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Yes-associated Protein (YAP) and its paralog Transcriptional Coactivator with PDZ-binding Motif (TAZ) are major regulators of gene transcription/expression, primarily controlled by the Hippo pathway and the cytoskeleton. Integrating an array of chemical and mechanical signals, they impact growth, differentiation, and regeneration. Accordingly, they also play key roles in tumorigenesis and metastasis formation. Their activity is primarily regulated by their localization, that is, Hippo pathway- and/or cytoskeleton-controlled cytosolic or nuclear sequestration. While many details of such prevailing retention models have been elucidated, much less is known about their actual nuclear traffic: import and export. Although their size is not far from the cutoff for passive diffusion through the nuclear pore complex (NPC), and they do not contain any classic nuclear localization (NLS) or nuclear export signal (NES), evidence has been accumulating that their shuttling involves mediated and thus regulatable/targetable processes. The aim of this review is to summarize emerging information/concepts about their nucleocytoplasmic shuttling, encompassing the relevant structural requirements (NLS, NES), nuclear transport receptors (NTRs, karyophererins), and NPC components, along with the potential transport mechanisms and their regulation. While dissecting retention vs. transport is often challenging, the emerging picture suggests that YAP/TAZ shuttles across the NPC via multiple, non-exclusive, mediated mechanisms, constituting a novel and intriguing facet of YAP/TAZ biology.

Signal-dependent transport into and out of the nucleus mediated by members of the importin (IMP) superfamily of nuclear transporters is critical to the eukaryotic function and a point of therapeutic intervention with the potential to limit disease progression and pathogenic outcomes. Although the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii both retain unique IMPα genes that are essential, a detailed analysis of their properties has not been performed. As a first step to validate apicomplexan IMPα as a target, we set out to compare the properties of P. falciparum and T. gondii IMPα (PfIMPα and TgIMPα, respectively) to those of mammalian IMPα, as exemplified by Mus musculus IMPα (MmIMPα). Close similarities were evident, with all three showing high-affinity binding to modular nuclear localisation signals (NLSs) from apicomplexans as well as Simian virus SV40 large tumour antigen (T-ag). PfIMPα and TgIMPα were also capable of binding to mammalian IMPβ1 (MmIMPβ1) with high affinity; strikingly, NLS binding by PfIMPα and TgIMPα could be inhibited by the mammalian IMPα targeting small molecules ivermectin and GW5074 through direct binding to PfIMPα and TgIMPα to perturb the α-helical structure. Importantly, GW5074 could be shown for the first time to resemble ivermectin in being able to limit growth of P. falciparum. The results confirm apicomplexan IMPα as a viable target for the development of therapeutics, with agents targeting it worthy of further consideration as an antimalarial.

The transport of host proteins into and out of the nucleus is key to host function. However, nuclear transport is restricted by nuclear pores that perforate the nuclear envelope. Protein intrinsic disorder is an inherent feature of this selective transport barrier and is also a feature of the nuclear transport receptors that facilitate the active nuclear transport of cargo, and the nuclear transport signals on the cargo itself. Furthermore, intrinsic disorder is an inherent feature of viral proteins and viral strategies to disrupt host nucleocytoplasmic transport to benefit their replication. In this review, we highlight the role that intrinsic disorder plays in the nuclear transport of host and viral proteins. We also describe viral subversion mechanisms of the host nuclear transport machinery in which intrinsic disorder is a feature. Finally, we discuss nuclear import and export as therapeutic targets for viral infectious disease.

We recently reported that HIV-1 cores that retained >94% of their capsid (CA) protein entered the nucleus and disassembled (uncoated) near their integration site <1.5 h before integration. However, whether the nuclear capsids lost their integrity by rupturing or a small loss of CA before capsid disassembly was unclear. Here, we utilized a previously reported vector in which green fluorescent protein is inserted in HIV-1 Gag (iGFP); proteolytic processing efficiently releases GFP, some of which remains trapped inside capsids and serves as a fluid phase content marker that is released when the capsids lose their integrity. We found that nuclear capsids retained their integrity until shortly before integration and lost their GFP content marker ∼1 to 3 min before loss of capsid-associated mRuby-tagged cleavage and polyadenylation specificity factor 6 (mRuby-CPSF6). In contrast, loss of GFP fused to CA and mRuby-CPSF6 occurred simultaneously, indicating that viral cores retain their integrity until just minutes before uncoating. Our results indicate that HIV-1 evolved to retain its capsid integrity and maintain a separation between macromolecules in the viral core and the nuclear environment until uncoating occurs just before integration. These observations imply that intact HIV-1 capsids are imported through nuclear pores; that reverse transcription occurs in an intact capsid; and that interactions between the preintegration complex and LEDGF/p75, and possibly other host factors that facilitate integration, must occur during the short time period between loss of capsid integrity and integration.

