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. 2002 Jul 19;298(2):258–270. doi: 10.1006/viro.2002.1495

Functional Properties of the Predicted Helicase of Porcine Reproductive and Respiratory Syndrome Virus

Elida M Bautista a,1, Kay S Faaberg b, Dan Mickelson b, Edward D McGruder a
PMCID: PMC7130902  PMID: 12127789

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the positive-strand RNA virus family Arteriviridae. Although considerable research has focused on this important pathogen, little is known about the function of most PRRSV proteins. To examine characteristics of putative nonstructural proteins (nsp) encoded in ORF1b, which have been identified by nucleotide similarity to domains of equine arteritis virus, defined genomic regions were cloned and expressed in the pRSET expression system. One region, nsp10, encoded a protein with a putative helicase domain and was further examined for functional helicase-like activities. PRRSV nsp10 was found to possess a thermolabile and pH-sensitive NTPase activity that was modulated by polynucleotides and to unwind dsRNA in a 5′ to 3′ polarity. These results provide the first evidence of the functional properties of PRRSV helicase and further support the finding that nidovirus helicases possess properties that distinguish them from other viral helicases.

Keywords: PRRSV, arterivirus, coronavirus, Nidovirales, RNA-dependent RNA polymerase, helicase, NTPase

References

REFERENCES

  • 1.Allende R., Lewis T.L., Lu Z., Rock D.L., Kutish G.F., Ali A., Doster A.R., Osorio F.A. North American and European porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions. J. Gen. Virol. 1999;80:307–315. doi: 10.1099/0022-1317-80-2-307. [DOI] [PubMed] [Google Scholar]
  • 2.Allende R., Kutish G.F., Laegreid W., Lu Z., Lewis T.L., Rock D.L., Friesen J., Galeota J.A., Doster A.R., Osorio F.A. Mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype. Arch. Virol. 2000;145:1149–1161. doi: 10.1007/s007050070115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Baker S.C., Yokomori K., Dong S., Carlisle R., Gorbalenya A.E., Koonin E.V., Lai M.M. Identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus. J. Virol. 1993;67:6056–6063. doi: 10.1128/jvi.67.10.6056-6063.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Bautista E.M., Meulenberg J.J., Choi C.S., Molitor T.W. Structural polypeptides of the American (VR-2332) strain of porcine reproductive and respiratory syndrome virus. Arch. Virol. 1996;141:1357–1365. doi: 10.1007/BF01718837. [DOI] [PubMed] [Google Scholar]
  • 5.Benfield D.A., Nelson E., Collins J.E., Harris L., Goyal S.M., Robison D., Christianson W.T., Morrison R.B., Gorcyca D., Chladek D. Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate ATCC VR-2332) J. Vet. Diagn. Invest. 1992;4:127–133. doi: 10.1177/104063879200400202. [DOI] [PubMed] [Google Scholar]
  • 6.Bisaillon M., Bergeron J., Lemay G. Characterization of the nucleoside triphosphate phosphohydrolase and helicase activities of the reovirus lambdal protein. J. Biol. Chem. 1997;272:18298–18303. doi: 10.1074/jbc.272.29.18298. [DOI] [PubMed] [Google Scholar]
  • 7.Brierley I., Digard P., Inglis S.C. Characterization of an efficient coronavirus ribosomal frameshifting signal: Requirement for an RNA pseudoknot. Cell. 1989;57:537–547. doi: 10.1016/0092-8674(89)90124-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Cavanagh D. Nidovirales: A new order comprising Coronaviridae and Arteriviridae. Arch. Virol. 1997;142:629–633. [PubMed] [Google Scholar]
  • 9.Collins J.E., Benfield D.A., Christianson W.T., Harris L., Hennings J.C., Shaw D.P., Goyal S.M., McCullough S., Morrison R.B., Joo H.S., Gorcyka D., Chladek D. Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs. J. Vet. Diagn. Invest. 1992;4:117–126. doi: 10.1177/104063879200400201. [DOI] [PubMed] [Google Scholar]
