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Nucleoporin NUP153 phenylalanine-glycine motifs engage a common binding pocket within the HIV-1 capsid protein to mediate lentiviral infectivity.

Matreyek KA, Yücel SS, Li X, Engelman A - PLoS Pathog. (2013)

Bottom Line: NUP153(C) fused to the effector domains of the rhesus Trim5α restriction factor (Trim-NUP153(C)) potently restricted HIV-1, providing an intracellular readout for the NUP153(C)-CA interaction during retroviral infection.Our results highlight similar mechanisms of binding for disparate host factors to the same region of HIV-1 CA during viral ingress.We conclude that a subset of lentiviral CA proteins directly engage FG-motifs present on NUP153 to affect viral nuclear import.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Department of Medicine, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Lentiviruses can infect non-dividing cells, and various cellular transport proteins provide crucial functions for lentiviral nuclear entry and integration. We previously showed that the viral capsid (CA) protein mediated the dependency on cellular nucleoporin (NUP) 153 during HIV-1 infection, and now demonstrate a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG)-repeat enriched NUP153 C-terminal domain (NUP153(C)). NUP153(C) fused to the effector domains of the rhesus Trim5α restriction factor (Trim-NUP153(C)) potently restricted HIV-1, providing an intracellular readout for the NUP153(C)-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV) bound NUP153(C) under these conditions, results that correlated with direct binding between purified proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 protein during virus infection. Mutagenesis experiments concordantly identified NUP153(C) and CA residues important for binding and lentiviral infectivity. Different FG motifs within NUP153(C) mediated binding to HIV-1 versus EIAV capsids. HIV-1 CA binding mapped to residues that line the common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74) inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6) protein, with Asn57 (Asp58 in EIAV) playing a particularly important role. PF74 and CPSF6 accordingly each competed with NUP153(C) for binding to the HIV-1 CA pocket, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153(C) expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicates that the NUP153(C)-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. Our results highlight similar mechanisms of binding for disparate host factors to the same region of HIV-1 CA during viral ingress. We conclude that a subset of lentiviral CA proteins directly engage FG-motifs present on NUP153 to affect viral nuclear import.

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NUP153C directly binds the HIV-1 CA N-terminal domain.(A) Schematic of NUP153 protein, with residue numbers of domain boundaries indicated. (B) Full length or truncated fragments of HA-tagged NUP153 extracted from 293T cells were tested for binding to HIV-1 CA-NC. Pelleted proteins were resolved by SDS-PAGE and visualized by western blotting with anti-HA antibody 3F10 (top), or by Coomassie stain (bottom). Input, 20% of binding reaction. CA-NC was included in the binding reactions as indicated. (C) Recombinant, tag-free NUP153C and GST purified from E. coli were similarly tested for binding to CA-NC; proteins were detected with Coomassie stain. (D) Recombinant NUP153C pulled down with full length his-tagged wild-type (WT) or W184A/M185A HIV-1 CA, and detected with Coomassie stain. (E) Recombinant NUP153C pulled down with his-tagged CAN, and detected with Coomassie stain. Each experiment was repeated at least 3 times, with a single representative result shown.
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ppat-1003693-g001: NUP153C directly binds the HIV-1 CA N-terminal domain.(A) Schematic of NUP153 protein, with residue numbers of domain boundaries indicated. (B) Full length or truncated fragments of HA-tagged NUP153 extracted from 293T cells were tested for binding to HIV-1 CA-NC. Pelleted proteins were resolved by SDS-PAGE and visualized by western blotting with anti-HA antibody 3F10 (top), or by Coomassie stain (bottom). Input, 20% of binding reaction. CA-NC was included in the binding reactions as indicated. (C) Recombinant, tag-free NUP153C and GST purified from E. coli were similarly tested for binding to CA-NC; proteins were detected with Coomassie stain. (D) Recombinant NUP153C pulled down with full length his-tagged wild-type (WT) or W184A/M185A HIV-1 CA, and detected with Coomassie stain. (E) Recombinant NUP153C pulled down with his-tagged CAN, and detected with Coomassie stain. Each experiment was repeated at least 3 times, with a single representative result shown.

Mentions: As we previously found CA to be the dominant viral determinant of the requirement for NUP153 during HIV-1 infection [19], we tested whether a physical interaction between NUP153 and HIV-1 CA exists. Our initial assay utilized a recombinant viral fusion protein consisting of HIV-1 CA and nucleocapsid (NC) proteins, which when assembled in vitro in the presence of high salt and single stranded nucleic acid forms large tube-like structures that readily pellet through cushions of sucrose [29]. In this way, CA-interacting proteins can co-sediment with the tube structures [18], [44]. Full length or various fragments of HA-tagged NUP153 expression constructs were transfected into 293T cells, and the resulting proteins were tested for their ability to co-sediment with CA-NC assemblies. Full-length NUP153 (residues 1–1475) pelleted through the sucrose cushion in a CA-NC dependent manner (Figure 1A and 1B). The NUP153 N-terminal domain (residues 1–650) failed to bind CA-NC under conditions that supported efficient NUP153C (residues 896–1475) binding. The C-terminal NUP153 deletion mutant comprised of residues 1–1198 failed to bind, confirming the importance of the NUP153 FG-repeat domain in binding, and mapping the interaction to residues 1199–1475 of the full length protein.


