Limits...
Trimeric HIV Env provides epitope occlusion mediated by hypervariable loops.

Moscoso CG, Xing L, Hui J, Hu J, Kalkhoran MB, Yenigun OM, Sun Y, Paavolainen L, Martin L, Vahlne A, Zambonelli C, Barnett SW, Srivastava IK, Cheng RH - Sci Rep (2014)

Bottom Line: A prominent basal quaternary location of V2 and V3' that challenges previous reports would facilitate gp41-independent gp120-gp120 interactions and suggests a quaternary mechanism of epitope occlusion facilitated by hypervariable loops.Deletion of V2 resulted in dramatic exposure of basal, membrane-proximal gp41 epitopes, consistent with its predicted basal location.The structural features of HIV-1 Env characterized here provide grounds for a paradigm shift in loop exposure and epitope occlusion, while providing substantive rationale for epitope display required for elicitation of broadly neutralizing antibodies, as well as substantiating previous pertinent literature disregarded in recent reports.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California, Davis, CA 95616.

ABSTRACT
Hypervariable loops of HIV-1 Env protein gp120 are speculated to play roles in the conformational transition of Env to the receptor binding-induced metastable state. Structural analysis of full-length Env-based immunogens, containing the entire V2 loop, displayed tighter association between gp120 subunits, resulting in a smaller trimeric diameter than constructs lacking V2. A prominent basal quaternary location of V2 and V3' that challenges previous reports would facilitate gp41-independent gp120-gp120 interactions and suggests a quaternary mechanism of epitope occlusion facilitated by hypervariable loops. Deletion of V2 resulted in dramatic exposure of basal, membrane-proximal gp41 epitopes, consistent with its predicted basal location. The structural features of HIV-1 Env characterized here provide grounds for a paradigm shift in loop exposure and epitope occlusion, while providing substantive rationale for epitope display required for elicitation of broadly neutralizing antibodies, as well as substantiating previous pertinent literature disregarded in recent reports.

Show MeSH

Related in: MedlinePlus

Structure of gp140-PG16 complex reveals basal V2 location, and deletion of V2 results in exposure of basal gp41 epitopes as detected by western blot and ELISA.(A) The gp140 trimer was incubated with PG16 Fab and imaged by cryoEM. Structure of the gp140-PG16 complex reveals a basal density emanating from the putative location of the V2 loop (red). Resolution is ~26 Å at 0.5 FSC. (B) Superimposition of the gp140 unliganded map (solid) and the gp140-PG16 complex (mesh) reveals that binding of PG16 occurs at the basal V2 density (red mesh). (C) Western blot of gp140 and gp140ΔV2 constructs labeled with antibodies 50–69 and 2F5, targeting apical and basal gp41 epitopes respectively, shows that exposure of the 50–69 epitope (targeting the intrahelical disulfide-containing loop on gp41) is unaffected by V2 deletion, while 2F5 epitope exposure (targeting the gp41 membrane-proximal external region) is greatly enhanced after V2 deletion. Blots shown are representative results. (D) ELISA of gp140 and gp140ΔV2 bound to 50–69 and 2F5 similarly reveal that while 50–69 epitope exposure is unaffected by V2 deletion, 2F5 epitope exposure is significantly enhanced by V2 deletion (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231788&req=5

f2: Structure of gp140-PG16 complex reveals basal V2 location, and deletion of V2 results in exposure of basal gp41 epitopes as detected by western blot and ELISA.(A) The gp140 trimer was incubated with PG16 Fab and imaged by cryoEM. Structure of the gp140-PG16 complex reveals a basal density emanating from the putative location of the V2 loop (red). Resolution is ~26 Å at 0.5 FSC. (B) Superimposition of the gp140 unliganded map (solid) and the gp140-PG16 complex (mesh) reveals that binding of PG16 occurs at the basal V2 density (red mesh). (C) Western blot of gp140 and gp140ΔV2 constructs labeled with antibodies 50–69 and 2F5, targeting apical and basal gp41 epitopes respectively, shows that exposure of the 50–69 epitope (targeting the intrahelical disulfide-containing loop on gp41) is unaffected by V2 deletion, while 2F5 epitope exposure (targeting the gp41 membrane-proximal external region) is greatly enhanced after V2 deletion. Blots shown are representative results. (D) ELISA of gp140 and gp140ΔV2 bound to 50–69 and 2F5 similarly reveal that while 50–69 epitope exposure is unaffected by V2 deletion, 2F5 epitope exposure is significantly enhanced by V2 deletion (n = 3).

Mentions: Conjugation of gold-labeled PG16 Fab′ fragments with trimeric gp140 followed by single particle reconstruction of gp140-PG16 complexes revealed a density protruding from the basal V2 location (Fig. 2A). The gp140-PG16 map confirmed the basal location of V2, as well as the interprotomeric location of the QNE. Superimposition of the gp140 unliganded map with the gp140-PG16 map confirmed the basal protruding density apportioned to V2, as well as additional density assigned to the PG16 Fab (Fig. 2B).


