Structural Basis of CD4 Downregulation by HIV-1 Nef

The HIV-1 Nef protein suppresses multiple immune surveillance mechanisms to promote viral pathogenesis and is an attractive target for the development of novel therapeutics. A key function of Nef is to remove the CD4 receptor from the cell surface by hijacking clathrin- and AP2-dependent endocytosis. However, exactly how Nef does this has been elusive. Here, we describe the underlying mechanism as revealed by a 3.0Å crystal structure of a fusion protein comprised of Nef and the cytoplasmic domain of CD4 bound to the tetrameric AP2 complex. An intricate combination of conformational changes occurs in both Nef and AP2 to enable CD4 binding and downregulation. A pocket on Nef previously identified as crucial for recruiting class I MHC is also responsible for recruiting CD4, revealing a potential approach to inhibit two of Nef’s activities and sensitize the virus to immune clearance.

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We look forward to seeing the revised manuscript and thank you for the opportunity to review your work. This is a solid and complete study in which functional experiments nicely support the structural data. The study clearly shows how Nef connects CD4 to AP-2, leading to CD4 endocytosis. Moreover, the work provides novel information into the sequence requirements for this Nef activity. In this regard, a significant contribution of the study was to map the interaction site of CD4 in Nef. It is shown that CD4 CD binds to a pocket in Nef that had been previously demonstrated by these authors to be involved in MHC-I recruitment. The authors propose that this common CD4/MHC-I interaction site in Nef could be useful for pharmacological targeting. However, the common Nef residues contacting both CD4 and MHC-I are not readily visible in the structures shown. Also, it was not experimentally demonstrated that specific residues in this pocket are required for the downregulation of both CD4 and MHC-I. Specific comments -It seems important to experimentally show the correlation between loss of CD4 downregulation and AP-2 binding capacity for the novel Nef mutants described in this study. In this regard, the authors could expand the GST pulldown assays presented in Figure 1 only for Nef WT.
-Did the authors consider that poor expression displayed by some Nef mutants (e.g., M79D, T138D, L37D, W57A-L58A) might result from instability caused by changes in global protein structure? This would challenge their interpretation that compromised activity is specifically due to loss of CD4/AP-2 interaction. Testing the capacity of these mutants to downregulate MHC-I would help to show if they still are biochemically active.
-It would be helpful to include a panel in Figure 4, highlighting the Nef residues contacting both CD4 and MHC-I. Also, to sustain their hypothesis of a shared interaction site, the authors should test whether Nef mutants predicted to lose interaction with both CD4 and MHC-I are defective in downregulate the two targets. If those common residues were among the ones already tested for CD4 downregulation (Figure 2), they should also be tested for MHC-I. This would sustain their hypothesis of using a small-molecule for Nef inhibition targeting a single interaction site.
-Nef is thought to prevent MHC-I targeting to the cell surface by hijacking AP-1 at the TGN, whereas Nef hijacks AP-2 to induce CD4 endocytosis (Collins and Collins PLoS Pathog. 2014 Jan; 10(1): e1003851). Based on their comparative structural analysis of MHC-I:Nef:AP-1 and CD4:Nef:AP-2 tripartite complexes, can the authors discuss the apparent specificity of AP-1 and AP-2 participation in these two similar but mechanistically distinct Nef functions? In other words, is there any structural basis for a Nef's preference in using AP-1 to downregulate MHC-I and AP-2 to downregulate CD4?
Minor: -In the Extended data Figure 8, It would be useful also to highlight the residues that are involved in MHC-I downregulation and mark the Nef residues corresponding to CD4/MHC-I binding pocket.
-In Figure 2f, the numbers for residues W57 e L58 are inverted.
Reviewer #2: Remarks to the Author: Hijacking of endogenous membrane trafficking systems seems to be a common feature of many viral infection cycles. In the case of HIV, the viral protein Nef has previously been shown to interact with both the AP1 and AP2 clathrin adaptor complexes. These complexes are central to the formation of clathrin-coated vesicles in the golgi (AP1) and the plasma membrane (AP2).
In this work, the authors determined an x-ray crystal structure of a portion of the AP2 complex bound to the HIV protein Nef and a portion of the CD4 receptor. This structure shows that while Nef engages host cargo (CD4 and MCH-I) using the same binding site, Nef interacts with AP1 and AP2 in distinct manners. To validate the biological importance of the interaction seen in the crystal structure, the authors make several mutations in various Nef binding pockets, which reduce the ability of Nef to downregulate CD4 levels at the plasma membrane. Additionally, the authors take a comprehensive approach to model building and to probing the dynamics of AP2 when engaged in the AP2:Nef:CD4 complex.
