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. 2000 Jul;6(7):1056–1068. doi: 10.1017/s1355838200000728

The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5'-to-3' polarity.

A Seybert 1, A Hegyi 1, S G Siddell 1, J Ziebuhr 1
PMCID: PMC1369980  PMID: 10917600

Abstract

The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5'-to-3' polarity. The DNA helicase activity of the enzyme preferentially unwound 5'-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases.

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Selected References

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  1. Baker T. A., Bell S. P. Polymerases and the replisome: machines within machines. Cell. 1998 Feb 6;92(3):295–305. doi: 10.1016/s0092-8674(00)80923-x. [DOI] [PubMed] [Google Scholar]
  2. Bayliss C. D., Smith G. L. Vaccinia virion protein I8R has both DNA and RNA helicase activities: implications for vaccinia virus transcription. J Virol. 1996 Feb;70(2):794–800. doi: 10.1128/jvi.70.2.794-800.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bird L. E., Subramanya H. S., Wigley D. B. Helicases: a unifying structural theme? Curr Opin Struct Biol. 1998 Feb;8(1):14–18. doi: 10.1016/s0959-440x(98)80004-3. [DOI] [PubMed] [Google Scholar]
  4. Buck K. W. Comparison of the replication of positive-stranded RNA viruses of plants and animals. Adv Virus Res. 1996;47:159–251. doi: 10.1016/S0065-3527(08)60736-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cho H. S., Ha N. C., Kang L. W., Chung K. M., Back S. H., Jang S. K., Oh B. H. Crystal structure of RNA helicase from genotype 1b hepatitis C virus. A feasible mechanism of unwinding duplex RNA. J Biol Chem. 1998 Jun 12;273(24):15045–15052. doi: 10.1074/jbc.273.24.15045. [DOI] [PubMed] [Google Scholar]
  6. Costa M., Ochem A., Staub A., Falaschi A. Human DNA helicase VIII: a DNA and RNA helicase corresponding to the G3BP protein, an element of the ras transduction pathway. Nucleic Acids Res. 1999 Feb 1;27(3):817–821. doi: 10.1093/nar/27.3.817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Czaplinski K., Weng Y., Hagan K. W., Peltz S. W. Purification and characterization of the Upf1 protein: a factor involved in translation and mRNA degradation. RNA. 1995 Aug;1(6):610–623. [PMC free article] [PubMed] [Google Scholar]
  8. Dé I., Sawicki S. G., Sawicki D. L. Sindbis virus RNA-negative mutants that fail to convert from minus-strand to plus-strand synthesis: role of the nsP2 protein. J Virol. 1996 May;70(5):2706–2719. doi: 10.1128/jvi.70.5.2706-2719.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fuller-Pace F. V., Nicol S. M., Reid A. D., Lane D. P. DbpA: a DEAD box protein specifically activated by 23s rRNA. EMBO J. 1993 Sep;12(9):3619–3626. doi: 10.1002/j.1460-2075.1993.tb06035.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gomez de Cedrón M., Ehsani N., Mikkola M. L., García J. A., Käriäinen L. RNA helicase activity of Semliki Forest virus replicase protein NSP2. FEBS Lett. 1999 Apr 1;448(1):19–22. doi: 10.1016/s0014-5793(99)00321-x. [DOI] [PubMed] [Google Scholar]
  11. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. Coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis. Nucleic Acids Res. 1989 Jun 26;17(12):4847–4861. doi: 10.1093/nar/17.12.4847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 1989 Jun 26;17(12):4713–4730. doi: 10.1093/nar/17.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gorbalenya A. E., Koonin E. V. Viral proteins containing the purine NTP-binding sequence pattern. Nucleic Acids Res. 1989 Nov 11;17(21):8413–8440. doi: 10.1093/nar/17.21.8413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gros C., Wengler G. Identification of an RNA-stimulated NTPase in the predicted helicase sequence of the Rubella virus nonstructural polyprotein. Virology. 1996 Mar 1;217(1):367–372. doi: 10.1006/viro.1996.0125. [DOI] [PubMed] [Google Scholar]
