Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2–V3 interaction and modulates neutralization sensitivity

Abstract

Elicitation of broadly neutralizing antibodies is essential for the development of a protective vaccine against HIV-1. However, the native HIV-1 envelope adopts a protected conformation that conceals highly conserved sites of vulnerability from antibody recognition. Although high-definition structures of the monomeric core of the envelope glycoprotein subunit gp120 and, more recently, of a stabilized soluble gp140 trimer have been solved, fundamental aspects related to the conformation and function of the native envelope remain unresolved. Here, we show that the conserved central region of the second variable loop (V2) of gp120 contains sulfated tyrosines (Tys173 and Tys177) that in the CD4-unbound prefusion state mediate intramolecular interaction between V2 and the conserved base of the third variable loop (V3), functionally mimicking sulfated tyrosines in CCR5 and anti-coreceptor-binding-site antibodies such as 412d. Recombinant gp120 expressed in continuous cell lines displays low constitutive levels of V2 tyrosine sulfation, which can be enhanced markedly by overexpression of the tyrosyl sulfotransferase TPST2. In contrast, virion-associated gp120 produced by primary CD4 + T cells is inherently highly sulfated. Consistent with a functional role of the V2 sulfotyrosines, enhancement of tyrosine sulfation decreased binding and neutralization of HIV-1 BaL by monomeric soluble CD4, 412d, and anti-V3 antibodies and increased recognition by the trimer-preferring antibodies PG9, PG16, CH01, and PGT145. Conversely, inhibition of tyrosine sulfation increased sensitivity to soluble CD4, 412d, and anti-V3 antibodies and diminished recognition by trimer-preferring antibodies. These results identify the sulfotyrosine-mediated V2-V3 interaction as a critical constraint that stabilizes the native HIV-1 envelope trimer and modulates its sensitivity to neutralization.T he development of a protective vaccine remains a high priority for the global control of the HIV/AIDS epidemic (1). However, the unique biological features of HIV-1 make this task extremely challenging. The main obstacles include the ability of the virus to integrate into the host chromosomes, a remarkable degree of genetic variability, and the cryptic, antibody-shielded conformation adopted by the viral envelope in the native spikes that protrude from the virion surface (2). These spikes are composed of homotrimers of heterodimers of the envelope glycoprotein subunits gp120 and gp41 maintained in an energetically unfavorable, metastable conformation (3, 4). Upon binding to CD4 and a coreceptor such as CCR5 or CXCR4, gp120 undergoes dramatic conformational changes that lead to a low-energy state, creating permissive conditions for activation of the gp41 fusogenic mechanism (3). In the prefusion conformation, gp120 effectively conceals its highly conserved receptor-and coreceptor-binding sites from antibody recognition, imposing a high-entropy penalty for interaction with CD4 or antibodies to the coreceptor-binding site such as 17b; in contrast, in the open, l...

HIV-1 gp41 Residues Modulate CD4-Induced Conformational Changes in the Envelope Glycoprotein and Evolution of a Relaxed Conformation of gp120

“…This possibility is supported by the position of these residues in the liganded trimer, where S306 is located directly in the 17b binding site, while T319 and E322 are within the unresolved portion of the V3 loop (40). These conclusions are consistent with previous work showing that it is possible to trigger V3 loop exposure independently of V1/V2 exposure and that V1/V2 likely acts as a clamp restraining the unliganded trimer in the closed state (42)(43)(44)(45)(46)(47)(48)(49).…”

Glycan modulation and sulfoengineering of anti–HIV-1 monoclonal antibody PG9 in plants

“…Although some plants have a TPST and can sulfate phytohormones (20), PG9 expressed in N. benthamiana did not contain detectable amounts of sulfotyrosines, indicating that mammalian-type sulfation does not occur naturally in N. benthamiana leaves. Overexpression of the hsTPSTs hsTPST1 and hsTPST2 (18) increased sulfation of recombinantly produced proteins in mammalian cells (6,28). However, expression of full-length hsTPST1 in N. benthamiana did not yield efficient levels of PG9 tyrosine sulfation.…”

“…It is of note that tyrosine sulfation also affects the antigenicity of gp120 itself. It has been reported that gp120 from CD4 + T cell-produced virions is more extensively sulfated than cell line-produced gp120 (28) and that V2 needs to be sulfated to stabilize V3. Thus, efficient tyrosine sulfation of gp120 vaccine candidates might be crucial for eliciting a sustained antibody response.…”

Antibodies Targeting the Envelope of HIV-1

“…Electron tomography, cryo-electron microscopy, and biochemical studies have shown that the V1V2 domain is localized at the apex of the trimeric HIV-1 Env structures, and therefore at least some of the V1V2 epitopes are accessible to Abs (75,76). V2 loop sequences differ in length, but the majority of amino acids are highly conserved, suggesting conserved structural elements (77).…”