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Summary

Deep mutational scanning (DMS) is a method formally introduced in 2010 [1] that uses next generation sequencing technology to assay millions of protein variants in a single experiment. Since then, hundreds of studies have applied this fundamental technique to engineer proteins [2], understand mutational tolerance [3], and reveal evolutionary trajectories in sequence space [4]. However, DMS is classically limited to studying only missense variants whereas nature can also sample from other mutations such as insertion-deletions (indels). While there is an appreciation for indels in areas such antibody maturation [5] or viral antibody antagonism [6], the effect on viral restriction factors has not been comprehensively assessed. In this work, Tenthorey et al. use both DMS and deep indel scanning (DIS) to investigate the evolution of an antiretroviral restriction factor known as TRIM5α. They convincingly show that for TRIM5α, indels can facilitate the evolution of novel restriction functions where individual missense mutations fail. 

Previous work by the same group was able to identify missense mutations that significantly increased human TRIM5α’s (HsT5) ability to restrict HIV-1 infection [7]. However, they discover here that while similar mutations in HsT5 can restrict other simian lentiviruses as well, no single missense mutation is sufficient to restrict a simian lentivirus endemic to sabaeus monkeys (SIVsab). A comparison to rhesus macaque TRIM5α (RhT5) revealed the presence of a small, but functionally significant indel that leads authors to test whether indels can confer restriction of SIVsab onto HsT5 by DIS. Their data conclusively shows that several small indels in the v1 loop of the HsT5 B30.2 domain drastically enhance restriction of SIVsab without even a single missense mutation. Alone, these results would be interesting, but to add additional biological relevance, the authors show that TRIM5α homologs in simian primates show a high incidence of indels in the same region of the protein essential for viral restriction: the v1 loop. By swapping the indels present in human, rhesus macaque, sabeaus monkey, and capuchin homologs of TRIM5α, they show naturally evolved indels can also enhance or abrogate viral restriction.

This study overall has few, if any, issues that should be addressed: it is well-written, data is succinctly and clearly presented, and conclusions are well-founded. Thus, we have only a few comments to make below.

Major comments

• Figure 2A shows an interesting result: while exchanging the v1 loop in HsT5 with that of RhT5 results in viral restriction being significantly increased compared to WT HsT5, it is still an order of magnitude less than WT RhT5. While this is consistent with the importance of the v1 loop in viral restriction as mentioned by the authors, does it not suggest the presence of epistasis? In other words, it appears as though other parts of the protein can also alter viral restriction. The only mention of these results is here: “TRIM5α into the human protein greatly enhances SIVsab restriction, although not to the same level as full-length rhesus TRIM5α (Fig. 2A)”. However, we believe this deserves additional attention, at least in the discussion. Reasons for this are due to the claim made in the title of the paper itself: “inaccessible”. If indeed there are missense mutations or indels in other parts of HsT5 that were not assayed here, but that may contribute to viral restriction, we feel that limitation should at the very least be addressed. Perhaps there is an additional missense mutation outside of the v1 loop that in combination with a single missense mutation within the v1 loop may potentially increase viral restriction of HsT5. If this were the case, it could make such an evolutionary scenario far more plausible.

Minor comments

• We thank the authors for generating a GitHub repo that contains the code and sequencing data. However, it would also be helpful to include a descriptive README file so that navigating the repository, more specifically its code, is easier for reproducibility purposes.

References

1. Fowler DM, Araya CL, Fleishman SJ, Kellogg EH, Stephany JJ, Baker D, et al. High-resolution mapping of protein sequence-function relationships. Nat Methods. 2010;7: 741–746.

2. Harris DT, Wang N, Riley TP, Anderson SD, Singh NK, Procko E, et al. Deep Mutational Scans as a Guide to Engineering High Affinity T Cell Receptor Interactions with Peptide-bound Major Histocompatibility Complex. J Biol Chem. 2016;291: 24566–24578.

3. Thyagarajan B, Bloom JD. The inherent mutational tolerance and antigenic evolvability of influenza hemagglutinin. Elife. 2014;3. doi:10.7554/eLife.03300

4. Starr TN, Picton LK, Thornton JW. Alternative evolutionary histories in the sequence space of an ancient protein. Nature. 2017;549: 409–413.

5. Lupo C, Spisak N, Walczak AM, Mora T. Learning the statistics and landscape of somatic mutation-induced insertions and deletions in antibodies. PLoS Comput Biol. 2022;18: e1010167.

6. Kim AS, Zimmerman O, Fox JM, Nelson CA, Basore K, Zhang R, et al. An Evolutionary Insertion in the Mxra8 Receptor-Binding Site Confers Resistance to Alphavirus Infection and Pathogenesis. Cell Host Microbe. 2020;27: 428–440.e9.

7. Tenthorey JL, Young C, Sodeinde A, Emerman M, Malik HS. Mutational resilience of antiviral restriction favors primate TRIM5α in host-virus evolutionary arms races. Elife. 2020;9. doi:10.7554/eLife.59988

Competing interests

The authors declare that they have no competing interests.