This work will be of much interest to the membrane trafficking community and researchers interested in HIV. This data likely explains the mechanism the cells use to internalize CD4 via AP2-mediated endocytosis.
I go into more detail below, but the major issue with this work is that the cell biology data is poorly organized, and the authors use a less-than-convincing approach to justify the significance of mutations in Nef that seem to destabilize the protein and/or reduce expression in cells. If the authors can address the concerns below, considering that the structural component of this work is solid, the manuscript should strongly be considered for publication.

Major issues
• The lack of an introduction section will make this study difficult for non-experts to understand. The only introduction or background information to be found is in the abstract, which nicely sets the stage. However, this should be expanded upon in the main text so that readers are better prepared to contextualize the results.
• The cell sorting data in Figure 2 and expanded upon in Extended figure 1 are hard to follow in the text, and the dose compensation assays are unconvincing. The assay requires co-transfection of a GFP-containing plasmid and a Nef-containing plasmid, and then cells are sorted using two colors: GFP signal (a proxy for Nef transfection) and antibody staining of surface-exposed CD4.
The first problem with this data is that it is poorly described in the text. There needs to be at least a single sentence describing the assay in the main text. Just a simple explanation would help readers be able to interpret Figure 2e.
Regarding Figure 2e, there are 8 separate mutants displayed. Four of these mutants are mentioned on page 2 line 32 (F121D, D123R, M79D, and T138D) when describing the CD4 binding pocket on Nef, two are mentioned on page 3 line 6 when describing the N-loop on Nef (W57A/L58A, L112D), and one is mentioned on page 4 line 15 when mentioning the beta binding pocket on Nef (L100A-I109A). This corresponds to seven mutants discussed throughout the text. However, there are 8 mutants in figure 2e. The L164A-L165A mutant is never described in the text, as far as I can tell.
To summarize, the various mutants described in figure 2e are described in a haphazard manner throughout the text. It makes following the functional assays difficult to the reader. In addition, several of the mutations seem to destabilize Nef, and this makes interpreting the various mutations more difficult as figure 2e is discussing mutations in several binding pockets around Nef.
• The dose-compensation assay shown in Extended Figure 1 and 6 does not seem like a good way to probe whether the defects of the mutation come from a simple destabilization of Nef or from an impaired interaction with CD4.
• For all of the flow-cytometry data, is there any sort of quantitative metric that can be used to say if a cell line has downregulated CD4? The difference between no Nef and WT plots are visually distinctive, but some of the mutants seem to not be so striking. For example, comparing L37D and L76D in Extended Data Fig 1 is difficult. One is suggested to break CD4 interaction (L37D) and the other is suggested to not (L76D). • I believe that the authors should re-arrange the figures by putting all of the Nef immunoblots in the supplement, and only put flow cytometry data for mutants that do not affect Nef expression/stability in the main text. A figure showing the multiple binding sites on Nef (CD4 site, Nef Loop, etc.) and highlighting the residues that when mutated abolish the ability to downregulate CD4 would be a powerful combination of the two techniques.
Essentially, I am not convinced that Nef mutants that show reduced Nef stability/expression can be claimed as vital to CD4 downregulation with confidence. If the authors simply stated that those mutations could not be fully analyzed because of their effects on protein stability, and instead focus only on the mutants that do not affect Nef stability, the results would be much clearer. • Related to my comment about the introduction above in "Major Issues", I think the manuscript is difficult to read without sub-headings to guide the reader. I'm unaware of the journal guidelines, but overall I found the paper difficult to read as it just blends together as a series of structural description. Breaking the main text up with sub-headings might re-enforce the various findings of the paper. Reviewer #3: Remarks to the Author: Kwon and coworkers report a structural analysis of a complex of a CD4-Nef fusion protein with AP-2. The problem is significant in that the mechanism of CD4 downregulation, a highly conserved activity of HIV-1 Nef, has not been fully described at a structural level. Prior studies had reported the structure of Nef bound to AP-2 but without CD4. In the current complex, the CD4 cytoplasmic tail, though contiguous with the Nef polypeptide, appears to be occupying its bona fide binding site in Nef, and AP-2 is bound in its open conformation. Interestingly, the CD4 binding site in Nef overlaps the binding site for MHCI, and the core domain of Nef binds differently to AP-2 and AP-1. The work appears to have been performed rigorously, and the functional data, including analysis of new Nef point mutants, supports the biological relevance of the structure. However, the functional data are devoid of information regarding replicates, and there is no statistical analysis, thereby casting doubt on the reproducibility of those experiments. The text of the paper is clear though somewhat terse. The prior literature is appropriately cited.