  • 10.Conzelmann K.K., Visser N., Van Woensel P., Thiel H.J. Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group. Virology. 1993;193:329–339. doi: 10.1006/viro.1993.1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.de Vries A.A., Glaser A.L., Raamsman M.J., de Haan C.A., Sarnataro S., Godeke G.J., Rottier P.J. Genetic manipulation of equine arteritis virus using full-length cDNA clones: separation of overlapping genes and expression of a foreign epitope. Virology. 2000;270:84–97. doi: 10.1006/viro.2000.0245. [DOI] [PubMed] [Google Scholar]
  • 12.de 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;65:2910–2920. doi: 10.1128/jvi.65.6.2910-2920.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.den Boon J.A., Faaberg K.S., Meulenberg J.J., Wassenaar A.L., Plagemann P.G., Gorbalenya A.E., Snijder E.J. Processing and evolution of the N-terminal region of the arterivirus replicase ORF1a protein: Identification of two papainlike cysteine proteases. J. Virol. 1995;69:4500–4505. doi: 10.1128/jvi.69.7.4500-4505.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.den Boon J.A., Kleijnen M.F., Spaan W.J., Snijder E.J. Equine arteritis virus subgenomic mRNA synthesis: Analysis of leader-body junctions and replicative-form RNAs. J. Virol. 1996;70:4291–4298. doi: 10.1128/jvi.70.7.4291-4298.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Godeny E.K., Chen L., Kumar S.N., Methven S.L., Koonin E.V., Brinton M.A. Complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase-elevating virus (LDV) Virology. 1993;194:585–596. doi: 10.1006/viro.1993.1298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.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;17:4847–4861. doi: 10.1093/nar/17.12.4847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Gwack Y., Yoo H., Song I., Choe J., Han J.H. RNA-stimulated ATPase and RNA helicase activities and RNA binding domain of hepatitis G virus nonstructural protein 3. J. Virol. 1999;73:2909–2915. doi: 10.1128/jvi.73.4.2909-2915.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Herold J., Siddell S., Ziebuhr J. Characterization of coronavirus RNA polymerase gene products. Methods Enzymol. 1996;275:68–89. doi: 10.1016/S0076-6879(96)75007-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.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;71:5631–5634. doi: 10.1128/jvi.71.7.5631-5634.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Jiang P., Chen P.Y., Dong Y.Y., Cai J.L., Cai B.X., Jiang Z.H. Isolation and genome characterization of porcine reproductive and respiratory syndrome virus in P.R. China. J. Vet. Diagn. Invest. 2000;12:156–158. doi: 10.1177/104063870001200211. [DOI] [PubMed] [Google Scholar]
  • 21.Kadare 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;70:8169–8174. doi: 10.1128/jvi.70.11.8169-8174.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Kadare G., Haenni A.L. Virus-encoded RNA helicases. J. Virol. 1997;71:2583–2590. doi: 10.1128/jvi.71.4.2583-2590.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Kim H.S., Kwang J., Yoon I.J., Joo H.S., Frey M.L. Enhanced replication of porcine reproductive and respiratory syndrome (Prrs) virus in a homogeneous subpopulation of Ma-104 cell line. Arch. Virol. 1993;133:477–483. doi: 10.1007/BF01313785. [DOI] [PubMed] [Google Scholar]
  • 24.Li H., Clum S., You S., Ebner K.E., Padmanabhan R. The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids. J. Virol. 1999;73:3108–3116. doi: 10.1128/jvi.73.4.3108-3116.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.McElroy W.D., DeLuca M.A. Firefly and bacterial luminescence: Basic science and applications. J. Appl. Biochem. 1983;5:197–209. [PubMed] [Google Scholar]
