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1.
Biochem Biophys Res Commun ; 586: 137-142, 2022 01 01.
Article in English | MEDLINE | ID: covidwho-1520712

ABSTRACT

Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.


Subject(s)
COVID-19/metabolism , COVID-19/virology , Host Microbial Interactions/physiology , Membrane Glycoproteins/metabolism , Nuclear Pore Complex Proteins/metabolism , RNA-Binding Proteins/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Active Transport, Cell Nucleus , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/virology , Gene Knockdown Techniques , HeLa Cells , Humans , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/genetics , Models, Biological , Nuclear Envelope/metabolism , Nuclear Envelope/virology , Nuclear Pore Complex Proteins/antagonists & inhibitors , Nuclear Pore Complex Proteins/genetics , RNA-Binding Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factor RelA/metabolism , Viral Nonstructural Proteins/genetics
2.
Viruses ; 13(10)2021 10 15.
Article in English | MEDLINE | ID: covidwho-1470998

ABSTRACT

Nuclear transport and vesicle trafficking are key cellular functions involved in the pathogenesis of RNA viruses. Among other pleiotropic effects on virus-infected host cells, ivermectin (IVM) inhibits nuclear transport mechanisms mediated by importins and atorvastatin (ATV) affects actin cytoskeleton-dependent trafficking controlled by Rho GTPases signaling. In this work, we first analyzed the response to infection in nasopharyngeal swabs from SARS-CoV-2-positive and -negative patients by assessing the gene expression of the respective host cell drug targets importins and Rho GTPases. COVID-19 patients showed alterations in KPNA3, KPNA5, KPNA7, KPNB1, RHOA, and CDC42 expression compared with non-COVID-19 patients. An in vitro model of infection with Poly(I:C), a synthetic analog of viral double-stranded RNA, triggered NF-κB activation, an effect that was halted by IVM and ATV treatment. Importin and Rho GTPases gene expression was also impaired by these drugs. Furthermore, through confocal microscopy, we analyzed the effects of IVM and ATV on nuclear to cytoplasmic importin α distribution, alone or in combination. Results showed a significant inhibition of importin α nuclear accumulation under IVM and ATV treatments. These findings confirm transcriptional alterations in importins and Rho GTPases upon SARS-CoV-2 infection and point to IVM and ATV as valid drugs to impair nuclear localization of importin α when used at clinically-relevant concentrations.


Subject(s)
Active Transport, Cell Nucleus/drug effects , Atorvastatin/pharmacology , COVID-19/drug therapy , Ivermectin/pharmacology , SARS-CoV-2/drug effects , alpha Karyopherins/metabolism , A549 Cells , Actin Cytoskeleton/drug effects , Animals , Antiviral Agents/pharmacology , Cell Line, Tumor , Chlorocebus aethiops , Drug Repositioning , HeLa Cells , Humans , NF-kappa B/metabolism , Vero Cells , rho GTP-Binding Proteins/metabolism
3.
Viruses ; 13(2)2021 02 10.
Article in English | MEDLINE | ID: covidwho-1395005

ABSTRACT

Since the discovery of HIV-1, the viral capsid has been recognized to have an important role as a structural protein that holds the viral genome, together with viral proteins essential for viral life cycle, such as the reverse transcriptase (RT) and the integrase (IN). The reverse transcription process takes place between the cytoplasm and the nucleus of the host cell, thus the Reverse Transcription Complexes (RTCs)/Pre-integration Complexes (PICs) are hosted in intact or partial cores. Early biochemical assays failed to identify the viral CA associated to the RTC/PIC, possibly due to the stringent detergent conditions used to fractionate the cells or to isolate the viral complexes. More recently, it has been observed that some host partners of capsid, such as Nup153 and CPSF6, can only bind multimeric CA proteins organized in hexamers. Those host factors are mainly located in the nuclear compartment, suggesting the entrance of the viral CA as multimeric structure inside the nucleus. Recent data show CA complexes within the nucleus having a different morphology from the cytoplasmic ones, clearly highlighting the remodeling of the viral cores during nuclear translocation. Thus, the multimeric CA complexes lead the viral genome into the host nuclear compartment, piloting the intranuclear journey of HIV-1 in order to successfully replicate. The aim of this review is to discuss and analyze the main discoveries to date that uncover the viral capsid as a key player in the reverse transcription and PIC maturation until the viral DNA integration into the host genome.


