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CPSF6 defines a conserved capsid interface that modulates HIV-1 replication.

ABSTRACT: The HIV-1 genome enters cells inside a shell comprised of capsid (CA) protein. Variation in CA sequence alters HIV-1 infectivity and escape from host restriction factors. However, apart from the Cyclophilin A-binding loop, CA has no known interfaces with which to interact with cellular cofactors. Here we describe a novel protein-protein interface in the N-terminal domain of HIV-1 CA, determined by X-ray crystallography, which mediates both viral restriction and host cofactor dependence. The interface is highly conserved across lentiviruses and is accessible in the context of a hexameric lattice. Mutation of the interface prevents binding to and restriction by CPSF6-358, a truncated cytosolic form of the RNA processing factor, cleavage and polyadenylation specific factor 6 (CPSF6). Furthermore, mutations that prevent CPSF6 binding also relieve dependence on nuclear entry cofactors TNPO3 and RanBP2. These results suggest that the HIV-1 capsid mediates direct host cofactor interactions to facilitate viral infection.

SUBMITTER: Price AJ

PROVIDER: S-EPMC3431306 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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CPSF6 defines a conserved capsid interface that modulates HIV-1 replication.

Price Amanda J AJ Fletcher Adam J AJ Schaller Torsten T Elliott Tom T Lee KyeongEun K KewalRamani Vineet N VN Chin Jason W JW Towers Greg J GJ James Leo C LC

PLoS pathogens 20120830 8

The HIV-1 genome enters cells inside a shell comprised of capsid (CA) protein. Variation in CA sequence alters HIV-1 infectivity and escape from host restriction factors. However, apart from the Cyclophilin A-binding loop, CA has no known interfaces with which to interact with cellular cofactors. Here we describe a novel protein-protein interface in the N-terminal domain of HIV-1 CA, determined by X-ray crystallography, which mediates both viral restriction and host cofactor dependence. The inte ...[more]

PMID: 22956906

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Project description:BackgroundDespite intensive investigation the mechanism by which HIV-1 reaches the host cell nucleus is unknown. TNPO3, a karyopherin mediating nuclear entry of SR-proteins, was shown to be required for HIV-1 infectivity. Some investigators have reported that TNPO3 promotes HIV-1 nuclear import, as would be expected for a karyopherin. Yet, an equal number of investigators have failed to obtain evidence that supports this model. Here, a series of experiments were performed to better elucidate the mechanism by which TNPO3 promotes HIV-1 infectivity.ResultsTo examine the role of TNPO3 in HIV-1 replication, the 2-LTR circles that are commonly used as a marker for HIV-1 nuclear entry were cloned after infection of TNPO3 knockdown cells. Potential explanation for the discrepancy in the literature concerning the effect of TNPO3 was provided by sequencing hundreds of these clones: a significant fraction resulted from autointegration into sites near the LTRs and therefore were not bona fide 2-LTR circles. In response to this finding, new techniques were developed to monitor HIV-1 cDNA, including qPCR reactions that distinguish 2-LTR circles from autointegrants, as well as massive parallel sequencing of HIV-1 cDNA. With these assays, TNPO3 knockdown was found to reduce the levels of 2-LTR circles. This finding was puzzling, though, since previous work has shown that the HIV-1 determinant for TNPO3-dependence is capsid (CA), an HIV-1 protein that forms a mega-dalton protein lattice in the cytoplasm. TNPO3 imports cellular splicing factors via their SR-domain. Attention was therefore directed towards CPSF6, an SR-protein that binds HIV-1 CA and inhibits HIV-1 nuclear import when the C-terminal SR-domain is deleted. The effect of 27 HIV-1 capsid mutants on sensitivity to TNPO3 knockdown was then found to correlate strongly with sensitivity to inhibition by a C-terminal deletion mutant of CPSF6 (R2?=?0.883, p?<?0.0001). TNPO3 knockdown was then shown to cause CPSF6 to accumulate in the cytoplasm. Mislocalization of CPSF6 to the cytoplasm, whether by TNPO3 knockdown, deletion of the CPSF6 nuclear localization signal, or by fusion of CPSF6 to a nuclear export signal, resulted in inhibition of HIV-1 replication. Additionally, targeting CPSF6 to the nucleus by fusion to a heterologous nuclear localization signal rescued HIV-1 from the inhibitory effects of TNPO3 knockdown. Finally, mislocalization of CPSF6 to the cytoplasm was associated with abnormal stabilization of the HIV-1 CA core.ConclusionTNPO3 promotes HIV-1 infectivity indirectly, by shifting the CA-binding protein CPSF6 to the nucleus, thus preventing the excessive HIV-1 CA stability that would otherwise result from cytoplasmic accumulation of CPSF6.

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.

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CPSF6 defines a conserved capsid interface that modulates HIV-1 replication.

Publications

CPSF6 defines a conserved capsid interface that modulates HIV-1 replication.

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