  15. Gross C. H., Shuman S. Mutational analysis of vaccinia virus nucleoside triphosphate phosphohydrolase II, a DExH box RNA helicase. J Virol. 1995 Aug;69(8):4727–4736. doi: 10.1128/jvi.69.8.4727-4736.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grötzinger C., Heusipp G., Ziebuhr J., Harms U., Süss J., Siddell S. G. Characterization of a 105-kDa polypeptide encoded in gene 1 of the human coronavirus HCV 229E. Virology. 1996 Aug 1;222(1):227–235. doi: 10.1006/viro.1996.0413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gwack Y., Kim D. W., Han J. H., Choe J. DNA helicase activity of the hepatitis C virus nonstructural protein 3. Eur J Biochem. 1997 Nov 15;250(1):47–54. doi: 10.1111/j.1432-1033.1997.00047.x. [DOI] [PubMed] [Google Scholar]
  18. Hall M. C., Matson S. W. Helicase motifs: the engine that powers DNA unwinding. Mol Microbiol. 1999 Dec;34(5):867–877. doi: 10.1046/j.1365-2958.1999.01659.x. [DOI] [PubMed] [Google Scholar]
  19. Henningsen U., Schliwa M. Reversal in the direction of movement of a molecular motor. Nature. 1997 Sep 4;389(6646):93–96. doi: 10.1038/38022. [DOI] [PubMed] [Google Scholar]
  20. Herold J., Raabe T., Schelle-Prinz B., Siddell S. G. Nucleotide sequence of the human coronavirus 229E RNA polymerase locus. Virology. 1993 Aug;195(2):680–691. doi: 10.1006/viro.1993.1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Heusipp G., Harms U., Siddell S. G., Ziebuhr J. Identification of an ATPase activity associated with a 71-kilodalton polypeptide encoded in gene 1 of the human coronavirus 229E. J Virol. 1997 Jul;71(7):5631–5634. doi: 10.1128/jvi.71.7.5631-5634.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Janda M., Ahlquist P. Brome mosaic virus RNA replication protein 1a dramatically increases in vivo stability but not translation of viral genomic RNA3. Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2227–2232. doi: 10.1073/pnas.95.5.2227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jankowsky E., Gross C. H., Shuman S., Pyle A. M. The DExH protein NPH-II is a processive and directional motor for unwinding RNA. Nature. 2000 Jan 27;403(6768):447–451. doi: 10.1038/35000239. [DOI] [PubMed] [Google Scholar]
  24. Kadaré G., David C., Haenni A. L. ATPase, GTPase, and RNA binding activities associated with the 206-kilodalton protein of turnip yellow mosaic virus. J Virol. 1996 Nov;70(11):8169–8174. doi: 10.1128/jvi.70.11.8169-8174.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kadaré G., Haenni A. L. Virus-encoded RNA helicases. J Virol. 1997 Apr;71(4):2583–2590. doi: 10.1128/jvi.71.4.2583-2590.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kim H. D., Choe J., Seo Y. S. The sen1(+) gene of Schizosaccharomyces pombe, a homologue of budding yeast SEN1, encodes an RNA and DNA helicase. Biochemistry. 1999 Nov 2;38(44):14697–14710. doi: 10.1021/bi991470c. [DOI] [PubMed] [Google Scholar]
  27. Kim J. L., Morgenstern K. A., Griffith J. P., Dwyer M. D., Thomson J. A., Murcko M. A., Lin C., Caron P. R. Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. Structure. 1998 Jan 15;6(1):89–100. doi: 10.1016/s0969-2126(98)00010-0. [DOI] [PubMed] [Google Scholar]
  28. Koonin E. V., Dolja V. V. 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. [DOI] [PubMed] [Google Scholar]
  29. Korolev S., Hsieh J., Gauss G. H., Lohman T. M., Waksman G. Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP. Cell. 1997 Aug 22;90(4):635–647. doi: 10.1016/s0092-8674(00)80525-5. [DOI] [PubMed] [Google Scholar]
  30. Korolev S., Yao N., Lohman T. M., Weber P. C., Waksman G. Comparisons between the structures of HCV and Rep helicases reveal structural similarities between SF1 and SF2 super-families of helicases. Protein Sci. 1998 Mar;7(3):605–610. doi: 10.1002/pro.5560070309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kroner P. A., Young B. M., Ahlquist P. Analysis of the role of brome mosaic virus 1a protein domains in RNA replication, using linker insertion mutagenesis. J Virol. 1990 Dec;64(12):6110–6120. doi: 10.1128/jvi.64.12.6110-6120.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kwong A. D., Kim J. L., Lin C. Structure and function of hepatitis C virus NS3 helicase. Curr Top Microbiol Immunol. 2000;242:171–196. doi: 10.1007/978-3-642-59605-6_9. [DOI] [PubMed] [Google Scholar]