Subject(s)
Capsid/metabolism , Cell Nucleus/virology , HIV-1/physiology , Active Transport, Cell Nucleus , Capsid/chemistry , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Cell Nucleus/metabolism , HIV-1/chemistry , HIV-1/metabolism , Models, Biological , Nuclear Pore Complex Proteins/metabolism , Reverse Transcription , Virus Integration , Virus Replication
4.
Front Immunol ; 12: 624293, 2021.
Article in English | MEDLINE | ID: covidwho-1394756

ABSTRACT

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, which interacts with a wide range of organic molecules of endogenous and exogenous origin, including environmental pollutants, tryptophan metabolites, and microbial metabolites. The activation of AHR by these agonists drives its translocation into the nucleus where it controls the expression of a large number of target genes that include the AHR repressor (AHRR), detoxifying monooxygenases (CYP1A1 and CYP1B1), and cytokines. Recent advances reveal that AHR signaling modulates aspects of the intrinsic, innate and adaptive immune response to diverse microorganisms. This review will focus on the increasing evidence supporting a role for AHR as a modulator of the host response to viral infection.


Subject(s)
Adaptive Immunity , Immunity, Innate , Receptors, Aryl Hydrocarbon/metabolism , Virus Diseases/virology , Viruses/immunology , Active Transport, Cell Nucleus , Animals , Gene Expression Regulation , Host-Pathogen Interactions , Humans , Ligands , Signal Transduction , Virus Diseases/genetics , Virus Diseases/immunology , Virus Diseases/metabolism , Viruses/genetics , Viruses/pathogenicity
5.
J Biol Chem ; 297(1): 100856, 2021 07.
Article in English | MEDLINE | ID: covidwho-1283409

ABSTRACT

The nuclear pore complex is the sole gateway connecting the nucleoplasm and cytoplasm. In humans, the nuclear pore complex is one of the largest multiprotein assemblies in the cell, with a molecular mass of ∼110 MDa and consisting of 8 to 64 copies of about 34 different nuclear pore proteins, termed nucleoporins, for a total of 1000 subunits per pore. Trafficking events across the nuclear pore are mediated by nuclear transport receptors and are highly regulated. The nuclear pore complex is also used by several RNA viruses and almost all DNA viruses to access the host cell nucleoplasm for replication. Viruses hijack the nuclear pore complex, and nuclear transport receptors, to access the nucleoplasm where they replicate. In addition, the nuclear pore complex is used by the cell innate immune system, a network of signal transduction pathways that coordinates the first response to foreign invaders, including viruses and other pathogens. Several branches of this response depend on dynamic signaling events that involve the nuclear translocation of downstream signal transducers. Mounting evidence has shown that these signaling cascades, especially those steps that involve nucleocytoplasmic trafficking events, are targeted by viruses so that they can evade the innate immune system. This review summarizes how nuclear pore proteins and nuclear transport receptors contribute to the innate immune response and highlights how viruses manipulate this cellular machinery to favor infection. A comprehensive understanding of nuclear pore proteins in antiviral innate immunity will likely contribute to the development of new antiviral therapeutic strategies.


Subject(s)
Immunity, Innate/genetics , Nuclear Pore Complex Proteins/genetics , Nuclear Pore/genetics , Virus Diseases/genetics , Active Transport, Cell Nucleus/genetics , Active Transport, Cell Nucleus/immunology , DNA Viruses/genetics , DNA Viruses/pathogenicity , Humans , Immune Evasion/genetics , Immune Evasion/immunology , NF-kappa B/genetics , Nuclear Pore/immunology , Nuclear Pore Complex Proteins/immunology , RNA Viruses/genetics , RNA Viruses/pathogenicity , Viral Nonstructural Proteins/genetics , Virus Diseases/immunology , Virus Diseases/virology , Virus Replication/genetics , Virus Replication/immunology
6.
PLoS One ; 16(6): e0253089, 2021.
Article in English | MEDLINE | ID: covidwho-1282298

