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

Colón-Thillet, Rossana; Hsieh, Edward J.; Graf, Laura; McLaughlin, Richard N.; Young, Janet M.; Kochs, Georg; Emerman, Michael; Malik, Harmit S.

doi:10.1101/557264

Cited by 4 publications

(3 citation statements)

References 36 publications

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“…Our DMS analyses of human TRIM5α revealed unexpected ease of gaining antiviral potency against HIV-1 and potentially other lentiviruses. However, gains in potency against one virus might be offset by a concomitant loss of function against other viruses, as previously seen for the antiviral protein MxA (Colón-Thillet et al, 2019). Such functional tradeoffs might partially .…”

Section: Resilience Of Antiviral Restriction Is a General Property Of Trim5α Adaptationmentioning

confidence: 92%

Mutational resilience of antiviral restriction favors primate TRIM5α in host-virus evolutionary arms races

Tenthorey

Young

Sodeinde

et al. 2020

Preprint

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1Host antiviral proteins engage in evolutionary arms races with viruses, in which both sides 2 rapidly evolve at interaction interfaces to gain or evade immune defense. For example, primate 3 TRIM5α uses its rapidly evolving "v1" loop to bind retroviral capsids, and single mutations in this 4 loop can dramatically improve retroviral restriction. However, it is unknown whether such gains 5 of viral restriction are rare, or if they incur loss of pre-existing function against other viruses. 6Using deep mutational scanning, we comprehensively measured how single mutations in the 7 TRIM5α v1 loop affect restriction of divergent retroviruses. Unexpectedly, we found that the 8 majority of mutations increase antiviral function. Moreover, most random mutations do not 9 disrupt potent viral restriction, even when it is newly acquired via single adaptive substitutions. 10Our results indicate that TRIM5α's adaptive landscape is remarkably broad and mutationally 11 resilient, maximizing its chances of success in evolutionary arms races with retroviruses. 13Here, we investigated the adaptive landscape of antiviral specificity conferred by the 1 rapidly evolving, capsid-binding v1 loop of TRIM5α. To our surprise, we found that, rather than 2 the evolutionary landscape of TRIM5α being narrowly constrained among all possible amino 3 acid substitutions, the majority of random mutations in the v1 loop resulted in gains of antiviral 4 restriction. We found that the primary v1 loop determinant for TRIM5α's restriction of HIV-1 and 5 other lentiviruses is its net electrostatic charge. Furthermore, both rhesus and human TRIM5α 6 proteins are highly resilient to mutation, in that they withstand more than half of all possible 7 single amino acid mutations in the v1 loop without compromising their antiviral restriction 8 abilities. This unexpectedly permissive landscape allows TRIM5α to sample a wide variety of 9 mutations to maximize its chances of success in arms races with retroviruses. 10 11 RESULTS 12 A deep mutational scan of the TRIM5α v1 loop 13 Despite their rapid evolution, primate TRIM5α orthologs have sampled relatively limited amino 14 acid diversity at rapidly evolving positions within the capsid-binding v1 loop (Figure 1A). For 15 example, although single amino acid changes at residue 332 are responsible for dramatic 16 differences in antiviral restriction (Y. Li et al., 2006), this residue repeatedly toggles between just 17 three amino acids. The limited diversity is not due to evolutionary inaccessibility, since most 18 amino acids that can be sampled with single nucleotide changes are not observed among19primate TRIM5α orthologs ( Figure 1B). There are two alternative explanations for this restricted 20 diversity. First, it might suggest that adaptive gain-of-function mutations in TRIM5α are rare, 21 with TRIM5α's evolutionary landscape mainly consisting of fitness valleys with only a few 22 mutational avenues to reach fitness peaks ( Figure 1C). Conversely, the limited diversity might 23 be a consequence of epistatic in...

