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.M easles is still a leading cause of vaccine-preventable death among children. The virus exhibits extremely high levels of infectivity, witnessed by its ability to infect the rare susceptible individuals present in highly vaccinated populations successfully (1). Measles virus (MV) is a single-stranded RNA virus with a genome of negative polarity, 15,894 nucleotides in length (2). It is a typical member of the subfamily Paramyxovirinae, with a genome that encodes six transcription units expressing six structural and at least two nonstructural proteins. The program of gene expression and the functions of the genes were well reviewed previously (3) and in essence do not differ from those of the wellstudied members of this group, such as Sendai virus and human parainfluenza virus 5 (hPIV5).The MV substitution rate has been much debated (4-7) because of the relative stability of MV in the field, but only a single paper has made an estimate of the spontaneous mutation rate in vitro (8). The high spontaneous mutation rates for RNA viruses result in the generation of a viral quasispecies consisting of a swarm of different viruses (9). This has important consequences for the properties of these viruses, especially in relation to evolutionary adaptability and their potential to make cross-species jumps (9). It is thus important to assess the mutation rate of these RNA viruses. Several methods have been employed to measure the mutation rates, but it has generally not been recognized that the determination of the actual mutation rates is impossible unless a number of parameters that are difficult to establish have been ...

A novel twelve class fluctuation test reveals higher than expected mutation rates for influenza A viruses

“…Our reversion to fluorescence assay has several additional advantages over ones that rely on phenotypic markers such as drug or antibody resistance (Zhang et al, 2013). First, the marker was selectively neutral, as the mutant GFP and revertant wild type GFP viruses had equal fitness.…”

The mutation rates and mutational bias of influenza A virus

“…Fluctuation tests are sensitive for rare mutational events and avoid many of the issues with sequencing assays (18, 20–22). Our reversion to fluorescence assay has several additional advantages over ones that rely on phenotypic markers such as drug or antibody resistance (26). First, the marker was selectively neutral, as the mutant GFP and revertant wild type GFP viruses had equal fitness.…”

“…Because nonsense mutations are lethal and generally not propagated, their frequencies approximate the mutation rate in the prior replication cycle (25). The second is a Luria-Delbrück fluctuation test that scores reversion to fluorescence in virally encoded green fluorescent protein (GFP) mutants (26). The GFP method enabled interrogation of all 12 substitution mutation classes independently and under distinct replication conditions.…”

Synergizing vaccinations with therapeutics for measles eradication

“…Although experimental assessment recently revealed that MeV mutations rates in vivo are in the order of 1.8×10 −6 per base and replication event (115) and thus several orders of magnitude lower than originally assumed (116), escape mutations mediating viral resistance to an inhibitor will certainly emerge in the field. The precedence set by antiretroviral therapy suggests, for instance, that allosteric reverse transcriptase inhibitors are predominantly associated with primary site resistance (117, 118), while escape from inhibition by the peptidic entry inhibitor enfuvirtide can be based on primary and secondary resistance mechanisms (119-121).…”