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic, infecting over 43 million people and claiming over 1 million lives, with these numbers increasing daily. Therefore, there is urgent need to understand the molecular mechanisms governing SARS-CoV-2 pathogenesis, immune evasion, and disease progression. Here, we show that SARS-CoV-2 can block IRF3 and NF-κB activation early during virus infection. We also identify that the SARS-CoV-2 viral proteins NSP1 and NSP13 can block interferon activation via distinct mechanisms. NSP1 antagonizes interferon signaling by suppressing host mRNA translation, while NSP13 downregulates interferon and NF-κB promoter signaling by limiting TBK1 and IRF3 activation, as phospho-TBK1 and phospho-IRF3 protein levels are reduced with increasing levels of NSP13 protein expression. NSP13 can also reduce NF-κB activation by both limiting NF-κB phosphorylation and nuclear translocation. Last, we also show that NSP13 binds to TBK1 and downregulates IFIT1 protein expression. Collectively, these data illustrate that SARS-CoV-2 bypasses multiple innate immune activation pathways through distinct mechanisms.


Subject(s)
Adaptor Proteins, Signal Transducing/immunology , COVID-19/immunology , Cell Nucleus/immunology , Interferon Regulatory Factor-3/immunology , RNA-Binding Proteins/immunology , SARS-CoV-2/immunology , Signal Transduction/immunology , Viral Nonstructural Proteins/immunology , Active Transport, Cell Nucleus/genetics , Active Transport, Cell Nucleus/immunology , Adaptor Proteins, Signal Transducing/genetics , COVID-19/genetics , Cell Nucleus/genetics , HeLa Cells , Humans , Interferon Regulatory Factor-3/genetics , NF-kappa B/genetics , NF-kappa B/immunology , Phosphorylation/genetics , Phosphorylation/immunology , /immunology , RNA-Binding Proteins/genetics , SARS-CoV-2/genetics , Signal Transduction/genetics , Viral Nonstructural Proteins/genetics
7.
Antiviral Res ; 192: 105115, 2021 08.
Article in English | MEDLINE | ID: covidwho-1275131

ABSTRACT

The novel coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the recent global pandemic. The nuclear export protein (XPO1) has a direct role in the export of SARS-CoV proteins including ORF3b, ORF9b, and nucleocapsid. Inhibition of XPO1 induces anti-inflammatory, anti-viral, and antioxidant pathways. Selinexor is an FDA-approved XPO1 inhibitor. Through bioinformatics analysis, we predicted nuclear export sequences in the ACE-2 protein and confirmed by in vitro testing that inhibition of XPO1 with selinexor induces nuclear localization of ACE-2. Administration of selinexor inhibited viral infection prophylactically as well as therapeutically in vitro. In a ferret model of COVID-19, selinexor treatment reduced viral load in the lungs and protected against tissue damage in the nasal turbinates and lungs in vivo. Our studies demonstrated that selinexor downregulated the pro-inflammatory cytokines IL-1ß, IL-6, IL-10, IFN-γ, TNF-α, and GMCSF, commonly associated with the cytokine storm observed in COVID-19 patients. Our findings indicate that nuclear export is critical for SARS-CoV-2 infection and for COVID-19 pathology and suggest that inhibition of XPO1 by selinexor could be a viable anti-viral treatment option.


Subject(s)
COVID-19/drug therapy , Hydrazines/pharmacology , SARS-CoV-2/drug effects , Triazoles/pharmacology , Active Transport, Cell Nucleus/drug effects , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antiviral Agents/pharmacology , COVID-19/virology , Chlorocebus aethiops , Cytokines , Ferrets , Humans , Karyopherins/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Respiratory System/drug effects , Respiratory System/virology , SARS-CoV-2/metabolism , Tumor Suppressor Proteins/metabolism , Vero Cells , Virus Replication
8.
Cells ; 10(6)2021 06 07.
Article in English | MEDLINE | ID: covidwho-1259432

ABSTRACT

The host nucleocytoplasmic trafficking system is often hijacked by viruses to accomplish their replication and to suppress the host immune response. Viruses encode many factors that interact with the host nuclear transport receptors (NTRs) and the nucleoporins of the nuclear pore complex (NPC) to access the host nucleus. In this review, we discuss the viral factors and the host factors involved in the nuclear import and export of viral components. As nucleocytoplasmic shuttling is vital for the replication of many viruses, we also review several drugs that target the host nuclear transport machinery and discuss their feasibility for use in antiviral treatment.