  33. Laxton C. D., McMillan D., Sullivan V., Ackrill A. M. Expression and characterization of the hepatitis G virus helicase. J Viral Hepat. 1998 Jan;5(1):21–26. doi: 10.1046/j.1365-2893.1998.00084.x. [DOI] [PubMed] [Google Scholar]
  34. Lee C. G., Hurwitz J. Human RNA helicase A is homologous to the maleless protein of Drosophila. J Biol Chem. 1993 Aug 5;268(22):16822–16830. [PubMed] [Google Scholar]
  35. Linder P., Daugeron M. C. Are DEAD-box proteins becoming respectable helicases? Nat Struct Biol. 2000 Feb;7(2):97–99. doi: 10.1038/72464. [DOI] [PubMed] [Google Scholar]
  36. Lohman T. M., Bjornson K. P. Mechanisms of helicase-catalyzed DNA unwinding. Annu Rev Biochem. 1996;65:169–214. doi: 10.1146/annurev.bi.65.070196.001125. [DOI] [PubMed] [Google Scholar]
  37. Nicol S. M., Fuller-Pace F. V. The "DEAD box" protein DbpA interacts specifically with the peptidyltransferase center in 23S rRNA. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11681–11685. doi: 10.1073/pnas.92.25.11681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. O'Day C. L., Chavanikamannil F., Abelson J. 18S rRNA processing requires the RNA helicase-like protein Rrp3. Nucleic Acids Res. 1996 Aug 15;24(16):3201–3207. doi: 10.1093/nar/24.16.3201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. O'Reilly E. K., Tang N., Ahlquist P., Kao C. C. Biochemical and genetic analyses of the interaction between the helicase-like and polymerase-like proteins of the brome mosaic virus. Virology. 1995 Dec 1;214(1):59–71. doi: 10.1006/viro.1995.9954. [DOI] [PubMed] [Google Scholar]
  40. O'Reilly E. K., Wang Z., French R., Kao C. C. Interactions between the structural domains of the RNA replication proteins of plant-infecting RNA viruses. J Virol. 1998 Sep;72(9):7160–7169. doi: 10.1128/jvi.72.9.7160-7169.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Osman T. A., Buck K. W. Complete replication in vitro of tobacco mosaic virus RNA by a template-dependent, membrane-bound RNA polymerase. J Virol. 1996 Sep;70(9):6227–6234. doi: 10.1128/jvi.70.9.6227-6234.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Petty I. T., French R., Jones R. W., Jackson A. O. Identification of barley stripe mosaic virus genes involved in viral RNA replication and systemic movement. EMBO J. 1990 Nov;9(11):3453–3457. doi: 10.1002/j.1460-2075.1990.tb07553.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pfister T., Wimmer E. Characterization of the nucleoside triphosphatase activity of poliovirus protein 2C reveals a mechanism by which guanidine inhibits poliovirus replication. J Biol Chem. 1999 Mar 12;274(11):6992–7001. doi: 10.1074/jbc.274.11.6992. [DOI] [PubMed] [Google Scholar]
  44. Phillips K., Dauter Z., Murchie A. I., Lilley D. M., Luisi B. The crystal structure of a parallel-stranded guanine tetraplex at 0.95 A resolution. J Mol Biol. 1997 Oct 17;273(1):171–182. doi: 10.1006/jmbi.1997.1292. [DOI] [PubMed] [Google Scholar]
  45. Preugschat F., Averett D. R., Clarke B. E., Porter D. J. A steady-state and pre-steady-state kinetic analysis of the NTPase activity associated with the hepatitis C virus NS3 helicase domain. J Biol Chem. 1996 Oct 4;271(40):24449–24457. doi: 10.1074/jbc.271.40.24449. [DOI] [PubMed] [Google Scholar]
  46. Rikkonen M., Peränen J., Käriäinen L. ATPase and GTPase activities associated with Semliki Forest virus nonstructural protein nsP2. J Virol. 1994 Sep;68(9):5804–5810. doi: 10.1128/jvi.68.9.5804-5810.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rodríguez P. L., Carrasco L. Poliovirus protein 2C has ATPase and GTPase activities. J Biol Chem. 1993 Apr 15;268(11):8105–8110. [PubMed] [Google Scholar]
  48. Rouleau M., Smith R. J., Bancroft J. B., Mackie G. A. Purification, properties, and subcellular localization of foxtail mosaic potexvirus 26-kDa protein. Virology. 1994 Oct;204(1):254–265. doi: 10.1006/viro.1994.1530. [DOI] [PubMed] [Google Scholar]
  49. Scheffner M., Knippers R., Stahl H. RNA unwinding activity of SV40 large T antigen. Cell. 1989 Jun 16;57(6):955–963. doi: 10.1016/0092-8674(89)90334-6. [DOI] [PubMed] [Google Scholar]
