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W4280562558 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Overlapping coding regions balance selective forces between multiple genes. One possible division of nucleotide sequence is that the predominant selective force on a particular nucleotide can be attributed to just one gene. While this arrangement has been observed in regions in which one gene is structured and the other is disordered, we sought to explore how overlapping genes balance constraints when both protein products are structured over the same sequence. We use a combination of sequence analysis, functional assays, and selection experiments to examine an overlapped region in HIV-1 that encodes helical regions in both Env and Rev. We find that functional segregation occurs even in this overlap, with each protein spacing its functional residues in a manner that allows a mutable non-binding face of one helix to encode important functional residues on a charged face in the other helix. Additionally, our experiments reveal novel and critical functional residues in Env and have implications for the therapeutic targeting of HIV-1. Editor's evaluation 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. https://doi.org/10.7554/eLife.72482.sa0 Decision letter Reviews on Sciety eLife's review process Introduction HIV-1 contains nine genes encoded within a relatively short genome of 10,000 nucleotides (Frankel and Young, 1998). Eight of these nine genes overlap with another on the DNA level and consequently encode protein products in different reading frames. HIV-1 Env overlaps with Vpu, Rev, and Tat, and some parts of Env overlap with both Rev and Tat. Despite the prevalence of overlapping genes, the evolution and function of overlapping genes remains understudied. However, evidence establishing the importance of overlaps continues to grow: the SARS-CoV-2 pandemic has demonstrated the importance of identifying and studying overlapping reading frames, as many immune epitopes targeting canonical and non-canonical overlapping ORFs (open reading frames) have been identified in patients (Nelson et al., 2020; Weingarten-Gabbay et al., 2020;; Finkel et al., 2021). We previously showed that, in the case of the essential regulatory genes tat and rev, HIV-1 dedicates the nucleotide sequence to one protein or the other so that critical functional residues do not overlap with one another (Fernandes et al., 2016). For tat and rev, this arrangement is possible because both proteins consist of functional motifs spaced by disordered linkers, and thus functional motifs of one protein can be encoded in regions that overlap with disordered linkers in the other. Although the vast majority of overlapped proteins are predicted to be disordered (Rancurel et al., 2009) and thus have the potential for this arrangement, it is possible that some overlapped regions have more stringent structure and function requirements. Indeed, HIV-1 contains an intriguing overlapping region, between the Rev and Env genes (Figure 1A), in which both protein segments are structured and functionally important. This region might represent a viral Achilles’ heel, vulnerable to therapeutic targeting, as the mutational landscape for escape mutations would be constrained by the simultaneous requirements of both proteins. Figure 1 Download asset Open asset Organization of Rev NES/Env LLP-2 overlap. (A) Genetic organization of the rev/env overlap. (Top) The full genome organization of HIV-1 (HXB2 numbering) demonstrates that the second exon of rev overlaps tat (second exon) and env. A detailed annotation of this overlap depicts the overlap between the quasi-helical NES of Rev with the helical LLP-2 of Env. The ARM (arginine-rich motif, nuclear localization sequence), OD (oligomerization domain), and NES (nuclear export sequence) are other functional domains of Rev that overlap with Env-gp41 (cytoplasmic tail), including the KE (Kennedy epitope) and two lentiviral lytic peptide motifs (LLP-3 in addition to LLP-2). (B) The structure of the Rev NES (red: binding residues, white: non-binding residues) bound to Crm1 (yellow) (PDB: 3nc0). A helical wheel diagram demonstrates how the arrangement of the 2D amino acid sequence creates a distinct binding surface in 3D. (C) Composite modeled structure (PDB: 6ujv) of the Env C-terminus trimer, including the first 21 residues of the LLP-2. The LLP-2 is labeled with the hydrophobic face in gray and the charged surface (positively charged residues in red and negative in blue). A corresponding helical wheel diagram derived from Murphy et al., 2017, for the Env LLP-2 is also shown with charged residues colored. Functionally important residues of Rev are labeled in red. Rev is required for the nuclear export of partially spliced and un-spliced viral RNAs that encode necessary late stage viral proteins