Advances in HIV-1 Assembly

Abstract

The assembly of HIV-1 particles is a concerted and dynamic process that takes place on the plasma membrane of infected cells. An abundance of recent discoveries has advanced our understanding of the complex sequence of events leading to HIV-1 particle assembly, budding, and release. Structural studies have illuminated key features of assembly and maturation, including the dramatic structural transition that occurs between the immature Gag lattice and the formation of the mature viral capsid core. The critical role of inositol hexakisphosphate (IP6) in the assembly of both the immature and mature Gag lattice has been elucidated. The structural basis for selective packaging of genomic RNA into virions has been revealed. This review will provide an overview of the HIV-1 assembly process, with a focus on recent advances in the field, and will point out areas where questions remain that can benefit from future investigation.

Keywords: Env trafficking; Gag protein; RNA packaging; capsid protein; envelope glycoprotein; inositol hexakisphosphate; matrix protein; maturation; nucleocapsid protein.

Figure 1

Schematic overview of HIV-1 assembly process. Gag and Env traffic via different routes to reach a common site of assembly on the PM. The path taken by Gag to reach the PM remains uncertain. Env travels through the secretory pathway to the PM, and then is rapidly endocytosed. Depicted here is a role for the recycling endosome in the outward movement of Env to the site of particle assembly. Following the assembly of the immature virion and packaging of the viral gRNA, cleavage of Gag occurs, leading to dramatic structural rearrangements and the formation of the mature virion. Adapted from HIV Replication Cycle by BioRender.com (2022).

Figure 2

Selection of gRNA for packaging versus use as mRNA for translation. Note that in the CAP3G RNA, the 5′ cap is accessible and the RNA is monomeric, while in the CAP1G form the 5′ cap is sequestered and RNA dimerizes for packaging. RNA structure schematics modeled from [28]. DIS = dimer initiation site; SD = major splice donor; PBS = primer binding site. Figure adapted from HIV Replication Cycle by BioRender.com (2022).

Figure 3

Gag polyprotein with cleavage products. (A) Schematic diagram showing cleavage products/domains in different colors. (B) MA trimer structure from PDB 1HIW [39]. (C) CA-SP1 structure, side view, with one CA_NTD-CA_CTD-SP1 colored in blue, from PDB 5L93 [40]. Note cyclophilin A (cypA) binding loop, CA_NTD and CA_CTD connected by flexible loop, and CA-SP1 helix. (D) NC structure with single-stranded oligonucleotide (ss nucleotide), from PDB 1BJ6 [41]. Zinc ions shown as spheres. (E) p6 structure from PDB 2C55 [42]. (F) Immature MA lattice, top view, with single trimer in red; from PDB 7OVQ [43]. (G) Immature CA-SP1 lattice, top view, with single hexamer colored blue, from PDB 4USN [44]. Structures rendered by the PyMOL Molecular Graphics System, Shrodinger, LLC. The arrangement of this figure was adapted from [45]. Figure compiled using BioRender.com (2022).

Figure 4

Role of IP6 in immature Gag lattice and in mature capsid core formation. (A) Schematic depiction of maturation of immature lattice to mature core formation, with structural model below showing position of IP6. Note that IP6 is coordinated by K359 and K290 rings in immature hexamer (left), while it interacts with R18 in the mature viral core. Figure adapted from findings of [189]. (B) Top and side views of IP6 in mature lattice, with positions of R18 residues indicated in yellow. IP6 is depicted in red. From PDB 6BHT [189]. Structures rendered by the PyMOL Molecular Graphics System, Shrodinger, LLC.

Figure 5

Host pathways implicated in Env trafficking and particle incorporation. Env is synthesized on ER-associated ribosomes, forming trimers in this organelle. As gp160 timers progress through the Golgi, they are glycosylated, and furin-mediated cleavage occurs in the TGN. The cleaved trimers reach the PM, followed rapidly by endocytosis into early endosomes. From here, multiple pathways are possible, including degradation in the late endosome/lysosome, retrograde transport to the TGN, and transport to the ERC. Evidence favors a role for outward recycling from the ERC in mediating Env incorporation into particles in a CT-dependent manner. Shown are roles for clathrin/AP-2, retromer, a potential role for AP-1, and outward recycling mediated by Rab11-FIP1C. Figure compiled using BioRender.com (2022).

Figure 6

Restriction factors acting along the HIV-1 assembly pathway. On the left is pictured the unrestricted assembly of particles with fully cleaved Env. GBP5 and MARCH8 act to reduce particle infectivity through effects on Env, with a reduction in furin-mediated cleavage (GBP5) or disruption of Env trafficking and incorporation (MARCH8). PSGL-1 reduces Env incorporation but also reduces attachment to target cells. IQGAP1 binds to Gag and modulates particle formation, with reduced production of particles upon overexpression. Tetherin acts to prevent the release of fully formed particles, forming a physical tether to the PM and also acting as an innate immune sensor to stimulate signaling leading to NF-κB activation.