Redox-Triggered Infection by Disulfide-Shackled Human Immunodeficiency Virus Type 1 Pseudovirions

Abstract

. Here we produced pseudovirions bearing the mutant envelope and a reporter gene to examine the mutant's infectious properties. These pseudovirions attach to cells expressing CD4 and coreceptor but infect only when triggered with reducing agent, implying that gp120-gp41 dissociation is necessary for infection. Further studies suggested that virus entry was arrested after CD4 and coreceptor engagement. By measuring the activities of various entry inhibitors against the arrested intermediate, we found that gp120-targeting inhibitors typically act prior to virus attachment, whereas gp41 inhibitors are able to act postattachment. Unexpectedly, a significant fraction of antibodies in HIV-1-positive sera neutralized virus postattachment, suggesting that downstream fusion events and structures figure prominently in the host immune response. Overall, this disulfide-shackled virus is a unique tool with potential utility in vaccine design, drug discovery, and elucidation of the HIV-1 entry process.Human immunodeficiency virus type 1 (HIV-1) enters susceptible target cells via a complex cascade of receptor-mediated events. A fine characterization of this process is complicated by the transient nature of the lipid and protein rearrangements involved. The envelope glycoprotein (Env) is responsible for viral attachment and fusion. Env consists of noncovalently associated trimers of heterodimers comprising gp120 surface and gp41 transmembrane glycoproteins (29, 39). During infection, gp120 attaches to the CD4 receptor and undergoes conformational changes that enable coreceptor binding (39). This leads to further changes in gp41 (22) to form a six-helix bundle consisting of three alpha-helical hairpins (7, 48) and culminates in lipid mixing and membrane fusion.The study of HIV-1 entry and the Env conformations involved has provided a rich source of targets for a new generation of antiretroviral therapies (6,16,18). The most clinically advanced HIV-1 entry inhibitor, the peptide T-20 (also known as DP178), blocks fusion at nanomolar concentrations (49) by binding to a structure known as the gp41 prehairpin intermediate that becomes available during the fusion process (22).Env represents the primary target for the neutralizing antibody response. Successful vaccines against many viral infections elicit neutralizing antibodies (4) but have been difficult to elicit against HIV-1. The virus evades host immunity by exposing hypervariable and heavily glycosylated regions on gp120, while the conserved domains that bind its cellular receptors are located in recessed cavities (29, 39). As a result, only a few monoclonal antibodies (MAbs) against Env isolated to date are both potently and broadly neutralizing (5,9,33,34,39,41,44,45,52). Since conserved domains and potential targets for neutralizing antibodies may become exposed after receptor binding, fusion intermediates may find utility in vaccine research (12,13,20,26,33,37,51).Until now, HIV-1 fusion intermediates have been generated by incubating virus or Env-expressing cells with tar...

Time-resolved Imaging of HIV-1 Env-mediated Lipid and Content Mixing between a Single Virion and Cell Membrane

“…Deletion of HIV Env's CT increases expression levels in cells and somewhat increases cell-cell fusion, without affecting the fundamental properties of the process . HIV-1 Env pseudotyped against an HIV core led to the same level of infectivity as when the CT was deleted (Binley et al, 2003). In short, deleting the CT has minimal consequences on the fusion process.…”

“…HIV-1 JRFL Env with truncated cytoplasmic tail in a pCAGGS vector (Binley et al, 2003) was a gift from Dr. J. Binley (Torrey Pines Institute for Molecular Studies, San Diego, CA). Vectors expressing MLV Gag-pol, Gag-GFP, and LTR lacZ plasmids (Sherer et al, 2003) were kindly provided by Dr. W. Mothes (Yale University, New Haven, CT).…”

Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation

“…We propose that the engineered SOS bond anchors the gp120 subunit to gp41 ECTO , not by creating a new site of interaction but by supporting the weaker, noncovalent linkages that form naturally and which may be stabilized to some extent by the membrane-spanning domain. Membrane-associated, full-length (i.e., gp160) SOS trimers are still fusion-competent, provided the disulfide bond is reduced after receptor engagement; the SOS bond does not compromise trimer structure and function (29). In contrast, the I559P change is incompatible with fusion, presumably because it impedes the transition(s) from the prefusion form to the fusion intermediate and/or postfusion forms of gp41 (14,30).…”

Antibody vs. HIV in a clash of evolutionary titans

“…The description of broadly neutralizing mAbs that recognize contiguous epitopes on HIV-1 suggests that the MPER could be a highly promising vaccine target (47). Some evidence has emerged that the epitopes of these two mAbs are accessible, and possibly more so, after CD4 binding to gp120 (54). Both mAbs appear to recognize linear gp41 epitopes in that they bind with relatively high affinity to short peptides corresponding to cognate gp41 sequences, and their neutralizing activity can be effectively inhibited by such peptides.…”