The Choreography of HIV-1 Proteolytic Processing and Virion Assembly

Abstract

HIV-1 has been the target of intensive research at the molecular and biochemical levels for >25 years. Collectively, this work has led to a detailed understanding of viral replication and the development of 24 approved drugs that have five different targets on various viral proteins and one cellular target (CCR5). Although most drugs target viral enzymatic activities, our detailed knowledge of so much of the viral life cycle is leading us into other types of inhibitors that can block or disrupt proteinprotein interactions. Viruses have compact genomes and employ a strategy of using a small number of proteins that can form repeating structures to enclose space (i.e. condensing the viral genome inside of a protein shell), thus minimizing the need for a large protein coding capacity. This creates a relatively small number of critical protein-protein interactions that are essential for viral replication. For HIV-1, the Gag protein has the role of a polyprotein precursor that contains all of the structural proteins of the virion: matrix, capsid, spacer peptide 1, nucleocapsid, spacer peptide 2, and p6 (which contains protein-binding domains that interact with host proteins during budding). Similarly, the Gag-Pro-Pol precursor encodes most of the Gag protein but now includes the viral enzymes: protease, reverse transcriptase (with its associated RNase H activity), and integrase. Gag and Gag-Pro-Pol are the substrates of the viral protease, which is responsible for cleaving these precursors into their mature and fully active forms (see Fig. 1A).The Gag and Gag-Pro-Pol precursors assemble at the plasma membrane of the cell, with the membrane ultimately being pinched off from the cell surface to create a membrane-bound virion with a diameter of ϳ120 nm, representing a volume of ϳ0.9 attoliters (Fig. 1). The host ESCRT (endosomal sorting complex required for transport) pathway that is subverted to drive the membrane fission event needed for virion budding has been reviewed in detail (1-3). The virion assembly process that takes place at the cell membrane results in a finite number of each viral protein within the particle. The budded particle has ϳ2400 Gag molecules embedded in the membrane via the N-terminal matrix (MA) 3 protein domain, which, in a 120-nm sphere, gives Gag a concentration of ϳ4.4 mM, with a crude estimate that the Gag molecules occupy 50 -60% of the volume of the sphere (4). There are also ϳ120 Gag-Pro-Pol molecules (5). The embedded protease (PR) must dimerize, release itself from the Gag-Pro-Pol precursor, and then cleave the other PR cleavage sites in Gag and Gag-Pro-Pol (6). From these cleaved products, the nucleocapsid (NC) condenses and stabilizes the viral dimeric RNA, and ϳ1500 copies of the processed capsid (CA) protein reform to make the mature conical capsid structure around viral RNA to create an infectious particle (7). In this minireview, we examine outstanding issues surrounding the HIV-1 PR, the role of protein processing and rearrangement in the assembly pathway, the impact of PR inhibi...

Genetic Changes in HIV-1 Gag-Protease Associated with Protease Inhibitor-Based Therapy Failure in Pediatric Patients

“…For 10 patients (5,7,11,13,(15)(16)(17)(18)(19)(20) there were no changes in protease at failure, although nine had baseline protease polymorphisms.…”

“…[4][5][6] PI drug resistance is mediated by an accumulation of major mutations in the protease gene resulting in reduced binding of the inhibitor to the protease (PR) enzyme and high level resistance. 7 In addition, minor PR mutations, which have little or no effect on PI susceptibility, contribute to reduced susceptibility in combination with major mutations. 8 Studies have shown that mutations in Gag cleavage sites (CS) also contribute to PI resistance development.…”

Three-Dimensional Structural Characterization of HIV-1 Tethered to Human Cells

“…Ultrastructural analysis of HIV-1 tethered to human cells has been limited chiefly to examinations of chemically fixed and heavy-metal-stained samples by either thin-section transmission electron microscopy (TEM) (1,9,23,32,(35)(36)(37)(38) or scanning electron microscopy (SEM) (2,23,39). In previous studies, clusters of HIV-1 virions retained on the surface of infected or transfected cells expressing tetherin were consistently observed.…”

Role of Mason-Pfizer Monkey Virus CA-NC Spacer Peptide-Like Domain in Assembly of Immature Particles

“…In addition, NC and genomic RNA (gRNA) undergo transition from an immature to a condensed mature conformation upon processing of Gag (70,71). Interestingly, the cleavage of SP1/NC is the first step of particle maturation, while the C terminus of NC is released (NC/SP2 cleavage) concurrently with the final step of Gag maturation, i.e., the C-terminal cleavage of CA (68). Thus, strictly controlled timing of the maturation process holds CA and NC/RNA in their immature conformations, and sequential release of SP domains controls transformation of all these components into the mature core.…”

“…Structural transition of CA from the immature particle conformation into that of the mature core requires proteolytic liberation of both termini. While the release of the HIV CA N terminus is the second proteolytic step, the release of the CA C terminus is the final step of Gag maturation (68). The formation of an N-terminal ␤-hairpin, a critical structural element of the mature CA-NTD conformation, not only takes place upon liberation of the N-terminal proline of CA, but also requires proteolytic action at the opposite site of CA, releasing CTD-CA from downstream SP-1 (69).…”