as well as the genomic RNA for encapsidation (Malim and Cullen, 1991; Pollard and Malim, 1998; Rausch and Le Grice, 2015). Notably, Rev contains a quasi-helical nuclear export sequence (NES), that binds to the NES-binding groove of the host nuclear export factor Crm1 (Fornerod et al., 1997; Booth et al., 2014). Env is expressed as a polyprotein, composed of the gp120 surface and gp41 transmembrane proteins, which forms a trimeric viral envelope that mediates viral particle binding to cell entry receptors (Checkley et al., 2011; Murphy et al., 2017). The cytoplasmic tail of gp41 is the most C-terminal portion of Env and is highly variable in size (Tedbury and Freed, 2015). In contrast to other retroviruses, lentiviruses such as HIV-1 have an extended cytoplasmic tail containing the Kennedy epitope (KE) followed by three membrane associated lentiviral lytic peptide motifs (LLP-1, LLP-2, and LLP-3) (Steckbeck et al., 2013; Figure 1A). HIV-1 LLP-1, and LLP-2 contain highly conserved arginine and lysine residues that are involved in cation-π interactions and have been suggested to stabilize the cytoplasmic tail α-helical structure (Murphy et al., 2017). LLPs are amphipathic α helices that embed in the virus membranes (Kalia et al., 2003; Steckbeck et al., 2011; Murphy et al., 2017). LLP-2 may be transiently exposed on the virus surface to interact with the gp41 core and regulate virus-cell membrane fusion (Lu et al., 2008; Murphy et al., 2017; Piai et al., 2020). A recent nuclear magnetic resonance spectroscopy study revealed that LLP-2 forms two amphipathic helices that wrap around the transmembrane domain of Env, arranging a ‘baseplate’ that supports the transmembrane domain and the rest of the Env trimer (Piai et al., 2020). Additional evidence suggests that the C-terminal tail makes critical contacts with the matrix protein (Alfadhli et al., 2019) to facilitate Env incorporation into virions. The Rev NES and a portion of the Env LLP-2 are encoded by the same nucleotides in the viral genome, with Env encoded in the +1 reading frame relative to Rev (Figure 1A). As both these domains have important functions in viral replication and are structured, they provide an ideal system to examine how viruses balance the evolution of these two genes with critical, overlapping structured motifs. Here, we perform conservation analyses, functional assays, and selection experiments to examine how the virus balances selection pressures within the Rev-Env overlap. Surprisingly, we find that functional residues are also segregated at the nucleotide level, as for the Rev-Tat overlap. In Rev-Env case, the two-dimensional segregation of nucleotides in the genome is mirrored in the three-dimensional functional surfaces of the proteins such that the nucleotides encoding a functional surface in the helix of one protein encode residues on the non-binding face of the overlapping helix. Furthermore, our analyses reveal three important charged residues on the charged surface of LLP-2 that are required for Env function. Results Conservation of chemical properties in the Rev NES and Env LLP-2 The Rev NES (residues 75–83) binds to Crm1 using several critical hydrophobic residues and non-canonical prolines that change the helical register and position hydrophobic side chains on one face of the helix to fit within the NES recognition groove (Dong et al., 2009; Güttler et al., 2010; Figure 1B). The Env LLP-2 forms an amphipathic helix of about 33 amino acids long (Eisenberg and Wesson, 1990; Kalia et al., 2003; Murphy et al., 2017) that spans residues 767–788 of Env (242–274 of gp41) (Figure 1A). Nine of the LLP-2 residues (767–776) overlap with the quasi-helical Rev NES (Figure 1B). Mutating charged residues within LLP-2 or hydrophobic residues in the Rev NES negatively impacts viral replication (Kalia et al., 2003; Newman et al., 2007; Güttler et al., 2010; Fernandes et al., 2016), prompting us to examine in detail the selective forces that guide the evolution of this overlapping region. In order to measure the relative conservation of these regions within HIV patient sequences, we first downloaded curated alignments of the Los Alamos Database (hiv.lanl.org) (Foley, 2013) and calculated Shannon entropies for each amino acid in both proteins within the NES/LLP-2 overlap (Pan and Deem, 2011; Fernandes et al., 2016). Sites with low entropy (0=perfect conservation) indicate strong conservation of the amino acid in that particular protein, but it is impossible to determine if the observed conservation is simply driven by codon constraints imposed by the alternative frame. To compare the amino acid conservation of corresponding sites in both proteins, we grouped amino acids that share two nucleotides in the genome and plotted their entropies against one another (Fernandes et al., 2016; Figure 2A). Notably, the one site that contributes