MHV-Wü18 is an RNA-negative, temperature-sensitive mutant of mouse coronavirus, strain murine hepatitis virus (MHV)-A59. We have previously identified the putative causal mutation of MHV-Wü18 as a C to U transition at codon 2446 in ORF1b, which results in a substitution of proline 12 with serine in non-structural protein 16. Here, we have used a vaccinia virus-based reverse genetic system to produce a recombinant virus, inf-MHV-Wü18 that encodes nsp16 serine 12 with AGC rather than UCU; a difference that facilitates the isolation of second-site revertants. Sequence analysis of nine inf-MHV-Wü18 revertant viruses suggests that their phenotype is most probably due to the intra-molecular substitution of amino acids in nsp16. However, the revertant viruses displayed different plaque sizes and whole genome sequencing of two revertants showed that they were isogenic apart from a mutation in nsp13. These results are discussed in the context of a model of coronavirus MHV nsp16 structure.


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vol. , part 1, pp. 122 - 127

Sequence-based alignment of the nsp16 2' -MTase proteins of representative alpha-, beta- and gammacoronaviruses

Secondary structure prediction of the MTase core domain of the MHV nsp16 protein

The accuracy of the FIPV and MHV nsp16 models was assessed by using verify3d

Sequence analysis of inf-MHV-Wü18(AGC) revertants [Single PDF file](101 KB)

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