SARS-CoV-2 Orf6 is positioned in the nuclear pore complex by Rae1 to inhibit nucleocytoplasmic transport.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) accessory protein Orf6 works as an interferon antagonist, in part, by inhibiting the nuclear import activated p-STAT1, an activator of interferon-stimulated genes, and the export of the poly(A) RNA. Insight into the transport regulatory function of Orf6 has come from the observation that Orf6 binds to the nuclear pore complex (NPC) components: Rae1 and Nup98. To gain further insight into the mechanism of Orf6-mediated transport inhibition, we examined the role of Rae1 and Nup98. We show that Rae1 alone is not necessary to support p-STAT1 import or nuclear export of poly(A) RNA. Moreover, the loss of Rae1 suppresses the transport inhibitory activity of Orf6. We propose that the Rae1/Nup98 complex strategically positions Orf6 within the NPC where it alters FG-Nup interactions and their ability to support nuclear transport. In addition, we show that Rae1 is required for normal viral protein production during SARS-CoV-2 infection presumably through its role in supporting Orf6 function.

SUMOylation at the inner nuclear membrane facilitates nuclear envelope biogenesis during mitosis.

As eukaryotic cells progress through cell division, the nuclear envelope (NE) membrane must expand to accommodate the formation of progeny nuclei. In Saccharomyces cerevisiae, closed mitosis allows visualization of NE biogenesis during mitosis. During this period, the SUMO E3 ligase Siz2 binds the inner nuclear membrane (INM) and initiates a wave of INM protein SUMOylation. Here, we show these events increase INM levels of phosphatidic acid (PA), an intermediate of phospholipid biogenesis, and are necessary for normal mitotic NE membrane expansion. The increase in INM PA is driven by the Siz2-mediated inhibition of the PA phosphatase Pah1. During mitosis, this results from the binding of Siz2 to the INM and dissociation of Spo7 and Nem1, a complex required for the activation of Pah1. As cells enter interphase, the process is then reversed by the deSUMOylase Ulp1. This work further establishes a central role for temporally controlled INM SUMOylation in coordinating processes, including membrane expansion, that regulate NE biogenesis during mitosis.

Nuclear pore complexes mediate subtelomeric gene silencing by regulating PCNA levels on chromatin.

The nuclear pore complex (NPC) physically interacts with chromatin and regulates gene expression. The Saccharomyces cerevisiae inner ring nucleoporin Nup170 has been implicated in chromatin organization and the maintenance of gene silencing in subtelomeric regions. To gain insight into how Nup170 regulates this process, we used protein-protein interactions, genetic interactions, and transcriptome correlation analyses to identify the Ctf18-RFC complex, an alternative proliferating cell nuclear antigen (PCNA) loader, as a facilitator of the gene regulatory functions of Nup170. The Ctf18-RFC complex is recruited to a subpopulation of NPCs that lack the nuclear basket proteins Mlp1 and Mlp2. In the absence of Nup170, PCNA levels on DNA are reduced, resulting in the loss of silencing of subtelomeric genes. Increasing PCNA levels on DNA by removing Elg1, which is required for PCNA unloading, rescues subtelomeric silencing defects in nup170Δ. The NPC, therefore, mediates subtelomeric gene silencing by regulating PCNA levels on DNA.

Phosphorylation-dependent mitotic SUMOylation drives nuclear envelope-chromatin interactions.

In eukaryotes, chromatin binding to the inner nuclear membrane (INM) and nuclear pore complexes (NPCs) contributes to spatial organization of the genome and epigenetic programs important for gene expression. In mitosis, chromatin-nuclear envelope (NE) interactions are lost and then formed again as sister chromosomes segregate to postmitotic nuclei. Investigating these processes in S. cerevisiae, we identified temporally and spatially controlled phosphorylation-dependent SUMOylation events that positively regulate postmetaphase chromatin association with the NE. Our work establishes a phosphorylation-mediated targeting mechanism of the SUMO ligase Siz2 to the INM during mitosis, where Siz2 binds to and SUMOylates the VAP protein Scs2. The recruitment of Siz2 through Scs2 is further responsible for a wave of SUMOylation along the INM that supports the assembly and anchorage of subtelomeric chromatin at the INM and localization of an active gene (INO1) to NPCs during the later stages of mitosis and into G1-phase.

Nodosome Inhibition as a Novel Broad-Spectrum Antiviral Strategy against Arboviruses, Enteroviruses, and SARS-CoV-2.