to both functional surfaces (H769 in Env and P76 in Rev) is relatively well conserved in both proteins whereas the one site that contributes to neither interface (L755 in Env and T82 in Rev) is weakly conserved (Figure 2A). Unexpectedly, two known essential Crm1-binding residues (L78 and L83) displayed low conservation (high entropy) in the Rev reading frame, but the consensus sequence (Figure 2B) indicates that chemically conservative isoleucine substitutions are frequently found, thus explaining the high entropy. Moreover, entropy analysis using a reduced amino acid alphabet in which the chemically similar amino acids I/L/V are grouped produced low entropy values for all Crm1-binding residues (Figure 2C). The use of chemically conservative side chains to form critical contacts drastically increases the number of ‘synonymous’ codons (e.g. the six leucine codons can substitute with the three isoleucine codons with essentially equal fitness) and may reflect the kinds of interaction motifs that are most suited to evolve in constrained overlapping regions. The three charged residues (Figure 2C; green dots) in LLP-2 were relatively strongly conserved (Figure 2B) but showed lower conservation (high entropy) than the Rev NES-binding residues, suggesting that Rev may more greatly influence the selection of amino acids in the overlapping region. Figure 2 Download asset Open asset Rev nuclear export sequence (NES) and Env LLP-2 conservation. (A) Shannon entropies (low entropy = high conservation) from alignments of patient sequences (see panel B). Residues in Rev and Env are grouped by positions that share two nucleotides of overlap. Red points contain Crm1 contact residues, green points are charged LLP-2 residues, and yellow points contain both. (B) Sequence logos of Rev NES and Env LLP-2 showing sequence conservation at each position computed from a sequence alignment of patient data from the Los Alamos Database (https://www.hiv.lanl.gov/content/index; 2018). Positively charged residues (KRH) are in blue; negatively charged residues (DE) are in red. Functionally important residues of Rev are marked with a star. (C) Shannon entropies recomputed for a simplified amino acid alphabet (D=E, R=K, I=L and V, M, F=W & Y, N=Q, S=T). Coloring is as in 2A. Figure 2—source data 1 Site-specific entropy values used to make graphs in Figure 2. https://cdn.elifesciences.org/articles/72482/elife-72482-fig2-data1-v2.xlsx Download elife-72482-fig2-data1-v2.xlsx Deep mutational scanning of non-overlapped viruses selects for important LLP-2 and NES residues In order to deconvolute the functional constraints for every residue in both proteins absent the overlap, we created libraries of viruses in which Rev and Env were decoupled from one another. Although HIV-1 env has been exposed to deep mutational scanning previously, the overlap with rev has prevented exploration of the LLP-2 (Haddox et al., 2016; Haddox et al., 2018). Briefly, we used the NL4-3 rev-in-nef virus in which rev is moved to the non-essential, single-frame nef locus (Fernandes et al., 2016), and the endogenous rev locus is ablated, thus allowing the endogenous Env LLP-2 region and the rev-in-nef NES region to evolve independently from one another. We individually randomized each of the LLP-2 residues in the endogenous env locus, creating 10 Env libraries that each represent 64 alleles (every possible codon). Next, we infected SupT1 cells at low MOI (multiplicity of infection) with each library individually and measured the change in allele frequencies after approximately six generations of viral spread (Figure 3A). We then combined analyses of these libraries with previously generated deep mutational scanning data produced for the Rev NES from a rev-in-nef virus (Fernandes et al., 2016). These fitness landscapes (Figure 3B and C) represent the amino acid preference of each gene when the overlap is removed. We note that, as these DMS datasets were performed independently of one another and display different strengths of selection as demonstrated in the difference in stop codon fitness, comparisons between Rev and Env enrichment ratios require normalization. To this end we provide rescaled enrichment ratios as described in Gray et al., 2017, and plot both the standard enrichments and rescaled data (Figure 3—figure supplement 1). Figure 3 with 1 supplement see all Download asset Open asset Deep mutational scanning of Rev nuclear export sequence (NES) and Env LLP-2. (A) Schematic of the experiment. Non-overlapped viruses with rev inserted in the nef locus were created and the endogenous LLP-2 region randomized to NNN to create 10 independent libraries. These libraries were then arrayed and allowed to spread (~6 generations of viral spread) and next-generation sequencing was used to determine frequencies of each variant in a replication competition experiment and