Effects of the SOS (A501C/T605C) and DS (I201C/A433C) Disulfide Bonds on HIV-1 Membrane Envelope Glycoprotein Conformation and Function

Abstract

Most broadly neutralizing antibodies and many entry inhibitors target the pretriggered (state 1) conformation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env). Here we examine two previously reported Env mutants designed to be stabilized in this conformation by the introduction of artificial disulfide bonds: A501C/T605C (called SOS) and I201C/A433C (called DS). SOS Env supported virus entry and cell-cell fusion only after exposure to a reducing agent, dithiothreitol (DTT). Deletion of the Env cytoplasmic tail improved the efficiency with which the SOS Env supported virus infection in a reducing environment. The antigenicity of the SOS Env was similar to that of the unmodified Env, except for greater sensitivity to some state 1-preferring ligands. In contrast, viruses with the DS Env were not infectious, even after DTT treatment. The proteolytic maturation of the DS Env on both cell surfaces and virions was severely compromised compared with that of the unmodified Env. The DS Env exhibited detectable cell-fusing activity when DTT was present. However, the profiles of cell-surface Env recognition and cell-cell fusion inhibition by antibodies differed for the DS Env and the unmodified Env. Thus, the DS Env appears to be stabilized in an off-pathway conformation that is nonfunctional on the virus. The SOS change exerted more subtle, context-dependent effects on Env conformation and function. IMPORTANCE The human immunodeficiency virus type 1 (HIV-1) envelope proteins (Envs) bind receptors on the host cell and change shape to allow the virus to enter the cell. Most virus-inhibiting antibodies and drugs recognize a particular shape of Env called state 1. Disulfide bonds formed by cysteine residues have been introduced into soluble forms of the flexible envelope proteins in an attempt to lock them into state 1 for use in vaccines and as research tools. We evaluated the effect of these cysteine substitutions on the ability of the membrane Env to support virus entry and on susceptibility to inhibition by antibodies and small molecules. We found that the conformation of the envelope proteins with the cysteine substitutions differed from that of the unmodified membrane envelope proteins. Awareness of these effects can assist efforts to create stable HIV-1 Env complexes that more closely resemble the state 1 conformation.

Functional and Highly Crosslinkable HIV-1 Envelope Glycoproteins Enriched in a Pretriggered Conformation

“…In a preliminary study, a total of 24 Env variants were analyzed for protein expression and processing, ability to support entry of a pseudotyped virus, and the sensitivity of the viral pseudotype to neutralization by the 19b antibody. The 19b antibody is a poorly neutralizing antibody that recognizes the gp120 V3 loop and serves as a sensitive indicator of HIV-1 Env transitions to State-2/3 conformations (45, 71–74, 137). With a few exceptions, most of the lysine substitutions were well tolerated with respect to HIV-1 AD8 Env processing, virus infectivity and sensitivity to 19b neutralization (data not shown).…”

“…Unfortunately, bNAbs have not been efficiently and consistently elicited in animals immunized with current HIV-1 vaccine candidates, including stabilized soluble gp140 (sgp140) SOSIP.664 trimers (61)(62)(63)(64)(65)(66)(67)(68)(69). Compared with functional membrane Envs, differences in the antigenicity, glycosylation and conformation of sgp140 SOSIP.664 trimers have been observed (70)(71)(72)(73)(74)(75)(76)(77), potentially contributing to the inefficiency of bNAb elicitation. Single-molecule FRET (smFRET) analysis indicates that the sgp140 SOSIP.664 trimers assume a State-2-like conformation (78).…”

SOS and IP Modifications Predominantly Affect the Yield but Not Other Properties of SOSIP.664 HIV-1 Env Glycoprotein Trimers

“…Even so, the SOS bond is compatible with membrane fusion provided it is broken by the addition of a reducing agent at an appropriate time (36). It has been reported elsewhere that the SOS and IP changes perturb the structure of trimers and create entities that are not appropriate mimics of "native Env proteins" (24)(25)(26)(27)(28). However, much of the methodology used to draw such conclusions is questionable.…”

“…Given an increasingly detailed body of information about how bNAbs emerge during HIV-1 infection, it is not surprising that a protein immunogen does not induce bNAbs efficiently by itself (1,3,8,(21)(22)(23). It has been suggested, however, that SOSIP trimers are not in the correct conformation to induce bNAbs, i.e., that the SOS and IP changes have detrimental effects on epitope presentation (24)(25)(26)(27)(28). The methods and data interpretation underlying these assertions have been questioned (see references 26 and 29 and the comment by J. P. Moore at https://www .nature.com/articles/s41586-019-1101-y#article-comments).…”

Quaternary Interaction of the HIV-1 Envelope Trimer with CD4 and Neutralizing Antibodies

“…Despite some lingering controversy [ 40 , 41 , 42 , 43 , 44 ], SOSIP trimers are generally accepted in the field as close proxies of the native Env form and, thereby, have offered an opportunity to explore the critical initial events in the interaction between the Env spike and the CD4 receptor. In 2017, using a combination of in silico docking, functional mutagenesis and cryo-EM analysis, we reported that this initial interaction has a quaternary nature, as it involves not only the primary CD4-BS, but also a previously unrecognized second CD4-BS (that we defined CD4-BS2) located in the outer layer (layer-1) of the inner domain of a neighboring gp120 protomer.…”