Subject(s)
Cell Nucleus/metabolism , Cell Nucleus/virology , SARS-CoV-2/physiology , Virus Physiological Phenomena , Virus Replication/physiology , Active Transport, Cell Nucleus/physiology , COVID-19/metabolism , COVID-19/virology , Host-Pathogen Interactions/physiology , Humans , Nucleocytoplasmic Transport Proteins/metabolism , Virus Internalization , Viruses/pathogenicity
10.
Front Immunol ; 12: 663586, 2021.
Article in English | MEDLINE | ID: covidwho-1190318

ABSTRACT

As of January 2021, SARS-CoV-2 has killed over 2 million individuals across the world. As such, there is an urgent need for vaccines and therapeutics to reduce the burden of COVID-19. Several vaccines, including mRNA, vector-based vaccines, and inactivated vaccines, have been approved for emergency use in various countries. However, the slow roll-out of vaccines and insufficient global supply remains a challenge to turn the tide of the pandemic. Moreover, vaccines are important tools for preventing the disease but therapeutic tools to treat patients are also needed. As such, since the beginning of the pandemic, repurposed FDA-approved drugs have been sought as potential therapeutic options for COVID-19 due to their known safety profiles and potential anti-viral effects. One of these drugs is ivermectin (IVM), an antiparasitic drug created in the 1970s. IVM later exerted antiviral activity against various viruses including SARS-CoV-2. In this review, we delineate the story of how this antiparasitic drug was eventually identified as a potential treatment option for COVID-19. We review SARS-CoV-2 lifecycle, the role of the nucleocapsid protein, the turning points in past research that provided initial 'hints' for IVM's antiviral activity and its molecular mechanism of action- and finally, we culminate with the current clinical findings.


Subject(s)
Active Transport, Cell Nucleus/drug effects , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Ivermectin/therapeutic use , SARS-CoV-2/drug effects , Animals , Cell Line , Chlorocebus aethiops , Coronavirus Nucleocapsid Proteins/antagonists & inhibitors , Coronavirus Nucleocapsid Proteins/metabolism , Drug Repositioning , Humans , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/metabolism , Protein Transport/drug effects , SARS-CoV-2/growth & development , Vero Cells , Virus Replication/drug effects , alpha Karyopherins/antagonists & inhibitors , beta Karyopherins/antagonists & inhibitors
11.
mBio ; 12(2)2021 04 13.
Article in English | MEDLINE | ID: covidwho-1183285

