Proton-Linked Dimerization of a Retroviral Capsid Protein Initiates Capsid Assembly

Abstract

In mature retroviral particles, the capsid protein (CA) forms a shell encasing the viral replication complex. Human immunodeficiency virus (HIV) CA dimerizes in solution, through its C-terminal domain (CTD), and this interaction is important for capsid assembly. In contrast, other retroviral capsid proteins, including that of Rous sarcoma virus (RSV), do not dimerize with measurable affinity. Here we show, using X-ray crystallography and other biophysical methods, that acidification causes RSV CA to dimerize in a fashion analogous to HIV CA, and that this drives capsid assembly in vitro. A pair of aspartic acid residues, located within the CTD dimer interface, explains why dimerization is linked to proton binding. Our results show that despite overarching structural similarities, the intermolecular forces responsible for forming and stabilizing the retroviral capsid differ markedly across retroviral genera. Our data further suggest that proton binding may regulate RSV capsid assembly, or modulate stability of the assembled capsid.

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

“…Expression and Purification of CA-Full-length RSV CA (Prague C strain, Genbank TM Accession Number V01197) was produced by heterologous expression in Escherichia coli and purified to homogeneity as previously described (18). Following purification the protein was dialyzed into a standard storage buffer (10 mM MOPS/KOH (pH 7.0), 50 mM NaCl, 0.5 mM sodium azide, 0.25 mM TCEP-HCl) and stored at 4°C.…”

“…RSV CA, in contrast, remains monomeric at neutral pH. It can however be induced to assemble in vitro either by mild acidification (18,22), or by the addition of anions such as phosphate (25,26). In the case of mild acidification, the assembly products are a mixture of planar sheets composed purely of CA hexamers; "T ϭ 1" icosahedra composed purely of CA pentamers; and Core-Like Particles (CLPs), which are inferred to contain both hexamers and pentamers (supplemental Figs.…”

“…Despite morphological differences, the mature capsid has a common molecular organization across all retroviral genera. It is built in large part from an array of hexameric CA (24,(27)(28)(29), where the hexameric rings of the N-terminal domain (NTD) are linked to neighboring rings via dimerization of the C-terminal domain (CTD) (18,30,31). The NTD thereby forms the capsid exterior, while the CTD is positioned underneath, facing the capsid interior.…”

“…Protonation of an aspartic acid residue within the CTD facilitates dimerization of CA (18), which in turn drives higher-order capsid assembly (Fig. 1).…”

“…Addition of sodium phosphate to RSV CA at neutral pH results in the formation of similar assembly products although the underlying mechanism is not completely defined. 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).…”