Experimental Evolution Reveals a Genetic Basis for Membrane-Associated Virus Release

Figures

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  Fig. 1.. Directed evolution of membrane-associated viral shedding. (A) Scheme of the evolution protocol. Free virions and fast-sedimenting MAVPs were separated by low-speed centrifugation. S (supernatant) or P (pellet) fractions were used for inoculating fresh cells, and this process was iterated 20 times. A different P selection regime consisted of treating P fractions with Triton X-100 to disrupt membranes and release free particles (bp lines). Furthermore, we explored a variant of the MAVP selection regime by using annexin V for purifying phosphatidylserine-rich particles from P fractions (p* and bp* lines, not shown). (B) Titers of the S and P fractions for the WT founder and each of the evolved lines. The dashed line indicates the P/S titer ratio of the founder. (C) P/S titer ratio for the founder virus and evolved lines. Error bars correspond to the standard error of the mean (SEM, n = 3 replicates). Asterisks indicate the statistical significance of a log-scale t-test against the founder (ns, nonsignificant; **P < 0.01).

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  Fig. 2.. Potential fitness costs of membrane-associated viral shedding. (A) Effect of Triton X-100 treatment on the titer of raw infection media, S and P fractions for the WT founder (D: detergent; ∅: untreated controls). Error bars correspond to the SEM (n = 3 replicates). Asterisks indicate the statistical significance of a log-scale t-test against the founder (ns, nonsignificant; ***P < 0.001). (B) Increase in the number of PFUs as a function of inoculum incubation time in standard plaque assays using S-fraction, P-fraction, and bP inocula. Regression lines are shown (a: slope; r2: coefficient of determination). Error bars correspond to the SEM (n = 3 replicates).

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  Fig. 3.. Genetic analysis of the evolved lines. The indicated mutations were introduced in a WT infectious cDNA clone. Mutations that emerged in s lines are shown in red and those evolved in p, bp, p*, or bp* lines are shown in blue. (A) Titers of the P and S fractions. The dashed line indicates the P/S titer ratio for the WT. (B) P/S titer ratio for the WT and each of the mutants. (C) P*/S titer ratio for the WT and N63H mutant. (D) Titer of the P fraction and of the P fraction after Triton X-100 treatment (bP). The dashed line indicates the bP/P titer ratio for the WT. (E) bP/P titer ratio for the WT and each of the mutants. Error bars correspond to the SEM (n = 3 replicates). Asterisks indicate the statistical significance of a log-scale t-test against the founder (ns, nonsignificant; *P < 0.05, **P < 0.01, and ***P < 0.001).

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  Fig. 4.. Effects of the N63H mutation on viral titer, spread, and fitness. (A) One-step growth curves initiated with 10 PFU/cell of S-fraction virus. (B) Cytotoxicity curve (10 PFU/cell of S fraction). (C) Viral spread in cultures inoculated at 0.001 PFU/cell, quantified by automated real-time fluorescence microscopy using CVB3-mCherry viruses (left: fluorescence-based growth curve; right: representative images taken at 4× magnification in the phase contrast and red channels). (D) Head-to-head competition assays between N63H and WT using eGFP and mCherry reporters (1:1 input ratio, 0.001 PFU/cell at inoculation). The red to total fluorescent areas at 20 hpi are shown for each competition. Error bars correspond to the SEM (n = 3 replicates). Asterisks indicate the statistical significance of a log-scale t-test against the founder (**P < 0.01 and ***P < 0.001). (E) Plaques in agar semisolidified medium with or without MgCl2.