  50. Schmid S. R., Linder P. D-E-A-D protein family of putative RNA helicases. Mol Microbiol. 1992 Feb;6(3):283–291. doi: 10.1111/j.1365-2958.1992.tb01470.x. [DOI] [PubMed] [Google Scholar]
  51. Soultanas P., Dillingham M. S., Velankar S. S., Wigley D. B. DNA binding mediates conformational changes and metal ion coordination in the active site of PcrA helicase. J Mol Biol. 1999 Jul 2;290(1):137–148. doi: 10.1006/jmbi.1999.2873. [DOI] [PubMed] [Google Scholar]
  52. Stahl H., Dröge P., Knippers R. DNA helicase activity of SV40 large tumor antigen. EMBO J. 1986 Aug;5(8):1939–1944. doi: 10.1002/j.1460-2075.1986.tb04447.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Subramanya H. S., Bird L. E., Brannigan J. A., Wigley D. B. Crystal structure of a DExx box DNA helicase. Nature. 1996 Nov 28;384(6607):379–383. doi: 10.1038/384379a0. [DOI] [PubMed] [Google Scholar]
  54. Suzich J. A., Tamura J. K., Palmer-Hill F., Warrener P., Grakoui A., Rice C. M., Feinstone S. M., Collett M. S. Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes. J Virol. 1993 Oct;67(10):6152–6158. doi: 10.1128/jvi.67.10.6152-6158.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Velankar S. S., Soultanas P., Dillingham M. S., Subramanya H. S., Wigley D. B. Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism. Cell. 1999 Apr 2;97(1):75–84. doi: 10.1016/s0092-8674(00)80716-3. [DOI] [PubMed] [Google Scholar]
  56. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Xu D., Nouraini S., Field D., Tang S. J., Friesen J. D. An RNA-dependent ATPase associated with U2/U6 snRNAs in pre-mRNA splicing. Nature. 1996 Jun 20;381(6584):709–713. doi: 10.1038/381709a0. [DOI] [PubMed] [Google Scholar]
  58. Yao N., Hesson T., Cable M., Hong Z., Kwong A. D., Le H. V., Weber P. C. Structure of the hepatitis C virus RNA helicase domain. Nat Struct Biol. 1997 Jun;4(6):463–467. doi: 10.1038/nsb0697-463. [DOI] [PubMed] [Google Scholar]
  59. Yao Z., Jones D. H., Grose C. Site-directed mutagenesis of herpesvirus glycoprotein phosphorylation sites by recombination polymerase chain reaction. PCR Methods Appl. 1992 Feb;1(3):205–207. doi: 10.1101/gr.1.3.205. [DOI] [PubMed] [Google Scholar]
  60. Zhang S., Grosse F. Nuclear DNA helicase II unwinds both DNA and RNA. Biochemistry. 1994 Apr 5;33(13):3906–3912. doi: 10.1021/bi00179a016. [DOI] [PubMed] [Google Scholar]
  61. Ziebuhr J., Herold J., Siddell S. G. Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity. J Virol. 1995 Jul;69(7):4331–4338. doi: 10.1128/jvi.69.7.4331-4338.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Ziebuhr J., Snijder E. J., Gorbalenya A. E. Virus-encoded proteinases and proteolytic processing in the Nidovirales. J Gen Virol. 2000 Apr;81(Pt 4):853–879. doi: 10.1099/0022-1317-81-4-853. [DOI] [PubMed] [Google Scholar]
  63. de la Cruz J., Kressler D., Linder P. Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families. Trends Biochem Sci. 1999 May;24(5):192–198. doi: 10.1016/s0968-0004(99)01376-6. [DOI] [PubMed] [Google Scholar]
  64. den Boon J. A., Snijder E. J., Chirnside E. D., de Vries A. A., Horzinek M. C., Spaan W. J. Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily. J Virol. 1991 Jun;65(6):2910–2920. doi: 10.1128/jvi.65.6.2910-2920.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. van Dinten L. C., Rensen S., Gorbalenya A. E., Snijder E. J. Proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and Is essential for virus replication. J Virol. 1999 Mar;73(3):2027–2037. doi: 10.1128/jvi.73.3.2027-2037.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. van Dinten L. C., Wassenaar A. L., Gorbalenya A. E., Spaan W. J., Snijder E. J. Processing of the equine arteritis virus replicase ORF1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains. J Virol. 1996 Oct;70(10):6625–6633. doi: 10.1128/jvi.70.10.6625-6633.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. van Dinten L. C., den Boon J. A., Wassenaar A. L., Spaan W. J., Snijder E. J. An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):991–996. doi: 10.1073/pnas.94.3.991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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