ABSTRACT

RNA viruses that replicate in the cytoplasm often disrupt nucleocytoplasmic transport to preferentially translate their own transcripts and prevent host antiviral responses. The Sarbecovirus accessory protein ORF6 has previously been shown to be a major inhibitor of interferon production in both severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we show SARS-CoV-2-infected cells display an elevated level of nuclear mRNA accumulation compared to mock-infected cells. We demonstrate that ORF6 is responsible for this nuclear imprisonment of host mRNA, and using a cotransfected reporter assay, we show this nuclear retention of mRNA blocks expression of newly transcribed mRNAs. ORF6's nuclear entrapment of host mRNA is associated with its ability to copurify with the mRNA export factors, Rae1 and Nup98. These protein-protein interactions map to the C terminus of ORF6 and can be abolished by a single amino acid mutation in Met58. Overexpression of Rae1 restores reporter expression in the presence of SARS-CoV-2 ORF6. SARS-CoV ORF6 also interacts with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly copurifies with Rae1 and Nup98 and results in significantly reduced expression of reporter proteins compared to SARS-CoV ORF6, a potential mechanism for the delayed symptom onset and presymptomatic transmission uniquely associated with the SARS-CoV-2 pandemic. We also show that both SARS-CoV and SARS-CoV-2 ORF6 block nuclear import of a broad range of host proteins. Together, these data support a model in which ORF6 clogs the nuclear pore through its interactions with Rae1 and Nup98 to prevent both nuclear import and export, rendering host cells incapable of responding to SARS-CoV-2 infection.IMPORTANCE SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), is an RNA virus with a large genome that encodes multiple accessory proteins. While these accessory proteins are not required for growth in vitro, they can contribute to the pathogenicity of the virus. We demonstrate that SARS-CoV-2-infected cells accumulate poly(A) mRNA in the nucleus, which is attributed to the accessory protein ORF6. Nuclear entrapment of mRNA and reduced expression of newly transcribed reporter proteins are associated with ORF6's interactions with the mRNA export proteins Rae1 and Nup98. SARS-CoV ORF6 also shows the same interactions with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly represses reporter expression and copurifies with Rae1 and Nup98 compared to SARS-CoV ORF6. Both SARS-CoV ORF6 and SARS-CoV-2 ORF6 block nuclear import of a wide range of host factors through interactions with Rae1 and Nup98. Together, our results suggest ORF6's disruption of nucleocytoplasmic transport prevents infected cells from responding to the invading virus.


Subject(s)
Cell Nucleus/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Nuclear Pore Complex Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , SARS-CoV-2/metabolism , Viral Proteins/metabolism , Active Transport, Cell Nucleus , Binding Sites , COVID-19/metabolism , COVID-19/virology , Cell Line , Gene Expression Regulation , Humans , Mutation , Nuclear Matrix-Associated Proteins/genetics , Nuclear Pore Complex Proteins/genetics , Nucleocytoplasmic Transport Proteins/genetics , Protein Binding , RNA, Messenger/metabolism , SARS-CoV-2/genetics , Viral Proteins/chemistry , Viral Proteins/genetics
12.
Sci Adv ; 7(6)2021 02.
Article in English | MEDLINE | ID: covidwho-1066794

ABSTRACT

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.


Subject(s)
COVID-19/metabolism , Gene Expression , Host Microbial Interactions/genetics , RNA, Messenger/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Virulence Factors/metabolism , Active Transport, Cell Nucleus/genetics , Animals , COVID-19/virology , Chlorocebus aethiops , HEK293 Cells , Humans , Nuclear Pore/metabolism , Nucleocytoplasmic Transport Proteins/genetics , Nucleocytoplasmic Transport Proteins/metabolism , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , SARS-CoV-2/chemistry , Transfection , Vero Cells , Viral Nonstructural Proteins/genetics
13.
Biochem Biophys Res Commun ; 536: 59-66, 2021 01 15.
Article in English | MEDLINE | ID: covidwho-971357

ABSTRACT

The novel human betacoronavirus SARS-CoV-2 has caused an unprecedented pandemic in the 21st century. Several studies have revealed interactions between SARS-CoV-2 viral proteins and host nucleoporins, yet their functions are largely unknown. Here, we demonstrate that the open-reading frame 6 (ORF6) of SARS-CoV-2 can directly manipulate localization and functions of nucleoporins. We found that ORF6 protein disrupted nuclear rim staining of nucleoporins RAE1 and NUP98. Consequently, this disruption caused aberrant nucleocytoplasmic trafficking and led to nuclear accumulation of mRNA transporters such as hnRNPA1. Ultimately, host cell nucleus size was reduced and cell growth was halted.