In the present report, we describe two small molecules with broad-spectrum antiviral activity. These drugs block the formation of the nodosome. The studies were prompted by the observation that infection of human fetal brain cells with Zika virus (ZIKV) induces the expression of nucleotide-binding oligomerization domain-containing protein 2 (NOD2), a host factor that was found to promote ZIKV replication and spread. A drug that targets NOD2 was shown to have potent broad-spectrum antiviral activity against other flaviviruses, alphaviruses, enteroviruses, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). Another drug that inhibits receptor-interacting serine/threonine protein kinase 2 (RIPK2), which functions downstream of NOD2, also decreased the replication of these pathogenic RNA viruses. The antiviral effect of this drug was particularly potent against enteroviruses. The broad-spectrum action of nodosome-targeting drugs is mediated in part by the enhancement of the interferon response. Together, these results suggest that further preclinical investigation of nodosome inhibitors as potential broad-spectrum antivirals is warranted.

Nsp1 protein of SARS-CoV-2 disrupts the mRNA export machinery to inhibit host gene expression.

The ongoing unprecedented severe acute respiratory syndrome caused by the SARS-CoV-2 outbreak worldwide has highlighted the need for understanding viral-host interactions involved in mechanisms of virulence. Here, we show that the virulence factor Nsp1 protein of SARS-CoV-2 interacts with the host messenger RNA (mRNA) export receptor heterodimer NXF1-NXT1, which is responsible for nuclear export of cellular mRNAs. Nsp1 prevents proper binding of NXF1 to mRNA export adaptors and NXF1 docking at the nuclear pore complex. As a result, a significant number of cellular mRNAs are retained in the nucleus during infection. Increased levels of NXF1 rescues the Nsp1-mediated mRNA export block and inhibits SARS-CoV-2 infection. Thus, antagonizing the Nsp1 inhibitory function on mRNA export may represent a strategy to restoring proper antiviral host gene expression in infected cells.

SARS-CoV-2 Orf6 hijacks Nup98 to block STAT nuclear import and antagonize interferon signaling.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.

Recruitment of an Activated Gene to the Yeast Nuclear Pore Complex Requires Sumoylation.

In addition to their role in regulating transport across the nuclear envelope, increasing evidence suggests nuclear pore complexes (NPCs) function in regulating gene expression. For example, the induction of certain genes (e.g., yeast INO1) is accompanied by their movement from the nuclear interior to NPCs. As sumoylation has been linked to the regulation of chromatin spatial organization and transcriptional activity, we investigated the role of sumoylation in the expression and NPC recruitment of the INO1 gene. We observed that induction of INO1 is accompanied by both increased and decreased sumoylation of proteins associated with specific regions along the INO1 locus. Furthermore, we show that the E3 ligase Siz2/Nfi1 is required for targeting the INO1 locus to the NPC where it interacts with the SUMO isopeptidase Ulp1. Our data suggest that this interaction is required for both the association of INO1 with the NPC and for its normal expression. These results imply that sumoylation is a key regulator of INO1 targeting to the NPC, and a cycle of sumoylation and NPC-associated desumoylation events contribute to the regulation of INO1 expression.

The Canadian Rare Diseases Models and Mechanisms (RDMM) Network: Connecting Understudied Genes to Model Organisms.

Advances in genomics have transformed our ability to identify the genetic causes of rare diseases (RDs), yet we have a limited understanding of the mechanistic roles of most genes in health and disease. When a novel RD gene is first discovered, there is minimal insight into its biological function, the pathogenic mechanisms of disease-causing variants, and how therapy might be approached. To address this gap, the Canadian Rare Diseases Models and Mechanisms (RDMM) Network was established to connect clinicians discovering new disease genes with Canadian scientists able to study equivalent genes and pathways in model organisms (MOs). The Network is built around a registry of more than 500 Canadian MO scientists, representing expertise for over 7,500 human genes. RDMM uses a committee process to identify and evaluate clinician-MO scientist collaborations and approve 25,000 Canadian dollars in catalyst funding. To date, we have made 85 clinician-MO scientist connections and funded 105 projects. These collaborations help confirm variant pathogenicity and unravel the molecular mechanisms of RD, and also test novel therapies and lead to long-term collaborations. To expand the impact and reach of this model, we made the RDMM Registry open-source, portable, and customizable, and we freely share our committee structures and processes. We are currently working with emerging networks in Europe, Australia, and Japan to link international RDMM networks and registries and enable matches across borders. We will continue to create meaningful collaborations, generate knowledge, and advance RD research locally and globally for the benefit of patients and families living with RD.

Passive diffusion through nuclear pore complexes regulates levels of the yeast SAGA and SLIK coactivator complexes.