to calculate experimental fitness. (B) Fitness (blue = unfit, white = neutral, and gold = fit) for every allele in the region of the Env LLP-2 that overlaps the Rev NES. Row headers denote the reference sequence while column headers specify the allele. (C) Fitness for every allele in the region of the Rev NES that overlaps the Env LLP-2. Functionally important residues of Rev are labeled in red. Figure 3—source data 1 Fold change values in allele frequency during deep mutational scanning experiment for Rev nuclear export sequence (NES) used to generate heatmaps. https://cdn.elifesciences.org/articles/72482/elife-72482-fig3-data1-v2.xlsx Download elife-72482-fig3-data1-v2.xlsx Figure 3—source data 2 Fold change values in allele frequency during deep mutational scanning experiment for Env LLP-2 used to generate heatmaps in Figure 3. https://cdn.elifesciences.org/articles/72482/elife-72482-fig3-data2-v2.xlsx Download elife-72482-fig3-data2-v2.xlsx The LLP-2 fitness landscape shows that while LLP-2 is generally highly mutable, it has a strong preference for a negatively charged side chain (D or E) at residue 773 (Figure 3B). Although the importance of positive charges in LLP-2 has been suggested previously (Kalia et al., 2003; Steckbeck et al., 2011), our experiment is the first to identify strong selection for a negative charge. The data also show strong selection against the ‘helix-breaker’ proline (Li et al., 1996) at all positions, further underscoring the importance of its helical structure. Stop codons are robustly selected against as well, demonstrating the importance of the cytoplasmic tail in HIV-1 replication. Additionally, L771, which is surrounded by two positively charged arginines, displays a strong preference against charged residues. The Rev NES fitness landscape is consistent with many mutational studies, showing selection for large hydrophobic side chains (ILV) at each Crm1 contacting residue. The patient isolates do not show such a striking preference (Figure 2B), perhaps reflecting constraints imposed by overlapping gene mutational paths (e.g. any transitional mutation from L to V would have to be fit in Rev and also in Env) or the existence of other in vivo pressures not captured in our experiments (e.g. immune or cell-type-specific responses). Unlike LLP-2, we do not observe selection against proline in the semi-helical Rev NES, consistent with the presence of prolines in its reference sequence. Functional assays confirm the importance of Rev NES hydrophobic residues and LLP-2 charged residues To confirm that our selection data reflect Rev functional requirements, we assayed individual alanine mutants at the nine NES residues in a p24 Rev-dependent RRE reporter assay (Smith et al., 1990). Mutation of any of the leucines (positions 75, 78, 81, and 83) or proline at position 76 resulted in loss of function (Figure 4A; high p24=active Rev), consistent with previous data of Crm1-binding residues (Figure 1C; Fornerod et al., 1997; Güttler et al., 2010; Booth et al., 2014; Jayaraman et al., 2019). Consistent with our selection data, mutation of the NES non-binding face had limited impact on Rev function. Figure 4 Download asset Open asset Functional dissection of Rev nuclear export sequence (NES) and Env LLPs. (A) Schematic of the Rev reporter assay and activities of individual NES alanine mutants. To monitor Rev export activity, an RRE-containing gag-pol reporter system that produces viral capsid (p24) in a Rev-dependent manner was used. Data are mean ± standard deviation (s.d.) of three biological replicates. Western blot shows expression levels of Strep-tagged Rev variants and GAPDH as loading controls. Functionally important residues of Rev are labeled in red. (B) Schematic of the Env reporter assay in the TZM-bL system and results with individual Env LLP-2 alanine mutants. Data are mean ± s.d. of three biological replicates. Western blots show Env incorporation (gp120 antibody) into pseudoviruses and p24 as loading controls. For these experiments, we ablated the env reading frame by mutating the start codon and introducing multiple downstream stop codons. Plasmids were co-transfected into 293T cells with each alanine mutant to generate Env pseudotyped viruses. The resulting virions were used to infect TZM-bL reporter cells which express the HIV-1 surface receptors CD4, CCR5, CXCR4, and produce luciferase under the control of the tat-dependent HIV-1 LTR (Sarzotti-Kelsoe et al., 2014). A functional Env protein permits binding and entry and expression of Tat and thus reflects the number of infectious viral particles in a single round of infection, but the integrated virus lacks the env gene and cannot initiate additional rounds of replication. Incorporation of Env mutants into virions was also measured by Western blot. Figure 4—source data 1 Reporter data used to generate graphs for