Subject(s)
Cell Nucleus Size , Nuclear Matrix-Associated Proteins/metabolism , Nuclear Pore Complex Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , Viral Proteins/metabolism , Active Transport, Cell Nucleus , Cell Nucleus/virology , HEK293 Cells , Heterogeneous Nuclear Ribonucleoprotein A1/metabolism , Humans , SARS-CoV-2
14.
PLoS One ; 15(11): e0241739, 2020.
Article in English | MEDLINE | ID: covidwho-934332

ABSTRACT

Due to the challenges for developing vaccines in devastating pandemic situations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), developing and screening of novel antiviral agents are peremptorily demanded. Herein, we developed EGYVIR as a potent immunomodulatory herbal extract with promising antiviral activity against SARS-CoV-2. It constitutes of a combination of black pepper extract with curcumin extract. The antiviral effect of EGYVIR extract is attributed to the two key phases of the disease in severe cases. First, the inhibition of the nuclear translocation of NF-kß p50, attenuating the SARS-CoV-2 infection-associated cytokine storm. Additionally, the EGYVIR extract has an in vitro virucidal effect for SARS-CoV-2. The in vitro study of EGYVIR extract against SARS-CoV-2 on Huh-7 cell lines, revealed the potential role of NF-kß/TNFα/IL-6 during the infection process. EGYVIR antagonizes the NF-kß pathway in-silico and in-vitro studies. Consequently, it has the potential to hinder the release of IL-6 and TNFα, decreasing the production of essential cytokines storm elements.


Subject(s)
Antiviral Agents/pharmacology , Immunologic Factors/pharmacology , Plant Extracts/pharmacology , SARS-CoV-2/drug effects , Active Transport, Cell Nucleus/drug effects , Animals , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Chlorocebus aethiops , Curcuma/chemistry , Humans , Interleukin-6/metabolism , Kinetics , NF-KappaB Inhibitor alpha/metabolism , NF-kappa B p50 Subunit/metabolism , Piper nigrum/chemistry , Tumor Necrosis Factor-alpha/metabolism , Vero Cells
15.
Proc Natl Acad Sci U S A ; 117(45): 28344-28354, 2020 11 10.
Article in English | MEDLINE | ID: covidwho-887237

ABSTRACT

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.


Subject(s)
COVID-19/metabolism , Interferons/metabolism , Nuclear Pore Complex Proteins/metabolism , Nuclear Pore/metabolism , STAT1 Transcription Factor/metabolism , STAT2 Transcription Factor/metabolism , Viral Proteins/metabolism , Active Transport, Cell Nucleus , Animals , Binding Sites , Chlorocebus aethiops , HEK293 Cells , Humans , Nuclear Matrix-Associated Proteins/chemistry , Nuclear Matrix-Associated Proteins/metabolism , Nucleocytoplasmic Transport Proteins/chemistry , Nucleocytoplasmic Transport Proteins/metabolism , Protein Binding , Signal Transduction , Vero Cells
16.
Virus Res ; 278: 197843, 2020 03.
Article in English | MEDLINE | ID: covidwho-833528

ABSTRACT

Swine acute diarrhea syndrome coronavirus (SADS-CoV), a newly emerging enteric coronavirus, is considered to be associated with swine acute diarrhea syndrome (SADS) which has caused significantly economic losses to the porcine industry. Interactions between SADS-CoV and the host innate immune response is unclear yet. In this study, we used IPEC-J2 cells as a model to explore potential evasion strategies employed by SADS-CoV. Our results showed that SADS-CoV infection failed to induce IFN-ß production, and inhibited poly (I:C) and Sendai virus (SeV)-triggered IFN-ß expression. SADS-CoV also blocked poly (I:C)-induced phosphorylation and nuclear translocation of IRF-3 and NF-κB. Furthermore, SADS-CoV did not interfere with the activity of IFN-ß promoter stimulated by IRF3, TBK1 and IKKε, but counteracted its activation induced by IPS-1 and RIG-I. Collectively, this study is the first investigation that shows interactions between SADS-CoV and the host innate immunity, which provides information of the molecular mechanisms underlying SASD-CoV infection.


