Evolutionary strategies for MxA antiviral proteins to overcome breadth-specificity tradeoffs in Orthomyxovirus restriction

Abstract

Antiviral restriction factors such as MxA (myxovirus resistance protein A) inhibit a broad range of viruses. However, they face the challenge of maintaining this breadth as viruses evolve to escape their defense. Viral escape drives restriction factors to evolve rapidly, selecting for amino acid changes at their virus-binding interfaces to regain defense. The human innate immune protein MxA has been well-characterized as a restriction factor of orthomyxoviruses. MxA restriction of orthomyxoviruses depends on its binding to viral nucleoproteins (NPs). However, other biochemical details of its restriction mechanism are still poorly understood. Previously, positive selection analyses helped narrow down rapidly evolving residues in MxA loop L4 as being critical for NP binding and viral restriction. Subsequently, combinatorial mutagenesis in human MxA loop L4 identified amino acid variations within the mutational landscape of MxA rapidly evolving residues that conferred enhanced ‘super-restriction’ of the Thogotovirus (THOV) orthomyxovirus. However, MxA’s gain of THOV restriction appeared to correlate with the loss of restriction of another orthomyxovirus, H5N1 avian influenza virus (IAV). This suggested a tradeoff between MxA’s antiviral specificity and breadth. Here, I employed a modified combinatorial mutagenesis strategy to readily identify super-restrictor MxA variants with over ten-fold enhanced restriction of the avian IAV strain H5N1. Consistent with previous studies, a gain of H5N1 restriction led to reduced THOV restriction. However, I discovered two evolutionary strategies that enable restriction factors, such as MxA, to increase their restriction of diverse viruses to overcome breadth-specificity tradeoffs. First, I found rare ‘generalist’ super-restrictors with enhanced restriction of both viruses. Second, a heterozygous combination of ‘specialist’ super-restrictors, one against THOV and the other against H5N1, could simultaneously enhance restriction against two viruses. The findings presented here contribute to our understanding of the evolutionary arms race of the MxA-NP interaction, lend insights into the structure and biochemistry of their arms race interface, and reveal strategies to overcome breath-specificity tradeoffs that may be pervasive in host-virus conflicts.