Structure of a Monomeric Mutant of the HIV-1 Capsid Protein

Abstract

The capsid protein (CA) of the HIV-1 virus plays a significant role in the assembly of the immature virion, and is the critical building block of its mature capsid. Thus, there has been a significant interest in the CA protein as a target in the design of inhibitors of early and late stage events in the HIV-1 virus replication cycle. However, due to its inherent flexibility from the inter-domain linker and the monomer-dimer equilibrium in solution, HIV-1 wild-type CA monomer has defied structural determinations by X-ray crystallography and NMR spectroscopy. Here we report the detailed solution structure of the full length HIV-1 CA using a monomeric mutant that, though non-infective, preserves many of the critical properties of the wild-type protein. The structure shows independently folded N-terminal (NTD) and C-terminal domains (CTD) joined by a flexible linker. The CTD domain shows some differences from that of the dimeric wild-type CTD structures. This study provides insights into the molecular mechanism of the wild-type CA dimerization critical for capsid assembly. The monomeric mutant allows investigation of interactions of CA with human cellular proteins exploited by the HIV-1 virus, directly in solution without the complications associated with the monomer-dimer equilibrium of the wild-type protein. This structure also permits the design of inhibitors directed at a novel target, viz., interdomain flexibility, as well as inhibitors that target multiple interdomain interactions critical for assembly and interactions of CA with host cellular proteins that play significant roles within the replication cycle of the HIV-1 virus.

Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication

“…The CA-CTD and the capsid mutant CA-CTD W184A/M185A (WAMA) were prepared in 25 mM phosphate (pH 6.5), 100 mM NaCl, 0.02% NaN 3 , and 10% D 2 O, with or without 5 mM dithiothreitol (DTT) to a final concentration of 1 mM and 330 M, respectively. Two-dimensional (2D) [ 1 H, 15 N] heteronuclear single-quantum coherence (HSQC) spectra were acquired at 298 K, with or without a 1.2-fold or 2.4-fold molar excess of ebselen prepared as a 50 or 100 mM stock in hexadeutero-DMSO (DMSO-d6). All samples contained the same amount of DMSO-d6 (2%).…”

“…We monitored the binding of ebselen to CTD via [ 1 H, 15 N] heteronuclear single-quantum coherence (HSQC) NMR on CA-CTD and on the capsid mutant CA-CTD W184A/M185A (WAMA) that bears amino acid substitutions that disrupt dimerization (13). Peak assignments were based on published HSQC chemical shifts for CA-CTD (24) and the WAMA mutant (25).…”

“…Structures of the full-length capsid, NTD, and CTD have been studied by crystallography, cryo-electron microscopy, and nuclear magnetic resonance (NMR) (12)(13)(14)(15)(16). The CA-NTD is composed of seven ␣-helices (CA helices 1 to 7), while the smaller CA-CTD is composed of a short 3 10 -helix followed by an extended strand and four ␣-helices (CA helices 8 to 11).…”

“…In solution, CA dimerization (dissociation constant [K d ] of 18 M) is mainly dependent on Trp184 and Met185 residues in helix 9 of the CTD (13). Mutation of these residues interferes with CA assembly in vitro and abolishes viral infectivity (15,17). This interface is therefore required for efficient assembly of both the mature and immature capsid lattice.…”

Contributions of Charged Residues in Structurally Dynamic Capsid Surface Loops to Rous Sarcoma Virus Assembly

“…This ␤-hairpin forms only after CA is cleaved from the upstream Gag domain. In the NTD, helices ␣1 to ␣4 and the last helix form a tight bundle, while the loops and short helices between ␣4 and the last helix comprise a surface-exposed flexible-loop (FL) region that is the least conserved region in the CA sequence and exhibits considerable structural variation across retroviral families (11)(12)(13)(14)(15)(16)(17)(18)(19)87).…”