-095130
• [87]Chang JM, Di Tommaso P, Taly JF, Notredame C. Accurate multiple sequence alignment of transmembrane proteins with PSI-Coffee. BMC Bioinformatics. 2012;13 Suppl 4:S1. doi:10.1186/1471-2105-13-S4-S1.
• [88]Jaroszewski L, Li Z, Cai XH, Weber C, Godzik A. FFAS server: novel features and applications. Nucleic Acids Res. 2011;39(Web Server issue):W38-44. doi:gkr441.
• [89]Remmert M, Biegert A, Hauser A, Soding J. HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nat Methods. 2012;9(2):173–5. doi:10.1038/Nmeth.1818.
• [90]Biegert A, Soding J: Sequence context-specific profiles for homology searching. Proc Natl Acad Sci U S A 2009, 106(10):3770-5. doi:10.1073/pnas.0810767106
• [91]Angermuller C, Biegert A, Soding J: Discriminative modelling of context-specific amino acid substitution probabilities. Bioinformatics 2012, 28(24):3240-7. doi:10.1093/bioinformatics/bts622
• [92]Biegert A, Mayer C, Remmert M, Soding J, Lupas AN. The MPI Bioinformatics Toolkit for protein sequence analysis. Nucleic Acids Res. 2006;34(Web Server issue):W335–9. doi:10.1093/nar/gkl217.
• [93]Di Tommaso P, Moretti S, Xenarios I, Orobitg M, Montanyola A, Chang JM, et al. T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res. 2011;39(Web Server issue):W13–7. doi:10.1093/nar/gkr245.
• [94]Katoh K, Frith MC: Adding unaligned sequences into an existing alignment using MAFFT and LAST. Bioinformatics 2012, 28(23):3144-6. doi:10.1093/bioinformatics/bts578
• [95]Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ: Jalview Version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics 2009, 25(9):1189-91. doi:10.1093/bioinformatics/btp033
• [96]Procter JB, Thompson J, Letunic I, Creevey C, Jossinet F, Barton GJ: Visualization of multiple alignments, phylogenies and gene family evolution. Nat Methods 2010, 7(3 Suppl):S16-25. doi:10.1038/nmeth.1434
• [97]Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, et al. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 2008;36(Web Server issue):W465–9. doi:10.1093/nar/gkn180.
• [98]Ishida T, Kinoshita K: Prediction of disordered regions in proteins based on the meta approach. Bioinformatics 2008, 24(11):1344-8. doi:10.1093/bioinformatics/btn195
• [99]Ferron F, Longhi S, Canard B, Karlin D: A practical overview of protein disorder prediction methods. Proteins 2006, 65(1):1-14. doi:10.1002/prot.21075
• [100]Wootton JC. Nonglobular domains in protein sequences - automated segmentation using complexity-measures. Comput Chem. 1994;18(3):269–85. doi:10.1016/0097-8485(94)85023-2.
• [101]Ooi HS, Kwo CY, Wildpaner M, Sirota FL, Eisenhaber B, Maurer-Stroh S, et al. ANNIE: integrated de novo protein sequence annotation. Nucleic Acids Res. 2009;37(Web Server issue):W435–40. doi:10.1093/nar/gkp254.
• [102]Eisenberg D, Weiss RM, Terwilliger TC: The helical hydrophobic moment: a measure of the amphiphilicity of a helix. Nature 1982, 299(5881):371-4.