HIV-1 assembly, budding, and maturation

Abstract

A defining property of retroviruses is their ability to assemble into particles that can leave producer cells and spread infection to susceptible cells and hosts. Virion morphogenesis can be divided into three stages: assembly, wherein the virion is created and essential components are packaged; budding, wherein the virion crosses the plasma membrane and obtains its lipid envelope; and maturation, wherein the virion changes structure and becomes infectious. All of these stages are coordinated by the Gag polyprotein and its proteolytic maturation products, which function as the major structural proteins of the virus. Here, we review our current understanding of the mechanisms of HIV-1 assembly, budding, and maturation, starting with a general overview and then providing detailed descriptions of each of the different stages of virion morphogenesis.

Figure 1.

HIV-1 assembly, budding, and maturation. (A) Schematic illustration showing the different stages of HIV-1 assembly, budding, and maturation. (B) Domain structure of the HIV-1 Gag protein; arrows denote the five sites that are cleaved by the viral PR during maturation. (C) Structural model of the HIV-1 Gag protein, created by combining structures of the isolated MA-CA_NTD (2GOL), CA_CTD (1BAJ), and NC (1MFS) proteins, with a helical model for SP1. (D) Schematic model showing the organization of the immature HIV-1 virion. (E) Schematic model showing the organization of the mature HIV-1 virion. (F) Central section from a cryo-EM tomographic reconstruction of an immature HIV-1 virion. (G) Central section from a tomographic reconstruction of a mature HIV-1 virion. (H) Structure of HIV-1 protease (PR, 3D3T). The two subunits in the dimer are shown in different shades of purple, the “flap” and dimerization interfaces are labeled, positions of the active site Asp25 residues are shown in red, and a bound peptide corresponding to the SP2-p6 cleavage site is shown as a stick model, with oxygen atoms in red and nitrogen atoms in blue.

Figure 2.

Myristoyl switch model for MA^Gag recognition of the plasma membrane (Saad et al. 2006). MA^Gag (yellow) proteins are shown with the aliphatic myristoyl group (brown) sequestered within the soluble protein (left, 1UPH), and with the myristoyl group extruded into the membrane when bound to the plasma membrane specific phosphatidyl inositide, PI(4,5)P₂, shown in red (right, 2H3F). The PI(4,5)P₂ inositol head group and unsaturated 2′-fatty acid bind within MA, allosterically inducing extrusion of the myristoyl group, whereas the saturated 1′-fatty acid of PI(4,5)P₂ remains embedded in the membrane. Basic residues on the membrane binding surface of MA^Gag are shown in blue.

Figure 3.

5′ Untranslated region (UTR) of the HIV-1 RNA genome and its interactions with the viral NC protein. Lower image shows a secondary structure model for the 5′UTR, highlighting the TAR stem loop structure (which binds the viral Tat protein), the polyadenylation site, the U5 element, the primer binding site (PBS, which anneals to the tRNA^Lys,3 primer), and four stem loops within the packaging site, which contain the dimer initiation site (DIS, stem-loop I, which forms a kissing loop structure that initiates association of the two copies of the genomic RNA), the splice donor (SD, stem loop II, which acts as the 5′ donor for splicing of subgenomic RNAs), the Psi site (ψ, stem loop III, which forms an essential part of the packaging signal), and the Gag start codon (AUG, stem loop IV, which contains the start site for Gag translation). Upper structures show three different complexes between the NC protein (red, with zinc atoms shown in grey and Zn-coordinating side chains shown explicitly) and viral RNAs (blue), corresponding to the U5 region (Spriggs et al. 2008), the SD stem loop (1F6U), and the ψ stem loop (1A1T).

Figure 4.

Assembly and structure of immature HIV-1 particles. (A) (Left) Central slice through a cryo-EM tomogram of a budding HIV-1 virion. (Right) Map of the Gag lattice in the budding virion. Positions of Gag hexagons are colored according to their hexagonal order, from low (brown) to high (green). (B) Cryo-EM tomogram of an immature HIV-1 virion, showing the structure of the immature HIV-1 Gag lattice. The surface was cut perpendicular to the membrane to reveal the two membrane leaflets, the two CA domains (orange and burnt orange), and the NC layer (red). (C) Schematic of the conformational rearrangements in the capsid lattice during maturation. (Left) Arrangement of the amino-terminal (orange) and carboxy-terminal (burnt orange) domains of CA in the immature lattice, viewed from outside the particle (upper), and rotated 90° around the horizontal axis (lower). Domains from neighboring hexamers are indicated in lighter colors, and sixfold lattice positions are marked by hexagons. (Right) Equivalent interactions of CA subunits within the mature HIV-1 capsid lattice.

Figure 5.

Summary of the essential core ESCRT machinery used in HIV-1 budding (with auxiliary factors shown in parentheses), illustrating a leading model for the budding mechanism. Late domain motifs within p6^Gag bind directly to the UEV domain of the TSG101 subunit of the heterotetrameric ESCRT-I complex (red, with bound ubiquitin in black, 1S1Q, 2P22) and the V domain of ALIX (blue, 2OEV). These interactions result in the recruitment of the ESCRT-III proteins of the CHMP1, CHMP2, and CHMP4 families (green, 2GD5), which apparently polymerize into a “dome” that promotes closure of the membrane neck (Peel et al. 2011). They also recruit the VPS4 ATPases (purple, 1XWI, 1YXR), which completes the membrane fission reaction and uses the energy of ATPase to release the ESCRT-III from the membrane and back into the cytoplasm. See text for details.

Figure 6.

Fullerene cone model for the HIV-1 capsid. (A) Molecular model of the HIV-1 capsid, with CA hexamers in orange and pentamers in tan (adapted from Pornillos et al. 2011). (B) Structure of the HIV-1 CA hexamer (3H47). (C) Structure of the HIV-1 CA pentamer (3P05). (D) Detailed structure of the CA_NTD–CA_NTD interface that stabilizes the hexameric ring. Hydrophobic residues that stabilize the interaction between CA helices 1, 2, and 3 are highlighted. (E) Detailed structure of the CA_NTD(orange)–CA_CTD(tan) interface that forms a “girdle” around the hexameric and pentameric rings. Interface residues are highlighted, as are a series of salt bridges and hydrogen bonds that stabilize the interface while allowing it to “swivel” in response to changes in lattice curvature. (F,G) Two alternative structures of the CA_CTD dimer, 1A43and 2KOD. Two key interface residues (W184 and M185) are shown in each case to emphasize the fact that similar CA surfaces are used in both dimers.