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Intracellular localization of the SARS coronavirus protein 9b: evidence of active export from the nucleus.

ABSTRACT: Open reading frame 9b (ORF 9b) encodes a 98 amino acid group-specific protein of severe acute respiratory syndrome (SARS) coronavirus (CoV). It has no homology with known proteins and its function in SARS CoV replication has not been determined. The N-terminal part of the 9b protein was used to raise polyclonal antibodies in rabbits, and these antibodies could detect 9b protein in infected cells. We analyzed the sub-cellular localization of recombinant 9b protein using fluorescence microscopy of live transfected cells and indirect immunofluorescence of transfected fixed cells. Our findings indicate that the 9b protein is exported outside of a cell nucleus and localizes to the endoplasmic reticulum. Our data also suggest that the 46-LRLGSQLSL-54 amino acid sequence of 9b functions as a nuclear export signal (NES).

SUBMITTER: Moshynskyy I

PROVIDER: S-EPMC7114319 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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Intracellular localization of the SARS coronavirus protein 9b: evidence of active export from the nucleus.

Moshynskyy Igor I Viswanathan Sathiyanarayanan S Vasilenko Natalia N Lobanov Vladislav V Petric Martin M Babiuk Lorne A LA Zakhartchouk Alexander N AN

Virus research 20070419 1

Open reading frame 9b (ORF 9b) encodes a 98 amino acid group-specific protein of severe acute respiratory syndrome (SARS) coronavirus (CoV). It has no homology with known proteins and its function in SARS CoV replication has not been determined. The N-terminal part of the 9b protein was used to raise polyclonal antibodies in rabbits, and these antibodies could detect 9b protein in infected cells. We analyzed the sub-cellular localization of recombinant 9b protein using fluorescence microscopy of ...[more]

PMID: 17448558

Similar Datasets

SARS-CoV 9b protein diffuses into nucleus, undergoes active Crm1 mediated nucleocytoplasmic export and triggers apoptosis when retained in the nucleus.

Project description:BACKGROUND: 9b is an accessory protein of the SARS-CoV. It is a small protein of 98 amino acids and its structure has been solved recently. 9b is known to localize in the extra-nuclear region and has been postulated to possess a nuclear export signal (NES), however the role of NES in 9b functioning is not well understood. PRINCIPAL FINDINGS/METHODOLOGY: In this report, we demonstrate that 9b in the absence of any nuclear localization signal (NLS) enters the nucleus by passive transport. Using various cell cycle inhibitors, we have shown that the nuclear entry of 9b is independent of the cell cycle. Further, we found that 9b interacts with the cellular protein Crm1 and gets exported out of the nucleus using an active NES. We have also revealed that this NES activity influences the half-life of 9b and affects host cell death. We found that an export signal deficient SARS-CoV 9b protein induces apoptosis in transiently transfected cells and showed elevated caspase-3 activity. CONCLUSION/SIGNIFICANCE: Here, we showed that nuclear shuttling of 9b and its interaction with Crm1 are essential for the proper degradation of 9b and blocking the nuclear export of this protein induces apoptosis. This phenomenon may be critical in providing a novel role to the 9b accessory protein of SARS-CoV.

| S-EPMC3103500 | biostudies-literature

The crystal structure of ORF-9b, a lipid binding protein from the SARS coronavirus.

Project description:To achieve the greatest output from their limited genomes, viruses frequently make use of alternative open reading frames, in which translation is initiated from a start codon within an existing gene and, being out of frame, gives rise to a distinct protein product. These alternative protein products are, as yet, poorly characterized structurally. Here we report the crystal structure of ORF-9b, an alternative open reading frame within the nucleocapsid (N) gene from the SARS coronavirus. The protein has a novel fold, a dimeric tent-like beta structure with an amphipathic surface, and a central hydrophobic cavity that binds lipid molecules. This cavity is likely to be involved in membrane attachment and, in mammalian cells, ORF-9b associates with intracellular vesicles, consistent with a role in the assembly of the virion. Analysis of ORF-9b and other overlapping genes suggests that they provide snapshots of the early evolution of novel protein folds.

| S-EPMC7126280 | biostudies-literature

Acquisition of new protein domains by coronaviruses: analysis of overlapping genes coding for proteins N and 9b in SARS coronavirus.

