To understand MxA target recognition, we recently used an alternative approach that leverages the evolutionary history of host-virus interactions [23
]. Interactions between viral proteins and intracellular defenses represent key molecular battlegrounds that significantly influence host resistance or susceptibility. Because successful engagement by one party comes at the detriment of the other, host virus protein-protein interactions rapidly evolve to establish or evade recognition (). We can use signatures of rapid evolution to gain substantial molecular insights into host-virus interactions. Rapid evolution can be formalized by the dN/dS statistic, which calculates the observed rate of non-synonymous (dN) relative to synonymous (dS) nucleotide substitutions in an alignment of orthogonal sequences. dN/dS ratios higher than one are indicative of positive selection, whether calculated as an average over the entire gene or on a per codon basis. These “hotspots” of positive selection predict residues that significantly impact the affinity of host-viral interactions [24
]. Such studies have identified key domains/residues that determine either escape of some host antiviral proteins from viral antagonism or those that allow host proteins to recognize altered viral epitopes to maintain their antiviral function (reviewed in [24
An arms race between MxA and targeted viral proteins
The broad antiviral activity of MxA suggests that it has been involved in multiple arms race conflicts throughout its history (). Such a conflict-ridden past would be predicted to result in particularly strong evolutionary signatures of positive selection in MxA. Moreover, since MxA manifests its antiviral action via specific recognition of viral epitopes, “hotspots” of positive selection could therefore be used to directly identify at least some of the key determinants of MxA target recognition. In an analysis of MxA orthologs from simian primates, we identified the disordered loop L4, which protrudes from the stalk domain (), as one such “hotspot” of positive selection. We found that variation in L4 explains differences in antiviral activity among primate MxA orthologs against THOV and influenza A viruses (FLUAV). Human L4 grafted onto mouse Mx1 conferred both gain of antiviral activity and concomitant binding of Mx1 to the THOV NP. Intriguingly, MxA antiviral specificity for THOV is largely governed by a single amino acid F561 in L4, which has been recurrently mutated throughout primate MxA evolution. Thus, the loop L4 is a key determinant of MxA interaction with NP proteins from orthomyxoviruses.
Although the large phenotypic effect of a single amino acid change in MxA seems extraordinary, other studies on the evolutionary dynamics between host antiviral genes and viruses have also uncovered occurrences of positively selected (adaptive) single residue changes with profound impacts on host-virus interactions (for example, [26
]). The MxA structure, in combination with the evolutionary analysis, may also point to how single amino acid changes can have large functional outcomes. As described above, molecular modeling predicts that MxA oligomers form a ring-like complex. Interestingly, the loop L4 is positioned inward from the ring’s inner surface (), suggesting that in its oligomeric state small changes elicited by single amino acids may act cooperatively to produce significant effects in viral target binding and consequently antiviral activity. Interestingly, another restriction factor TRIMCyp recognizes markedly different viral epitopes by interconverting a disordered surface loop between multiple conformations [30
]. A similar mechanism may also afford MxA the flexibility to recognize divergent targets by virtue of the disordered nature of L4. MxA oligomerization and L4 flexibility may help explain how single residue changes in MxA L4 manifest such large effect phenotypes.
The specificity encoded by individual amino acids in MxA L4 provides a model to explain MxA recognition of multiple targets. Not only does the residue at 561 determine MxA antiviral specificity for THOV, but this specificity is also unperturbed by changes at distal or even neighboring amino acids that are also evolving under positive selection. This is intriguing since the MxA target interface is likely much broader than a single residue. Nonetheless, this finding does suggest a means by which MxA might maintain binding specificities to multiple targets. For example, whereas residue 561 may specify MxA specificity for THOV, other “hotspots” may coincide with distinct pathogen preferences that are independently specified. Moreover, other positively selected sites outside L4 may similarly act as specificity determinants for other viruses [24
]. Indeed, we predict that differences in MxA antiviral activity against non-orthomyxoviruses
could map outside L4.
The antiviral utility might be expected to be short-lived for proteins like MxA that recognize a specific target against rapidly evolving viruses. Insight into how MxA potentially circumvents this problem comes from the recent mapping of Mx-resistance residues on the influenza virus NP [6
]. Surface exposed residues were identified in the NP of human influenza viruses that were necessary and sufficient to confer protection against an Mx-sensitive avian H5N1 virus. Importantly, the number of residues required to gain Mx-resistance varied from 10 to four depending on whether amino acids were derived from the 2009 or 1918 pandemic H1N1 influenza virus strains. However, introducing MxA resistant mutations in the NP of avian H5N1 viruses caused significant attenuation of viral growth in the absence of MxA. Although this result is consistent with the more recent avian origin of the 1918 virus, it also highlights the possibility that several changes in the NP and the viral polymerase are required to epistatically compensate for compromised function in NP proteins that acquired MxA-resistance. Therefore, MxA evasion and efficient replication may independently shape the viral fitness landscape that constrains NP evolution and influences influenza virus host range. These studies suggest that the fitness cost to the virus that results from MxA evasion may narrow the gap in ‘evolvability’ between host and viral proteins (). It is possible that natural selection has honed MxA recognition onto those surfaces of the virus that are evolutionarily constrained by, for example, epistatic interactions. A similar strategy is utilized by viral antagonists that mimic highly constrained host proteins to subvert cellular processes [31
]. In this context, virus adaptation in an intermediate host, in which the NP protein is not under tight surveillance by MxA, may represent an important evolutionary transition stage in allowing enough NP variation to overcome MxA restriction via a single evolutionary transition ().