Nucleoporin NUP153 phenylalanine-glycine motifs engage a common binding pocket within the HIV-1 capsid protein to mediate lentiviral infectivity.

Matreyek KA, Yücel SS, Li X, Engelman A - PLoS Pathog. (2013)

NUP153C directly binds the HIV-1 CA N-terminal domain.(A) Schematic of NUP153 protein, with residue numbers of domain boundaries indicated. (B) Full length or truncated fragments of HA-tagged NUP153 extracted from 293T cells were tested for binding to HIV-1 CA-NC. Pelleted proteins were resolved by SDS-PAGE and visualized by western blotting with anti-HA antibody 3F10 (top), or by Coomassie stain (bottom). Input, 20% of binding reaction. CA-NC was included in the binding reactions as indicated. (C) Recombinant, tag-free NUP153C and GST purified from E. coli were similarly tested for binding to CA-NC; proteins were detected with Coomassie stain. (D) Recombinant NUP153C pulled down with full length his-tagged wild-type (WT) or W184A/M185A HIV-1 CA, and detected with Coomassie stain. (E) Recombinant NUP153C pulled down with his-tagged CAN, and detected with Coomassie stain. Each experiment was repeated at least 3 times, with a single representative result shown.
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getmorefigures.php?uid=PMC3795039&req=5

ppat-1003693-g001: NUP153C directly binds the HIV-1 CA N-terminal domain.(A) Schematic of NUP153 protein, with residue numbers of domain boundaries indicated. (B) Full length or truncated fragments of HA-tagged NUP153 extracted from 293T cells were tested for binding to HIV-1 CA-NC. Pelleted proteins were resolved by SDS-PAGE and visualized by western blotting with anti-HA antibody 3F10 (top), or by Coomassie stain (bottom). Input, 20% of binding reaction. CA-NC was included in the binding reactions as indicated. (C) Recombinant, tag-free NUP153C and GST purified from E. coli were similarly tested for binding to CA-NC; proteins were detected with Coomassie stain. (D) Recombinant NUP153C pulled down with full length his-tagged wild-type (WT) or W184A/M185A HIV-1 CA, and detected with Coomassie stain. (E) Recombinant NUP153C pulled down with his-tagged CAN, and detected with Coomassie stain. Each experiment was repeated at least 3 times, with a single representative result shown.
Mentions: As we previously found CA to be the dominant viral determinant of the requirement for NUP153 during HIV-1 infection [19], we tested whether a physical interaction between NUP153 and HIV-1 CA exists. Our initial assay utilized a recombinant viral fusion protein consisting of HIV-1 CA and nucleocapsid (NC) proteins, which when assembled in vitro in the presence of high salt and single stranded nucleic acid forms large tube-like structures that readily pellet through cushions of sucrose [29]. In this way, CA-interacting proteins can co-sediment with the tube structures [18], [44]. Full length or various fragments of HA-tagged NUP153 expression constructs were transfected into 293T cells, and the resulting proteins were tested for their ability to co-sediment with CA-NC assemblies. Full-length NUP153 (residues 1–1475) pelleted through the sucrose cushion in a CA-NC dependent manner (Figure 1A and 1B). The NUP153 N-terminal domain (residues 1–650) failed to bind CA-NC under conditions that supported efficient NUP153C (residues 896–1475) binding. The C-terminal NUP153 deletion mutant comprised of residues 1–1198 failed to bind, confirming the importance of the NUP153 FG-repeat domain in binding, and mapping the interaction to residues 1199–1475 of the full length protein.

Bottom Line: NUP153(C) fused to the effector domains of the rhesus Trim5α restriction factor (Trim-NUP153(C)) potently restricted HIV-1, providing an intracellular readout for the NUP153(C)-CA interaction during retroviral infection.Our results highlight similar mechanisms of binding for disparate host factors to the same region of HIV-1 CA during viral ingress.We conclude that a subset of lentiviral CA proteins directly engage FG-motifs present on NUP153 to affect viral nuclear import.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Department of Medicine, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Lentiviruses can infect non-dividing cells, and various cellular transport proteins provide crucial functions for lentiviral nuclear entry and integration. We previously showed that the viral capsid (CA) protein mediated the dependency on cellular nucleoporin (NUP) 153 during HIV-1 infection, and now demonstrate a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG)-repeat enriched NUP153 C-terminal domain (NUP153(C)). NUP153(C) fused to the effector domains of the rhesus Trim5α restriction factor (Trim-NUP153(C)) potently restricted HIV-1, providing an intracellular readout for the NUP153(C)-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV) bound NUP153(C) under these conditions, results that correlated with direct binding between purified proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 protein during virus infection. Mutagenesis experiments concordantly identified NUP153(C) and CA residues important for binding and lentiviral infectivity. Different FG motifs within NUP153(C) mediated binding to HIV-1 versus EIAV capsids. HIV-1 CA binding mapped to residues that line the common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74) inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6) protein, with Asn57 (Asp58 in EIAV) playing a particularly important role. PF74 and CPSF6 accordingly each competed with NUP153(C) for binding to the HIV-1 CA pocket, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153(C) expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicates that the NUP153(C)-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. Our results highlight similar mechanisms of binding for disparate host factors to the same region of HIV-1 CA during viral ingress. We conclude that a subset of lentiviral CA proteins directly engage FG-motifs present on NUP153 to affect viral nuclear import.

Show MeSH
Related in: MedlinePlus