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

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.

A DNA-origami nuclear pore mimic reveals nuclear entry mechanisms of HIV-1 capsid

“…It is well established that the CA tube is a good mimic of the flat side of the capsid (Zhao et al, 2011). Additionally, it was reported that the N21C/A22C CA mutant can assemble into CA spheres (30-45 nm) that mimic the highly curved capsid tips (Zhang et al, 2018;Zhao et al, 2011) (Figure 4A and S4). We assembled N21C/A22C CA spheres and confirmed that these bound well to the NUP153 NuPOD, mostly residing in the middle of the NuPOD rings.…”

“…CA sphere Following a previously established protocol (Zhang et al, 2018), purified N21C/A22C CA proteins (1 mg/mL) were dialyzed into a 50 mM Tris-HCl, 1 M NaCl, pH 8.0 buffer overnight at 4°C.…”

“…CA can be assembled through various disulfide bonds into standalone hexamers or pentamers (Pornillos et al, 2009;Pornillos et al, 2011), or multi-hexamer/pentamer units as well as a hexamer-2 structure that mimics the interface of three hexamers in the assembled capsid (Summers et al, 2019). Further assembly of higher-order structures such as CA tubes (Lopez et al, 2011), spheres (Zhang et al, 2018), and stable cones are also achievable (Dick et al, 2018) ( Figure 1A). However, these assemblies have not yet been applied to exploring interactions with the NPC as there are few in vitro platforms that can mimic the dimensions and complexity of the NPC.…”

Visualizing HIV-1 Capsid and Its Interactions with Antivirals and Host Factors

“…Christensen and colleagues demonstrated that reverse transcription takes place inside an intact or nearly intact capsid, using capsids derived from wild-type virions in an in vitro system [44]. The newly reverse-transcribed DNA bulged out of the capsid as it broke down over the course of 8-10 h. This offered support for the concept that progressive uncoating is driven at least in part by increased pressure due to the completion of reverse transcription [16,29,45].…”

“…CryoEM has implications for the design of therapeutics as well. One report of an engineered CA protein discussed its self-assembly into hexamers containing stabilizing disulfide bonds and then into icosahedral particles with a 40 nm diameter [ 45 ]. They contained 12 pentamers and 30 hexamers as determined by cryoEM reconstruction.…”