Nuclear pore complexes (NPCs) control gene expression by regulating the bi-directional exchange of proteins and RNAs between nuclear and cytoplasmic compartments, including access of transcriptional regulators to the nucleoplasm. Here, we show that the yeast (Saccharomyces cerevisiae) nucleoporin Nup170, in addition to binding and silencing subtelomeric genes, supports transcription of genes regulated by the SAGA transcriptional activator complex. Specifically, we show that a lower amount of SAGA complex is bound to target genes in the absence of Nup170. Consistent with this observation, levels of the SAGA complex are decreased in cells lacking Nup170, while those of the SAGA-related SLIK complexes are increased. This change in the ratio of SAGA to SLIK complexes is due to increased nuclear activity of Pep4, a protease responsible for production of the SLIK complex. Further analyses of various nucleoporin mutants revealed that the increased nuclear entry of Pep4 observed in the nup170Δ mutant likely occurs as the consequence of an increase in the sieving limits of the NPC diffusion channel. On the basis of these results, we propose that changes in passive diffusion rates represent a mechanism for regulating SAGA- and SLIK complex-mediated transcriptional events.

Mutant huntingtin interacts with the sterol regulatory element-binding proteins and impairs their nuclear import.

Brain cholesterol homeostasis is altered in Huntington's disease (HD), a neurodegenerative disorder caused by the expansion of a CAG nucleotide repeat in the HTT gene. Genes involved in the synthesis of cholesterol and fatty acids were shown to be downregulated shortly after the expression of mutant huntingtin (mHTT) in inducible HD cells. Nuclear levels of the transcription factors that regulate lipid biogenesis, the sterol regulatory element-binding proteins (SREBP1 and SREBP2), were found to be decreased in HD models compared to wild-type, but the underlying causes were not known. SREBPs are synthesized as inactive endoplasmic reticulum-localized precursors. Their mature forms (mSREBPs) are generated upon transport of the SREBP precursors to the Golgi and proteolytic cleavage, and are rapidly imported into the nucleus by binding to importin ß. We show that, although SREBP2 processing into mSREBP2 is not affected in YAC128 HD mice, mSREBP2 is mislocalized to the cytoplasm. Chimeric mSREBP2-and mSREBP1-EGFP proteins are also mislocalized to the cytoplasm in immortalized striatal cells expressing mHTT, in YAC128 neurons and in fibroblasts from HD patients. We further show that mHTT binds to the SREBP2/importin ß complex required for nuclear import and sequesters it in the cytoplasm. As a result, HD cells fail to upregulate cholesterogenic genes under sterol-depleted conditions. These findings provide mechanistic insight into the downregulation of genes involved in the synthesis of cholesterol and fatty acids in HD models, and have potential implications for other pathways modulated by SREBPs, including autophagy and excitotoxicity.

Nucleoplasmic Nup98 controls gene expression by regulating a DExH/D-box protein.

The nucleoporin Nup98 has been linked to the regulation of transcription and RNA metabolism, 1-3 but the mechanisms by which Nup98 contributes to these processes remains largely undefined. Recently, we uncovered interactions between Nup98 and several DExH/D-box proteins (DBPs), a protein family well-known for modulating gene expression and RNA metabolism. 4-6 Analysis of Nup98 and one of these DBPs, DHX9, showed that they directly interact, their association is facilitated by RNA, and Nup98 binding stimulates DHX9 ATPase activity. 7 Furthermore, these proteins were dependent on one another for their proper association with a subset of gene loci to control transcription and modulate mRNA splicing. 7 On the basis of these observations, we proposed that Nup98 functions to regulate DHX9 activity within the nucleoplasm. 7 Since Nup98 is associated with several DBPs, regulation of DHX9 by Nup98 may represent a paradigm for understanding how Nup98, and possibly other FG-Nup proteins, could direct the diverse cellular activities of multiple DBPs.

Yeast silencing factor Sir4 and a subset of nucleoporins form a complex distinct from nuclear pore complexes.

Interactions occurring at the nuclear envelope (NE)-chromatin interface influence both NE structure and chromatin organization. Insights into the functions of NE-chromatin interactions have come from the study of yeast subtelomeric chromatin and its association with the NE, including the identification of various proteins necessary for tethering subtelomeric chromatin to the NE and the silencing of resident genes. Here we show that four of these proteins-the silencing factor Sir4, NE-associated Esc1, the SUMO E3 ligase Siz2, and the nuclear pore complex (NPC) protein Nup170-physically and functionally interact with one another and a subset of NPC components (nucleoporins or Nups). Importantly, this group of Nups is largely restricted to members of the inner and outer NPC rings, but it lacks numerous others including cytoplasmically and nucleoplasmically positioned Nups. We propose that this Sir4-associated Nup complex is distinct from holo-NPCs and that it plays a role in subtelomeric chromatin organization and NE tethering.

