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.

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.

During the mating of yeast Saccharomyces cerevisiae, two haploid nuclei fuse to produce a diploid nucleus. This process requires the functions of BiP/Kar2p, a member of the Hsp70 family in the endoplasmic reticulum, and its partner protein, Jem1p. To investigate further the role of BiP and Jem1p in nuclear fusion, we screened for partner proteins for Jem1p by the yeast two-hybrid system and identified Nep98p. Nep98p is an essential integral membrane protein of the nuclear envelope and is enriched in the spindle pole body (SPB), the sole microtubule-organizing center in yeast. Temperature-sensitive nep98 mutant cells contain abnormal SPBs lacking the half-bridge, suggesting the essential role of Nep98p in the organization of the normal SPB. Additionally, nep98 mutant cells show defects in mitotic nuclear division and nuclear fusion during mating. Because Jem1p is not required for nuclear division, Nep98p probably has dual functions in Jem1p-dependent karyogamy and in Jem1p-independent nuclear division.In the sexual phase of yeast Saccharomyces cerevisiae, haploid cells of opposite mating types mate to produce diploid cells. After cell fusion, the two haploid nuclei fuse to form a diploid nucleus (1). This process, karyogamy, can be dissected into two steps: nuclear congression and nuclear fusion (1, 2). Analyses of yeast mutants defective in nuclear fusion have revealed the involvement of BiP/Kar2p, an Hsp70 molecular chaperone in the endoplasmic reticulum (ER) 1 (3). BiP also performs functions including protein import into the ER and ER-related protein degradation (4, 5). BiP has three functional partner proteins in the DnaJ family: Scj1p, Sec63p, and Jem1p (6 -8), and the functions of BiP are specified by different DnaJ-like proteins. Protein import into the ER lumen is mediated by BiP and Sec63p, and protein aggregation is prevented in the ER lumen by BiP with Scj1p and Jem1p (9). Nuclear fusion is facilitated by the Sec63p complex and Jem1p; the zygotes of kar2, sec63, and jem1 mutants contain two closely opposed haploid nuclei that do not fuse (2,8,10).As a step toward understanding the mechanism of nuclear membrane fusion by BiP with Jem1p, we have screened for proteins that interact with Jem1p using the yeast two-hybrid system. The identified protein, Nep98p, is an essential integral membrane protein of the nuclear envelope and is enriched in the spindle pole body (SPB), the sole microtubule-organizing center of budding yeast and a functional homologue of the centrosome in mammalian cells (11). The temperature-sensitive (ts) nep98 mutant cells have abnormal SPBs lacking the half-bridge and show defects in both mitotic nuclear division and karyogamy, suggesting that Nep98p is essential for SPB organization and function. ura3 leu2 trp1 his3 lys2 suc2) and SEY6211 (MATa ura3 leu2 trp1 his3 ade2 suc2) (12) were used as the parental strains for the mutants constructed in this study. SEY621D was constructed by mating SEY6210 with SEY6211. PJ69-4A (MATa ura3 leu2 trp1 his3 gal4 gal80 GAL2-ADE2 LYS2::GAL1-HIS3 me...

We have established that two homologous nucleoporins, Nup170p and Nup157p, play an essential role in the formation of nuclear pore complexes (NPCs) in Saccharomyces cerevisiae. By regulating their synthesis, we showed that the loss of these nucleoporins triggers a decrease in NPCs caused by a halt in new NPC assembly. Preexisting NPCs are ultimately lost by dilution as cells grow, causing the inhibition of nuclear transport and the loss of viability. Significantly, the loss of Nup170p/Nup157p had distinct effects on the assembly of different architectural components of the NPC. Nucleoporins (nups) positioned on the cytoplasmic face of the NPC rapidly accumulated in cytoplasmic foci. These nup complexes could be recruited into new NPCs after reinitiation of Nup170p synthesis, and may represent a physiological intermediate. Loss of Nup170p/Nup157p also caused core and nucleoplasmically positioned nups to accumulate in NPC-like structures adjacent to the inner nuclear membrane, which suggests that these nucleoporins are required for formation of the pore membrane and the incorporation of cytoplasmic nups into forming NPCs.

Mitochondria import most of their resident proteins from the cytosol, and the import receptor Tom20 of the outer-membrane translocator TOM40 complex plays an essential role in specificity of mitochondrial protein import. Here we analyzed the effects of Tom20 binding on NMR spectra of a long mitochondrial presequence and found that it contains two distinct Tom20-binding elements. In vitro import and cross-linking experiments revealed that, although the N-terminal Tom20-binding element is essential for targeting to mitochondria, the C-terminal element increases efficiency of protein import in the step prior to translocation across the inner membrane. Therefore Tom20 has a dual role in protein import into mitochondria: recognition of the targeting signal in the presequence and tethering the presequence to the TOM40 complex to increase import efficiency.protein's function relies on its correct subcellular location. Newly synthesized proteins are delivered to their sites of actions by cellular protein transport systems. Because subcellular compartments are bounded by biological membrane(s) in eukaryotic cells, the protein transport needs to start with insertion into or translocation across the target membrane. Therefore most protein transport systems involve a targeting signal on the cargo protein and a multiprotein complex on the target membrane called a translocator (1).Mitochondria are essential organelles in eukaryotic cells that consist of the outer and inner membranes and two aqueous compartments, the intermembrane space (IMS) and the matrix. Most mitochondrial proteins are encoded in the nuclear genome, synthesized in the cytosol, and subsequently imported into mitochondria. Mitochondrial protein import is mediated by translocators in the outer and inner membranes, including two TOM (translocase of the outer mitochondrial membrane) complexes, the TOM40 complex and the TOB (topogenesis of outer-membrane β-barrel proteins)/SAM (sorting and assembly machinery) complex, and two TIM (translocase of the inner mitochondrial membrane) complexes, the TIM23 complex and the TIM22 complex (2-5). The import pathway generally starts from the TOM40 complex and then branches out into several distinct intramitochondrial sorting pathways involving other translocators.The targeting information for mitochondria is contained in the N-terminal cleavable presequence or within the mature part of precursor proteins. The targeting information as well as intramitochondrial sorting information is recognized by several receptor subunits of the TOM and TIM complexes along the import pathways. Among them, Tom20, a peripheral subunit of the TOM40 complex, is a general import receptor that recognizes mitochondrial targeting signals contained in presequences. Tom20 is anchored to the outer membrane by the N-terminal hydrophobic segment and exposes a receptor domain to the cytosol. The NMR and X-ray structures of the receptor domain of rat Tom20 in a complex with a presequence peptide showed that the bound presequence forms an amphiphilic helix...