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Structural basis of HIV-1 capsid recognition by PF74 and CPSF6.

ABSTRACT: Upon infection of susceptible cells by HIV-1, the conical capsid formed by ?250 hexamers and 12 pentamers of the CA protein is delivered to the cytoplasm. The capsid shields the RNA genome and proteins required for reverse transcription. In addition, the surface of the capsid mediates numerous host-virus interactions, which either promote infection or enable viral restriction by innate immune responses. In the intact capsid, there is an intermolecular interface between the N-terminal domain (NTD) of one subunit and the C-terminal domain (CTD) of the adjacent subunit within the same hexameric ring. The NTD-CTD interface is critical for capsid assembly, both as an architectural element of the CA hexamer and pentamer and as a mechanistic element for generating lattice curvature. Here we report biochemical experiments showing that PF-3450074 (PF74), a drug that inhibits HIV-1 infection, as well as host proteins cleavage and polyadenylation specific factor 6 (CPSF6) and nucleoporin 153 kDa (NUP153), bind to the CA hexamer with at least 10-fold higher affinities compared with nonassembled CA or isolated CA domains. The crystal structure of PF74 in complex with the CA hexamer reveals that PF74 binds in a preformed pocket encompassing the NTD-CTD interface, suggesting that the principal inhibitory target of PF74 is the assembled capsid. Likewise, CPSF6 binds in the same pocket. Given that the NTD-CTD interface is a specific molecular signature of assembled hexamers in the capsid, binding of NUP153 at this site suggests that key features of capsid architecture remain intact upon delivery of the preintegration complex to the nucleus.

SUBMITTER: Bhattacharya A

PROVIDER: S-EPMC4284599 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Structural basis of HIV-1 capsid recognition by PF74 and CPSF6.

Bhattacharya Akash A Alam Steven L SL Fricke Thomas T Zadrozny Kaneil K Sedzicki Jaroslaw J Taylor Alexander B AB Demeler Borries B Pornillos Owen O Ganser-Pornillos Barbie K BK Diaz-Griffero Felipe F Ivanov Dmitri N DN Yeager Mark M

Proceedings of the National Academy of Sciences of the United States of America 20141217 52

Upon infection of susceptible cells by HIV-1, the conical capsid formed by ∼250 hexamers and 12 pentamers of the CA protein is delivered to the cytoplasm. The capsid shields the RNA genome and proteins required for reverse transcription. In addition, the surface of the capsid mediates numerous host-virus interactions, which either promote infection or enable viral restriction by innate immune responses. In the intact capsid, there is an intermolecular interface between the N-terminal domain (NTD ...[more]

PMID: 25518861

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Project description:Cleavage and polyadenylation specificity factor 6 (CPSF6) is a human protein that binds HIV-1 capsid and mediates nuclear transport and integration targeting of HIV-1 preintegration complexes. Truncation of the protein at its C-terminal nuclear-targeting arginine/serine-rich (RS) domain produces a protein, CPSF6-358, that potently inhibits HIV-1 infection by targeting the capsid and inhibiting nuclear entry. To understand the molecular mechanism behind this restriction, the interaction between CPSF6-358 and HIV-1 capsid was characterized using in vitro and in vivo assays. Purified CPSF6-358 protein formed oligomers and bound in vitro-assembled wild-type (WT) capsid protein (CA) tubes, but not CA tubes containing a mutation in the putative binding site of CPSF6. Intriguingly, binding of CPSF6-358 oligomers to WT CA tubes physically disrupted the tubular assemblies into small fragments. Furthermore, fixed- and live-cell imaging showed that stably expressed CPSF6-358 forms cytoplasmic puncta upon WT HIV-1 infection and leads to capsid permeabilization. These events did not occur when the HIV-1 capsid contained a mutation known to prevent CPSF6 binding, nor did they occur in the presence of a small-molecule inhibitor of capsid binding to CPSF6-358. Together, our in vitro biochemical and transmission electron microscopy data and in vivo intracellular imaging results provide the first direct evidence for an oligomeric nature of CPSF6-358 and suggest a plausible mechanism for restriction of HIV-1 infection by CPSF6-358.IMPORTANCE After entry into cells, the HIV-1 capsid, which contains the viral genome, interacts with numerous host cell factors to facilitate crucial events required for replication, including uncoating. One such host cell factor, called CPSF6, is predominantly located in the cell nucleus and interacts with HIV-1 capsid. The interaction between CA and CPSF6 is critical during HIV-1 replication in vivo Truncation of CPSF6 leads to its localization to the cell cytoplasm and inhibition of HIV-1 infection. Here, we determined that truncated CPSF6 protein forms large higher-order complexes that bind directly to HIV-1 capsid, leading to its disruption. Truncated CPSF6 expression in cells leads to premature capsid uncoating that is detrimental to HIV-1 infection. Our study provides the first direct evidence for an oligomeric nature of truncated CPSF6 and insights into the highly regulated process of HIV-1 capsid uncoating.

Dataset Information

Structural basis of HIV-1 capsid recognition by PF74 and CPSF6.

Publications

Structural basis of HIV-1 capsid recognition by PF74 and CPSF6.

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