Anindita Varshneya, Jordan Detamore, Colin Wikholm, Isai Lopez Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J., & Hendrickson, W. A. (1998). Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature, 393(6686), 648-659. DOI: 10.1038/31405 Anindita Varshneya, Jordan Detamore, Colin Wikholm, Isai Lopez BIOL 368: Bioinfomatics Laboratory Department of Biology Loyola Marymount University October 25, 2016 Anu

Outline HIV-1 infection of host cells involves primary complexing of gp120 with CD4, secondary chemokine receptor binding, and cell fusion. Understanding conformational changes in complexed viral-cell structures may explain function and possible treatments. Kwong et al. (1998) studied gp120/CD4/NAb for structural determination and function. They identified the ternary complex structure, binding interfaces, and modes of immune evasion. Future studies should analyze other intermediates to address fusion mechanisms, alternate glycoprotein states, and other areas. Anu Note: Let me know if font seems too small SIMPLER LANGUAGE + V3 STUFF!!!! Especially in methods

Outline HIV-1 infection of host cells involves primary complexing of gp120 with CD4, secondary chemokine receptor binding, and cell fusion. Understanding conformational changes in complexed viral-cell structures may explain function and possible treatments. Kwong et al. (1998) studied gp120/CD4/NAb for structural determination and function. They identified the ternary complex structure, binding interfaces, and modes of immune evasion. Future studies should analyze other intermediates to address fusion mechanisms, alternate glycoprotein states, and other areas. Anu Note: Let me know if font seems too small SIMPLER LANGUAGE + V3 STUFF!!!! Especially in methods

The HIV-1 Expresses a Series of Surface Proteins for Infection of Host Cells The gp160 is cleaved into gp120 and gp41, which together form the viral spike. Colin “A structure-based schematic representation of the HIV-1 envelope glycoprotein trimer interacting with the primary receptor, CD4, and the co-receptor, CCR5 present on the target cell. The gp120 exterior envelope glycoprotein is modeled as a trimer in blue in non-covalent association with the gp41 trimeric transmembrane glycoprotein in brown. The protruding gp120 third major variable loop (V3) is shown in cyan” This is what HIV looks like http://people.mpi-inf.mpg.de/~fmueller/thesis/thesis_mueller.pdf

HIV-1 Infection of Host Cell Involves Primary Binding, Secondary Binding, and Membrane Fusion The gp120 binds to CD4, inducing conformational change in gp120. The gp120 binding sites are exposed and CXCR4 binds to gp120. The gp41 glycoprotein initiates fusion of virus and cell membranes. Colin “Overview of HIVentry. To deliver the viral payload into cells, HIV Env, comprised of gp120 and gp41 subunits (1), first attaches to the host cell, binding CD4 (2). This causes conformational changes in Env, allowing coreceptor binding, which is mediated in part by the V3 loop of Env (3). This initiates the membrane fusion process as the fusion peptide of gp41 inserts into the target membrane, followed by six-helix bundle formation and complete membrane fusion (4).” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3405824/

HIV-1 Infection of Host Cell Involves Primary Binding, Secondary Binding, and Membrane Fusion Leads to chemokine receptor binding -OR- Neutralization by CD4i NAb (CD4 induced neutralizing antibodies) Colin “Overview of HIVentry. To deliver the viral payload into cells, HIV Env, comprised of gp120 and gp41 subunits (1), first attaches to the host cell, binding CD4 (2). This causes conformational changes in Env, allowing coreceptor binding, which is mediated in part by the V3 loop of Env (3). This initiates the membrane fusion process as the fusion peptide of gp41 inserts into the target membrane, followed by six-helix bundle formation and complete membrane fusion (4).” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3405824/

Outline HIV-1 infection of host cells involves primary complexing of gp120 with CD4, secondary chemokine receptor binding, and cell fusion. Understanding conformational changes in complexed viral-cell structures may explain function and possible treatments. Kwong et al. (1998) studied gp120/CD4/NAb for structural determination and function. They identified the ternary complex structure, binding interfaces, and modes of immune evasion. Future studies should analyze other intermediates to address fusion mechanisms, alternate glycoprotein states, and other areas. Anu Note: Let me know if font seems too small SIMPLER LANGUAGE + V3 STUFF!!!! Especially in methods

The gp120 binding of CD4 Induces Conformational Changes that Allow for CD4i NAb Binding Structure is important for function. Understanding the distinctive structures of the tetramer is thus important for understanding function and possible treatments. Colin http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1002797

Outline HIV-1 infection of host cells involves primary complexing of gp120 with CD4, secondary chemokine receptor binding, and cell fusion. Understanding conformational changes in complexed viral-cell structures may explain function and possible treatments. Kwong et al. (1998) studied gp120/CD4/NAb for structural determination and function. They identified the ternary complex structure, binding interfaces, and modes of immune evasion. Future studies should analyze other intermediates to address fusion mechanisms, alternate glycoprotein states, and other areas. Anu Note: Let me know if font seems too small SIMPLER LANGUAGE + V3 STUFF!!!! Especially in methods