The cell cycle-dependent nucleocytoplasmic transport of proteins is predominantly regulated by CDK kinase activities; however, it is currently difficult to predict the proteins thus regulated, largely because of the low prediction efficiency of the motifs involved. Here, we report the successful prediction of CDK1-regulated nucleocytoplasmic shuttling proteins using a prediction system for nuclear localization signals (NLSs). By systematic amino acid replacement analyses in budding yeast, we created activity-based profiles for different classes of importin-α-dependent NLSs that represent the functional contributions of different amino acids at each position within an NLS class. We then developed a computer program for prediction of the classical importin-α/β pathway-specific NLSs (cNLS Mapper, available at http//nls-mapper.iab.keio.ac.jp/) that calculates NLS activities by using these profiles and an additivity-based motif scoring algorithm. This calculation method achieved significantly higher prediction accuracy in terms of both sensitivity and specificity than did current methods. The search for NLSs that overlap the consensus CDK1 phosphorylation site by using cNLS Mapper identified all previously reported and 5 previously uncharacterized yeast proteins (Yen1, Psy4, Pds1, Msa1, and Dna2) displaying CDK1- and cell cycle-regulated nuclear transport. CDK1 activated or repressed their nuclear import activity, depending on the position of CDK1-phosphorylation sites within NLSs. The application of this strategy to other functional linear motifs should be useful in systematic studies of protein-protein networks.

HIV-1 accesses the nuclear DNA of interphase cells via a poorly defined process involving functional interactions between the capsid protein (CA) and nucleoporins (Nups). Here, we show that HIV-1 CA can bind multiple Nups, and that both natural and manipulated variation in Nup levels impacts HIV-1 infection in a manner that is strikingly dependent on cell-type, cell-cycle, and cyclophilin A (CypA). We also show that Nups mediate the function of the antiviral protein MX2, and that MX2 can variably inhibit non-viral NLS function. Remarkably, both enhancing and inhibiting effects of cyclophilin A and MX2 on various HIV-1 CA mutants could be induced or abolished by manipulating levels of the Nup93 subcomplex, the Nup62 subcomplex, NUP88, NUP214, RANBP2, or NUP153. Our findings suggest that several Nup-dependent ‘pathways’ are variably exploited by HIV-1 to target host DNA in a cell-type, cell-cycle, CypA and CA-sequence dependent manner, and are differentially inhibited by MX2.

The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)–rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.

