Abstract
Substantial controversy surrounds the membrane topology of the HIV-1 gp41 C-terminal tail (CTT). While few studies have been designed to directly address the topology of the CTT, results from envelope (Env) protein trafficking studies suggest that the CTT sequence is cytoplasmically localized, as interactions with intracellular binding partners are required for proper Env targeting. However, previous studies from our lab demonstrate the exposure of a short CTT sequence, the Kennedy epitope, at the plasma membrane of intact Env-expressing cells, the exposure of which is not observed on viral particles. To address the topology of the entire CTT sequence, we serially replaced CTT sequences with a VSV-G epitope tag sequence and examined reactivity of cell- and virion-surface Env to an anti-VSV-G monoclonal antibody. Our results demonstrate that the majority of the CTT sequence is accessible to antibody binding on the surface of Env expressing cells, and that the CTT-exposed Env constitutes 20–50% of the cell-surface Env. Cell surface CTT exposure was also apparent in virus-infected cells. Passive transfer of Env through cell culture media to Env negative (non-transfected) cells was not responsible for the apparent cell surface CTT exposure. In contrast to the cell surface results, CTT-exposed Env was not detected on infectious pseudoviral particles containing VSV-G-substituted Env. Finally, a monoclonal antibody directed to the Kennedy epitope neutralized virus in a temperature-dependent manner in a post-attachment neutralization assay. Collectively, these results suggest that the membrane topology of the HIV gp41 CTT is more complex than the widely accepted intracytoplasmic model.
Highlights
The envelope (Env) protein of HIV, which is the major virallyencoded protein present on the surface of the virion, is the primary target of the humoral immune response [1]
The HA epitope tag was inserted into the V3 region of gp120 to allow for detection of the relative levels of cell surface Env in cells transfected with the various VSV-G substituted Envs
It was determined by separate staining for HA and VSV-G reactivity in aliquots from the same transfection that the anti-VSV-G (CTT) monoclonal antibody (MAb) was apparently inhibiting the binding of the anti-HA MAb
Summary
The envelope (Env) protein of HIV, which is the major virallyencoded protein present on the surface of the virion, is the primary target of the humoral immune response [1]. The gp120 protein and the gp ectodomain have been extensively studied, both structurally and functionally, as they appear to be the important targets of the antibody response in infected individuals [1]. The CTT, on the other hand, has largely been studied at a functional level, and has been demonstrated to play a role in viral Env incorporation [9,10,11,12], virion maturation [13,14,15,16], cellular Env trafficking [17,18], and more recently, as a modulator of Env gp120 conformation on both the cell and virion surfaces [19,20]. Relatively little is known about the structure of the CTT aside from characterizations of peptide analogs of CTT subdomains, known as the lentivirus lytic peptides (LLPs), that have been demonstrated to be predominantly helical in membrane and membrane-mimetic environments [21,22,23]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