Project description:Acquisition of new proteins by viruses usually occurs through horizontal gene transfer or through gene duplication, but another, less common mechanism is the usage of completely or partially overlapping reading frames. A case of acquisition of a completely new protein through introduction of a start codon in an alternative reading frame is the protein encoded by open reading frame (orf) 9b of SARS coronavirus. This gene completely overlaps with the nucleocapsid (N) gene (orf9a). Our findings indicate that the orf9b gene features a discordant codon-usage pattern. We analyzed the evolution of orf9b in concert with orf9a using sequence data of betacoronavirus-lineage b and found that orf9b, which encodes the overprinting protein, evolved largely independent of the overprinted orf9a. We also examined the protein products of these genomic sequences for their structural flexibility and found that it is not necessary for a newly acquired, overlapping protein product to be intrinsically disordered, in contrast to earlier suggestions. Our findings contribute to characterizing sequence properties of newly acquired genes making use of overlapping reading frames.

| S-EPMC7089080 | biostudies-literature

SARS coronavirus series

Project description:Set of microarray experiments used to identify an unknown coronavirus in a viral culture derived from a patient with SARS. March 2003. Keywords = SARS Keywords = coronavirus Keywords = viral discovery Keywords = viruses Keywords = respiratory infection

2010-06-05 | E-GEOD-546 | biostudies-arrayexpress

Structure and intracellular targeting of the SARS-coronavirus Orf7a accessory protein.

Project description:The open reading frame (ORF) 7a of the SARS-associated coronavirus (SARS-CoV) encodes a unique type I transmembrane protein of unknown function. We have determined the 1.8 A resolution crystal structure of the N-terminal ectodomain of orf7a, revealing a compact seven-stranded beta sandwich unexpectedly similar in fold and topology to members of the Ig superfamily. We also demonstrate that, in SARS-CoV- infected cells, the orf7a protein is expressed and retained intracellularly. Confocal microscopy studies using orf7a and orf7a/CD4 chimeras implicate the short cytoplasmic tail and transmembrane domain in trafficking of the protein within the endoplasmic reticulum and Golgi network. Taken together, our findings provide a structural and cellular framework in which to explore the role of orf7a in SARS-CoV pathogenesis.

| S-EPMC7125549 | biostudies-literature

Nuclear/nucleolar localization properties of C-terminal nucleocapsid protein of SARS coronavirus.

Project description:A novel coronavirus (CoV) has recently been identified as the aetiological agent of severe acute respiratory syndrome (SARS). Nucleocapsid (N) proteins of the Coronaviridae family have no discernable homology, but they share a common nucleolar-cytoplasmic distribution pattern. There are three putative nuclear localization signal (NLS) motifs present in the N. To determine the role of these putative NLSs in the intracellular localization of the SARS-CoV N, we performed a confocal microscopy analysis using rabbit anti-N antisera. In this report, we show that the wild type N was distributed mainly in the cytoplasm. The N-terminal of the N, which contains the NLS1 (aa38-44), was localized to the nucleus. The C-terminus of the N, which contains both NLS2 (aa257-265) and NLS3 (aa369-390) was localized to the cytoplasm and the nucleolus. Results derived from analysis of various deletion mutations show that the region containing amino acids 226-289 is able to mediate nucleolar localization. The deletion of two hydrophobic regions that flanked the NLS3 recovered its activity and localized to the nucleus. Furthermore, deletion of leucine rich region (220-LALLLLDRLNRL) resulted in the accumulation of N to the cytoplasm and nucleolus, and when fusing this peptide to EGFP localization was cytoplasmic, suggesting that the N may act as a shuttle protein. Differences in nuclear/nucleolar localization properties of N from other members of coronavirus family suggest a unique function for N, which may play an important role in the pathogenesis of SARS.

| S-EPMC7114095 | biostudies-literature

SARS-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the MAVS/TRAF3/TRAF6 signalosome.

