Coronaviruses (CoVs) are large single-stranded positive RNA viruses of the order Nidovirales, family Coronaviridae, genus Coronavirus [4, 32]. They are generally associated with respiratory and enteric infections and have long been recognized as important pathogens of livestock and companion animals[4, 24, 28]. The recent emergence of the severe acute respiratory syndrome coronavirus (SARS-CoV) and an increased awareness of the extent of human coronavirus-associated disease have renewed interest in this group of viruses [10, 22].
Coronaviruses are positive-stranded RNA viruses with genomes ranging in size from 27 to 32 kb. 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 S-adenosyl-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.
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.
Compared with the wild-type MHV-A59 virus, the ts mutant Wu"-ts18 failed to form plaques or synthesize viral RNA when infection was initiated and maintained at the non-permissive temperature 39.5℃. A single nucleotide change (C20880 to U, ) which induced the amino acid proline (Pro: CCU) change to Serine (Ser: UCU) of Nsp16 at position 12 was identified as the mutation responsible for the ts mutant phenotype. In order to identify the determinants which were important in the function of Nsp16. We first constructed the recombinant ts mutants Wu"-ts18(cd), which have the same amino acid change as Wu"-ts18. Three nucleotides (C20880 to A, C20881 to G, U20882 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 1-3×109 PFU/mL as the wild-type virus inf-MHV-A59 at the permissive temperature 33℃ and 37℃ (Fig. 1). However, at the non-permissive temperature 39.5℃, compared with the large plaque size formed by inf-MHV-A59, the plaque morphology formed by Wu"-ts18(cd) were heterogenous in plaque size, ranging from almost wild type (6-7mm in diameter), to intermediate (5-6mm in diameter) and small (2-3mm in diameter). The titer of the passage one virus stock was about 1~5×103PFU/ mL, far below that of wild-type virus inf-MHV-A59 (1-5×107PFU/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 highest titer (~108PFU/mL) at 12 h p.i., which were delayed somewhat compared with results at 37℃, which reached the highest titer (3-5×107PFU/mL) at 7 h p.i.. However, at the non-permissive temperature 39.5℃, inf-MHV-A59 had the normal titer, whereas Wu"-ts18 (cd) had a titer of 3-5×102 PFU/mL.
To analyze the phenotype of Wu"-ts18 (cd) in more detail, the efficiency of plating (EOP) of the two recombinant viruses were calculated by dividing the titer at 39.5℃ by the titer at 33℃ (Table 1). The EOP for inf-MHV-A59 virus was 1.25 which was indistinguishable from the wild type MHV-A59 virus (1.0), confirming that there was no impairment of growth at 39.5℃. In contrast, at 33℃, the recom-binant virus Wu"-ts18(cd) stock had a titer of 3-5×108 PFU/mL and at 39.5℃ had a titer of 1-2×102 PFU/mL, which was an EOP of ~10-7 which was similar to the EOP obtained with the original Wu-ts18 ts mutant. So as expected, the recombinant virus Wu"-ts18 (cd) was indistinguishable from the ts mutant Wu"-ts18 in terms of temperature sensitivity.
Table 1. Temperature sensitivity of recombinant Wu"-ts18(cd)
To obtain the revertant of ts mutants Wu"-ts18 (cd), 17C1-1cells were infected with serially diluted Wu"-ts18 (cd) at the non-permissive temperature (39.5℃), 48 h later, revertants were produced, causing large-sized plaques and revertants with noticeably smaller plaques. We picked several plaques into separate wells (including large, intermediate and small size) for plaque purification and after three rounds of plaque purification at the non-permissive temperature (39.5℃), some large (wild-type sized) plaque size revertants consistently formed large wild-type plaques (R1, R3, R4, R7, R8, Fig. 3A); some intermediate and small plaque size revertants reverted to large wild-type plaques (R2, R5, R6), and only one small plaque size revertant consistently formed small plaques (R9, Fig. 3B) which was clearly distinguishable from the wild-type plaques.
Figure 3. Plaque assay of revertant virus R8 and R9 of Wu"-ts18 (cd) at the non-permissive (39.5℃) temperatures. After 2 days incubated at 39.5℃, R8 plaques were 5-7 mm in diameter, and R9 plaques were 2-3mm in diameter.
After three rounds plaque purification, the revertant plaques were then expanded at 39.5℃ in T25 flasks, and total cellular RNA was obtained. Each isolated virus was subjected to RT-PCR and the entire coding region of the replicase genes (ORF1a and ORF1b) was sequenced as described. Sequencing results (Table 2) showed that all the revertants had retained the introduced Nsp16 position 12 mutation. The most frequent amino acid change that reverted from the ts virus to non-ts phenotype was Nsp16 position 43 (Asn to Ser). Seven of nine revertants had the same amino acid change at Nsp16 position 43 and the presence of this mutation alone reverted from the ts mutant Wu"-ts18 (cd) to non-ts phenotype (such as R1). However, the single amino acid change at Nsp16 position 76 (Lys to Glu) or position 130 (Asp to Asn) produced the same result (revertants R2 and R3). We also obtained revertants with the position 43 mutation plus another mutation in Nsp16, such as R4, R5, R6 and R7. The R8 and R9 had the same mutation at position 43 (Asn to Ser), but they formed large plaque size and small plaque size respectively. Finally, the whole genome of R8 and R9 were sequenced, the results showed there was only one amino acid difference between the revertant R8 and R9. The small plaque isolate R8 revealed an additional mutation in Nsp13 at position 115 (Thr to Ile).
Table 2. Sequence analysis of the Wu"-ts18 (cd) revertants
The functionally uncharacterized Nsp16, has previously been predicted to be a S-adenosyl-Lmethionine (AdoMet)-dependent RNA nucleoside-2'-O-methyltransferase (2'O-MTase) [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 and sequence comparisons of the 2'O-MTases suggest that the conserved K-D-K-E tetrad formed the active site for the 2'-O methyl transfer reaction. Sequence alignment results showed the amino acid K46-D130-K170-E203 of MHV-A59 Nsp16 corresponded to the conserved K-D-K-E active site. It was notable that the most mutations of the revertants were at the position 43 of Nsp16, this site was very close to the conserved catalytic site position 46. Though the revertants R4, R5, R6 and R7 had the mutation at position 43 (Asn to Ser) plus another mutation within Nsp16. There was no reason to attribute these additional changes to the revertant phenotype, especially as they were all well away from catalytic positions. The revertants R3 had a single mutation at Nsp16 postion 130, this was a catalytic residue which formed the conserved catalytic tetrad (K-D-K-E)[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.
Together, these sequencing results identified the single mutation at Nsp16 position 43 (Asn to Ser) or position 76 (Lys to Glu) and position 130 (Asp to Asn) had the ability to revert from the ts mutant Wu"-ts18(cd) to non-ts phenotype, and formed large wild-type plaques; an additional independent mutation in Nsp13 (superfamily 1 helicase) at position 115 (Thr to Ile) could also play an important role on plaque size, being associated with the small plaque phenotype. To date, this is the first report of a Nsp12 ts mutant virus and this mutant can be used to study the role of Nsp13 in MHV virus replication.