Combinatorial mutagenesis of rapidly evolving residues yields super-restrictor antiviral proteins

Abstract

Antagonistic interactions drive host-virus evolutionary arms races, which often manifest as recurrent amino acid changes (i.e., positive selection) at their protein-protein interaction interfaces. Here, we investigated whether combinatorial mutagenesis of positions under positive selection in a host antiviral protein could enhance its restrictive properties. We tested approximately 700 variants of human MxA, generated by combinatorial mutagenesis, for their ability to restrict Thogotovirus (THOV). We identified MxA super-restrictors with increased binding to the THOV nucleoprotein (NP) target protein and 10-fold higher anti-THOV restriction relative to wild-type human MxA, the most potent naturally occurring anti-THOV restrictor identified. Our findings reveal a means to elicit super-restrictor antiviral proteins by leveraging signatures of positive selection. Although some MxA super-restrictors of THOV were impaired in their restriction of H5N1 influenza A virus (IAV), other super-restrictor variants increased THOV restriction without impairment of IAV restriction. Thus, broadly acting antiviral proteins such as MxA mitigate breadth-versus-specificity trade-offs that could otherwise constrain their adaptive landscape.

Fig 1. Combinatorial random mutagenesis of rapidly evolving sites in the MxA L4 alters antiviral potency against THOV.

(A) A positive selection–based mutagenesis scheme to obtain MxA combinatorial variants of the five rapidly evolving sites (arrowheads) in L4 (N = any nucleotide, S = G or C). (B). Random MxA variants are presented in order of increasing THOV fold restriction, with their sequence features summarized to the right. Nonrestrictive (“NR”) variants with lower restriction than wtMxA are shown below the dashed green line. These contain very diverse residues in the randomized L4 positions. Approximately 6% of the variants have equivalent restriction to wtMxA (green restrictors, or “R,”) shown between the pink and green dashed lines, all possess an F, Y, or W residue at position 561. L4 sequences of four variants that restrict THOV better than wtMxA (pink super-restrictors, or “SRs”) are shown above the pink dashed line. (C) Fold restriction of MxA variants recovered with F, Y, or W residues at position 561. All data points represent averages of minireplicon experiments done in triplicate for each variant; data available in S1 Data. Super-restrictors are shown in pink, restrictors are shown in green, and nonrestrictors are shown in gray. aa, amino acid; L4, L4 loop; NR, nonrestrictor; SR, super-restrictor; THOV, Thogotovirus; wtMxA, wild-type human MxA.

Fig 2. A modified mutagenesis strategy yields potent THOV super-restrictor variants.

(A) A modified combinatorial mutagenesis strategy with fixed F561 and four randomized L4 positions. Nonrestrictor (“NR”), restrictor (“R”), and super-restrictor (“SR”) variants are presented in order of increasing restriction activity (x-axis). Super-restrictors are shown in pink, restrictors are shown in green, and nonrestrictors are shown in gray. (B) Retesting of anti-THOV restriction activity of super-restrictor MxA variants (relative to the empty vector) obtained from the original five-site screen (pink) and the modified 4-site screen (black). The three most potent super-restrictors (blue) have 7- to 10-fold higher anti-THOV restriction activity relative to wtMxA. Data are presented as mean of three experiments (error bars ± SEM). For super-restrictor variants, restriction values were compared to the wtMxA control in the experiment *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (Mann-Whitney U test). Western blots indicate levels of expression of transfected MxA variants relative to a loading control (β-actin). (C) Dose responsiveness of super-restriction activity for MxA variants versus the wtMxA. Western blots indicate levels of expression of each of transfected MxA variants at various plasmid concentrations (top row) relative to a loading control (β-tubulin, bottom row). The data underlying this figure can be found in S1 Data, and original western blots are available in S1 Raw Data. L4, L4 loop; NR, nonrestrictor; SR, super-restrictor; THOV, Thogotovirus.

Fig 3. Restriction and biochemical properties of MxA variants in THOV-infected cells.

(A) Using THOV infection of Huh7 cells expressing different MxA variants, we compared THOV restriction provided by wtMxA, MxA super-restrictors, or MxA nonrestrictors (PFFSS or ΔL4) relative to a catalytically inactive MxA (T103A) mutant. Statistical significance of increased restriction of super-restrictors relative to wtMxA was ascertained using a one-way ANOVA test (*p ≤ 0.05). Western blots show the levels of MxA variants, THOV NP protein, and a loading control (β-actin). (B) Coimmunoprecipitation studies of viral NP target protein with different MxA variants using cell lysates of THOV-infected 293T cells transfected with the FLAG-tagged MxA expression constructs. MxA super-restrictors pull down more THOV NP protein than wtMxA does, whereas MxA nonrestrictors (PFFSS or ΔL4) pull down significantly lower amounts of THOV NP protein. Western blots of the FLAG-tagged MxA variants and THOV NP protein are shown. The data underlying this figure can be found in S1 Data, and original western blots are available in S1 Raw Data. IP, immunoprecipitation; L4, L4 loop; NP, nucleoprotein; THOV, Thogotovirus; wtMxA, wild-type human MxA.

Fig 4. Molecular basis of MxA super-restriction.

(A) Comparison of anti-THOV activity of Q540 (super-restrictor) to Q540G MxA variants. A G540Q mutation in wtMxA resulted in increased restriction (white versus green bar), whereas Q-to-G changes reduced super-restrictor activity of three super-restrictors (black versus gray bars). (B) Effect of all single-residue G540X mutations in wtMxA (green) on anti-THOV restriction relative to the empty vector. The catalytically inactive MxA, T103A, is a negative control. (C) Anti-THOV activity of super-restrictor QFQSM MxA variant compared to QFLSM and QFVSM MxA variants demonstrates the contribution of residue 564. (D) Effect of single-residue X540A changes in three MxA super-restrictor variants: HFSGR, TFAMC, and VFRSV. All variants are compared to wtMxA. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (Mann-Whitney U test). The data underlying this figure can be found in S1 Data, and original western blots are available in S1 Raw Data. n.s., not significant; THOV, Thogotovirus; wtMxA, wild-type human MxA.

Fig 5. MxA variant restriction of IAV and THOV.

(A, B) MxA “nonrestrictor” variants still encoding a necessary aromatic residue at position 561 were retested in minireplicon assays to measure restriction against THOV (A) and H5N1 IAV (B) viruses. Western blots showing the expression levels of FLAG-tagged MxA variants and a loading control (β-tubulin are presented). (C) Restriction activity of MxA five-site super-restrictor variants (pink) and four-site super-restrictor variants (black, blue) against the H5N1 strain of IAV in a minireplicon assay. MxA variants are grouped according to whether they were observed in the five-site (pink) or four-site (black, blue) screens and presented in order of increased anti-THOV restriction (from Fig 2B). In all panels, data are presented as means of three experiments (error bars ± SEM), and the IAV restriction of MxA variants is compared to wtMxA based on Mann-Whitney U tests (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). Fig 2B reports the expression levels for various MxA variants. (D) There is a weak inverse correlation between the THOV and IAV restriction activities for the three most potent anti-THOV super-restrictors—VFRSV, TFAMC, and QFAYS (blue circles)—compared to wtMxA (dashed line; Pearson linear correlation r² = 0.7559, p = 0.13), but this inverse correlation is not observed when comparing all MxA super-restrictors (solid line; r² = 0.01132, p = 0.61). The data underlying this figure can be found in S1 Data, and original western blots are available in S1 Raw Data. IAV, influenza A virus; n.s., not significant; THOV, Thogotovirus; wtMxA, wild-type human MxA.