T = 4 Icosahedral HIV-1 Capsid As an Immunogenic Vector for HIV-1 V3 Loop Epitope Display.

Zhiqing Zhang,Jie Jiang,Shuangquan Gao,Qingbing Zheng,Xiaodong Yan,Ying Gu,Maozhou He,Feng Zhang,Shimeng Bai,Shaowei Li,Ningshao Xia

doi:10.3390/v10120667

Abstract

The HIV-1 mature capsid (CA) assumes an amorphous, fullerene conical configuration due to its high flexibility. How native CA self-assembles is still unclear despite having well-defined structures of its pentamer and hexamer building blocks. Here we explored the self-assembly of an engineered capsid protein built through artificial disulfide bonding (CA N21C/A22C) and determined the structure of one fraction of the globular particles. CA N21C/A22C was found to self-assemble into particles in relatively high ionic solutions. These particles contained disulfide-bonding hexamers as determined via non-reducing SDS-PAGE, and exhibited two major components of 57.3 S and 80.5 S in the sedimentation velocity assay. Particles had a globular morphology, approximately 40 nm in diameter, in negative-staining TEM. Through cryo-EM 3-D reconstruction, we determined a novel T = 4 icosahedral structure of CA, comprising 12 pentamers and 30 hexamers at 25 Å resolution. We engineered the HIV-1 V3 loop to the CA particles, and found the resultant particles resembled the morphology of their parental particles in TEM, had a positive reaction with V3-specific neutralizing antibodies, and conferred neutralization immunogenicity in mice. Our results shed light on HIV CA assembly and provide a particulate CA for epitope display.

Highlights

The human immunodeficiency virus type 1 (HIV-1) Gag precursor protein is the major structural protein coded by the gag gene
The pure CA N21C/A22C protein was dialyzed into assembly buffer (50 mM Tris-HCl, pH 8.0, 1 M NaCl) overnight at 4 ◦ C
CA N21C/A22C assembled at 10 mg/mL in assembly buffer (50 mM Tris-HCl, pH 8.0, 1 M NaCl and 20 mM β-ME) and resolved only with 5-mer bands and no 6-mer bands in gel

Summary

Introduction

The human immunodeficiency virus type 1 (HIV-1) Gag precursor protein is the major structural protein coded by the gag gene. Gag is cleaved into three major structural proteins—matrix (MA), capsid (CA) and nucleocapsid (NC)—and undergoes a dramatic morphological rearrangement [1,2]. The CA protein contains two independent and highly helical domains, the N-terminal domain (NTD) and C-terminal domain (CTD), which are connected by a short flexible linker [3]. The structures of CA and its isolated domains have been solved by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy [3,4,5,6,7]. The HIV-1 capsid has an inherent structural variability that facilitates its spontaneous assembly into different conformations in vitro [8]. In 2010, Pornillos et al [9,10,11] adopted a disulfide crosslinking

Methods

Results

Conclusion