Abstract
Targeted RNA recombination was the first reverse genetics system devised for coronaviruses at a time when it was not clear whether the construction of full-length infectious cDNA clones would become possible. In its current state targeted RNA recombination offers a versatile and powerful method for the site-directed mutagenesis of the downstream third of the coronavirus genome, which encodes all the viral structural proteins. The development of this system is described, with an emphasis on recent improvements, and multiple applications of this technique to the study of coronavirus molecular biology and pathogenesis are reviewed. Additionally, the relative strengths and limitations of targeted RNA recombination and infectious cDNA systems are contrasted.
Keywords
- Infectious Bronchitis Virus
- Mouse Hepatitis Virus
- Reverse Genetic System
- Defective Interfere
- Murine Coronavirus
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References
Almazán F, González JM, Pánzes Z, Izeta A, Calvo E, Plana-Durán J, Enjuanes L (2000) Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci USA 97:5516–5521
Banner LR, Keck JG, Lai MMC (1990) A clustering of RNA recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus. Virology 175:548–555
Banner LR, Lai MMC (1991) Random nature of coronavirus RNA recombination in the absence of selective pressure. Virology 185:441–445
Baric RS, Fu K, Schaad MC, Stohlman SA (1990) Establishing a genetic recombination map for murine coronavirus strain A59 complementation groups. Virology 177:646–656
Brian DA, Spaan WJM (1997) Recombination and coronavirus defective interfering RNAs. Semin Virol 8:101–111
Casais R, Thiel V, Siddell SG, Cavanagh D, Britton P (2001) Reverse genetics system for the avian coronavirus infectious bronchitis virus. J Virol 75:12359–12369
Cavanagh D, Davis P, Cook J, Li D (1990) Molecular basis of the variation exhibited by avian infectious bronchitis coronavirus (IBV). Adv Exp Med Biol 276:369–372
Chang R-Y, Hofmann MA, Sethna PB, Brian DA (1994) A cis-acting function for the coronavirus leader in defective interfering RNA replication. J Virol 68:8223–8231
Curtis KM, Yount B, Baric RS (2002) Heterologous gene expression from transmissible gastroenteritis virus replicon particles. J Virol 76:1422–1434.
Das Sarma J, Fu L, Tsai JC, Weiss SR, Lavi E (2000) Demyelination determinants map to the spike glycoprotein gene of coronavirus mouse hepatitis virus. J Virol 74:9206–9213
Das Sarma J, Scheen E, Seo SH, Koval M, Weiss SR (2002) Enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system. J Neurovirol 8:381–391
deHaan CAM, Kuo L, Masters PS, Vennema H, Rottier PJM (1998) Coronavirus particle assembly: primary structure requirements of the membrane protein. J Virol 72:6838–6850
de Haan CAM, Masters PS, Shen X, Weiss S, Rottier PJM (2002a) The group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host. Virology 296:177–189
de Haan CAM, Volders H, Koetzner CA, Masters PS, Rottier PJM (2002b) Coronaviruses maintain viability despite dramatic rearrangements of the strictly conserved genome organization. J Virol 76:12491–12502
de Haan CAM, van Genne L, Stoop JN, Volders H, Rottier PJM (2003) Coronaviruses as vectors: position dependence of foreign gene expression. J Virol 77:11312–11323
Fischer F, Peng D, Hingley ST, Weiss SR, Masters PS (1997a) The internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication. J Virol 71:996–1003
Fischer F, Stegen CF, Koetzner CA, Masters PS (1997b) Analysis of a recombinant mouse hepatitis virus expressing a foreign gene reveals a novel aspect of coronavirus transcription. J Virol 71:5148–5160
Fischer F, Stegen CF, Masters PS, Samsonoff WA (1998) Analysis of constructed E gene mutants of mouse hepatitis virus confirms a pivotal role for E protein in coronavirus assembly. J Virol 72:7885–7894
Fu K, Baric RS (1994) Map locations of mouse hepatitis virus temperature-sensitive mutants: confirmation of variable rates of recombination. J Virol 68:7458–7466
