Evolution-guided design of super-restrictor antiviral proteins reveals a breadth-versus-specificity tradeoff

Background:

Mammalian cells encode a wide variety of antiviral proteins that exploit various mechanisms to impair viral infection. A single host species (for example, humans) must contend with a range of viral pathogens, providing a strong evolutionary pressure on immune proteins to maintain activity in the face of rapidly evolving viral substrates. This “evolutionary arms race” drives the rapid evolution of sequences at key interaction sites in viral and immune proteins, as viruses mutate to avoid immune restriction and immune proteins evolve to regain restrictive capacity. These key interaction sites can therefore be identified by computational analysis of substitution rates among orthologues of an immune gene. If the substitution rate of nucleotides that results in amino acid changes is higher than synonymous (amino-acid preserving) substitutions, the site is under “positive selection”, while those where synonymous changes are more frequent are under “negative” or “purifying” selection. Previous work had identified sites of positive selection in the antiviral protein MxA, and substitutions at one of these sites explained species differences in viral restrictive capacity of different MxA orthologues [Mitchell and Patzina 2012]. Here, Colón-Thillet et al use deeper combinatorial mutagenesis studies to further characterize the roles of positively selected sites in MxA antiviral activity.

 

Key Findings:

• Most combinations of substitutions impair MxA activity, but a few have “super-restrictor” activity

The authors use a combinatorial mutagenesis approach to screen multiple simultaneous substitutions in the L4 loop of MxA, rather than individual amino acid substitutions. Using a cell-based assay for replication of Thogoto virus (THOV), they showed that the vast majority (95%) of variants had reduced THOV restriction activity. This is consistent with the suggestion from positive selection that these sites are important for antiviral activity, and so many mutations should reduce activity. Nonetheless, several amino acid combinations had increased restriction activity relative to the already extremely potent wild-type MxA – the authors call these “super-restrictors”.

• Super-restrictors are dependent on epistatic interactions between different residues

The few super-restrictors found in the initial screen all shared an aromatic amino acid at position 561, but aromatic variants at 561 were not always sufficient in other combinations to enhance activity. This suggested that other residues may contribute to enhanced activity. Indeed, by fixing position 561 and re-varying the other positions, the authors identified further super-restrictors. However, no individual substitution tested was sufficient to enhance activity to the extent of the combinatorial super-restrictors. In fact, some (the authors explore the WT-enhancing 540A substitution as an example) could decrease the activity of super-restrictors, despite enhanced activity in isolation. This demonstrated that equally potent super-restrictors would be unlikely to be discovered by testing single substitutions and combining favorable results from those simpler screens.

• Increased specificity of super-restrictors suggests a breadth-vs-specificity tradeoff in MxA evolution

MxA restricts many viruses beyond THOV. The authors tested whether super-restrictor activity towards THOV would result in similarly increased activity against another virus, Influenza A (IAV). While some variants that had impaired THOV restriction were still capable of restricting IAV, none of the super-restrictors showed IAV restriction activity as potent as wild-type. Thus, there is a tradeoff between the ability of MxA to potently neutralize one virus and its breadth to serve as a “multi-tasking” antiviral protein. It seems likely that MxA is under evolutionary pressure to maintain breadth, rather than specializing to target individual viruses.

 

Importance:

This preprint illustrates the balance of evolutionary pressures acting on a key antiviral protein, MxA. By demonstrating the combinatorial activity of multiple positively selected sites for determining potency of MxA, the authors show how evolution-guided design may give even deeper insights into the ongoing arms race between the immune systems and pathogens. The breadth-specificity tradeoff identified suggests the importance of a broad antiviral repertoire and reveals how evolution has maximized the response to the varying pressures acting on the host immune system at MxA. This may serve as a broader paradigm for understanding the constraints on evolution of antiviral immunity in general.

 

Moving Forward / Questions for Authors

• Many interesting extensions of this approach to studying epistatic interactions and combinatorial effects of antiviral immunity might rely on more high-throughput approaches. For example, use of a fluorescent reporter for antiviral activity instead of luciferase would enable FACS-based selections that could expand throughput to enable screening of all possible L4 amino acid combinations (3.2 million total). Orthogonal reporters for different viruses (e.g. GFP for THOV and RFP for IAV) would enable exploration of the overall shape of the breadth-specificity tradeoff curve. However, not all assays are readily amenable to scale-up of this nature. It will be exciting to see if this viral restriction activity assay may be expanded to enable larger scale examination of activity.
• In some examples of protein evolution, an ancient “epistatic ratchet” constrains subsequent evolution [Bridgham 2009]. Is it possible that the sites available for positive selection have been “set” by an ancient evolutionary epistatic ratchet? For example, what are the relationships between MxA L4 residues and the positively selected loop in MxB and their respective “evolvabilities” for viral specificity and breadth? What about in other antiviral families, such as the TRIM or APOBEC families? One may imagine that members of a family have different breadth-specificity tradeoffs due to ancient epistatic sites that now no longer show as positively selected.
• Super-restrictors show decreased breadth against distinct viruses. Does this tradeoff hold for variants of a single virus? For example, is the minimal set of mutations in THOV to evade MxA restriction different for super-restrictors compared to wtMxA?

 

References:

• Mitchell P.S., Patzina C. et al. Evolution-Guided Identification of Antiviral Specificity Determinants in the Broadly Acting Interferon-Induced Innate Immunity Factor MxA. Cell Host Microbe 12(4) 598-604
• Cridgham J.T. et al. An epistatic ratchet constrains the direction of glucocorticoid receptor evolution. Nature 461(7263) 515-519

 

doi: https://doi.org/10.1242/prelights.9402

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