Viral interference of nucleocytoplasmic transport

Figures

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  Figure 1. Overview of viral strategies subverting nucleocytoplasmic transport.

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  Figure 2. Architecture of the human nuclear pore complex.A, the near-atomic composite structure of the intact human NPC, determined by quantitatively docking high-resolution nucleoporin and nucleoporin complex structures, solved by X-ray crystallography and single-particle cryo-EM, alongside a small number of homology and artificial intelligence (AI)–generated models, into an ∼12 Å cryo-ET map of the human NPC (41, 42). The nuclear envelope is visualized as a gray isosurface, with the individual nucleoporins and subcomplexes colored according to the legend. The cytoplasmic filaments (red) are modeled projecting into the cytoplasm based on available X-ray crystallography structures spanning domains flexibly attached within NUP358 homopentameric bundles in complex with their RAN, SUMO•RANGAP1, and UBC9 binding partners (41). The nuclear basket is displayed as an electron density map (pink) (100). B, nucleoporin nomenclature for the Saccharomyces cerevisiae (sc) and Homo sapiens (hs) NPCs, categorized by subcomplex. Predicted nucleoporin copy number per NPC is indicated in parentheses. Asterisks (∗) denote that NUP62 and its fungal homologs are also found in the cytoplasmic filaments. Hash…

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  Figure 3. The karyopherin mediated nuclear transport system.A, overview of karyopherin transport factors identified in Homo sapiens and Saccharomyces cerevisiae, alongside prominent viral interaction partners. Functionally grouped by α-karyopherin adaptors (green), nuclear import β-karyopherins (yellow), bidirectional β-karyopherins (light blue), and nuclear export β-karyopherins (dark blue). Alternative nomenclature indicated in parentheses. B, RAN GTPase cycle. Nuclear RAN(GTP) is incorporated into β-karyopherin complexes to trigger cargo release from importins or to promote export complex formation. Upon exiting the nucleus, the NUP358-associated GTPase-activating protein RANGAP1 stimulates RAN’s intrinsic GTP hydrolysis activity, triggering dissociation of RAN(GDP) from β-karyopherins and the release of export cargoes. Cytoplasmic NTF2 homodimers recognize free RAN(GDP) forming a heterotetrametric nuclear import complex. Upon entering the nucleus, the NPCs' nuclear basket constituent NUP153 presents four zinc finger domains capable of binding RAN(GDP). Restarting the cycle, nucleosome-bound RAN guanine exchange factor (GEF), RCC1, promotes GDP release from RAN and exchange for GTP.…

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  Figure 4. The FG–repeat diffusion barrier is a hot spot for viral proteases.A, the near-atomic composite structure of the intact human NPC (41, 42), the nuclear envelope is depicted as a gray isosurface, eightfold symmetric spokes are represented in transparent cartoon surface format (alternating cyan/purple). Nucleoporin locations on the NPC scaffold where FG–repeat regions project from are colored as indicated in the legend. B, crystal structure of inactive enterovirus-A71 2Apro in complex with a canonical substrate peptide derived from the VP1-2A cleavage junction (PDB ID: 4FVD) (498). C, domain architectures of NUP98, NUP62, and NUP153, human FG–nucleoporins targeted by viral 2Apro and 3Cpro, red lines indicate biochemically mapped 2Apro cleavage sites (264, 267). Domain abbreviations: APD, autoproteolytic domain; CC, coiled-coil domain; FG, FG–repeat region; G, GLEBS domain; ZnF, zinc finger domain; U, unstructured. D, crystal structure of rhinovirus-A2 3Cpro bound to the AG7088 inhibitor (PDB ID: 1CQQ) (499). CFNC, cytoplasmic filament nucleoporin complex; cryo-ET, cryo-electron tomography; FG, phenylalanine–glycine; INM, inner nuclear membrane; NE, nuclear envelope; NPC, nuclear…

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  Figure 5. Viral entry to the nucleus. To scale cutaway cartoon views of (A) the dilated NPC’s architecture and (B) viral capsids. (C–H) EM visualization of viral capsids (red) traversing the nuclear envelope (NE; green). C, thin section EM of digitonin-permeabilized HeLa cells, supplemented with purified partially uncoated adenovirus capsids, NPC densities corresponding to the cytoplasmic filaments are indicated (white arrows) (304). D, thin section EM of herpes simplex virus-1 (HSV-1) capsids associated with the cytoplasmic face of the NPC. HSV-1 infected Vero cells, DNA loaded capsid approaching NPC (top). In vitro–purified nuclei incubated with uncoated HSV-1 capsids (bottom), NPC densities corresponding to the cytoplasmic filaments are visualized contacting the capsid vertices (white arrows) (309). E, thin section EM of Xenopus laevis oocytes microinjected with purified hepatitis B virus (HBV) capsids demonstrates that the capsid can translocate the NPC intact (326, 500). F, slice through a cryo-ET reconstruction of SupT1-R5 cells infected with HIV-1 (top) (344). Template matching of an NPC’s inner ring derived from cryo-ET reconstructions of human monocyte–derived macrophages infected…

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  Figure 6. Viral disruption of karyopherin function.A, crystal structures of α-karyopherin cargo adapters in complex with viral proteins containing classical nuclear localization signals (NLSs) highlight their strategic use of the classical nuclear import pathway to either force entry into the nucleus or competitively inhibit the import of host cargoes, including key effectors of the innate immune response and other nuclear regulators: influenza A virus (IAV) PB2 in complex with KAPα4 (PDB ID: 4UAE) (371); Hendra virus W protein in complex with mmKapα2 (PDB ID: 6BW1) and KAPα4 (PDB ID: 6BW9) (406); Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4B in complex with KAPα4 (PDB ID: 7RFY) (410); Dengue virus-3 (DENV-3) NS5 in complex with mmKapα2 (PDB ID: 5FC8) (428). B, crystal structures of viral proteins exhibiting nonclassical interactions with α-karyopherin cargo adapters: IAV NP in complex with mmKapα2 (PDB ID: 4ZDU) (370); HIV-1 Vpr in complex with mmKapα2 (PDB ID: 5B56) (413); ebola virus VP24 in complex with a C-terminal fragment of KAPα5 (PDB ID: 4U2X) (415), N-terminal region sourced from the AlphaFold3 repository (gray) (502, 503). C, crystal structure of the nonclassical…

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  Figure 7. Viral interference of mRNA export.A, schematic representation of mRNP assembly and export. In the nucleus: mRNA is transcribed by RNA polymerase II, and a 7-methylguanylate cap is deposited at the 5′ end of the transcript recruiting the cap-binding complex (CBC). Nascent RNA is then compacted by heterogeneous nuclear ribonucleoprotein (hnRNP)-C heterotetramers, which are required for efficient splicing. Following splicing exon junction complexes (EJCs) are deposited downstream from splice sites and the TRanscription EXport (TREX) complex is loaded. The mRNP is then “licensed” for export to the cytoplasm by the DEAD-box helicase, UAP56, which binds TREX and facilitates loading of the p15•TAP export factors to produce an mRNP. The mRNP is subsequently exported through the NPC and remodeled at the cytoplasmic face, where the helicase activity of the DEAD-box helicase DDX19 removes p15•TAP preventing futile import of mRNA into the nucleus. B, crystal structures of viral virulence factors bound at the RNA binding pocket of the cytoplasmic filament nucleoporin complex RAE1•NUP98: vesicular stomatitis virus (VSV)-M protein in complex with RAE1•NUP98GLEBS (PDB ID: 4OWR) (444); severe…