Construction and genetic analysis of murine hepatitis virus strain A59 Nsp16 temperature sensitive mutant and the revertant virus
Tóm tắt
Coronaviruses (CoVs) are generally associated with respiratory and enteric infections and have long been recognized as important pathogens of livestock and companion animals. Mouse hepatitis virus (MHV) is a widely studied model system for Coronavirus replication and pathogenesis. In this study, we created a MHV-A59 temperature sensitive (ts) mutant Wu”-ts18(cd) using the recombinant vaccinia reverse genetics system. Virus replication assay in 17C1-1 cells showed the plaque phenotype and replication characterization of constructed Wu”-ts18(cd) were indistinguishable from the reported ts mutant Wu”-ts18. Then we cultured the ts mutant Wu”-ts18(cd) at non-permissive temperature 39.5°C, which “forced” the ts recombinant virus to use second-site mutation to revert from a ts to a non-ts phenotype. Sequence analysis showed most of the revertants had the same single amino acid mutation at Nsp16 position 43. The single amino acid mutation at Nsp16 position 76 or position 130 could also revert the ts mutant Wu”-ts18 (cd) to non-ts phenotype, an additional independent mutation in Nsp13 position 115 played an important role on plaque size. The results provided us with genetic information on the functional determinants of Nsp16. This allowed us to build up a more reasonable model of CoVs replication-transcription complex.
Tài liệu tham khảo
Ahola T, Laakkonen P, Vihinen H, et al. 1997. Critical residues of Semliki Forest Virus RNA capping enzyme involved in methyltransferase and guanylyltransferase-like activities. J Virol, 71: 392–397.
Almazan F, Dediego M L, Galán C, et al. 2006. Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis. J Virol, 80: 10900–10906.
Benarroch D, Selisko B, Locatelli G A, et al. 2004. The RNA helicase, nucleotide 5′-triphosphatase and RNA 5′-triphosphatase activities of Dengue virus protein NS3 are Mg2+ dependent and require a functional Walker B motif in the helicase catalytic core. Virology, 328: 208–218.
Brian D A, Baric R S. 2005. Coronavirus genome structure and replication. Curr Top Microbiol Immunol, 287: 1–30.
Brockway S M, Denison M R. 2005. Mutagenesis of the murine hepatitis virus nsp1-coding region identifies residues important for protein processing, viral RNA synthesis, and viral replication. Virology, 340: 209–223.
Bujnicki J M, Rychlewski L. 2002. In silico identification, structure prediction and phylogenetic analysis of the 2′-O-ribose (cap 1) methyltransferase domain in the large structural protein of ssRNA negative-strand viruses. Protein Eng, 15: 101–108.
Casais R, Thiel V, Siddell S G, et al. 2001. Reverse genetics system for the avian coronavirus infectious bronchitis virus. J Virol, 75: 12359–12369.
Coley S E, Lavi E, Sawicki S G, et al. 2005. Recombinant mouse hepatitis virus strain A59 from cloned, full-length cDNA replicates to high titers in vitro and is fully pathogenic in vivo. J Virol, 79: 3097–3106.
Decroly E, Imbert I, Coutard B, et al. 2008. Coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (Nucleoside-2′O)-methyltransferase activity. J Virol, 82: 8071–8084.
Drosten C, Gunther S, Preiser W, et al. 2003. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med, 348: 1967–1976.
Eckerle L D, Lu X, Sperry S M, L, et al. 2007. High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants. J Virol, 81: 12135–12144.
Egloff M P, Benarroch D, Selisko B, et al. 2002. An RNA cap (nucleoside-2′O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization. EMBO J, 21: 2757–2768.
Egloff M P, Decroly E, Malet H, et al. 2007. Structural and functional analysis of methylation and 5′ RNA sequence requirements of short capped RNAs by the methyltransferase domain of dengue virus NS5. J Mol Biol, 372: 723–736.
Egloff M P, Ferron F, Campanacci V, et al. 2004. The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world. Proc Natl Acad Sci USA, 101: 3792–3796.
