Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2–V3 interaction and modulates neutralization sensitivity Raffaello Cimbroa,1, Thomas R. Gallanta, Michael A. Dolanb, Christina Guzzoa, Peng Zhanga, Yin Lina, Huiyi Miaoa, Donald Van Ryka, James Arthosa, Inna Gorshkovac, Patrick H. Brownc, Darrell E. Hurtb, and Paolo Lussoa,2 aLaboratory of Immunoregulation and bBioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and cBiomedical Engineering and Physical Science Shared Resource, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892 Edited by Dennis R. Burton, The Scripps Research Institute, La Jolla, CA, and Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, and accepted by the Editorial Board January 13, 2014 (received for review August 5, 2013) Elicitation of broadly neutralizing antibodies is essential for the receptor- and coreceptor-binding sites is believed to be a primary development of a protective vaccine against HIV-1. However, the mechanism of immune evasion by HIV-1 (4). native HIV-1 envelope adopts a protected conformation that con- The inherent conformational flexibility of gp120, along with ceals highly conserved sites of vulnerability from antibody recognition. the extensive N-linked glycosylation that covers most of the ex- Although high-definition structures of the monomeric core of the en- posed surface of the glycoprotein, has severely hampered attempts velope glycoprotein subunit gp120 and, more recently, of a stabilized to elucidate the native structure of the HIV-1 envelope spike. As soluble gp140 trimer have been solved, fundamental aspects re- a consequence, most of the available high-definition structures of lated to the conformation and function of the native envelope gp120 have been obtained with deglycosylated, variable loop- remain unresolved. Here, we show that the conserved central re- truncated core monomers in complex with stabilizing ligands such gion of the second variable loop (V2) of gp120 contains sulfated as soluble CD4 (sCD4) (6–10). Important information regarding tyrosines (Tys173 and Tys177) that in the CD4-unbound prefusion the overall conformation and ligand interactions of the trimeric state mediate intramolecular interaction between V2 and the con- spike at intermediate resolution has emerged from the use of in- MICROBIOLOGY served base of the third variable loop (V3), functionally mimicking creasingly refined cryo-electron microscopy (cryo-EM) technologies sulfated tyrosines in CCR5 and anti–coreceptor-binding-site anti- (11–17). Moreover, the crystal structure of a stabilized, soluble, bodies such as 412d. Recombinant gp120 expressed in continuous cleaved gp140 trimer (BG505 SOSIP.664) at 4.7-Å resolution was cell lines displays low constitutive levels of V2 tyrosine sulfation, reported recently (18). However, despite these advances, many which can be enhanced markedly by overexpression of the tyrosyl critical aspects related to the structural mechanisms of HIV-1 im- sulfotransferase TPST2. In contrast, virion-associated gp120 pro- mune vulnerability and evasion remain unresolved. In particular, + duced by primary CD4 T cells is inherently highly sulfated. Con- the fine molecular details of the interaction between the second and sistent with a functional role of the V2 sulfotyrosines, enhancement third variable loops (V2 and V3, respectively) of gp120, which are of tyrosine sulfation decreased binding and neutralization of HIV-1 believed to play a critical role in stabilizing the prefusion envelope BaL by monomeric soluble CD4, 412d, and anti-V3 antibodies and structure (19–21), are elucidated only partially. Functionally, V2 increased recognition by the trimer-preferring antibodies PG9, PG16, and V3 cooperate in the formation of quaternary epitopes targeted CH01, and PGT145. Conversely, inhibition of tyrosine sulfation in- creased sensitivity to soluble CD4, 412d, and anti-V3 antibodies Significance and diminished recognition by trimer-preferring antibodies. These results identify the sulfotyrosine-mediated V2–V3 interaction as a Despite intensive efforts, the structure of the native HIV-1 critical constraint that stabilizes the native HIV-1 envelope trimer envelope trimer—the sole relevant target for vaccine design— and modulates its sensitivity to neutralization. has remained elusive. Our work identifies a key structural con- straint that stabilizes the native envelope conformation and he development of a protective vaccine remains a high pri- modulates its sensitivity to neutralization. We show that this Tority for the global control of the HIV/AIDS epidemic (1). constraint is established by previously unrecognized sulfated However, the unique biological features of HIV-1 make this task tyrosines within the second variable loop (V2) of the envelope extremely challenging. The main obstacles include the ability of glycoprotein subunit gp120, which mediate intramolecular the virus to integrate into the host chromosomes, a remarkable interaction with the base of the third