Human Nup98 regulates the localization and activity of DExH/D-box helicase DHX9.

Beyond their role at nuclear pore complexes, some nucleoporins function in the nucleoplasm. One such nucleoporin, Nup98, binds chromatin and regulates gene expression. To gain insight into how Nup98 contributes to this process, we focused on identifying novel binding partners and understanding the significance of these interactions. Here we report on the identification of the DExH/D-box helicase DHX9 as an intranuclear Nup98 binding partner. Various results, including in vitro assays, show that the FG/GLFG region of Nup98 binds to N- and C-terminal regions of DHX9 in an RNA facilitated manner. Importantly, binding of Nup98 stimulates the ATPase activity of DHX9, and a transcriptional reporter assay suggests Nup98 supports DHX9-stimulated transcription. Consistent with these observations, our analysis revealed that Nup98 and DHX9 bind interdependently to similar gene loci and their transcripts. Based on our results, we propose that Nup98 functions as a co-factor that regulates DHX9 and, potentially, other RNA helicases.

SUMO and Nucleocytoplasmic Transport.

The transport of proteins between the nucleus and cytoplasm occurs through nuclear pore complexes and is facilitated by numerous transport factors. These transport processes are often regulated by post-translational modification or, reciprocally, transport can function to control post-translational modifications through regulated transport of key modifying enzymes. This interplay extends to relationships between nucleocytoplasmic transport and SUMO-dependent pathways. Examples of protein sumoylation inhibiting or stimulating nucleocytoplasmic transport have been documented, both through its effects on the physical properties of cargo molecules and by directly regulating the functions of components of the nuclear transport machinery. Conversely, the nuclear transport machinery regulates the localization of target proteins and enzymes controlling dynamics of sumoylation and desumoylation thereby affecting the sumoylation state of target proteins. These inter-relationships between SUMO and the nucleocytoplasmic transport machinery, and the varied ways in which they occur, are discussed.

Nucleoporins and chromatin metabolism.

Mounting evidence has implicated a group of proteins termed nucleoporins, or Nups, in various processes that regulate chromatin structure and function. Nups were first recognized as building blocks for nuclear pore complexes, but several members of this group of proteins also reside in the cytoplasm and within the nucleus. Moreover, many are dynamic and move between these various locations. Both at the nuclear envelope, as part of nuclear pore complexes, and within the nucleoplasm, Nups interact with protein complexes that function in gene transcription, chromatin remodeling, DNA repair, and DNA replication. Here, we review recent studies that provide further insight into the molecular details of these interactions and their role in regulating the activity of chromatin modifying factors.

The Nuclear Transport Factor Kap121 Is Required for Stability of the Dam1 Complex and Mitotic Kinetochore Bi-orientation.

The karyopherin (Kap) family of nuclear transport factors facilitates macromolecular transport through nuclear pore complexes (NPCs). The binding of Kaps to their cargos can also regulate, both temporally and spatially, the interactions of the cargo protein with interacting partners. Here, we show that the essential yeast Kap, Kap121, binds Dam1 and Duo1, components of the microtubule (MT)-associated Dam1 complex required for linking dynamic MT ends with kinetochores (KTs). Like mutations in the Dam1 complex, loss of Kap121 function compromises the formation of normal KT-MT attachments during mitosis. We show that the stability of the Dam1 complex in vivo is dependent on its association with Kap121. Furthermore, we show that the Kap121/Duo1 complex is maintained in the presence of RanGTP but Kap121 is released by the cooperative actions of RanGTP and tubulin. We propose that Kap121 stabilizes the Dam1 complex and participates in escorting it to spindle MTs.

The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites.

Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication.

Functional characterization of nuclear localization and export signals in hepatitis C virus proteins and their role in the membranous web.

The hepatitis C virus (HCV) is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS) and nuclear export signals (NES) have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC) proteins (termed nucleoporins or Nups) are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin α5 (IPOA5/kapα1), importin ß3 (IPO5/kap ß3), and exportin 1 (XPO1/CRM1) both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication.

Assessing regulated nuclear transport in Saccharomyces cerevisiae.

Regulated protein transport between the cytoplasm and the nucleoplasm occurs through nuclear pore complexes and is critical to the function of numerous biological pathways. Saccharomyces cerevisiae has been used as a model system to probe the underlying mechanisms of nuclear transport and how they regulate various physiological processes. This has been facilitated, in part, by studies that couple the microscopic observation of a fluorescently tagged transport cargo's in vivo localization with numerous genetic and biochemical tools available to yeast researchers. Here, we describe some of these methods as they pertain to studies on regulated nuclear transport.