The authors must indicate the number of times each of the following experiments was performed, and provide mean values and standard deviations for the data, along with appropriate statistical analysis: We sincerely thank the reviewers for their critical insights and thoughtful suggestions. We found them to be very helpful in identifying weaknesses in our earlier manuscript and pointing us to the right directions for improvement. We have addressed each of these concerns and suggestions as detailed below.
Reviewer #1: Major issues: "It seems important to experimentally show the correlation between loss of CD4 downregulation and AP-2 binding capacity for the novel Nef mutants described in this study. In this regard, the authors could expand the GST pulldown assays presented in Figure 1 only for Nef WT." This is a great suggestion. Following it, we have analyzed the effect of these novel Nef mutations using an in vitro fluorescence polarization assay (Fig. 2e). Here, binding of a fluorescence tag-labeled CD4 tail to the Nef/AP2 complex was characterized for both WT and mutant Nefs. This assay goes a step further than the GST pulldown assay in that it allowed a quantitative comparison of CD4-binding abilities. As expected, correlation was observed between the loss of CD4 downregulation ( Fig. 2f-h) and in vitro CD4binding abilities for each Nef mutation tested.
"Did the authors consider that poor expression displayed by some Nef mutants (e.g., M79D, T138D, L37D, W57A-L58A) might result from instability caused by changes in global protein structure? This would challenge their interpretation that compromised activity is specifically due to loss of CD4/AP-2 interaction. Testing the capacity of these mutants to downregulate MHC-I would help to show if they still are biochemically active." We agree that poor expression of these Nef mutants could be due to their reduced stability. Testing the capacity of these mutants to downregulate MHC-I was a great idea. However, in our experience, poorly expressed Nef mutants are uniformly impaired in their ability to downregulate MHC-I. We have instead addressed this concern by following reviewer #2's suggestion: we left these poorly expressed mutants out and focused only on well-expressed ones ( Fig. 2f-h). Results from the latter, we believe, sufficiently validate the structural observations. "It would be helpful to include a panel in Figure 4, highlighting the Nef residues contacting both CD4 and MHC-I. Also, to sustain their hypothesis of a shared interaction site, the authors should test whether Nef mutants predicted to lose interaction with both CD4 and MHC-I are defective in downregulate the two targets. If those common residues were among the ones already tested for CD4 downregulation ( Figure  2), they should also be tested for MHC-I. This would sustain their hypothesis of using a small-molecule for Nef inhibition targeting a single interaction site." Surprisingly, there is only one Nef residue, Asp123, that is critical for both mechanisms. How this residue makes contact with CD4 is illustrated in Fig. 2b. The effect of mutating D123 on CD4 binding and CD4 downregulation is shown in Fig. 2e-h. Such effect has also been observed in the past (references 28, 29). This same Asp123 residue contributes to MHC-I downregulation by participating in a three-way electrostatic interaction, as we have previously reported (reference 35). There, Asp123 forms salt bridges with two Arg residues of Mu1, both of which then form salt bridges with Asp327 of MHC-I (reference 35, Figure 3d). Although Nef Asp123 does not make direct contact with MHC-I, mutation at this site completely abolishes MHC-I downregulation. This effect has been observed repeatedly in the past including in our own publication (references 35-37). We, therefore, did not test this mutation on MHC-I downregulation again here. Due to the different ways that Asp123 contributes to binding in each case, we find it difficult to structurally illustrate how Asp123 contacts both CD4 and MHC-I in the overlaid structures. We therefore did not include such a panel. We did provide more details on the requirement of Asp123 in the main text. We hope the reviewer will find these changes adequate and acceptable. Importantly, although only Asp123 is dually critical for both mechanisms, residues that form this dual-functional pocket are conserved likely because both mechanisms are highly conserved functions of Nef (Extended Data Fig. 5).