variable loop, V3. Strik- degree of genetic variability, and the cryptic, antibody-shielded ingly, the V2 sulfotyrosines functionally mimic those present in conformation adopted by the viral envelope in the native spikes the N terminus of the CCR5 coreceptor, which bind to the same that protrude from the virion surface (2). These spikes are com- V3 region. Our results shed light on the mechanisms adopted posed of homotrimers of heterodimers of the envelope gly- by HIV-1 to elude immunologic control and open new per- coprotein subunits gp120 and gp41 maintained in an energetically spectives for vaccine design. unfavorable, metastable conformation (3, 4). Upon binding to CD4 and a coreceptor such as CCR5 or CXCR4, gp120 undergoes Author contributions: R.C. and P.L. designed research; R.C., T.R.G., M.A.D., C.G., P.Z., Y.L., H.M., D.V.R., I.G., P.H.B., D.E.H., and P.L. performed research; R.C., M.A.D., J.A., D.E.H., and dramatic conformational changes that lead to a low-energy state, P.L. analyzed data; and R.C. and P.L. wrote the paper. creating permissive conditions for activation of the gp41 fusogenic The authors declare no conflict of interest. mechanism (3). In the prefusion conformation, gp120 effectively This article is a PNAS Direct Submission. D.R.B. is a guest editor invited by the Editorial conceals its highly conserved receptor- and coreceptor-binding Board. sites from antibody recognition, imposing a high-entropy penalty 1Present address: Division of Rheumatology, The Johns Hopkins School of Medicine, Baltimore, for interaction with CD4 or antibodies to the coreceptor-binding MD 21224. site such as 17b; in contrast, in the open, low-energy conformation, 2To whom correspondence should be addressed. E-mail: [email protected]. gp120 interacts with CD4 and 17b with minimal thermodynamic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. changes (4, 5). This conformational masking of the vulnerable 1073/pnas.1314718111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1314718111 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 by some of the most potent and broadly neutralizing mAbs hitherto identified (22, 23), and V2 effectively masks neutralization epitopes AB in V3 (24–27). Cryo-EM studies have provided evidence that in the prefusion conformation V2 and V3 are spatially contiguous and account for most of the density at the apex of the trimeric envelope spike (12–17). Although various fragments of V2 and V3 were crystallized separately using antibody-complexed synthetic peptides (28, 29), scaffolded chimeric constructs of the first and second variable loops (V1V2) (30, 31), or a V3-containing gp120 core monomer (8), the only study in which the two loops were visualized simultaneously is the recent report of the BG505 SOSIP.664 trimer crystal structure (18). In this artificially stabilized trimer, which CD displays several antigenic features of the native envelope (32), V2 and V3 appear to interact directly at the trimer apex with the V3 β-hairpin extensively buried under the V1V2 four-stranded Greek- key β-sheet (18). In the present study, we provide evidence that the conserved central region of the gp120 V2 loop contains previously un- recognized sulfated tyrosines that, in the CD4-unbound pre- Fig. 1. The V2 domain of HIV-1 gp120 contains sulfated tyrosines. (A)Se- fusion state, mediate intramolecular interaction between V2 and quence alignment of the CCR5 N-terminal domain and the conserved central the CCR5-binding site at the base of V3. Our results suggest that region of the V2 domain of HIV-1 gp120 (consensus sequence for subtype B). the sulfotyrosine-bolstered interaction between V2 and V3 is Two colinear conserved tyrosine residues present in CCR5 and gp120 V2 are a key structural constraint that stabilizes the native conformation highlighted in green. (B) Detection of sulfated tyrosines by Western blot in gp120 purified from HeLa cells expressing WT HIV-1 BaL gp160 or a partially of the HIV-1 envelope trimer. V2-deleted mutant (Δ164–190) lacking Tyr173 and Tyr177 by vaccinia tech- nology. gp120 was immunoprecipitated from the cell surface and analyzed Results by Western blot using a specific mAb for sulfated tyrosines (1C-A2); a murine The Conserved Central Region of the V2 Loop of HIV-1 gp120 Contains anti-gp120 mAb (b13) was tested in parallel as a loading control (gp120). (C) Sulfated Tyrosines. Despite their definition as variable loops, the Detection of sulfated tyrosines by autoradiography in metabolically labeled V2 and V3 regions of gp120 contain highly conserved domains HEK293 cells expressing HIV-1 BaL WT or Δ164–190 gp160 by vaccinia (33). Because several lines of evidence suggest that V3 estab- technology. The cells were labeled with free [35S]sulfate or [35S]cysteine/[35S] – methionine by overnight culture in sulfate-free or cysteine/methionine-free lishes direct interaction with V2 (12 21), and the conserved base 35 of V3 is the binding site for the N-terminal region of the CCR5 medium, respectively.