A Two-Pronged Structural Analysis of Retroviral Maturation Indicates that Core Formation Proceeds by a Disassembly-Reassembly Pathway Rather than a Displacive Transition

Abstract

cRetrovirus maturation involves sequential cleavages of the Gag polyprotein, initially arrayed in a spherical shell, leading to formation of capsids with polyhedral or conical morphology. Evidence suggests that capsids assemble de novo inside maturing virions from dissociated capsid (CA) protein, but the possibility persists of a displacive pathway in which the CA shell remains assembled but is remodeled. Inhibition of the final cleavage between CA and spacer peptide SP1/SP blocks the production of mature capsids. We investigated whether retention of SP might render CA assembly incompetent by testing the ability of Rous sarcoma virus (RSV) CA-SP to assemble in vitro into icosahedral capsids. Capsids were indeed assembled and were indistinguishable from those formed by CA alone, indicating that SP was disordered. We also used cryo-electron tomography to characterize HIV-1 particles produced in the presence of maturation inhibitor PF-46396 or with the cleavage-blocking CA5 mutation. Inhibitor-treated virions have a shell that resembles the CA layer of the immature Gag shell but is less complete. Some CA protein is generated but usually not enough for a mature core to assemble. We propose that inhibitors like PF-46396 bind to the Gag lattice where they deny the protease access to the CA-SP1 cleavage site and prevent the release of CA. CA5 particles, which exhibit no cleavage at the CA-SP1 site, have spheroidal shells with relatively thin walls. It appears that this lattice progresses displacively toward a mature-like state but produces neither conical cores nor infectious virions. These observations support the disassembly-reassembly pathway for core formation.

Distribution and Redistribution of HIV-1 Nucleocapsid Protein in Immature, Mature, and Integrase-Inhibited Virions: a Role for Integrase in Maturation

“…In order to assemble a core, Gag and Gag-Pol must be dissected by PR, with their component domains being separated in a tightly regulated cleavage program (70). Finally, CA is released from the maturing Gag shell into a soluble pool, from where it assembles to form the mature core, leaving about 50% unassembled (4,26,48,49). During core assembly, NC together with the vRNA and IN and RT enzymes becomes situated into the mature core (see above).…”

“…We refer to this feature as an "eccentric condensate." It follows that ALLINIs can be useful probes to help understand how IN contributes to maturation, in a similar way that maturation inhibitors (which prevent the scission of the CA-SP1 junction within Gag, the final proteolytic cleavage that leads to the generation of a mature core [25,26]), or mutations that prevent the cleavage of the Pol polyprotein (27,28), have provided structural insights.…”

How disordered is my protein and what is its disorder for? A guide through the “dark side” of the protein universe

“…60,61 Finally, the spectacular rise of cryo-EM in the last decade 62 presages of an increasing number of examples where protein flexibility will be documented by this powerful, fast-growing structural technique (for 2 such examples see refs. 63,64 ). While there are IDPs/IDPRs that are able to perform their function while remaining completely disordered (e.g.…”

Potential Role for CA-SP in Nucleating Retroviral Capsid Maturation

“…1) (2,10,(20)(21)(22)(23). The initial cleavages at the N termini of CA and NC free NC to begin the formation of the RNP and also yield immature capsid proteins that bear a short spacer peptide (SP) at the C terminus.…”

“…However, if CA-SP1 is fully cleaved from Gag, but the subsequent trimming of the SP sequence from the CA C terminus is prevented, the Gag lattice remains partially intact, and only limited protein patches that resemble the mature capsid lattice are seen. The formation of complete capsids and the acquisition of infectivity are blocked (9,11,21,22). Thus, trimming of SP from CA is essential for successful completion of maturation.…”