Lower-resolution EM reconstructions suggest that membrane Envs exhibit a hollow architecture with a cavity along the trimer axis (34,C38, 43), in contrast to the more compact architecture of sgp140 SOSIP.664, which is stabilized by a triple-helical gp41 coiled coil along the trimer axis (28,C32, 62). only after CD4 binding, the sgp140 SOSIP.664 HR1 coiled coil was accessible to the gp41 HR2 peptide even in the absence of CD4. Our results delineate differences in both gp120 and gp41 subunits between functional membrane Env and the sgp140 SOSIP.664 trimer and provide distance constraints that can assist validation of candidate structural models of the native HIV-1 Env trimer. IMPORTANCE HIV-1 envelope glycoprotein spikes mediate the entry of the virus into host cells and are a major target for vaccine-induced antibodies. Soluble forms of the envelope glycoproteins that are stable and easily produced have been characterized extensively and are being considered as vaccines. Here, we present evidence that these stabilized soluble envelope glycoproteins differ in multiple respects from the natural HIV-1 envelope glycoproteins. By pinpointing these differences, our results can guide the improvement of envelope glycoprotein preparations to achieve greater similarity to the viral envelope glycoprotein spike, potentially increasing their effectiveness as a vaccine. lectin affinity chromatography. The gel was Western blotted with a rabbit Ipragliflozin anti-gp120-HRP antibody. The results indicate that Env712 is efficiently processed in HOS cells, BS3 cross-linking is effective, and Env712 Ipragliflozin is largely a trimer. (B) Cell-based ELISA of efficiently cleaved HIV-1AD8 Env712 displayed on the surface of HOS cells. The Env712-expressing cells were untreated or incubated with 20?M BMS-806. Half of the cell cultures were then cross-linked with 5?mM BS3. After washing, the cells were analyzed by cell-based ELISA with the indicated primary antibodies. The results are shown relative to the values seen for the 2G12 anti-gp120 antibody (set at 100% for each condition). The means and standard deviations from two experiments are shown. Recognition of untreated and BS3-cross-linked Env712 by the antibodies did not significantly differ (independent samples test). (C) HOS cells expressing the HIV-1AD8 Env712 glycoprotein were treated with 5?mM BS3 for 30 min at 25C. Env712 was purified from cell membranes and analyzed by SDS-PAGE. The gel was stained with Coomassie brilliant blue. Subsaturating concentrations of cross-linkers have been shown to yield optimal XL-MS results (47, 53, 87, 88). In preparation for the XL-MS analysis, we identified subsaturating concentrations of BS3 for cross-linking HIV-1AD8 Env712 glycoproteins transiently expressed on the HOS cell surface (Fig. 6C). XL-MS analysis of the unliganded HIV-1AD8 membrane Env. HOS cells transiently expressing the HIV-1AD8 Env712 glycoprotein were incubated with an isotopically coded Ipragliflozin BS3 cross-linker under physiological conditions. Note that because BS3 is water soluble and membrane impermeable, cross-linking and XL-MS analysis in this case is limited to Env712 glycoproteins on the cell surface. After quenching the cross-linking reaction, the BS3-cross-linked Env712 glycoproteins were purified from detergent-solubilized membranes, deglycosylated, protease digested, and subjected to analysis by mass spectrometry. Eleven cross-links in the Env712 glycoproteins were identified by XL-MS (Fig. 7A and ?andBB and Table S3). Three of these occurred between the Env712 protomers, and one linked gp120 and gp41. The sequence of HIV-1AD8 Env712 was aligned with that of sgp140 SOSIP.664 (Fig. 8). The identified cross-links were mapped onto the crystal structures of sgp140 SOSIP.664 trimers from multiple HIV-1 strains to evaluate the similarity of these structures to the conformation of the membrane Env712 trimer on the HOS cell surface (Fig. 7C and ?andDD and Table 3). As noted above, despite the significant primary sequence diversity of the HIV-1 strains of origin (from phylogenetic clades A, B, C, AG, and G), these Ipragliflozin sgp140 SOSIP.664 structural models are highly Rabbit Polyclonal to Notch 2 (Cleaved-Asp1733) similar (30, 62,C66). Moreover, the sgp140 SOSIP.664 trimers, whether bound to different broadly neutralizing antibodies or in a ligand-free state, have similar structures. For example, the C root mean square deviation between an unliganded sgp140 SOSIP.664 trimer (4ZMJ) and a sgp140 SOSIP.664 trimer bound to two antibody Fabs.