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  1. Author Response:

    Joint Public Review:

    Fernandes et al. ask the question: "What are the evolutionary constraints on genomic sequence that encode two different proteins?" To this end, they compare the functional constraints on mutations in HIV Rev and Env, which are encoded in different reading frames from the same region of the viral genome. Interestingly, residues that are functionally constrained in one protein are, for the most part, not as constrained in the other. The elegance of this solution is attractive and will be of interest to the protein evolution and structure communities.

    To address their questions, the authors (1) examined amino acid conservation in patient HIV sequences for both proteins, (2) performed deep mutational scanning of HIV Env to compare to published data on Rev, and (3) dissected the functional impact of key mutations for both proteins. This approach leads them to propose a model in which functionally important residues in one protein do not overlap with functionally important residues in the other protein.

    While this approach and data generally support this model, there are two residues in Env (Y768 and L771) that are conserved, relatively mutationally intolerant, and overlap with functionally important residues in Rev. Because these residues are not found on the charged Env helical face, they are not considered critical residues in the proposed model. However, the authors should discuss the possibility that other constraints on protein evolution, such as stability and folding, could also affect their definition of 'critical'. On balance, however, their interpretations are reasonable.

    As the reviewers note this brings into question how constraints should be classified. For instance, the hydrophobic residue in the LLP-2 do not appear, from our data, to contribute to functional interfaces as their side chains can be ablated to alanine with no effect. However, L771 shows strong selection against many types of side chains (nonhydrophobic) suggesting that unwanted chemical properties can disrupt function, possibly via stability or folding. These distinctions are important in considering the evolutionary space in which a residue can sample productively, though whether these residues are “functionally critical” is a matter of perspective. We have added additional discussion of this point.

    We also note that the analysis comparing patient conservation to the DMS dataset performed above further suggests that Rev is an important selective force at these sites as the patient data displays greater conservation than the DMS data suggests.

    Based on these experiments, it is concluded that part of each helix is mutable, while encoding important functional "constrained" residues in the other helix. The study is well done and the data of good quality and convincing. The conclusions are justified and of potential importance for future therapeutic strategies. These studies could facilitate the interpretation of genome evolution in other viruses, such as SARS-CoV-2, that encode open-readingframe overlaps. However, some parts of the manuscript need clarification and potential extension.

    1. To measure the relative conservation of Env and Rev, the authors downloaded curated alignments of the Los Alamos Database. Information should be provided as to how many sequences were compared and whether this included viruses from all the different subtypes. This reviewer assumes they only looked at HIV 1 group M, but this needs to be clarified.
    1. In the Rev reporter assays, the authors employ pCMV-GagPol-RRE, which contains an RRE from the pNL4-3 "lab" virus. Recent studies have shown that different Rev/RRE combinations can have different activities. The authors should discuss this information and its relevance to their findings.

    In this study we focused on the NL4-3 HIV-1 genotype (for both Rev and RRE) as it is well-studied, contains intact ORFs of all canonical gene products, and allows us to pair our reporter and viral assays. As the reviewer notes, Rev-RRE activities have a wide range of activities outside this single genotype. We believe that the virus's ability to adjust and tune this activity is a pliable feature that likely also helps accommodate the fact that both Rev and RRE have evolved in overlapped regions. We have added this discussion point to the text, but note that our results do not offer direct support of this model, and we look forward to future studies such as Jackson et al, that explore this idea more fully (Jackson et al., 2016).

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  2. Evaluation Summary:

    This study should be of broad interest to all virologists and many students of molecular genetics. It examines the constraints in a part of the HIV 1 genome that encodes important functional regions of two proteins, Rev and Env, in overlapping reading frames. It is convincingly shown that functional segregation occurs in a part of the overlap region that is critical for both proteins, which has important implications for HIV biology and may aid in the design of future HIV therapies.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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  3. Joint Public Review:

    Fernandes et al. ask the question: "What are the evolutionary constraints on genomic sequence that encode two different proteins?" To this end, they compare the functional constraints on mutations in HIV Rev and Env, which are encoded in different reading frames from the same region of the viral genome. Interestingly, residues that are functionally constrained in one protein are, for the most part, not as constrained in the other. The elegance of this solution is attractive and will be of interest to the protein evolution and structure communities.

    To address their questions, the authors (1) examined amino acid conservation in patient HIV sequences for both proteins, (2) performed deep mutational scanning of HIV Env to compare to published data on Rev, and (3) dissected the functional impact of key mutations for both proteins. This approach leads them to propose a model in which functionally important residues in one protein do not overlap with functionally important residues in the other protein.

    While this approach and data generally support this model, there are two residues in Env (Y768 and L771) that are conserved, relatively mutationally intolerant, and overlap with functionally important residues in Rev. Because these residues are not found on the charged Env helical face, they are not considered critical residues in the proposed model. However, the authors should discuss the possibility that other constraints on protein evolution, such as stability and folding, could also affect their definition of 'critical'. On balance, however, their interpretations are reasonable.

    Based on these experiments, it is concluded that part of each helix is mutable, while encoding important functional "constrained" residues in the other helix. The study is well done and the data of good quality and convincing. The conclusions are justified and of potential importance for future therapeutic strategies. These studies could facilitate the interpretation of genome evolution in other viruses, such as SARS-CoV-2, that encode open-readingframe overlaps. However, some parts of the manuscript need clarification and potential extension.

    1. To measure the relative conservation of Env and Rev, the authors downloaded curated alignments of the Los Alamos Database. Information should be provided as to how many sequences were compared and whether this included viruses from all the different subtypes. This reviewer assumes they only looked at HIV 1 group M, but this needs to be clarified.

    2. In the Rev reporter assays, the authors employ pCMV-GagPol-RRE, which contains an RRE from the pNL4-3 "lab" virus. Recent studies have shown that different Rev/RRE combinations can have different activities. The authors should discuss this information and its relevance to their findings.

    Was this evaluation helpful?