Mutational analysis of the human being immunodeficiency virus type 1 gene product proteolytic cleavage site. and cell membranes (7C13). In the infected cell, the HIV-1 Env trimer is definitely synthesized like a precursor of approximately 850 amino acid residues and undergoes transmission peptide cleavage, folding, trimerization, and glycosylation in the endoplasmic reticulum (14C16). The producing gp160 Env precursor is definitely transported to the Golgi apparatus, where proteolytic cleavage by sponsor furin-like proteases generates the gp120 and gp41 subunits (17C23). The proteolytically processed, adult Env trimers are transferred to the infected cell surface and integrated into budding virions. The Env spike is the only virus-specific target for neutralizing antibodies, which have been shown in passive protection studies to interrupt the acquisition of HIV-1-like viruses in monkeys (24, 25). The HIV-1 Envs have developed to elicit neutralizing antibodies inefficiently, creating difficulties for the development of vaccines (26). A minority of HIV-1-infected individuals, after several years of illness, generate antibodies that potently neutralize a variety of HIV-1 geographic isolates (27C31). These broadly neutralizing antibodies are directed against the CD4-binding site (CD4BS) (32C37), carbohydrate-dependent epitopes on gp120 (38C41), or peptide epitopes in the membrane-proximal external region (MPER) of gp41 (32C47). Acknowledgement of the adult Env trimer is definitely a prerequisite for HIV-1 neutralization by antibodies (26, 33C35, 48C57). Even though global conformations of proteolytically cleaved and uncleaved HIV-1 Env trimers are indistinguishable at 20-? resolution (56, 58C60), proteolytic cleavage can influence the binding of antibodies to the HIV-1 Env trimer (33, 35, 48, 61C63). For example, potent and broadly neutralizing CD4BS antibodies have been shown to recognize the proteolytically cleaved Env trimer as well as or better than the uncleaved Env trimer, whereas weakly neutralizing CD4BS antibodies efficiently recognize only the uncleaved Env trimer (33, 35, 37, 48, 62, 64). Nonneutralizing antibodies realizing the immunodominant cluster 1 and cluster 2 epitopes within the gp41 ectodomain bound better to cleavage-deficient Env trimers than ISRIB (trans-isomer) cleaved Env trimers (48). In the same study, the anti-MPER antibodies 2F5 and 4E10 identified the uncleaved Env trimer better than the proteolytically processed Env Rabbit Polyclonal to WEE2 trimer in the unliganded state. CD4 binding resulted in a large increase in the binding of the antibodies directed against the cluster 1 and cluster 2 gp41 epitopes, probably as a result of gp120 dropping. In contrast, CD4 binding only minimally improved the binding of the neutralizing 2F5 and 4E10 anti-MPER antibodies (48), although virion-binding assays suggest that these antibodies require recognition of a downstream Env conformation to accomplish neutralization of HIV-1 (42, 43, 45, 49, 55, 65, 66). Therefore, the conformational effects of Env proteolytic processing influence in important ways the access of antibodies to their epitopes. We investigated the consequences of HIV-1 gp160 proteolytic cleavage within the conformation and antigenicity of the Env trimer by expressing the cleavage-competent (cl+) wild-type and cleavage-deficient (cl?) mutant Envs from two main viruses, HIV-1JR-FL and HIV-1AD8. The cl? mutants have R508S and R511S changes at the site of gp160 proteolytic cleavage (17, 19). The full-length cl+ and cl? Envs were transiently indicated in HOS cells, in which we have found proteolytic ISRIB (trans-isomer) cleavage of the wild-type Envs to be highly efficient compared with that in additional cell types (observe Fig. S1 in the supplemental material). The binding of a panel of anti-Env monoclonal antibodies (MAbs) and CD4-Ig to the cl+ and cl? Env variants within the cell surface was measured by a cell-based enzyme-linked immunosorbent assay (ELISA) (67, 68). In parallel experiments, cells expressing the wild-type AD8 and JR-FL Envs were treated with glutaraldehyde (GA) prior to measurement of ligand binding. Env-expressing cells were fixed by incubation with 5 mM glutaraldehyde for 15 min at space temperature. Glutaraldehyde activity was then halted using 25 mM glycine. Cells were consequently washed and assayed for antibody binding ISRIB (trans-isomer) using.