Godeke GJ, de Haan CA, Rossen JW, Vennema H, Rottier PJM (2000) Assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein. J Virol 74:1566–1571
Goebel SJ, Hsue B, Dombrowski TF, Masters PS (2004) Characterization of the RNA components of a putative molecular switch in the 3′ untranslated region of the murine coronavirus genome. J Virol 78:669–682
Haijema BJ, Volders H, Rottier PJM (2003) Switching species tropism: an effective way to manipulate the feline coronavirus genome. J Virol 77:4528–4538
Herrewegh AA, Vennema H, Horzinek MC, Rottier PJM, de Groot RJ (1995) The molecular genetics of feline coronaviruses: comparative sequence analysis of the ORF7a/7b transcription unit of different biotypes. Virology 212:622–631
Herrewegh AA, Smeenk I, Horzinek MC, Rottier PJM, de Groot RJ (1998) Feline coronavirus type II strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type I and canine coronavirus. J Virol 72:4508–4514
Hingley ST, Leparc-Goffart I, Seo SH, Tsai JC, Weiss SR (2002) The virulence of mouse hepatitis virus strain A59 is not dependent on efficient spike protein cleavage and cell-to-cell fusion. J Neurovirol 8:400–410
Hsue B, Masters PS (1997) A bulged stem-loop structure in the 3′ untranslated region of the genome of the coronavirus mouse hepatitis virus is essential for replication. J Virol 71:7567–7578
Hsue B, Masters PS (1999) Insertion of a new transcriptional unit into the genome of mouse hepatitis virus. J Virol 73:6128–6135
Hsue B, Hartshorne T, Masters PS (2000) Characterization of an essential RNA secondary structure in the 3′ untranslated region of the murine coronavirus genome. J Virol 74:6911–6921
Jia W, Karaca K, Parrish CR, Naqi SA (1995) A novel variant of avian infectious bronchitis virus resulting from recombination among three different strains. Arch Virol 140:259–271
Keck JG, Stohlman SA, Soe LH, Makino S, Lai MMC (1987) Multiple recombination sites at the 5′-end of murine coronavirus RNA. Virology 156:331–341
Keck JG, Matsushima GK, Makino S, Fleming JO, Vannier DM, Stohlman SA, Lai MMC (1988a) In vivo RNA-RNA recombination of coronavirus in mouse brain. J Virol 62:1810–1813
Keck JG, Soe LH, Makino S, Stohlman SA, Lai MMC (1988b) RNA recombination of murine coronaviruses: recombination between fusion-positive mouse hepatitis virus A59 and fusion-negative mouse hepatitis virus 2. J Virol 62:1989–1998
Kirkegaard K, Baltimore D (1986) The mechanism of RNA recombination in poliovirus. Cell 47:433–443
Koetzner CA, Parker MM, Ricard CS, Sturman LS, Masters PS (1992) Repair and mutagenesis of the genome of a deletion mutant of the coronavirus mouse hepatitis virus by targeted RNA recombination. J Virol 66:1841–1848
Kottier SA, Cavanagh D, Britton P (1995) Experimental evidence of recombination in coronavirus infectious bronchitis virus. Virology 213:569–580
Kuo L, Godeke G-J, Raamsman MJB, Masters PS, Rottier PJM (2000) Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier. J Virol 74:1393–1406
Kuo L, Masters PS (2002) Genetic evidence for a structural interaction between the carboxy termini of the membrane and nucleocapsid proteins of mouse hepatitis virus. J Virol 76:4987–4999
Kuo L, Masters PS (2003) The small envelope protein E is not essential for murine coronavirus replication. J Virol 77:4597–4608
Kusters JG, Jager EJ, Niesters HGM, van der Zeijst BAM (1990) Sequence evidence for RNA recombination in field isolates of avian coronavirus infectious bronchitis virus. Vaccine 8:605–608
Lai MMC, Baric RS, Makino S, Keck JG, Egbert J, Leibowitz JL, Stohlman SA (1985) Recombination between nonsegmented RNA genomes of murine coronaviruses. J Virol 56:449–456
Lai MMC (1992) RNA recombination in animal and plant viruses. Microbiol Rev 56:61–79
Lai MMC (1996) Recombination in large RNA viruses: coronaviruses. Semin Virol 7:381–388
Ledinko N (1963) Genetic recombination with poliovirus type 1: studies of crosses between a normal horse serum-resistant mutant and several guanidine-resistant mutants of the same strain. Virology 20:107–119