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Section: Resilience Of Antiviral Restriction Is a General Property Of Trim5α Adaptationmentioning

confidence: 92%

Mutational resilience of antiviral restriction favors primate TRIM5α in host-virus evolutionary arms races

Tenthorey

Young

Sodeinde

et al. 2020

Preprint

Self Cite

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“…Perhaps many of the giant gene families described above will contract in the future, either due to genetic drift, or because of the detrimental effects of these large families. For example, it is important to remember that hosts may be battling multiple types of pathogens, and in some cases resistance to one pathogen can cause susceptibility to another pathogen [29,65–67]. And while viruses have extreme pressures to reduce copy number after there has been expansion and mutation to generate a more beneficial gene variant, other organisms have genomes that can be more tolerant of additional copy number in their genomes.…”

Section: Concluding Thoughts About Shared Evolutionary Themes In Host–pathogen Battlesmentioning

confidence: 99%

Conservation lost: host‐pathogen battles drive diversification and expansion of gene families

Lažetić

Troemel

2020

The FEBS Journal

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One of the strongest drivers in evolution is the struggle to survive a hostpathogen battle. This pressure selects for diversity among the factors directly involved in this battle, including virulence factors deployed by pathogens, their corresponding host targets, and host immune factors. A logical outcome of this diversification is that over time, the sequence of many immune factors will not be evolutionarily conserved across a broad range of species. Thus, while universal sequence conservation is often hailed as the hallmark of the importance of a particular gene, the immune system does not necessarily play by these rules when defending against coevolving pathogens. This loss of sequence conservation is in contrast to many signaling pathways in development and basic cell biology that are not targeted by pathogens. In addition to diversification, another consequence of host-pathogen battles can be an amplification in gene number, thus leading to large gene families that have sequence relatively specific to a particular strain, species, or clade. Here we highlight this general theme across a variety of pathogen virulence factors and host immune factors. We summarize the wide range and number across species of these expanded, lineage-specific host-pathogen factors including ubiquitin ligases, nucleotide-binding leucine-rich repeat receptors, GTPases, and proteins without obvious biochemical function but that nonetheless play key roles in immunity.

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“…Super restriction factors are defined as evolution-guided variants of natural restriction factors with improved properties based on previous work done on the restriction factor MXA (21). Super restriction factors provide a unique tool to study how restriction factors work and the evolutionary compromises for paths that have yet to be sampled in nature.…”

Section: Introductionmentioning

confidence: 99%

APOBEC3C tandem domain proteins create super restriction factors against HIV-1

McDonnell

Crawford

Dingens

et al. 2020

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AbstractHumans encode proteins, called restriction factors, that inhibit replication of viruses like HIV-1. One family of antiviral proteins, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3, shortened to A3) acts by deaminating cytidines to uridines during the reverse transcription reaction of HIV-1. The A3 locus encodes seven genes, named A3A-A3H. These genes either have one or two cytidine deaminase domains and several of these A3s potently restrict HIV-1. A3C, which has only a single cytidine deaminase domain, however, inhibits HIV-1 only very weakly. We tested novel double domain protein combinations by genetically linking two A3C genes to make a synthetic tandem domain protein. This protein created a “super restriction factor” that had more potent antiviral activity than the native A3C protein, which correlated with increased packaging into virions. Furthermore, disabling one of the active sites of the synthetic tandem domain protein results in an even greater increase in the antiviral activity—recapitulating a similar evolution seen in A3F and A3G (double domain A3s that only use a single catalytically active deaminase domain). These A3C tandem domain proteins do not have an increase in mutational activity, but instead inhibit formation of reverse transcription products which correlates with their ability to form large higher order complexes in cells. Finally, the A3C-A3C super restriction factor largely escaped antagonism by the HIV-1 viral protein, Vif.ImportanceAs a part of the innate immune system, humans encode proteins that inhibit viruses like HIV-1. These broadly acting antiviral proteins do not protect humans from viral infections because viruses encode proteins that antagonize the host antiviral proteins to evade the innate immune system. One such example of a host antiviral protein is APOBEC3C (A3C), which weakly inhibits HIV-1. Here, we show that we can improve the antiviral activity of A3C by duplicating the DNA sequence to create a synthetic tandem domain, and furthermore, are relatively resistant to the viral antagonist, Vif. Together, these data give insights about how nature has evolved a defense against viral pathogens like HIV.

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

Cited by 4 publications

References 36 publications

Mutational resilience of antiviral restriction favors primate TRIM5α in host-virus evolutionary arms races

Mutational resilience of antiviral restriction favors primate TRIM5α in host-virus evolutionary arms races

Conservation lost: host‐pathogen battles drive diversification and expansion of gene families

APOBEC3C tandem domain proteins create super restriction factors against HIV-1

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