Structural studies of human immunodeficiency virus type 1 by cryo-electron tomography

Abstract

Gag, the major structural component of the type 1 human immunodeficiency virus (HIV-1), comprises the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 proteins, as well as the SP1 and SP2 spacer peptides. In the immature HIV-1 virion, the domains of Gag are arranged radially with the amino-terminus of MA at the membrane. Mature viral particles are formed when Gag is proteolytically cleaved, allowing CA to reassemble into the viral core, which contains NC bound to genomic RNA. While the structures of nearly every HIV-1 protein are known in atomic detail from X-ray crystallography and NMR spectroscopy, many questions remain about the intermolecular interactions in both the immature and mature particles. We have obtained three-dimensional structures of individual immature and mature HIV-1 virus-like particles by cryoelectron tomography. Reconstructions of the mature particles revealed diverse core morphologies with a preference for conical shapes consistent with 5,7 fullerene cones. Uniform positioning of the wide end of the cores and an internal density likely to be the NC/RNA complex were also observed, as were multiple and nested viral cores. Our results support the fullerene cone model for the core structure and suggest that specific interactions may occur between the CA, MA, and NC layers in the mature virion. These experiments also aided the characterization of a new cryostage allowing routine collection of dual-axis tomograms. Tomograms of the immature virions revealed patches of hexagonally ordered Gag molecules interspersed with regions of disordered or absent Gag. We developed novel tools for analysis of locally ordered lattices embedded in curved structures, including a method for locating and averaging the Gag unit cell in situ. The unit cell average revealed that the CA domains and the SP1 spacer peptides were organized in a hexagonal lattice with ring-to-ring spacing of 8 nm in the CA layer and 7.5 nm in SP1. No regular lattice was found in the MA or NC layers. Based on the averaged Gag unit cell, we proposed a pseudoatomic model for the CA and SP1 domains in the immature virion.