The Maturational Refolding of the β-Hairpin Motif of Equine Infectious Anemia Virus Capsid Protein Extends Its Helix α1 at Capsid Assembly Locus*

Abstract

Background: The function of the maturational refolded N-terminal β-hairpin in retroviral capsid remains unknown.

Results: Folding the β-hairpin of equine infectious anemia virus (EIAV) capsid extends its downstream helix α1 at the N terminus, which forms the oligomerization core of retroviral capsids.

Conclusion: The β-hairpin structures helix α1, which could be necessary for capsid assembly.

Significance: Solution NMR revealed the function of the puzzling β-hairpin motif in retroviral capsid.

Abstract

A retroviral capsid (CA) protein consists of two helical domains, CA ^N and CA ^C, which drive hexamer and dimer formations, respectively, to form a capsid lattice. The N-terminal 13 residues of CA refold to a β-hairpin motif upon processing from its precursor polyprotein Gag. The β-hairpin is essential for correct CA assembly but unexpectedly it is not within any CA oligomeric interfaces. To understand the β-hairpin function we studied the full-length CA protein from equine infectious anemia virus (EIAV), a lentivirus sharing the same cone-shaped capsid core as HIV-1. Solution NMR spectroscopy is perfectly suited to study EIAV-CA that dimerizes weaker than HIV-1-CA. Comparison between the wild-type (wt) EIAV-CA and a variant lacking the β-hairpin structure demonstrated that folding of the β-hairpin specifically extended the N terminus of helix α1 from Tyr ²⁰ to Pro ¹⁷. This coil to helix transition involves the conserved sequence of Thr ¹⁶-Pro ¹⁷-Arg ¹⁸ (Ser ¹⁶-Pro ¹⁷-Arg ¹⁸ in HIV-1-CA). The extended region of helix α1 constituted an expanded EIAV-CA ^N oligomeric interface and overlapped with the HIV-1-CA hexamer-core residue Arg ¹⁸, helical in structure and pivotal in assembly. Therefore we propose the function of the maturational refolding of the β-hairpin in CA assembly is to extend helix α1 at the N terminus to enhance the CA ^N oligomerization along the capsid assembly core interface. In addition, NMR resonance line broadening indicated the presence of micro-millisecond exchange kinetics due to the EIAV-CA ^N domain oligomerization, independent to the faster EIAV-CA ^C domain dimerization.