Rev and Env alanine scans in Figure 4. https://cdn.elifesciences.org/articles/72482/elife-72482-fig4-data1-v2.xlsx Download elife-72482-fig4-data1-v2.xlsx Figure 4—source data 2 Unedited Western blot for Figure 4. https://cdn.elifesciences.org/articles/72482/elife-72482-fig4-data2-v2.pdf Download elife-72482-fig4-data2-v2.pdf To confirm that our selection data reflect LLP-2 functional requirements, we measured the infectivity of virions generated from pseudotyped env-deficient viruses complemented with Env LLP-2 alanine mutants in trans. The D773A mutation produced the most drastic phenotype (15% of wild-type Env), consistent with our selection data (Figure 4B). Previous work noted the conservation of arginines within the LLPs (Kalia et al., 2005; Steckbeck et al., 2011) and our data confirms that the first arginine in LLP-2 (R770) is also important for Env function, in addition to D773. Charged LLP-2 residues facilitate Env function in virus replication assays As D773 in LLP-2 is clearly an important residue based on the selection and reporter data, we examined the effects of several substitutions at this position on viral replication. D773E, a conservative change predicted to maintain function based on our fitness data, showed ~50% of wild-type activity in the reporter assay, but substantially higher than the non-conservative D773A and D773N substitutions (Figure 5A). These results correlated well with the replication of rev-in-nef viruses engineered with the mutations (Figure 5A). It is interesting that D773A viruses still can replicate, albeit poorly, despite its very low activity; these results suggest that the virus can partially compensate for the loss of function, particularly when not competing with the wild-type sequence. The weakly active D773N allele is observed in ~2.6% of patient isolates (Figure 2B), further suggesting that the virus has alternative mechanisms to achieve the function normally performed by the negative charge at this position. Figure 5 Download asset Open asset Importance of LLP-2 charged residues for replication. (A) Left: Env functional assays as described in Figure 4 with different D773 mutations. Right: Viral replication kinetics in spreading assays using NL4-3 rev-in-nef proviral plasmids (Fernandes et al., 2016) engineered with Env D773 mutations. (B) Left: Env functional assays showing the role of three charged residues. Western blots show incorporation of Env mutants in virions (dark boxes represent a break in the lane loading presentation, with all samples loaded on a single gel). Right: Viral replication kinetics of viruses engineered with the mutations. R1D→ AA is a double mutant with R770/D773 changed to alanine, R2D→ AA is a double mutant with R772/D773 changed to alanine, and RRD→ AAA a triple mutant R770/R772/D773 changed to alanine. (C) Left: Env functional assays showing effects of positive charge mutations with corresponding Western blots of Env incorporation in virions. Right: Viral replication kinetics of viruses engineered with the mutations. Replication assays are represented on a log scale. All data are mean ± standard deviation (s.d.) of biological triplicates. Figure 5—source data 1 Reporter data for Env mutants in Figures 5 and 6. https://cdn.elifesciences.org/articles/72482/elife-72482-fig5-data1-v2.xlsx Download elife-72482-fig5-data1-v2.xlsx Figure 5—source data 2 Viral spread data used to generate graphs for Env mutants in Figures 5 and 6. https://cdn.elifesciences.org/articles/72482/elife-72482-fig5-data2-v2.xlsx Download elife-72482-fig5-data2-v2.xlsx Figure 5—source data 3 Unedited Western blot for Figure 5. https://cdn.elifesciences.org/articles/72482/elife-72482-fig5-data3-v2.pdf Download elife-72482-fig5-data3-v2.pdf Figure 6 Download asset Open asset Importance of LLP-2 structure and charge for Env activity. (A) Left: Env functional assays as described in Figure 4 when LLP-2 helical structure was disrupted by replacing 10 residues (767–776) with alternating glycine/serine residues (10GS) to create a flexible region. Western blots show expression of Env mutants (dark boxes represent a break in the lane loading presentation, with all samples loaded on a single gel). Right: Viral replication kinetics of wild-type (WT), 10GS, and 10GS + D262 viruses. (B) Left: Env functional assays with charge mutants within a 10-alanine (10A) context designed to maintain a helical structure with corresponding Western blots of Env incorporation in virions. Right: Viral replication kinetics with helical structure mutant viruses. Data points in both panels are mean ± s.d. of biological triplicates. Replication assays are represented on a log scale. Figure 6—source data 1 Unedited Western blot for Figure 6. https://cdn.elifesciences.org/articles/72482/elife-72482-fig6-data1-v2.pdf Download elife-72482-fig6-data1-v2.pdf Given the importance of D773 and