Project description:Coronaviruses (CoV) have recently emerged as potentially serious pathogens that can cause significant human morbidity and death. The severe acute respiratory syndrome (SARS)-CoV was identified as the etiologic agent of the 2002-2003 international SARS outbreak. Yet, how SARS evades innate immune responses to cause human disease remains poorly understood. In this study, we show that a protein encoded by SARS-CoV designated as open reading frame-9b (ORF-9b) localizes to mitochondria and causes mitochondrial elongation by triggering ubiquitination and proteasomal degradation of dynamin-like protein 1, a host protein involved in mitochondrial fission. Also, acting on mitochondria, ORF-9b targets the mitochondrial-associated adaptor molecule MAVS signalosome by usurping PCBP2 and the HECT domain E3 ligase AIP4 to trigger the degradation of MAVS, TRAF3, and TRAF 6. This severely limits host cell IFN responses. Reducing either PCBP2 or AIP4 expression substantially reversed the ORF-9b-mediated reduction of MAVS and the suppression of antiviral transcriptional responses. Finally, transient ORF-9b expression led to a strong induction of autophagy in cells. The induction of autophagy depended upon ATG5, a critical autophagy regulator, but the inhibition of MAVS signaling did not. These results indicate that SARS-CoV ORF-9b manipulates host cell mitochondria and mitochondrial function to help evade host innate immunity. This study has uncovered an important clue to the pathogenesis of SARS-CoV infection and illustrates the havoc that a small ORF can cause in cells.

| S-EPMC4179872 | biostudies-literature

Inhibitors of Protein Glycosylation Are Active against the Coronavirus Severe Acute Respiratory Syndrome Coronavirus SARS-CoV-2.

Project description:Repurposing clinically available drugs to treat the new coronavirus disease 2019 (COVID-19) is an urgent need in the course of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2) pandemic, as very few treatment options are available. The iminosugar Miglustat is a well-characterized drug for the treatment of rare genetic lysosome storage diseases, such as Gaucher and Niemann-Pick type C, and has also been described to be active against a variety of enveloped viruses. The activity of Miglustat is here demonstrated in the micromolar range for SARS-CoV-2 in vitro. The drug acts at the post-entry level and leads to a marked decrease of viral proteins and release of infectious viruses. The mechanism resides in the inhibitory activity toward α-glucosidases that are involved in the early stages of glycoprotein N-linked oligosaccharide processing in the endoplasmic reticulum, leading to a marked decrease of the viral Spike protein. Indeed, the antiviral potential of protein glycosylation inhibitors against SARS-CoV-2 is further highlighted by the low-micromolar activity of the investigational drug Celgosivir. These data point to a relevant role of this approach for the treatment of COVID-19.

| S-EPMC8144969 | biostudies-literature

SARS coronavirus series

2003-10-19 | GSE546 | GEO

Importin/karyopherin protein family members required for mRNA export from the nucleus.

Project description:The yeast Saccharomyces cerevisiae contains three proteins (Kap104p, Pse1p, and Kap123p) that share similarity to the 95-kDa beta subunit of the nuclear transport factor importin (also termed karyopherin and encoded by KAP95/RSL1 in yeast). Proteins that contain nuclear localization sequences are recognized in the cytoplasm and delivered to the nucleus by the heterodimeric importin complex. A second importin-related protein, transportin, delivers a subset of heterogeneous nuclear ribonucleoproteins (hnRNPs) to the nucleoplasm. We now show that in contrast to loss of importin beta (Kap95p/Rsl1p) and transportin (Kap104p), conditional loss of Pse1p in a strain lacking Kap123p results in a specific block of mRNA export from the nucleus. Overexpression of Sxm1p, a protein related to Cse1p in yeast and to the human cellular apoptosis susceptibility protein, relieves the defects of cells lacking Pse1p and Kap123p. Thus, a major role of Pse1p, Kap123p, and Sxm1p may be nuclear export rather than import, suggesting a symmetrical relationship between these processes.

| S-EPMC23031 | biostudies-literature

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