"Nef is thought to prevent MHC-I targeting to the cell surface by hijacking AP-1 at the TGN, whereas Nef hijacks AP-2 to induce CD4 endocytosis (Collins and Collins PLoS Pathog. 2014 Jan; 10(1): e1003851). Based on their comparative structural analysis of MHC-I:Nef:AP-1 and CD4:Nef:AP-2 tripartite complexes, can the authors discuss the apparent specificity of AP-1 and AP-2 participation in these two similar but mechanistically distinct Nef functions? In other words, is there any structural basis for a Nef's preference in using AP-1 to downregulate MHC-I and AP-2 to downregulate CD4?" For MHC-I downregulation, the structural basis of the preference of Nef for μ1 over μ2 has been addressed in our earlier publication (reference 35). For CD4 downregulation, structurally, there is no clear indication whether Nef prefers AP2 over AP1. We believe the choice of AP2 over AP1 here is more of a biological reason: Nef needs to target CD4 resident at the cell surface (newly synthesized CD4 is targeted by Vpu) and therefore hijacks the AP2-and clathrin-mediated endocytosis. On the other hand, Nef needs to target MHC-I at the TGN to divert complexes newly loaded with viral peptides from ever reaching the cell surface. Regarding how and why AP1 is used for downregulation of MHC-I while AP2 is used for CD4, the structural aspects of this distinction are based on a molecular switch mechanism involving the alternative binding of the Nef N-terminal helix vs. the β2 N-terminal helix to the hydrophobic crevice on the Nef core in the two complexes. We have tried to more clearly explain this mechanism in the revised text.

Minor issues:
"In the Extended data Figure 8, It would be useful also to highlight the residues that are involved in MHC-I downregulation and mark the Nef residues corresponding to CD4/MHC-I binding pocket." We have included additional labels in this figure (now Extended Data Fig. 5), which specified residues important for MHC-I downregulation (orange texts) as well as residues corresponding to the binding pocket (black texts).
"In Figure 2f, the numbers for residues W57 e L58 are inverted." This error has been corrected.
Reviewer #2: Major issues: "The lack of an introduction section will make this study difficult for non-experts to understand. The only introduction or background information to be found is in the abstract, which nicely sets the stage. However, this should be expanded upon in the main text so that readers are better prepared to contextualize the results." We have added an introduction section and expanded on the background knowledge of both Nef and clathrin adaptor protein complexes.
"The cell sorting data in Figure 2 and expanded upon in Extended figure 1 are hard to follow in the text, and the dose compensation assays are unconvincing. The assay requires co-transfection of a GFPcontaining plasmid and a Nef-containing plasmid, and then cells are sorted using two colors: GFP signal (a proxy for Nef transfection) and antibody staining of surface-exposed CD4.
The first problem with this data is that it is poorly described in the text. There needs to be at least a single sentence describing the assay in the main text. Just a simple explanation would help readers be able to interpret Figure 2e.
Regarding Figure 2e, there are 8 separate mutants displayed. Four of these mutants are mentioned on page 2 line 32 (F121D, D123R, M79D, and T138D) when describing the CD4 binding pocket on Nef, two are mentioned on page 3 line 6 when describing the N-loop on Nef (W57A/L58A, L112D), and one is mentioned on page 4 line 15 when mentioning the beta binding pocket on Nef (L100A-I109A). This corresponds to seven mutants discussed throughout the text. However, there are 8 mutants in figure 2e. The L164A-L165A mutant is never described in the text, as far as I can tell.
To summarize, the various mutants described in figure 2e are described in a haphazard manner throughout the text. It makes following the functional assays difficult to the reader. In addition, several of the mutations seem to destabilize Nef, and this makes interpreting the various mutations more difficult as figure 2e is discussing mutations in several binding pockets around Nef." This is a great suggestion. We have re-arranged these flow cytometry data and consolidated them into one figure for CD4 (Fig. 2f-h). The corresponding text is also consolidated into one paragraph. We have included a brief description of the CD4 downregulation assay. We opted not to show the data on the L164A:165A mutant here in Figure 2, although we do show it as a negative control in Figure 4. The doseresponse assays, which were intended to address the poor expression of certain Nef mutants, have been removed, following reviewer #2's other suggestion (below).
"The dose-compensation assay shown in Extended Figure 1 and 6 does not seem like a good way to probe whether the defects of the mutation come from a simple destabilization of Nef or from an impaired interaction with CD4." Again, we agree that the concern over the stability of these mutants is valid. We have removed these data and focused only on well-expressed Nef mutants for validation of the structural observations. "For all of the flow-cytometry data, is there any sort of quantitative metric that can be used to say if a cell line has downregulated CD4? The difference between no Nef and WT plots are visually distinctive, but some of the mutants seem to not be so striking. For example, comparing L37D and L76D in Extended Data Fig 1 is difficult. One is suggested to break CD4 interaction (L37D) and the other is suggested to not (L76D)." We have included a statistical representation of the flow-cytometry data (Fig. 2g). The data for both L37D and L76D mutants are removed due to the concern associated with their poor expression. Data for this mutant, although supportive of the molecular switch model, has been removed. This change, however, does not affect our interpretation as the remaining mutagenesis data sufficiently support this model (Fig. 4bc).