Trimeric HIV Env provides epitope occlusion mediated by hypervariable loops.

Moscoso CG, Xing L, Hui J, Hu J, Kalkhoran MB, Yenigun OM, Sun Y, Paavolainen L, Martin L, Vahlne A, Zambonelli C, Barnett SW, Srivastava IK, Cheng RH - Sci Rep (2014)

Structure of gp140-PG16 complex reveals basal V2 location, and deletion of V2 results in exposure of basal gp41 epitopes as detected by western blot and ELISA.(A) The gp140 trimer was incubated with PG16 Fab and imaged by cryoEM. Structure of the gp140-PG16 complex reveals a basal density emanating from the putative location of the V2 loop (red). Resolution is ~26 Å at 0.5 FSC. (B) Superimposition of the gp140 unliganded map (solid) and the gp140-PG16 complex (mesh) reveals that binding of PG16 occurs at the basal V2 density (red mesh). (C) Western blot of gp140 and gp140ΔV2 constructs labeled with antibodies 50–69 and 2F5, targeting apical and basal gp41 epitopes respectively, shows that exposure of the 50–69 epitope (targeting the intrahelical disulfide-containing loop on gp41) is unaffected by V2 deletion, while 2F5 epitope exposure (targeting the gp41 membrane-proximal external region) is greatly enhanced after V2 deletion. Blots shown are representative results. (D) ELISA of gp140 and gp140ΔV2 bound to 50–69 and 2F5 similarly reveal that while 50–69 epitope exposure is unaffected by V2 deletion, 2F5 epitope exposure is significantly enhanced by V2 deletion (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231788&req=5

f2: Structure of gp140-PG16 complex reveals basal V2 location, and deletion of V2 results in exposure of basal gp41 epitopes as detected by western blot and ELISA.(A) The gp140 trimer was incubated with PG16 Fab and imaged by cryoEM. Structure of the gp140-PG16 complex reveals a basal density emanating from the putative location of the V2 loop (red). Resolution is ~26 Å at 0.5 FSC. (B) Superimposition of the gp140 unliganded map (solid) and the gp140-PG16 complex (mesh) reveals that binding of PG16 occurs at the basal V2 density (red mesh). (C) Western blot of gp140 and gp140ΔV2 constructs labeled with antibodies 50–69 and 2F5, targeting apical and basal gp41 epitopes respectively, shows that exposure of the 50–69 epitope (targeting the intrahelical disulfide-containing loop on gp41) is unaffected by V2 deletion, while 2F5 epitope exposure (targeting the gp41 membrane-proximal external region) is greatly enhanced after V2 deletion. Blots shown are representative results. (D) ELISA of gp140 and gp140ΔV2 bound to 50–69 and 2F5 similarly reveal that while 50–69 epitope exposure is unaffected by V2 deletion, 2F5 epitope exposure is significantly enhanced by V2 deletion (n = 3).
Mentions: Conjugation of gold-labeled PG16 Fab′ fragments with trimeric gp140 followed by single particle reconstruction of gp140-PG16 complexes revealed a density protruding from the basal V2 location (Fig. 2A). The gp140-PG16 map confirmed the basal location of V2, as well as the interprotomeric location of the QNE. Superimposition of the gp140 unliganded map with the gp140-PG16 map confirmed the basal protruding density apportioned to V2, as well as additional density assigned to the PG16 Fab (Fig. 2B).

Bottom Line: A prominent basal quaternary location of V2 and V3' that challenges previous reports would facilitate gp41-independent gp120-gp120 interactions and suggests a quaternary mechanism of epitope occlusion facilitated by hypervariable loops.Deletion of V2 resulted in dramatic exposure of basal, membrane-proximal gp41 epitopes, consistent with its predicted basal location.The structural features of HIV-1 Env characterized here provide grounds for a paradigm shift in loop exposure and epitope occlusion, while providing substantive rationale for epitope display required for elicitation of broadly neutralizing antibodies, as well as substantiating previous pertinent literature disregarded in recent reports.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California, Davis, CA 95616.

ABSTRACT
Hypervariable loops of HIV-1 Env protein gp120 are speculated to play roles in the conformational transition of Env to the receptor binding-induced metastable state. Structural analysis of full-length Env-based immunogens, containing the entire V2 loop, displayed tighter association between gp120 subunits, resulting in a smaller trimeric diameter than constructs lacking V2. A prominent basal quaternary location of V2 and V3' that challenges previous reports would facilitate gp41-independent gp120-gp120 interactions and suggests a quaternary mechanism of epitope occlusion facilitated by hypervariable loops. Deletion of V2 resulted in dramatic exposure of basal, membrane-proximal gp41 epitopes, consistent with its predicted basal location. The structural features of HIV-1 Env characterized here provide grounds for a paradigm shift in loop exposure and epitope occlusion, while providing substantive rationale for epitope display required for elicitation of broadly neutralizing antibodies, as well as substantiating previous pertinent literature disregarded in recent reports.

Show MeSH
Related in: MedlinePlus