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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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The importance of FG motifs for Trim-NUP153C mediated inhibition of HIV-1 and EIAV infection.To-scale schematics (A and D), normalized infection data (B and E), and western blotting (C and F) as described for Figure 4. Infection data are the geometric mean of at least 4 experiments, with error bars denoting 95% confidence intervals. Inverted grey triangle (panels A and D) denotes area of missense mutation.
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ppat-1003693-g005: The importance of FG motifs for Trim-NUP153C mediated inhibition of HIV-1 and EIAV infection.To-scale schematics (A and D), normalized infection data (B and E), and western blotting (C and F) as described for Figure 4. Infection data are the geometric mean of at least 4 experiments, with error bars denoting 95% confidence intervals. Inverted grey triangle (panels A and D) denotes area of missense mutation.

Mentions: We next focused on the initial quarter of NUP153C for its importance in mediating restriction of EIAV infection. Stable cell lines expressing only the first quarter of NUP153C fused to the Trim RBCC, as well as smaller derivatives of the NUP153C sequence, were generated (Figure 5A). Residues 896–949, which yielded the smallest construct capable of restricting EIAV infection (Figure 5B), harbored only two of the 29 FG motifs present within NUP153C. The importance of these FG motifs in mediating EIAV restriction was tested by substituting four consecutive alanine residues for each corresponding FKFG sequence. The combination octa-alanine 903A/924A Trim-NUP153C mutant construct lost its ability to inhibit EIAV infection despite being expressed at a level equal to or greater than unmodified Trim-NUP153C (Figure 5B and 5C). The 903A/924A mutant moreover retained potent HIV-1 restriction. Separate mutation of each motif revealed 924-FKFG-927 as the dominant FG sequence for mediating EIAV restriction.


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)

The importance of FG motifs for Trim-NUP153C mediated inhibition of HIV-1 and EIAV infection.To-scale schematics (A and D), normalized infection data (B and E), and western blotting (C and F) as described for Figure 4. Infection data are the geometric mean of at least 4 experiments, with error bars denoting 95% confidence intervals. Inverted grey triangle (panels A and D) denotes area of missense mutation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3795039&req=5

ppat-1003693-g005: The importance of FG motifs for Trim-NUP153C mediated inhibition of HIV-1 and EIAV infection.To-scale schematics (A and D), normalized infection data (B and E), and western blotting (C and F) as described for Figure 4. Infection data are the geometric mean of at least 4 experiments, with error bars denoting 95% confidence intervals. Inverted grey triangle (panels A and D) denotes area of missense mutation.
Mentions: We next focused on the initial quarter of NUP153C for its importance in mediating restriction of EIAV infection. Stable cell lines expressing only the first quarter of NUP153C fused to the Trim RBCC, as well as smaller derivatives of the NUP153C sequence, were generated (Figure 5A). Residues 896–949, which yielded the smallest construct capable of restricting EIAV infection (Figure 5B), harbored only two of the 29 FG motifs present within NUP153C. The importance of these FG motifs in mediating EIAV restriction was tested by substituting four consecutive alanine residues for each corresponding FKFG sequence. The combination octa-alanine 903A/924A Trim-NUP153C mutant construct lost its ability to inhibit EIAV infection despite being expressed at a level equal to or greater than unmodified Trim-NUP153C (Figure 5B and 5C). The 903A/924A mutant moreover retained potent HIV-1 restriction. Separate mutation of each motif revealed 924-FKFG-927 as the dominant FG sequence for mediating EIAV restriction.

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