Characterization of the In Vitro HIV-1 Capsid Assembly Pathway

Abstract

SummaryDuring morphogenesis of mature HIV-1 cores, the viral capsid (CA) proteins assemble conical or tubular shells around the viral ribonucleoprotein complexes. This assembly step is mimicked in vitro through reactions in which CA proteins oligomerize to form long tubes, and this process can be modeled as consisting of a slow nucleation period followed by a rapid phase of tube growth. We have developed a novel fluorescence microscopy approach to monitor in vitro assembly reactions and have employed it, along with electron microscopy analysis, to characterize the assembly process. Our results indicate that temperature, salt concentration, and pH changes have differential effects on tube nucleation and growth steps. We also demonstrate that assembly can be unidirectional or bidirectional, that growth can be capped, and that proteins can assemble onto the surfaces of tubes, yielding multiwalled or nested structures. Finally, experiments show that a peptide inhibitor of in vitro assembly also can dismantle pre-existing tubes, suggesting that such reagents may possess antiviral effects against both viral assembly and uncoating. Our investigations help establish a basis for understanding the mechanism of mature HIV-1 core assembly, and avenues for antiviral inhibition.

Proton-driven Assembly of the Rous Sarcoma Virus Capsid Protein Results in the Formation of Icosahedral Particles

“…However, both protondriven and phosphate-driven assembly of RSV CA seem to involve a nucleation step (18,25). This also appears to be the case for in vitro assembly of HIV-1 CA (35).…”

Analysis of Small Molecule Ligands Targeting the HIV-1 Matrix Protein-RNA Binding Site

“…For NMR studies, the proteins were dialyzed against 50 mM sodium phosphate (pH 5.5), 100 mM NaCl, and 5 mM dithiothreitol (DTT) and then concentrated by Millipore ultrafiltration concentrators against the same buffer. Protein purities were evaluated after fractionation by SDS-PAGE (32,33,(57)(58)(59)(60)(61) by Coomassie Blue staining (32,(57)(58)(59). The HIV NC peptide was prepared commercially as reported previously (61).…”

Analysis of Human Immunodeficiency Virus Type 1 Matrix Binding to Membranes and Nucleic Acids

“…The proteins were desalted by buffer exchange in Sephadex G25 spin columns in 10 mM sodium phosphate (pH 7.8), supplemented with ␤-mercaptoethanol (1 mM final concentration), aliquoted, and stored at Ϫ80°C under nitrogen gas. Protein purities were evaluated after fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1,3,50,51,58) by Coomassie blue staining (1,3,58) and immunoblotting using a primary anti-HIV CA monoclonal antibody (Hy183; kindly provided by Bruce Chesebro, Rocky Mountain Laboratory) or sheep antiserum (no. 286; lot IDV-010) to MA obtained from Michael Phelan via the NIAID AIDS Research and Reference Program.…”