"I believe that the authors should re-arrange the figures by putting all of the Nef immunoblots in the supplement, and only put flow cytometry data for mutants that do not affect Nef expression/stability in the main text. A figure showing the multiple binding sites on Nef (CD4 site, Nef Loop, etc.) and highlighting the residues that when mutated abolish the ability to downregulate CD4 would be a powerful combination of the two techniques.
Essentially, I am not convinced that Nef mutants that show reduced Nef stability/expression can be claimed as vital to CD4 downregulation with confidence. If the authors simply stated that those mutations could not be fully analyzed because of their effects on protein stability, and instead focus only on the mutants that do not affect Nef stability, the results would be much clearer." We agree with the reviewer here. Following this suggestion, we have re-arranged the flow cytometry data as well as the corresponding immunoblots. We have now focused only on the well-expressed mutants and presented the mutagenesis data concerning both the CD4-binding and N-loop-binding sites on Nef into one figure (Fig. 2f-h). We have featured these important residues structurally in Fig. 2bd. Correspondingly, we have removed the flow data of poorly expressed Nef mutants. Again, we agree that these data cannot be interpreted with confidence and removing them does not diminish the validation of the structural observations. This label has now been added.
"Related to my comment about the introduction above in "Major Issues", I think the manuscript is difficult to read without sub-headings to guide the reader. I'm unaware of the journal guidelines, but overall I found the paper difficult to read as it just blends together as a series of structural description. Breaking the main text up with sub-headings might re-enforce the various findings of the paper." This was a weakness of our earlier manuscript. We have now divided the Results into sub-sections with a sub-heading added to each. We agree with the reviewer that the updated format reinforces our findings and we hope make reading easier. This is another great suggestion. Comparison between the current structure and the AP2:Tetherin:Nef structure reveals sophisticated ways that Nef proteins manipulate and remodel the β2 N-terminus to target different host proteins. We have now included a paragraph in the Discussion and elaborated on this finding.
"The work appears to have been performed rigorously, and the functional data, including analysis of new Nef point mutants, supports the biological relevance of the structure. However, the functional data are devoid of information regarding replicates, and there is no statistical analysis, thereby casting doubt on the reproducibility of those experiments.…………..
The authors must indicate the number of times each of the following experiments was performed, and provide mean values and standard deviations for the data, along with appropriate statistical analysis: We recognize the reviewer's concern on the lack of information on replicates and statistical analysis. Following the reviewer's suggestion, we have added this information as noted above (Fig 2g).
"The text of the paper is clear though somewhat terse." We have addressed this issue. We added an Introduction and Discussion. We divided the results into sub-sections and added sub-headings for them. We also incorporated more descriptions to facilitate the delivery of the messages and improve the fluency of the text. These additions and modifications will hopefully make the manuscript easier to read and follow.

Decision Letter, first revision:
5th May 2020 Dear Xiaofei, Thank you again for submitting your manuscript "Structural Basis of CD4 Downregulation by HIV-1 Nef". The reports of one of the original referees are below, and based on these comments, we are happy to accept your paper, in principle, for publication as an Article in Nature Structural & Molecular Biology, on the condition that you revise your manuscript in response to our editorial requirements.
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Please use the following link for uploading these materials: [REDACTED] We ask that you aim to return your revised paper within 14 days. If you have any further questions, please feel free to contact me. We sincerely thank the reviewers for approving our resubmission and for the editors for providing the guidance in preparing the final submission. We have addressed each of these concerns and suggestions as detailed below.
We have previously deposited the crystal structure at the PDB database. The coordinates will be released upon the publication of this manuscript. Additionally, we have also deposited the XL-MS dataset at the ProteomeXchange database and the integrative structure model at PDB-Dev. The Data Availability Statement has been updated accordingly.
We have provided the source data and uncropped gels (Supplementary Figure 1). An updated Reporting Summary has been provided.
We have added necessary descriptions in the abstract to make it more informative.
PDB ID has been added to Table 1.
Information about the type of replicates for Figure 2e and 2g has been included in the legend.
Additional information for cell lines has been added in the Methods. We did not create a separate section for this. Rather, we included such information in each of the two assays (CD4 downregulation and MHC-I downregulation) where cell lines were used.

Final Decision Letter:
16th Jun 2020 Dear Xiaofei, We are now happy to accept your revised paper "Structural Basis of CD4 Downregulation by HIV-1 Nef" for publication as a Article in Nature Structural & Molecular Biology.
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