Mutational Analysis of Measles Virus Suggests Constraints on Antigenic Variation of the Glycoproteins

Abstract

Measles virus undergoes error-prone replication like other RNA viruses, but over time, it has remained antigenically monotypic. The constraints on the virus that prevent the emergence of antigenic variants are unclear. As a first step in understanding this question, we subjected the measles virus genome to unbiased insertional mutagenesis, and viruses that could tolerate insertions were rescued. Only insertions in the nucleoprotein, phosphoprotein, matrix protein, as well as intergenic regions were easily recoverable. Insertions in the glycoproteins of measles virus were severely under-represented in our screen. Host immunity depends on developing neutralizing antibodies to the hemagglutinin and fusion glycoproteins; our analysis suggests that these proteins occupy very little evolutionary space and therefore have difficulty changing in the face of selective pressures. We propose that the inelasticity of these proteins prevents the sequence variation required to escape antibody neutralization in the host, allowing for long-lived immunity after infection with the virus.

Figure 1. Insertional mutagenesis of MeV genome

(A) Mutant viruses were rescued by transfecting BSR-T7 cells with the transposon library and then co-culturing the cells with A549s. The resulting viruses were passaged on A549 cells and viral RNA was sequenced. (B–D) The input library and both passages were subjected to deep sequencing. The total sequencing coverage of the genome is shown on the left panels, while the number of reads containing transposon insertions are indicated on the right. The numbers along the x-axis of the graphs indicate the genomic nucleotide position. The red bars under the genome diagrams in B–D indicate the individual RT-PCR products amplified for Illumina sequencing. Dashed lines indicate a threshold of 0.01% of the total reads.

Figure 2. MeV glycoproteins and polymerase genes are intolerant of insertions

(A) Plots representing individual insertion sites. Individual viruses in the input and the passages are represented by different colors; the thickness of the lines is representative of the proportion in the population. The MeV genomes are drawn either to scale (I), distorted to represent the actual coverage of insertions in the input (II), or distorted to represent the total percentage of reads in each region after the second passage (III). (B) Percent of reads in a region divided by the size of that region to give the fold over predicted values (as if there was no biological selection). Under-represented areas are displayed as negative values (red) while over-represented areas are displayed as positive values (green). (C) Individual insertion sites were cloned into a MeV+3 GFP construct to validate the sequencing results. 2x Stop indicates the presence of an additional stop codon after the normal stop codon of the N gene. (D) Top hits for the screen, plus the top hits for each region were cloned and rescued. For each panel title, the letter represents the genomic region while the number represents the genomic nucleotide position preceding the insertion. Scale bar=400μm. (E) Growth curves of viruses with insertions in the indicated sites. Values and error bars represent the mean and standard error of the mean, respectively. Green filled circles and line=Parental MeV.

Figure 3. Comparative analysis reveals MeV glycoproteins are exceptionally resistant to insertional mutation

The total number of sites that could tolerate insertions in each region were normalized to region size and graphed. Red columns indicate the major viral surface glycoproteins. (A) Influenza A virus. The HA1 head domain and HA2 stalk domain of the influenza A virus hemagglutinin are separated in the insert. (B) VEEV (C) HCV. The hyper-variable region of E2 is separated from the rest of the E2 protein in the insert. (D) MeV. All values are out of an arbitrary value of 1.