On the Role of the SP1 Domain in HIV-1 Particle Assembly: a Molecular Switch?

Abstract

Expression of a retroviral protein, Gag, in mammalian cells is sufficient for assembly of immature virus-like particles (VLPs). VLP assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We have investigated the role of SP1, a spacer between CA and NC in HIV-1 Gag, in VLP assembly. Mutational analysis showed that even subtle changes in the first 4 residues of SP1 destroy the ability of Gag to assemble correctly, frequently leading to formation of tubes or other misassembled structures rather than proper VLPs. We also studied the conformation of the CA-SP1 junction region in solution, using both molecular dynamics simulations and circular dichroism. Consonant with nuclear magnetic resonance (NMR) studies from other laboratories, we found that SP1 is nearly unstructured in aqueous solution but undergoes a concerted change to an ␣-helical conformation when the polarity of the environment is reduced by addition of dimethyl sulfoxide (DMSO), trifluoroethanol, or ethanol. Remarkably, such a coil-to-helix transition is also recapitulated in an aqueous medium at high peptide concentrations. The exquisite sensitivity of SP1 to mutational changes and its ability to undergo a concentration-dependent structural transition raise the possibility that SP1 could act as a molecular switch to prime HIV-1 Gag for VLP assembly. We suggest that changes in the local environment of SP1 when Gag oligomerizes on nucleic acid might trigger this switch.

Higher-Order Structure of the Rous Sarcoma Virus SP Assembly Domain

“…4C). SPApep became more ␣-helical at a concentration similar to what was previously reported using the HIV-1 SP1 peptide (52). Our finding suggests that the SPA helix region becomes more structured within an assembling virus particle, where the local Gag concentration might reach values around 4 mM, as roughly calculated using an average immature viral diameter of 129 nm and 2,500 Gag proteins per virion (2).…”

“…Previously published work showed that the CD spectra of a similar peptide based on the HIV-1 SP1 region became more helical as peptide concentration was increased (52). Due to the structural similarity between HIV-1 and RSV VLPs (2, 32), we predicted that SPApep should exhibit similar behavior when its concentration is increased.…”

“…This pillar-like density was originally hypothesized by Wright et al to be formed by a six-helix bundle (6HB) that contributes to the stability of an immature Gag hexamer (35). For both RSV and HIV-1, secondary structure predictions assign amino acids including and immediately flanking the spacer peptide domain as a single amphipathic helix (32,(49)(50)(51)(52). The prediction that sequences downstream of the known structured region of CA CTD form an alpha helix is not limited to alpharetroviruses and lentiviruses.…”

“…A more recent study using a different HIV-1 peptide encompassing the last eight amino acids of CA and the N-terminal two-thirds of SP1 showed a clear shift in the circular dichroism (CD) spectrum from unstructured peptide to alpha helix when the peptide concentration was increased to a level comparable to the high local protein concentration found in an assembling virion. The authors interpreted that finding to mean that SP1 acts as a conformational switch that is relatively unstructured until viral assembly increases the local concentration of Gag, at which point SP1 adopts a more helical conformation to form essential interacting surfaces that promote assembly (52).…”

HIV-1 Matrix-31 Membrane Binding Peptide Interacts Differently with Membranes Containing PS vs. PI(4,5)P 2

“…Mutating the 2 nd to 5 th residues after glycine also reduces membrane binding and virus particle production, indicating that these residues are required for N-myristoyltransferase recognition [16][17][18]. Thin section electron microscopy has more recently shown that the G2A mutation causes roughly spherical particles to assemble within the cytoplasm [19].…”