Lee CW, Jackwood MW (2000) Evidence of genetic diversity generated by recombination among avian coronavirus IBV. Arch Virol 145:2135–2148
Lee CW, Jackwood MW (2001) Spike gene analysis of the DE072 strain of infectious bronchitis virus: origin and evolution. Virus Genes 22:85–91
Leparc-Goffart I, Hingley ST, Chua MM, Phillips J, Lavi E, Weiss SR (1998) Targeted recombination within the spike gene of murine coronavirus mouse hepatitis virus-A59: Q159 is a determinant of hepatotropism. J Virol 72:9628–9636
Li K, Chen Z, Plagemann P (1999) High-frequency homologous genetic recombination of an arterivirus, lactate dehydrogenase-elevating virus, in mice and evolution of neuropathogenic variants. Virology 258:73–83
Luytjes W, Bredenbeek PJ, Noten AFH, Horzinek MC, Spaan WJM (1988) Sequence of mouse hepatitis virus A59 mRNA2: indications for RNA recombination between coronaviruses and influenza C virus. Virology 166:415–422
Makino S, Fleming JO, Keck JG, Stohlman SA, Lai MMC (1987) RNA recombination of coronaviruses: localization of neutralizing epitopes and neuropathogenic determinants on the carboxyl terminus of peplomers. Proc Natl Acad Sci USA 84:6567–6571
Masters PS, Koetzner CA, Kerr CA, Heo Y (1994) Optimization of targeted RNA recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis virus. J Virol 68:328–337
Masters PS (1999) Reverse genetics of the largest RNA viruses. Adv Virus Res 53:245–264
Méndez A, Smerdou C, Izeta A, Gebauer F, Enjuanes L (1996) Molecular characterization of transmissible gastroenteritis coronavirus defective interfering genomes: packaging and heterogeneity. Virology 217:495–507
Motokawa K, Hohdatsu T, Aizawa C, Koyama H, Hashimoto H (1995) Molecular cloning and sequence determination of the peplomer protein gene of feline infectious peritonitis virus type I. Arch Virol 140:469–480
Nagy PD, Simon A (1997) New insights into the mechanisms of RNA recombination. Virology 235:1–9
Navas S, Seo S-H, Chua MM, Das Sarma J, Lavi E, Hingley ST, Weiss SR (2001) Murine coronavirus spike protein determines the ability of the virus to replicate in the liver and cause hepatitis. J Virol 75:2452–2457
Neuman B, Cavanagh D, Britton P (2001) Use of defective RNAs containing reporter genes to investigate targeted recombination for avian infectious bronchitis virus. Adv Exp Med Biol 494:513–518
Ontiveros E, Kuo L, Masters PS, Perlman S (2001) Inactivation of expression of gene 4 of mouse hepatitis virus strain JHM does not affect virulence in the murine CNS. Virology 289:230–238
Ortego J, Escors D, Laude H, Enjuanes L (2002) Generation of a replication-competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome. J Virol 76:11518–11529
Parker MM, Masters PS (1990) Sequence comparison of the N genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein. Virology 179:463–468
Pasternak AO, van den Born E, Spaan WJM, Snijder EJ (2001) Sequence requirements for RNA strand transfer during nidovirus discontinuous subgenomic RNA synthesis. EMBO J 20:7220–7228
Peng D, Koetzner CA, Masters PS (1995a) Analysis of second-site revertants of a murine coronavirus nucleocapsid protein deletion mutant and construction of nucleocapsid protein mutants by targeted RNA recombination. J Virol 69:3449–3457
Peng D, Koetzner CA, McMahon T, Zhu Y, Masters PS (1995b) Construction of murine coronavirus mutants containing interspecies chimeric nucleocapsid proteins. J Virol 69:5475–5484
Phillips JJ, Chua MM, Lavi E, Weiss SR (1999) Pathogenesis of chimeric MHV4/MHV-A59 recombinant viruses: the murine coronavirus spike protein is a major determinant of neurovirulence. J Virol 73:7752–7760
Phillips JJ, Chua M, Seo SH, Weiss SR (2001) Multiple regions of the murine coronavirus spike glycoprotein influence neurovirulence. J Neurovirol 7:421–431
Phillips JJ, Chua MM, Rall GF, Weiss SR (2002) Murine coronavirus spike glycoprotein mediates degree of viral spread, inflammation, and virus-induced immunopathology in the central nervous system. Virology 301:109–120
