Existing Host Range Mutations Constrain Further Emergence of RNA Viruses

Abstract

RNA viruses are capable of rapid host shifting, typically due to a point mutation that confers expanded host range. As additional point mutations are necessary for further expansions, epistasis among host range mutations can potentially affect the mutational neighborhood and frequency of niche expansion. We mapped the mutational neighborhood of host range expansion using three genotypes of the double-stranded RNA (dsRNA) bacteriophage φ6 (wild type and two isogenic host range mutants) on the novel host Pseudomonas syringae pv. atrofaciens. Both Sanger sequencing of 50 P. syringae pv. atrofaciens mutant clones for each genotype and population Illumina sequencing revealed the same high-frequency mutations allowing infection of P. syringae pv. atrofaciens. Wild-type φ6 had at least nine different ways of mutating to enter the novel host, eight of which are in p3 (host attachment protein gene), and 13/50 clones had unchanged p3 genes. However, the two isogenic mutants had dramatically restricted neighborhoods: only one or two mutations, all in p3. Deep sequencing revealed that wild-type clones without mutations in p3 likely had changes in p12 (morphogenic protein), a region that was not polymorphic for the two isogenic host range mutants. Sanger sequencing confirmed that 10/13 of the wild-type φ6 clones had nonsynonymous mutations in p12, and 2 others had point mutations in p9 and p5. None of these genes had previously been associated with host range expansion in φ6. We demonstrate, for the first time, epistatic constraint in an RNA virus due to host range mutations themselves, which has implications for models of serial host range expansion.IMPORTANCE RNA viruses mutate rapidly and frequently expand their host ranges to infect novel hosts, leading to serial host shifts. Using an RNA bacteriophage model system (Pseudomonas phage φ6), we studied the impact of preexisting host range mutations on another host range expansion. Results from both clonal Sanger and Illumina sequencing show that extant host range mutations dramatically narrow the neighborhood of potential host range mutations compared to that of wild-type φ6. This research suggests that serial host-shifting viruses may follow a small number of molecular paths to enter additional novel hosts. We also identified new genes involved in φ6 host range expansion, expanding our knowledge of this important model system in experimental evolution.

Keywords: RNA virus; entropy; epistasis; host range mutations.

FIG 1

Two-dimensional schematic representing mutational neighborhoods of φ6 P3. Circles represent P3 mutational neighborhoods of the mutants, which are the centers of circles. The geometric shapes are known P3 mutants. It is assumed that they are distributed on the mutational neighborhood (circles) in a nonrandom way. The arrows are events, such as host range expansion. PA, P. syringae pv. atrofaciens; PT, P. syringae pv. tomato.

FIG 2

Change in Shannon entropy in the medium segment of φ6-WT, φ6-E8G, and φ6-G515S. Positions labeled correspond to amino acid positions in P3. Coding regions of the medium segment are aligned to the graphs. The x axis corresponds to the nucleotide positions on the medium segment.

FIG 3

Change in Shannon entropy in the small segment of φ6-WT, φ6-E8G, and φ6-G515S. Coding regions of the small segment are aligned to the graphs. Positions labeled correspond to amino acid positions in aligned genes. The x axis corresponds to the nucleotide positions on the small segment.

FIG 4

Change in Shannon entropy in large segment of φ6-WT, φ6-E8G, and φ6-G515S. Coding regions of the large segment are aligned to the graphs. The x axis corresponds to the nucleotide positions on the large segment.

FIG 5

P. syringae pv. atrofaciens host range mutation frequency of φ6-WT (4 h), φ6-WT, φ6-E8G, and φ6-G515S. Values are measured from four purified single plaques. Plaques used for mutational neighborhood mapping are in black.

FIG 6

Relative fitness of host range mutants on P. syringae pv. phaseolicola. Same-color genotypes share the same genetic background (φ6-WT, gray; φ6-E8G, yellow; φ6-G515S, green). Values are averages of six replicates each; error bars are standard deviations.