Enjuanes L, Sola I, Alonso S, et al. 2005. Coronavirus reverse genetics and development of vectors for gene expression. Curr Top Microbiol Immunol, 287: 161–197.
Ginalski K, Godzik A, Rychlewski L. 2006. Novel SARS unique AdoMet-dependent methyltransferase. Cell Cycle, 5: 2414–2416.
Guarino L A, Bhardwaj K, Dong W, et al. 2005. Mutational analysis of the SARS virus Nsp15 endoribonuclease, identification of residues affecting hexamer formation. J Mol Biol, 353: 106–1117.
Harcourt B H, Jukneliene D, Kanjanahaluethai A, et al. 2004. Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity. J Virol, 78: 13600–13612.
Ivanov K A, Ziebuhr J. 2004. Human coronavirus 229E nonstructural protein 13, characterization of duplex-unwinding, nucleoside triphosphatase, and RNA 5′-triphosphatase activities. J Virol, 78: 7833–7838.
Roth-Cross J K, Stokes H, Chang G, et al. 2009. Organ specific attenuation of Murine Hepatitis Virus Strain A59 by replacement of catalytic residues in the putative viral cyclic phosphodiesterase ns2. J Virol, 83: 3743–53.
Kozbial P Z, Mushegian A R. 2005. Natural history of S-adenosylmethionine-binding proteins. BMC Struct Biol, 5: 19.
Ksiazek T G, Erdman D, Goldsmith C S, et al. 2003. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med, 348: 1953–1966.
Martin J L, McMillan F M. 2002. SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold. Curr Opin Struct Biol, 12: 783–793.
Masters P S. 2006. The molecular biology of coronaviruses. Adv Virus Res, 66: 193–292.
Masters P S, Rottier P J M. 2005. Coronavirus reverse genetics by targeted RNA recombination. Curr Top Microbiol Immunol, 287: 133–159.
Putics A, Filipowicz W, Hall J, et al. 2005. ADP-ribose-1-monophosphatase, a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture. J Virol, 79: 12721–12731.
Ray D, Shah A, Tilgner M, et al. 2006. West Nile virus 5′-cap structure is formed by sequential guanine N-7 and ribose 2′-O methylations by nonstructural protein 5. J Virol 80: 8362–8370.
Sawicki S G, Sawicki D L, Siddell S G. 2007. A contemporary view of coronavirus transcription. J Virol, 81: 20–29.
Sawicki S G, Sawicki D L, Younker D, et al. 2005. Functional and genetic analysis of coronavirus replicase transcriptase proteins. PLoS Pathogens, 1: e39.
Sawicki D, Wang T, Sawicki S. 2001. The RNA structures engaged in replication and transcription of the A59 strain of mouse hepatitis virus. J Gen Virol, 82: 385–396.
Schelle B, Karl N, Ludewig B, et al. 2005. Selective replication of coronavirus genomes that express nucleocapsid protein. J Virol, 79: 6620–6630.
Siddell S G, Ziebuhr J, Snijder E J. 2005. Coronaviruses, toroviruses, and arteriviruses. In: Virology (Mahy B W J and ter Meulen V ed.), Hodder Arnold, London, United Kingdom: Topley & Wilson’s microbiology and microbial infections. p823–856.
Sturman L S, Eastwood C, Frana M F, et al. 1987. Temperature-sensitive mutants of MHV-A59. Adv Exp Med Biol, 218: 159–168.
Thiel V. 2007. Reverse genetic analysis of coronavirus replication. In: Coronaviruses: molecular and cellular biology (Thiel V. ed). Norfolk, United Kingdom: Caister Academic Press, p109–132.
Thiel V, Herold J, Schelle B, et al. 2001. Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome. J Gen Virol, 82: 1273–1281.
Thiel V, Siddell S G. 2005. Reverse genetics of coronaviruses using vaccinia virus vectors. Curr Top Microbiol Immunol, 287: 199–227.
Von Grotthuss M, Wyrwicz L S, Rychlewski L. 2003. mRNA cap-1-methyltransferase in the SARS genome. Cell, 113: 701–702.