Plyusnin A, Kukkonen SK, Plyusnina A, Vapalahti O, Vaheri A (2002) Transfection-mediated generation of functionally competent Tula hantavirus with recombinant S RNA segment. EMBO J 21:1497–1503
Rottier PJM (1995) The coronavirus membrane glycoprotein. In: Siddell SG (ed) The Coronaviridae. Plenum Press, New York, pp 115–139
Sánchez CM, Izeta A, Sánchez-Morgado JM, Alonso S, Sola I, Balasch M, Plana-Durán J, Enjuanes L (1999) Targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence. J Virol 73:7607–7618
Sethna PB, Hung S-L, Brian DA (1989) Coronavirus subgenomic minus-strand RNAs and the potential for mRNA replicons. Proc Natl Acad Sci USA 86:5626–5630
Shen X, Masters PS (2001) Evaluation of the role of heterogeneous nuclear ribonucleoprotein A1 as a host factor in murine coronavirus discontinuous transcription and genome replication. Proc Natl Acad Sci USA 98:2717–2722
Sturman LS, Eastwood C, Frana MF, Duchala C, Baker F, Ricard CS, Sawicki SG, Holmes KV (1987) Temperature-sensitive mutants of MHV-A59. Adv Exp Med Biol 218:159–168
Thiel V, Herold J, Schelle B, Siddell SG (2001) Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus. J Gen Virol 82:1273–1281
Tijms MA, van Dinten LC, Gorbalenya AE, Snijder EJ (2001) A zinc finger-containing papain-like protease couples subgenomic mRNA synthesis to genome translation in a positive-stranded RNA virus. Proc Natl Acad Sci USA 98:1889–1894
van der Most RG, Heijnen L, Spaan WJM, de Groot RJ (1992) Homologous RNA recombination allows efficient introduction of site-specific mutations into the genome of coronavirus MHV-A59 via synthetic co-replicating RNAs. Nucl Acids Res 20:3375–3381
van Dinten LC, den Boon JA, Wassenaar ALM, Spaan WJM, Snijder EJ (1997) An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription. Proc Natl Acad Sci USA 94:991–996
van Vugt JJ, Storgaard T, Oleksiewicz MB, Botner A (2001) High frequency RNA recombination in porcine reproductive and respiratory syndrome virus occurs preferentially between parental sequences with high similarity. J Gen Virol 82:2615–2620
Vennema H, Poland A, Floyd-Hawkins K, Pedersen NC (1995) A comparison of the genomes of FECVs and FIPVs and what they tell us about the relationships between feline coronaviruses and their evolution. Feline Pract 23:40–44
Vennema H, Godeke G-J, Rossen JWA, Voorhout WF, Horzinek MC, Opstelten D-J E, Rottier PJM (1996) Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes. EMBO J 15:2020–2028
Vennema H (1999) Genetic drift and genetic shift during feline coronavirus evolution. Vet Microbiol 69:139–141
Wang L, Junker D, Collisson EW (1993) Evidence of natural recombination within the S1 gene of infectious bronchitis virus. Virology 192:710–716
Williams GD, Chang RY, Brian DA (1999) A phylogenetically conserved hairpin-type 3′ untranslated region pseudoknot functions in coronavirus RNA replication. J Virol 73:8349–8355
Yount B, Curtis KM, Baric RS (2000) Strategy for systematic assembly of large RNA and DNA genomes: transmissible gastroenteritis virus model. J Virol 74:10600–10611
Yount B, Denison MR, Weiss SR, Baric RS (2002) Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59. J Virol 76:11065–11078
Yount B, Curtis KM, Fritz EA, Hensley LE, Jahrling PB, Prentice E, Denison MR, Geisbert TW, Baric RS (2003) Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci USA 100:12995–13000
Yuan S, Nelsen CJ, Murtaugh MP, Schmitt BJ, Faaberg KS (1999) Recombination between North American strains of porcine reproductive and respiratory syndrome virus. Virus Res 61:87–98
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Masters, P.S., Rottier, P.J.M. (2005). Coronavirus Reverse Genetics by Targeted RNA Recombination. In: Enjuanes, L. (eds) Coronavirus Replication and Reverse Genetics. Current Topics in Microbiology and Immunology, vol 287. Springer, Berlin, Heidelberg. https://doi.org/10.1007/3-540-26765-4_5
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