Nuclear entry of HIV-1 replication complexes through intact nuclear pore complexes is critical for successful infection. The host protein cleavage-and-polyadenylation-specificity-factor-6 (CPSF6) has been implicated in different stages of early HIV-1 replication. Applying quantitative microscopy of HIV-1 reverse-transcription and pre-integration-complexes (RTC/PIC), we show that CPSF6 is strongly recruited to nuclear replication complexes but absent from cytoplasmic RTC/PIC in primary human macrophages. Depletion of CPSF6 or lack of CPSF6 binding led to accumulation of HIV-1 subviral complexes at the nuclear envelope of macrophages and reduced infectivity. Two-color stimulated-emission-depletion microscopy indicated that under these circumstances HIV-1 complexes are retained inside the nuclear pore and undergo CA-multimer dependent CPSF6 clustering adjacent to the nuclear basket. We propose that nuclear entry of HIV-1 subviral complexes in macrophages is mediated by consecutive binding of Nup153 and CPSF6 to the hexameric CA lattice. Viruses are miniscule parasites that hijack the resources of a cell to make more of themselves. For many, this involves getting inside the nucleus, the fortress that protects the cell’s genetic information. To do so, viruses need to first find a way through a double-layered membrane called the nuclear envelope, which only opens up when a cell divides. Yet, the human immunodeficiency virus type 1 (HIV-1) can infect cells that no longer divide, and in which the nucleus’ walls never come down. The virus cores then head for the nuclear pores, heavily guarded holes in the nuclear envelope that allow the cell's own molecules to go in and out of the nucleus. But HIV-1 is too big to fit through, as its genetic information is encased in a capsid, a coat made of a complex assembly of proteins. However, research shows that these capsid proteins can bind to host proteins at the pore or even inside the nucleus. For example, the capsid protein can recognize the pore protein Nup153, or the nuclear protein CPSF6. These interactions could help the virus make its way in, but how these events unfold is still unclear. To explore this, Bejarano, Peng et al. attached fluorescent labels to HIV-1 and watched as it infected non-dividing cells. Rather than completely get rid of their capsid before they crossed the pores, the virus particles hung on to a large part of their lattice. This remaining coat then attached to CPSF6; when this protein was missing or could not bind to capsid proteins, the viral complexes got stuck in the nuclear pores. This suggests that the capsid lattice could first interact with Nup153 inside the pores: then, CPSF6 would take over, knocking Nup153 away and pulling HIV-1 into the nucleus. Armed with this knowledge, virologists and drug developers could try to block HIV-1 from entering the cell’s nucleus; they could also start to dissect how drugs that target the HIV-1 capsid work. Ultimately, HIV-1 may serve as a model to unravel how large objects can pass the nuclear pore, which may help us understand how molecules are constantly trafficked in and out of the nucleus.

IMPORTIN-4, the primary nuclear import receptor of core histones H3 and H4, binds the H3–H4 dimer and histone chaperone ASF1 prior to nuclear import. However, how H3–H3–ASF1 is recognized for transport cannot be explained by available crystal structures of IMPORTIN-4–histone tail peptide complexes. Our 3.5-Å IMPORTIN-4–H3–H4–ASF1 cryoelectron microscopy structure reveals the full nuclear import complex and shows a binding mode different from suggested by previous structures. The N-terminal half of IMPORTIN-4 clamps the globular H3–H4 domain and H3 αN helix, while its C-terminal half binds the H3 N-terminal tail weakly; tail contribution to binding energy is negligible. ASF1 binds H3–H4 without contacting IMPORTIN-4. Together, ASF1 and IMPORTIN-4 shield nucleosomal H3–H4 surfaces to chaperone and import it into the nucleus where RanGTP binds IMPORTIN-4, causing large conformational changes to release H3–H4–ASF1. This work explains how full-length H3–H4 binds IMPORTIN-4 in the cytoplasm and how it is released in the nucleus.

Nuclear effector proteins released by bacteria, oomycete, nematode, and fungi burden the global environment and crop yield. Microbial effectors are key weapons in the evolutionary arms race between plants and pathogens, vital in determining the success of pathogenic colonization. Secreted effectors undermine a multitude of host cellular processes depending on their target destination. Effectors are classified by their localization as either extracellular (apoplastic) or intracellular. Intracellular effectors can be further subclassified by their compartment such as the nucleus, cytoplasm or chloroplast. Nuclear effectors bring into question the role of the plant nucleus' intrinsic defence strategies and their vulnerability to effector‐based manipulation. Nuclear effectors interfere with multiple nuclear processes including the epigenetic regulation of the host chromatin, the impairment of the trans‐kingdom antifungal RNAi machinery, and diverse classes of immunity‐associated host proteins. These effector‐targeted pathways are widely conserved among different hosts and regulate a broad array of plant cellular processes. Thus, these nuclear sites constitute meaningful targets for effectors to subvert the plant defence system and acquire resources for pathogenic propagation. This review provides an extensive and comparative compilation of diverse plant microbe nuclear effector libraries, thereby highlighting the distinct and conserved mechanisms these effectors employ to modulate plant cellular processes for the pathogen's profit. This review provides an extensive comparison of plant microbe nuclear effectors, highlighting the mechanisms effectors employ to modulate plant cellular processes for the pathogen's benefit.