  • 26.Meulenberg J.J., Hulst M.M., de Meijer E.J., Moonen P.L., den Besten A., de Kluyver E.P., Wensvoort G., Moormann R.J. Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV. Virology. 1993;192:62–72. doi: 10.1006/viro.1993.1008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Meulenberg J.J., Bos-de Ruijter J.N., van de Graaf R., Wensvoort G., Moormann R.J. Infectious transcripts from cloned genome-length cDNA of porcine reproductive and respiratory syndrome virus. J. Virol. 1998;72:380–387. doi: 10.1128/jvi.72.1.380-387.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Motulski, H. J. 1999, Analyzing Data with GraphPad Prism, GraphPad Software Inc. San Diego, CA.
  • 29.Moyer J.D., Henderson J.F. Nucleoside triphosphate specificity of firefly luciferase. Anal. Biochem. 1983;131:187–189. doi: 10.1016/0003-2697(83)90152-5. [DOI] [PubMed] [Google Scholar]
  • 30.Murtaugh M.P., Elam M.R., Kakach L.T. Comparison of the structural protein coding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus. Arch. Virol. 1995;140:1451–1460. doi: 10.1007/BF01322671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Murtaugh M.P., Faaberg K.S., Laber J., Elam M., Kapur V. Genetic variation in the PRRS virus. Adv. Exp. Med. Biol. 1998;440:787–794. doi: 10.1007/978-1-4615-5331-1_102. [DOI] [PubMed] [Google Scholar]
  • 32.Nelsen C.J., Murtaugh M.P., Faaberg K.S. Porcine reproductive and respiratory syndrome virus comparison: Divergent evolution on two continents. J. Virol. 1999;73:270–280. doi: 10.1128/jvi.73.1.270-280.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Nielsen H.S., Storgaard T., Oleksiewicz M.B. Analysis of ORF 1 in European porcine reproductive and respiratory syndrome virus by long RT-PCR and restriction fragment length polymorphism (RFLP) analysis. Vet. Microbiol. 2000;76:221–228. doi: 10.1016/s0378-1135(00)00258-3. [DOI] [PubMed] [Google Scholar]
  • 34.Plagemann P.G., Moennig V. Lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: A new group of positive-strand RNA viruses. Adv. Virus Res. 1992;41:99–192. doi: 10.1016/S0065-3527(08)60036-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.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;271:24449–24457. doi: 10.1074/jbc.271.40.24449. [DOI] [PubMed] [Google Scholar]
  • 36.Rikkonen M., Peranen J., Kaariainen L. ATPase and GTPase activities associated with Semliki Forest virus nonstructural protein nsP2. J. Virol. 1994;68:5804–5810. doi: 10.1128/jvi.68.9.5804-5810.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Sambrook J. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press; Plainview: 1989. [Google Scholar]
  • 38.Seybert A., van Dinten L.C., Snijder E.J., Ziebuhr J. Biochemical characterization of the equine arteritis virus helicase suggests a close functional relationship between arterivirus and coronavirus helicases. J. Virol. 2000;74:9586–9593. doi: 10.1128/jvi.74.20.9586-9593.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Seybert A., Hegyi A., Siddell S.G., Ziebuhr J. The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5′-to-3′ polarity. RNA. 2000;6:1056–1068. doi: 10.1017/s1355838200000728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Shen S., Kwang J., Liu W., Liu D.X. Determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the Nsp2 gene with a unique insertion. Arch. Virol. 2000;145:871–883. doi: 10.1007/s007050050680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Smith P.K., Krohn R.I., Hermanson G.T., Mallia A.K., Gartner F.H., Provenzano M.D., Fujimoto E.K., Goeke N.M., Olson B.J., Klenk D.C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985;150:76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  • 42.Snijder E.J., Wassenaar A.L., Spaan W.J. The 5′ end of the equine arteritis virus replicase gene encodes a papainlike cysteine protease. J. Virol. 1992;66:7040–7048. doi: 10.1128/jvi.66.12.7040-7048.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Snijder E.J., Wassenaar A.L., Spaan W.J. Proteolytic processing of the N-terminal region of the equine arteritis virus replicase. Adv. Exp. Med. Biol. 1993;342:227–732. doi: 10.1007/978-1-4615-2996-5_36. [DOI] [PubMed] [Google Scholar]
