Abstract
BackgroundWe previously described the construction of an HIV-1 envelope glycoprotein complex (Env) that is stabilized by an engineered intermolecular disulfide bond (SOS) between gp120 and gp41. The modified Env protein antigenically mimics the functional wild-type Env complex. Here, we explore the effects of the covalent gp120 – gp41 interaction on virus replication and evolution.ResultsAn HIV-1 molecular clone containing the SOS Env gene was only minimally replication competent, suggesting that the engineered disulfide bond substantially impaired Env function. However, virus evolution occurred in cell culture infections, and it eventually always led to elimination of the intermolecular disulfide bond. In the course of these evolution studies, we identified additional and unusual second-site reversions within gp41.ConclusionsThese evolution paths highlight residues that play an important role in the interaction between gp120 and gp41. Furthermore, our results suggest that a covalent gp120 – gp41 interaction is incompatible with HIV-1 Env function, probably because this impedes conformational changes that are necessary for fusion to occur, which may involve the complete dissociation of gp120 from gp41.
Highlights
We previously described the construction of an HIV-1 envelope glycoprotein complex (Env) that is stabilized by an engineered intermolecular disulfide bond (SOS) between gp120 and gp41
We conclude that the SOS Env protein does not support virus replication, consistent with previous studies using a cell-cell fusion assay or Env-pseudotyped viruses in a single-cycle infection protocol [11,12]
The initial goal of our forced evolution studies was to generate SOS Env variants that could replicate despite having an intermolecular disulfide bond between gp120 and gp41
Summary
We previously described the construction of an HIV-1 envelope glycoprotein complex (Env) that is stabilized by an engineered intermolecular disulfide bond (SOS) between gp120 and gp. The trimeric HIV-1 envelope glycoprotein complex (Env) mediates viral entry into susceptible target cells. The surface subunit (SU; gp120) attaches to the receptor (CD4) and the coreceptor (CCR5 or CXCR4) on the cell surface, and subsequent conformational changes within the Env complex lead to membrane fusion mediated by the transmembrane subunit (TM; gp41) [1,2,3,4]. After intracellular cleavage of the precursor gp160 protein, three gp120 subunits stay non-covalently associated with three gp subunits. These non-covalent interactions are weak and gp120 dissociates from gp, a process that, if it occurs spontaneously and prematurely, inactivates the Env complex and leads to the exposure of non-neutralizing, immune-decoy epitopes on both gp120 and gp41 [57]. Uncleaved Env proteins, like the dissociated subunits, expose non-neutralizing epitopes [5,6,7,8,9]
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