R-value = 21.0% (5–2.5 Å data > 2σ, R-free = 30.3%) X-ray Crystallography of the gp120/CD4/NAb Complex Allowed for Reliable Modeling of Overall Ternary Structure R-value = 21.0% (5–2.5 Å data > 2σ, R-free = 30.3%) gp120 CD4 Fab 17b dark blue = heavy chain light blue = light chain ISAI LOPEZ “The R value is used to assess progress in the refinement of a model from X-ray crystallographic data, and can be used as one factor in evaluating the quality of a model. R is a measure of error between the observed intensities from the diffraction pattern and the predicted intensities that are calculated from the model. R values of 0.20 or less are taken as evidence that the model is reliable. As a rule of thumb, models with R values substantially exceeding (resolution/10) should be treated with caution. Thus, if the resolution of a model is 2.5 Å, that model's R value should not exceed 0.25. Free R value: a novel statistical quantity for assessing the accuracy of crystal structures “Despite stereochemical restraints2, it is possible to overfit or 'misfit' the diffraction data: an incorrect model can be refined to fairly good R values as several recent examples have shown… As examples show, there is a high correlation between R free T and the accuracy of the atomic model phases. This is useful because experimental phase information is usually inaccurate, incomplete or unavailable.” Kwong et al. 1998

Structural Views of the gp120 Protein Indicate Binding Sites for CD4 ISAI LOPEZ Kwong et al. 1998

Structural Views of the gp120 Protein Indicate Binding Sites for CD4 ISAI LOPEZ Residues that interact directly with CD4 are marked Kwong et al. 1998

Direct Contact Between CD4 and gp120 was Diagrammed Instead of Loss of Solvent-Accessible Surfaces ISAI LOPEZ Sequence alignment of amino acids among 3 different HIV-1 strains, 1 HIV-2 strain, and a strain of SIV. Dashes at the bottom denote level of conservation across all strains where 1 dash is most conserved and 5 dashes is most variable. It was noted that the inner domains had a higher degree of conservation that did the outside domains (Compare with Fig. 2a) Kwong et al. 1998

The gp120-CD4 Protein Complex Creates a Phe43 Cavity Essential to Viral Entry Anu Phe43 Kwong et al. 1998

The gp120 and CD4 Bind and Cause a Conformational Change That Create a Cavity Near the Phe 43 Residue Anu Kwong et al. 1998

CD4 Residues Phe43 and Arg59 Come in Direct Contact With the gp120 Anu Kwong et al. 1998

Electrostatic Diagram The 17b Antibody Binds with CD4i NAb and gp120, but the Interface Between the Proteins is Relatively Small d e Anu Electrostatic Diagram V3 Region Kwong et al. 1998

A Series of Conformational Changes are Induced Through gp120-CD4 Binding, and Chemokine Receptor Binding Structural analysis clarified details of mechanisms that lead to fusion: Conformational change occurs when CD4 binds Inner/outer domain shift-Phe43 cavity forms Alters the orientation of the N and C termini-priming gp120 core Chemokine binds to V3 loop Orientational shift occurs Further changes are triggered, ultimately leading to fusion Jordan Structural analysis of THIS study suggests the following details occur: “Thus CD4 binding not only orients the gp120 surface implicated in chemokine receptor binding to face the target cell, it also forms and exposes the site itself. These changes may all happen in a single, concerted shift in the relative orientation of the inner and outer domains. This conformational shift may alter the orientation of the N and C termini, at the proximal end of the inner domain, perhaps partially destabilizing the oligomeric gp120/gp41 interface21. Such a shift would also alter the relative placement of the V1/V2 stem (in the CD4i site), which emanates from the inner domain, and the V3 loop, which emanates from the outer domain. Perhaps relevant to this, mutations that permit an adaptation of HIV-1 to CD4-independent entry using CXCR4 involve sequence changes in both the V1/V2 stem and the V3 loop42.” Structural analysis from THIS study contributes to the fusion model in the followinhg way: “This conformational shift may alter the orientation of the N and C termini, at the proximal end of the inner domain, perhaps partially destabilizing the oligomeric gp120/gp41 interface21. Such a shift would also alter the relative placement of the V1/V2 stem (in the CD4i site), which emanates from the inner domain, and the V3 loop, which emanates from the outer domain.” initiation of gp120 fusion, gp120 V1 and V2 bind to CD4 causing a conformational change causing both the inner and outer domains of the gp120 protein to move to allow for an opening near the Phe43 residue, chemokine receptor attaches to the bridging sheet and V3 loop which causes another shift of the gp120 protein ultimately allowing the viral membrane to fuse with the cellular membrane Kwong et al. 1998

Summary HIV-1 infection of host cells involves a primary receptor binding, co-receptor binding, and subsequent cell fusion. Understanding HIV-1 structures is important in explaining function and discovering treatments. Kwong et al. (1998) studied the structure and function of a HIV-1 ternary complexe containing gp120, CD4, and a neutralizing antibody. They modeled several structures of the gp120/CD4/NAb complex and determined how they aid in immune evasion and host cell infection. Future studies should analyze the structures of other stages of HIV-1 infection in order to better understand viral mechanisms. Jordan

LMU Department of Biology Acknowledgments Jordan Dr. Kam D. Dahlquist LMU Department of Biology

References Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J., & Hendrickson, W. A. (1998). Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature, 393(6686), 648-659. DOI: 10.1038/31405 Müller, F. (2009). Assessing Antibody Neutralization of HIV-1 as an Initial Step in the Search for gp160-based Immunogens (Doctoral dissertation, Universität des Saarlandes Saarbrücken). Tran, E. E., Borgnia, M. J., Kuybeda, O., Schauder, D. M., Bartesaghi, A., Frank, G. A., ... & Subramaniam, S. (2012). Structural mechanism of trimeric HIV-1 envelope glycoprotein activation. PLoS Pathog, 8(7), e1002797. http://dx.doi.org/10.1371/journal.ppat.1002797 Wilen CB, Tilton JC, Doms RW. HIV: Cell Binding and Entry. Cold Spring Harbor Perspectives in Medicine. 2012;2(8):a006866. doi:10.1101/cshperspect.a006866.