Determination of spontaneous mutation frequencies in measles virus under nonselective conditions

Abstract

There is a paradox between the remarkable genetic stability of measles virus (MV) in the field and the high mutation rates implied by the frequency of the appearance of monoclonal antibody escape mutants generated when the virus is pressured to revert in vitro (S. J. Schrag, P. A. Rota, and W. J. Bellini, J. Virol. 73:51-54, 1999). We established a highly sensitive assay to determine frequencies of various categories of mutations in large populations of wild-type and laboratory-adapted MVs using recombinant viruses containing an additional transcription unit (ATU) encoding enhanced green fluorescent protein (EGFP). Single and double mutations were made in the fluorophore of EGFP to ablate fluorescence. The frequencies of reversion mutants in the population were determined by measuring the appearance of fluorescence indicating a revertant virus. This allows mutation rates to be measured under nonselective conditions, as phenotypic reversion to fluorescence requires only either a single- or a double-nucleotide change and amino acid substitution, which does not affect the length of the nonessential reporter protein expressed from the ATU. Mutation rates in MV are the same for wild-type and laboratory-adapted viruses, and they are an order of magnitude lower than the previous measurement assessed under selective conditions. The actual mutation rate for MV is approximately 1.8 × 10(-6) per base per replication event.

Fig 1

Design of EGFP^KO viruses. (A) Schematic representation of the genomes of the recombinant wild-type viruses encoding EGFP in an additional transcription unit in position 1, i.e., before the gene encoding the nucleocapsid (N) protein. EGFP^KO viruses for the laboratory-adapted strain expressed modified EGFP proteins from an ATU in the same position. The genome order from the 3′ end to the 5′ end is as follows: EGFP-nucleocapsid protein gene (N)-phosphoprotein gene (P/V/C)-matrix protein gene (M)-fusion protein gene (F)-hemagglutinin protein (H)-large protein (L). (B) Nucleotide (nt) and protein sequences of the fluorophore in the EGFP^KO viruses. aa, amino acids. (C) False-color images of green fluorescence detection in cell monolayers infected with wild-type, EGFP^KO, and revertant viruses. (D) Nucleotide and protein sequences of the fluorophore and genetic tag sequences of the wild type and a revertant virus.

Fig 2

Replication kinetics of the wild type and a representative EGFP^KO virus. Virus titers (50% tissue culture infective dose [TCID₅₀]) were measured in the supernatant (SN) and cell-associated (CA) fractions of Vero-cCD150 cells infected with wild-type rMV^IC323EGFP and the EFGP^KO virus rMV^IC323EGFP^a315c.

Fig 3

Development of the assay. (A) Effect of the fusion-inhibitory peptide (FIP). In the absence of FIP, syncytia form in Vero-cCD150 cell monolayers, which prevents enumeration of the revertant viruses. With FIP, the dynamic range for the measurement of revertant frequency is 1 to 1,000. At a higher magnification, paired green cells (the result of cell division) are detected. These are counted as single events in the measurement of mutation frequency in virus populations. (B) Establishment of the maximum MOI that results in discrete, countable infectious centers, reflecting the actual number of PFU used for the initial infection. Wells of a 96-well plate containing approximately 40,000 cells per well were infected at the indicated MOIs.

Fig 4

Mutation frequencies. (A) Mutation frequencies of specific transitions and transversions. Closed circles refer to the rates found with the rMV^IC323EGFP^KO viruses. Open circles refer to mutation frequencies established for the rMV^EdtagEGFP^stop viruses. (B) Tabulated data for reversion of a range of EGFP^KO mutant viruses.