Existing host range mutations constrain further emergence of RNA viruses

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 6 (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 6 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 6 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 6. 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.