Capsid Lattice Destabilization Leads to Premature Loss of the Viral Genome and Integrase Enzyme during HIV-1 Infection

Abstract

The human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein forms a conical lattice around the viral ribonucleoprotein complex (vRNP) consisting of a dimeric viral genome and associated proteins, together constituting the viral core. Upon entry into target cells, the viral core undergoes a process termed uncoating, during which CA molecules are shed from the lattice. Although the timing and degree of uncoating are important for reverse transcription and integration, the molecular basis of this phenomenon remains unclear. Using complementary approaches, we assessed the impact of core destabilization on the intrinsic stability of the CA lattice in vitro and fates of viral core components in infected cells. We found that substitutions in CA can impact the intrinsic stability of the CA lattice in vitro in the absence of vRNPs, which mirrored findings from assessment of CA stability in virions. Altering CA stability tended to increase the propensity to form morphologically aberrant particles, in which the vRNPs were mislocalized between the CA lattice and the viral lipid envelope. Importantly, destabilization of the CA lattice led to premature dissociation of CA from vRNPs in target cells, which was accompanied by proteasomal-independent losses of the viral genome and integrase enzyme. Overall, our studies show that the CA lattice protects the vRNP from untimely degradation in target cells and provide the mechanistic basis of how CA stability influences reverse transcription. IMPORTANCE The human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein forms a conical lattice around the viral RNA genome and the associated viral enzymes and proteins, together constituting the viral core. Upon infection of a new cell, viral cores are released into the cytoplasm where they undergo a process termed “uncoating”, i.e. shedding of CA molecules from the conical lattice. Although proper and timely uncoating has been shown to be important for reverse transcription, the molecular mechanisms that link these two events remain poorly understood. In this study, we show that destabilization of the CA lattice leads to premature dissociation of CA from viral cores, which exposes the viral genome and the integrase enzyme for degradation in target cells. Thus, our studies demonstrate that the CA lattice protects the viral ribonucleoprotein complexes from untimely degradation in target cells and provide the first causal link between how CA stability affects reverse transcription.

HIV-1 Capsid Uncoating Is a Multistep Process That Proceeds through Defect Formation Followed by Disassembly of the Capsid Lattice

“…In contrast, markedly accelerated uncoating was observed for K203A CA* mutant capsids known to form highly unstable cores that rapidly disassemble after viral fusion or lysis (Figure H and Figure S4A). Interestingly, a small fraction of the K203A CA*/untagged K203A mutant cores (∼10%) exhibited delayed uncoating over the course of 30 min (Figure S4B). Approximately 15% of mature particles retained very weak but detectable levels of CA* fluorescence by 30 min after saponin lysis (Figure S4C).…”

“…The HIV-1 capsid is a large (∼60 nm wide end, ∼40 nm narrow end, ∼100 nm length) proteinaceous structure that is comprised of ∼250 capsid protein (CA) hexamers and exactly 12 pentamers to form the conical capsid lattice. − Fusion of HIV-1 with the cell membrane releases the capsid into the cytosol where it interacts with a multitude of cellular dependency and restriction factors. Interactions with host dependency factors promote microtubule transport, import through the nuclear pore complex (NPC), and translocation to nuclear speckles for integration within the speckle-associated genomic domains. ,− Capsid disassembly, referred to as uncoating, is required for the release of the HIV-1 preintegration complex, but the extent and cellular sites of CA loss remain controversial. ,, HIV-1 capsid stability is tightly regulated by multiple host factors, such as IP6, Sec24C, Nup153, and several molecular motors. − Optimal core stability is essential for nuclear import and delivery of viral complexes to the sites of integration, as evidenced by the compromised infectivity of HIV-1 containing CA mutations that hyperstabilize or destabilize the capsid lattice. ,− Thus, timely HIV-1 uncoating is critical for productive infection.…”

A molecular switch modulates assembly and host factor binding of the HIV-1 capsid

“…Substitutions for Val59 and Gly60 within the TVGG motif result in non-viable or severely impaired viruses [21][22][23][24] . Mutations in the Pro38-Met39 pawl in helix 2 abolish CA assembly 25 or drastically lower the intrinsic stability of the capsid 26,27 . Asn57 and the Met66 gate modulate binding of the assembled capsid to host factors that are important for nuclear import and integration, as well as binding to one class of capsid-targeting inhibitors [28][29][30][31][32][33][34][35][36][37] .…”

A snapshot on HIV-1 evolution through the identification of phylogenetic-specific properties of HIV-1 integrases M/O

“…The longer permanence of the reverse transcription complex in the core that is observed in Jurkat cells, might account for the higher increase in 3’ processing for this cell type ( Fig 6B ). Indeed, since 3’ processing occurs right after reverse transcription, when a longer time is allotted to this process before the capsid is dismantled, it could benefit from a confined environment that keeps a high concentration of the components of the reaction, as it was previously observed for reverse transcription [ 49 – 51 ].…”