Characterization of a stable HIV-1 B/C recombinant, soluble, and trimeric envelope glycoprotein (Env) highly resistant to CD4-induced conformational changes

Rajesh Kumar,Gabriel Ozorowski,Vivek Kumar,Lauren G Holden,Tripti Shrivastava,Shilpa Patil,Suprit Deshpande,Andrew B Ward,Jayanta Bhattacharya

doi:10.1074/jbc.m117.803056

Abstract

The HIV-1 envelope (Env) is a glycoprotein consisting of a trimer of heterodimers containing gp120 and gp41 subunits that mediates virus entry and is a major target of broadly neutralizing antibodies (bnAbs) developed during infection in some individuals. The engagement of the HIV-1 gp120 glycoprotein to the host CD4 protein triggers conformational changes in gp120 that allow its binding to co-receptors and is necessary for virus entry to establish infection. Native-like HIV-1 Env immunogens representing distinct clades have been proposed to improve immunogenicity. In the present study, we examined the basis of resistance of an HIV-1 B/C recombinant Env (LT5.J4b12C) to non-neutralizing antibodies targeting CD4-induced Env epitopes in the presence of soluble CD4 (sCD4). Using native polyacrylamide gel shift assay and negative-stain EM, we found that the prefusion conformational state of LT5.J4b12C trimeric Env was largely unaffected in the presence of excess sCD4 with most Env trimers appearing to be in a ligand-free state. This resistance to CD4-induced conformational changes was associated with a lower affinity for CD4. Moreover, the LT5.J4b12C trimeric Env preferentially bound to the neutralizing antibodies compared with non-neutralizing antibodies. Taken together, we report on an HIV-1 B/C recombinant, native-like trimeric Env protein that is highly resistant to CD4-induced conformational changes but displays epitopes recognized by a diverse array of bnAbs. Such features make this B/C recombinant trimeric Env a useful addition to the pool of other recently identified native-like HIV-1 Env trimers suitable for use as antigenic bait for bnAb isolation, structural studies, and use as potential immunogens.

Highlights

The HIV-1 envelope (Env) is a glycoprotein consisting of a trimer of heterodimers containing gp120 and gp41 subunits that mediates virus entry and is a major target of broadly neutralizing antibodies developed during infection in some individuals
We report on an HIV-1 B/C recombinant, native-like trimeric Env protein that is highly resistant to CD4-induced conformational changes but displays epitopes recognized by a diverse array of bnAbs
Efficient furin cleavage was confirmed by reducing SDS-polyacrylamide gel electrophoresis (PAGE) (Fig. 1B), and LT5.J4b12C SOSIP.664 was found to remain stable at 37 °C for at least 72 h (Fig. 1C)

Summary

Clade mAb

Sensitivity of Env-pseudotyped viruses to bnAbs Values indicate concentration of IgG (␮g/ml) conferring 50% virus neutralization in TZM-bl cells. Our data substantiate our previous observation and further indicated that, due to its intrinsic resistance to sCD4, the LT5.J4b12C Env was unable to undergo conformational changes required for exposure of CD4i epitopes. Pseudoviruses expressing LT5.J4b12C Env were found to be neutralized by a number of bnAbs with distinct specificities such as b12, VRC03, PG9, PGT121, and PGT151 (Table 2). This observation along with the resistance to non-neutralizing antibodies such as 17b and 3074 noted in the present and previous [25] studies made LT5.J4b12C an attractive Env to explore further and probe whether the properties are retained when expressed as a soluble trimeric protein

Results

Discussion

Experimental procedures

Neutralization assay

Molecular modeling