key: cord-0978999-7gytw8j4 authors: Chang, Guo-hui; Luo, Bao-jun; Lu, Pin; Lin, Lei; Wu, Xiao-yan; Li, Jing; Hu, Yi; Zhu, Qing-yu title: Construction and genetic analysis of murine hepatitis virus strain A59 Nsp16 temperature sensitive mutant and the revertant virus date: 2011-02-18 journal: Virol Sin DOI: 10.1007/s12250-011-3145-x sha: 60bc70262b3b7b861fa20bc136b558d50d8f0893 doc_id: 978999 cord_uid: 7gytw8j4 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. Approximately two-thirds of the Coronaviruses genome encodes the viral nonstructural proteins (Nsp) that are involved in viral RNA synthesis. The majority of these proteins are encoded in two 5 '-proximal overlapping open reading frames, ORF1a and ORF1b, translated as polyproteins, pp1a and pp1ab, which are then processed by virus-encoded proteinases into Nsp16 [4, 28, 32, 34] . Many of the Coronavirus Nsps have been shown or are predicted to have enzymatic functions [5, 11, 18, 26] . Nsp16 is predicted to be a Sadenosyl-methionine-dependent 2'-O-methyl transferase which is involved in the formation of viral 5'-cap structures [9, 16] . Whereas the exact role of Nsp16 during CoV replication is still unknown, its functional importance is supported by mutagenesis experiments using a SARS-CoV replicon system. The deletion of the Nsp16 coding sequence blocked RNA synthesis, whereas a single mutation in the catalytic tetrad reduced replicon driven mRNA synthesis to about 10% of the level for the wild type [37] . Mouse hepatitis virus (MHV) is a widely studied model system for Coronavirus replication and pathogenesis. The development of MHV reverse genetics, in particular the method based on the use of vaccinia virus cloning vectors, makes it suitable for analyzing Coronavirus RNA replication and transcription [2, 7, 8, 15, 25, 36] . In this study, we first used reverse genetics to create a MHV-A59 temperature sensitive (ts) mutant Wu"-ts18 (cd), which had the same amino acid change at Nsp16 position 12 (Pro to Ser) as ts mutant Wu"-ts18 [29, 33] . Then we cultured the ts mutant Wu"-ts18 (cd) at non-permissive temperatures which "forced" the ts recombinant virus to use second-site mutation to revert from a ts to a non-ts phenotype. Sequence analysis of the revertant provided us genetic information on the functional determinants of Nsp16. This allowed us to build up a more complete model of the functional replication -transcription complex. Mouse 17 clone 1 fibroblast cells (17Cl-1) were cultured at 37 ℃ in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL). BHK-MHV-N cells, kindly provided by professor Stuart G. Siddell [8, 31] , were cultured in minimal essential medium (MEM) supplemented with HEPES (25 mmol/L), 5% FBS, and antibiotics. Recombinant vaccinia virus (vv inf-MHV-A59) contained a full-length MHV-A59 cDNA (GenBank accession number AY700211) [8] . Wu"-ts18 was a reported ts mutant of wild type MHV-A59 [28, 33] . Wu"-ts18(cd) In order to obtain the revertants of ts mutants, we first constructed the recombinant ts mutants Wu"-ts18 (cd), which had the same amino acid change as Wu"-ts18. Three nucleotide changes (C 20880 to A, C 20881 to G, U 20882 to C) were made to reduce the likelihood of reversion to the wild-type sequence. Mutagenesis was done using a reverse genetics system described previously [8, 20, 36] . Briefly, two rounds of In vitro transcription from purified, EagI-cleaved Wu"-ts18(cd) and the parental virus vv inf-MHV-A59 using bacteriophage T7 RNA polymerase in the presence of an m7G(5')ppp(5')G cap was done as described previously [8, 19, 20, 35] . The full-length MHV Recombinant virus were analyzed for replication by using plaque assay as described previously [8, 20, 35] . Briefly, 17Cl-1 cells were infected with viruses at an multiplicity of infection (MOI) of 10 PFU/cell. The PCR reaction products were purified by ethanol precipitation using ammonium acetate. Finally, sequence analysis was done using primers P1-P121 in the Invitrogen Company (Invitrogen Beijing, China). Computer-assisted analysis of sequence data was done using the Lasergene bio-computing software (DNASTAR). Three nucleotides (C 20880 to A, C 20881 to G, U 20882 to C) were mutated to reduce the likelihood of reversion to the wild-type sequence, which "forced" the virus to use second-site mutations to revert from a ts phenotype to a non-ts one. To determine if the constructed mutant viruses Wu"-ts18 (cd) were ts phenotype, the diluted passage one stocks of Wu"-ts18 (cd) which came from one plaque were used to infect 17Cl-1 cells at permissive (33℃ or 37℃) and nonpermissive (39.5℃) temperature. After culturing for 2 or 3 days, the Wu"-ts18 (cd) virus had the same plaque size (6-7mm in diameter) and plaque morphology as the