the neighboring positively charged R770 and R772 (Figure 4B), we sought to establish the importance of charge and spacing along the LLP-2 helix, with the goal of understanding what evolutionary constraints – absent the overlap – affect this region. We generated two double mutants with D773/R770 or D773/R772 changed to alanine (R1D→AA or R2D→AA) as well as the triple mutant (RRD→AAA) and found that mutation of R770 was particularly detrimental, in combination with D773 which is about one helical turn away (Figure 5B). Both of the double mutants showed significantly decreased virus replication (Figure 5B), again suggesting loss of function can be partially compensated in the viral context for the loss of function. The triple mutant was highly defective in both reporter and replication assays indicating the importance of these charged residues. Western blots of these virions indicated decreased Env protein stability for R1D. We constructed a set of additional charged residue mutants, including H769 as well as substitutions with lysine, to determine if their contributions could be attributed solely to charge. The results show no functional phenotype for H769K mutant in both reporter and replication assays and RR→KK outcomes confirm the importance of R770 and R773 (Figure 5C). While we did not examine the mutations of R770K and R773K independently out, deep mutational scanning of Env LLP-2 (Figure 3B) reveals selection against negative charged residues (D and E) and even K for both arginines, a bias is not observed in H769, suggesting that H769 is less constrained than the two arginines. It is interesting that substitution of both arginine with lysine is insufficient to maintain function or replication, and indeed the (RR→KK) mutant showed the most severe replication defect. It is possible that lysine substitutions might serve as a target for host restriction factors (Zhang et al., 2021) or introduce ubiquitination sites leading to protein turnover. Regardless of the mechanism, the data suggest that these particular residues are subject to significant evolutionary constraints. The results are also consistent with analyses of the Env cytoplasmic tail, which has shown enrichment for arginine and depletion for lysine when compared to the rest of Env and all other proteins found in the Uniprot database (Steckbeck et al., 2011). Helical requirement for LLP-2 function LLP-2 forms two helices (Murphy et al., 2017) and our deep mutational scanning data suggest strong selection against helix-breaking prolines, implying a structural constraint during evolution. To better understand the extent of this constraint, we replaced residues 767–776 with glycines and serines (10GS) to create a flexible linker. As anticipated, the 10GS variant had no activity in the reporter assay and was unable to replicate even when D773 was reintroduced (Figure 6A). We next replaced residues 767–776 with alanines (10A) which are often used to form rigid helical linkers (Chen et al., 2013). This 10A variant was similarly defective (Figure 6B). However, in this case, reintroducing important charged residues (R770 or D773 or the five-residue HRLRD segment) resulted in substantial restoration of activity and rescue of virus replication, particularly in the cases where D773 was present (Figure 6B). These data further confirm the importance of charge and helical structure in LLP-2 function (Kalia et al., 2005; Boscia et al., 2013; Steckbeck et al., 2014). Functional segregation of residues in overlapping structured segments Our experiments establish the functional restrictions in Env LLP-2 without competing selection pressure from Rev, permitting us to map the constraints of each reading frame onto the nucleotide sequence of the virus (Figure 7). Specifically, our analysis showed that in Env, D773 has a strong preference for negative charge, R770 and R772 have an intermediate preference for arginine side chains, H769 has a weak preference for a positive charge, and L771 selects against charged side chains. When combined with the preferences of the Rev NES Crm1-binding residues (Fernandes et al., 2016), it is apparent that the functional surfaces of each domain are positioned so as to minimize negative consequences of the overlap. The non-binding face of the Rev NES largely corresponds to the charged surface of the Env LLP-2 and the hydrophobic surface of the LLP-2 corresponds to the binding residues of the Rev NES (Figure 7). The majority of overlaps between opposing critical residues mostly involve mutable wobble bases, with the sole exception being the Env H769/Rev P76 overlap. However, in this case Rev appear" @default.
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W4280562558 title "Author response: Functional and structural segregation of overlapping helices in HIV-1" @default.
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