  • 44.Snijder E.J., Horzinek M.C., Spaan W.J. The coronaviruslike superfamily. Adv. Exp. Med. Biol. 1993;342:235–244. doi: 10.1007/978-1-4615-2996-5_37. [DOI] [PubMed] [Google Scholar]
  • 45.Snijder E.J., Wassenaar A.L., Spaan W.J. Proteolytic processing of the replicase ORF1a protein of equine arteritis virus. J. Virol. 1994;68:5755–5764. doi: 10.1128/jvi.68.9.5755-5764.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Snijder E.J., Wassenaar A.L., Spaan W.J., Gorbalenya A.E. The arterivirus Nsp2 protease. An unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases. J. Biol. Chem. 1995;270:16671–16676. doi: 10.1074/jbc.270.28.16671. [DOI] [PubMed] [Google Scholar]
  • 47.Snijder E.J., Wassenaar A.L., van Dinten L.C., Spaan W.J., Gorbalenya A.E. The arterivirus nsp4 protease is the prototype of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases. J. Biol. Chem. 1996;271:4864–4871. doi: 10.1074/jbc.271.9.4864. [DOI] [PubMed] [Google Scholar]
  • 48.Snijder E.J. The arterivirus replicase. The road from RNA to protein(s), and back again. Adv. Exp. Med. Biol. 1998;440:97–108. [PubMed] [Google Scholar]
  • 49.Snijder E.J., Meulenberg J.J.M. The molecular biology of arteriviruses. J. Gen. Virol. 1998;79:961–979. doi: 10.1099/0022-1317-79-5-961. [DOI] [PubMed] [Google Scholar]
  • 50.Tamura J.K., Warrener P., Collett M.S. RNA-stimulated NTPase associated with the P80 protein of the pestivirus bovine viral diarrhea virus. Virology. 1993;193:1–10. doi: 10.1006/viro.1993.1097. [DOI] [PubMed] [Google Scholar]
  • 51.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;70:6625–6633. doi: 10.1128/jvi.70.10.6625-6633.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.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. USA. 1997;94:991–996. doi: 10.1073/pnas.94.3.991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.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;73:2027–2037. doi: 10.1128/jvi.73.3.2027-2037.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.van Dinten L.C., van Tol H., Gorbalenya A.E., Snijder E.J. The predicted metal-binding region of the arterivirus helicase protein is involved in subgenomic mRNA synthesis, genome replication, and virion biogenesis. J. Virol. 2000;74:5213–5223. doi: 10.1128/jvi.74.11.5213-5223.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.van Marle G., van Dinten L.C., Spaan W.J., Luytjes W., Snijder E.J. Characterization of an equine arteritis virus replicase mutant defective in subgenomic mRNA synthesis. J. Virol. 1999;73:5274–5281. doi: 10.1128/jvi.73.7.5274-5281.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Wassenaar A.L., Spaan W.J., Gorbalenya A.E., Snijder E.J. Alternative proteolytic processing of the arterivirus replicase ORF1a polyprotein: Evidence that NSP2 acts as a cofactor for the NSP4 serine protease. J. Virol. 1997;71:9313–9322. doi: 10.1128/jvi.71.12.9313-9322.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Wootton S., Yoo D., Rogan D. Full-length sequence of a Canadian porcine reproductive and respiratory syndrome virus (PRRSV) isolate. Arch. Virol. 2000;145:2297–2323. doi: 10.1007/s007050070022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Yuan S., Nelsen C.J., Murtaugh M.P., Schmitt B.J., Faaberg K.S. Recombination between North American strains of porcine reproductive and respiratory syndrome virus. Virus Res. 1999;61:87–98. doi: 10.1016/S0168-1702(99)00029-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Yuan S., Mickelson D., Murtaugh M.P., Faaberg K.S. Complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains. Virus Res. 2001;74:99–110. doi: 10.1016/S0168-1702(00)00250-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Ziebuhr J., Snijder E.J., Gorbalenya A.E. Virus-encoded proteinases and proteolytic processing in the Nidovirales. J. Gen. Virol. 2000;4:853–879. doi: 10.1099/0022-1317-81-4-853. [DOI] [PubMed] [Google Scholar]

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