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. Author manuscript; available in PMC: 2020 May 1.
Published in final edited form as: Nat Microbiol. 2019 Jul 22;4(11):1781–1789. doi: 10.1038/s41564-019-0513-7

Figure 4 |. Inter-segment RNA interactions drive IAV segment co-segregation during reassortment.

Figure 4 |

a, Inter-segment RNA interaction map for the Udorn virus, with the interaction between PB1 and NA segments highlighted in dark blue. Structure prediction for the highlighted PB1-NA interaction is shown on the right. Circled nucleotides highlight the bases that differ between the Udorn and Wyo03 strains. b, Competitive reverse-engineering of influenza viruses. Six plasmids encoding H1N1 background segments are transfected together with an H3N2 NA segment-encoding plasmid and PB1 segment-encoding plasmids from both H1N1 and H3N2 strains. The origin of the PB1 segment in the progeny viruses is determined using RT-qPCR. c, Co-segregation between H1N1 or H3N2 PB1 segments and H3N2 NA segments; P values as indicated, ANOVA with Sidak correction for multiple testing, n = 5 (Ud-NA), n = 3 (Mem71-NA and PC73-NA), and n = 8 (Wyo03-NA) biologically independent experiments; bar plot centre represents the mean, error bars indicate SEM. d, Preferential Wyo03 PB1 and NA segment co-segregation is recovered by substituting the 4 nucleotides that differ in Wyo03 NA from those in the PB1-interacting region of Udorn NA (highlighted in a) for a Udorn-like sequence; P values as indicated, ANOVA with Sidak correction for multiple testing, n = 7 (PR8 PB1 vs. Wyo03 PB1 competitions) and n = 8 (PR8 PB1 vs. Udorn PB1 competitions) biologically independent experiments; bar plot centre represents the mean, error bars indicate SEM. e, Substitution of the 4 Udorn-like nucleotides regenerates a strong inter-segment RNA interaction between the H3N2-origin PB1 and NA segments in reassortant viruses. Mem71, A/Memphis/1/71; Wyo03, A/Wyoming/3/03; PC73, A/Port Chalmers/73; Udorn, A/Udorn/307/72; PR8, A/Puerto Rico/8/34.