Native Conformation and Canonical Disulfide Bond Formation Are Interlinked Properties of HIV-1 Env Glycoproteins

Abstract

We investigated whether there is any association between a native-like conformation and the presence of only the canonical (i.e., native) disulfide bonds in the gp120 subunits of a soluble recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein. We used a mass spectrometry (MS)-based method to map the disulfide bonds present in nonnative uncleaved gp140 proteins and native-like SOSIP.664 trimers based on the BG505 env gene. Our results show that uncleaved gp140 proteins were not homogeneous, in that substantial subpopulations (20 to 80%) contained aberrant disulfide bonds. In contrast, the gp120 subunits of the native-like SOSIP.664 trimer almost exclusively retained the canonical disulfide bond pattern. We also observed that the purification method could influence the proportion of an Env protein population that contained aberrant disulfide bonds. We infer that gp140 proteins may always contain a variable but substantial proportion of aberrant disulfide bonds but that the impact of this problem can be minimized via design and/or purification strategies that yield native-like trimers. The same factors may also be relevant to the production and purification of monomeric gp120 proteins that are free of aberrant disulfide bonds. IMPORTANCEIt is widely thought that a successful HIV-1 vaccine will include a recombinant form of the Env protein, a trimer located on the virion surface. To increase yield and simplify purification, Env proteins are often made in truncated, soluble forms. A consequence, however, can be the loss of the native conformation concomitant with the virion-associated trimer. Moreover, some soluble recombinant Env proteins contain aberrant disulfide bonds that are not expected to be present in the native trimer. To assess whether these observations are linked, to determine the extent of disulfide bond scrambling, and to understand why scrambling occurs, we determined the disulfide bond profiles of two soluble Env proteins with different designs that are being assessed as vaccine candidates. We found that uncleaved gp140 forms heterogeneous mixtures in which aberrant disulfide bonds abound. In contrast, BG505 SOSIP.664 trimers are more homogeneous, native-like entities that contain predominantly the native disulfide bond profile. The first human immunodeficiency virus type 1 (HIV-1) vaccine trial (designated RV144) to yield one statistically significant measure of efficacy was reported in 2009 (1). Since then, attempts to substantially improve on those initial results have been ongoing. Many vaccine design strategies are focused on the HIV-1 Env protein, which is the trimeric antigen present on the viral surface. Some Env immunogens, including proteins used in the RV144 trial, are based on a soluble monomeric form of the gp120 subunit of the trimer (1-7). An alternative approach involves soluble oligomeric gp140 proteins that contain both gp120 and the external component of the gp41 subunit (8-13). The concept underlying the use of gp140 proteins is that they may ...

Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers

“…The glycosylation profile of the C.1086 gp120 protein was not highly divergent from the trimeric consensus, and we hypothesize that here the genotype may drive an atypically favorable glycosylation profile even without employing conformation-specific purification methods (e.g., bNAb columns). For the proteins with less-optimal glycosylation profiles than C.1086 gp120, some of the divergence is likely attributable to protein misfolding (59), which potentially can be overcome by using a bNAb column, or other methods, to select the most appropriately folded population. These various concepts could be tested experimentally in appropriately designed studies that take into account advances in construct design and Env purification procedures.…”

“…The BG505 SOSIP.664 construct was expressed in a stable CHO cell line, as described previously (59). The protein was purified first by using a 2G12-affinity column and then by SEC (59,61,62).…”

“…The BG505 SOSIP.664 construct was expressed in a stable CHO cell line, as described previously (59). The protein was purified first by using a 2G12-affinity column and then by SEC (59,61,62). The C97ZA.012 SOSIP.664 trimer and the C97ZA.012 gp140(-)FT were expressed in 293F cells by transient transfection, as described previously (56).…”

Molecular Architecture of the Cleavage-Dependent Mannose Patch on a Soluble HIV-1 Envelope Glycoprotein Trimer

“…While this site is exclusively dominated by oligomannose-type glycans on SOSIP.664, it has a mixed composition (ϳ50% complex glycans) on the pseudotrimer, although Man 9 GlcNAc 2 still remains the most prominent oligomannose-type structure. One explanation may be that more than one folding state exists within the total population of pseudotrimers, for example, there may be a subpopulation in which aberrant disulfide bonds have formed (64). The gp41 glycan that differs most between the SOSIP.664 trimer and the pseudotrimer is N637, which resides near the protomer interface, very close to N276.…”

Range of CD4-Bound Conformations of HIV-1 gp120, as Defined Using Conditional CD4-Induced Antibodies

“…The formation of oligomeric forms of gp120 in transfected HEK 293T cells due to aberrant disulfide bridges is well documented (55,58,(79)(80)(81). Oligomerization can result specifically from rearranged disulfides in the V2 loop region.…”