wild-type virus inf-MHV-A59 in 17Cl-1 cells, and it obtained nearly the same high titer of 1-3×10 9 PFU/mL as the wild-type virus inf-MHV-A59 at the permissive temperature 33℃ and 37℃ (Fig.1) (1-5×10 7 PFU/mL). After three rounds of plaque purification, the replication characterization of mutants Wu"-ts18 (cd) at different temperatures were tested on 17Cl-1 cells with an MOI=10. At permissive temperature 33℃ or 37℃, the Wu"-ts18 (cd) mutant was found to be indistinguishable from the inf-MHV-A59 with respect to replication kinetics (Fig.2) ; the supernatants from tissue cultures infected with Wu"-ts18 (cd) had reached titers that were equal to or greater than the titers of supernatants from the tissue cultures infected with the parental virus inf-MHV-A59. At 33℃, the Wu"-ts18 (cd) and inf-MHV-A59 were manifested as exponential growth until about 5-6 h p.i., reached the The data for the viruses (MHV-A59 and Wu"-ts18) were taken from reference [8, 36] . The functionally uncharacterized Nsp16, has previously been predicted to be a S-adenosyl- [9, 16, 28, 30] possessing the highly conserved catalytic tetrad (K-D-K-E) that is a hallmark of RNA 2'O-MTases [1, 6, 12, 16] . Structure [21, 23] . Research on West Nile virus (WNV) showed all residues within the K-D-K-E tetrad of the WNV MTase were essential for 2'-O methylation activityl residue D was more critical than other tetrad residues, mutants with a mutation at position 146 (D to E) were viable but exhibited small plaques [ 27] . TS mutants and revertants had been described for a large number of positive-strand RNA viruses [5, 11, 14, 17, 33] , the major advantage of using ts mutants is that [3] and VSV [6, 12, 13] . For WNV MTase, it has been shown that mutations abolishing the 2'O-Mtase activity had a detrimental effect on the replication of a luciferase expressing RNA replicon [27] . Mutagenesis Critical residues of Semliki Forest Virus RNA capping enzyme involved in methyltransferase and guanylyltransferaselike activities Construction coronavirus RNA synthesis The RNA helicase, nucleotide 5'-triphosphatase and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg 2+ dependent and require a functional Walker B motif in the helicase catalytic core Coronavirus genome structure and replication Mutagenesis of the murine hepatitis virus nsp1-coding region identifies residues important for protein processing, viral RNA synthesis, and viral replication 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 Reverse genetics system for the avian coronavirus infectious bronchitis virus Recombinant mouse hepatitis virus strain A59 from cloned, full-length cDNA replicates to high titers in vitro and is fully pathogenic in vivo Coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (Nucleoside-2'O)-methyltransferase activity Identification of a novel coronavirus in patients with severe acute respiratory syndrome High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants An RNA cap (nucleoside-2'O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization Structural and functional analysis of methylation and 5' RNA sequence requirements of short capped RNAs by the methyltransferase domain of dengue virus NS5 The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world Coronavirus reverse genetics and development of vectors for gene expression Novel SARS unique AdoMet-dependent methyltransferase Mutational analysis of the SARS virus Nsp15 endoribonuclease, identification of residues affecting hexamer formation Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity Human coronavirus 229E nonstructural protein 13, characterization of duplexunwinding, nucleoside triphosphatase, and RNA 5'-triphosphatase activities Organ specific attenuation of Murine Hepatitis Virus Strain A59 by replacement of catalytic residues in the putative viral cyclic phosphodiesterase ns2 Natural history of S-adenosylmethionine-binding proteins A novel coronavirus associated with severe acute respiratory syndrome SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold The molecular biology of coronaviruses genetics by targeted RNA recombination ADP-ribose-1-monophosphatase, a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture West Nile virus 5'-cap structure is formed by sequential guanine N-7 and ribose 2'-O methylations by nonstructural protein 5 A contemporary view of coronavirus transcription Functional and genetic analysis of coronavirus replicase transcriptase proteins The RNA structures engaged in replication and transcription of the A59 strain of mouse hepatitis virus Selective replication of coronavirus genomes that express nucleocapsid protein The authors thank Volker Thiel and S.G Siddel. for helpful discussions and excellent technical assistance.