Subject(s)
Alphacoronavirus/physiology , Coronavirus Infections/immunology , DEAD Box Protein 58/antagonists & inhibitors , Interferon-beta/antagonists & inhibitors , Active Transport, Cell Nucleus , Animals , Cell Line , Cell Nucleus/metabolism , Coronavirus Infections/virology , DEAD Box Protein 58/metabolism , Host-Pathogen Interactions/immunology , Immunity, Innate , Interferon Regulatory Factor-3/metabolism , Interferon-beta/genetics , Interferon-beta/metabolism , NF-kappa B/metabolism , Phosphorylation , Promoter Regions, Genetic , Signal Transduction , Swine
17.
Biomolecules ; 10(9)2020 08 26.
Article in English | MEDLINE | ID: covidwho-822256

ABSTRACT

In Trichomonas vaginalis (T. vaginalis), cyclophilins play a vital role in dislodging Myb proteins from the membrane compartment and leading them to nuclear translocation. We previously reported that TvCyP1 cyclophilin from T. vaginalis forms a dimer and plays an essential role in moving the Myb1 transcription factor toward the nucleus. In comparison, TvCyP2 containing an extended segment at the N-terminus (N-terminal segment) formed a monomer and showed a different role in regulating protein trafficking. Four X-ray structures of TvCyP2 were determined under various conditions, all showing the N-terminal segment interacting with the active site of a neighboring TvCyP2, an unusual interaction. NMR study revealed that this particular interaction exists in solution as well and also the N-terminal segment seems to interact with the membrane. In vivo study of TvCyP2 and TvCyP2-∆N (TvCyP2 without the N-terminal segment) indicated that both proteins have different subcellular localization. Together, the structural and functional characteristics at the N-terminal segment offer valuable information for insights into the mechanism of how TvCyP2 regulates protein trafficking, which may be applied in drug development to prevent pathogenesis and disease progression in T. vaginalis infection.


Subject(s)
Cyclophilins/chemistry , Cyclophilins/metabolism , Protozoan Proteins/metabolism , Trichomonas vaginalis/metabolism , Active Transport, Cell Nucleus , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Cyclophilins/genetics , Endoplasmic Reticulum/metabolism , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Protein Stability , Protein Transport , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Transcription Factors/metabolism , Trichomonas vaginalis/genetics
18.
J Chin Med Assoc ; 83(8): 712-718, 2020 08.
Article in English | MEDLINE | ID: covidwho-733326

ABSTRACT

Recently, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was quickly identified as the causal pathogen leading to the outbreak of SARS-like illness all over the world. As the SARS-CoV-2 infection pandemic proceeds, many efforts are being dedicated to the development of diverse treatment strategies. Increasing evidence showed potential therapeutic agents directly acting against SARS-CoV-2 virus, such as interferon, RNA-dependent RNA polymerase inhibitors, protease inhibitors, viral entry blockers, neuraminidase inhibitor, vaccine, antibody agent targeting the SARS-CoV-2 RNA genome, natural killer cells, and nucleocytoplasmic trafficking inhibitor. To date, several direct anti-SARS-CoV-2 agents have demonstrated promising in vitro and clinical efficacy. This article reviews the current and future development of direct acting agents against SARS-CoV-2.


Subject(s)
Antiviral Agents/therapeutic use , Betacoronavirus , Coronavirus Infections/drug therapy , Drug Development , Pneumonia, Viral/drug therapy , Active Transport, Cell Nucleus/drug effects , Antibodies, Monoclonal/therapeutic use , Betacoronavirus/genetics , COVID-19 , Genome, Viral , Humans , Killer Cells, Natural/immunology , Pandemics , SARS-CoV-2
19.
Drug Discov Today ; 25(10): 1775-1781, 2020 10.
Article in English | MEDLINE | ID: covidwho-611872

ABSTRACT

Coronavirus 2019 (COVID-19; caused by Severe Acute Respiratory Syndrome Coronavirus 2; SARS-CoV-2) is a currently global health problem. Previous studies showed that blocking nucleocytoplasmic transport with exportin 1 (XPO1) inhibitors originally developed as anticancer drugs can quarantine key viral accessory proteins and genomic materials in the nucleus of host cell and reduce virus replication and immunopathogenicity. These observations support the concept of the inhibition of nuclear export as an effective strategy against an array of viruses, including influenza A, B, and SARS-CoV. Clinical studies using the XPO1 inhibitor selinexor as a therapy for COVID-19 infection are in progress.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , Cell Nucleus/drug effects , Drug Design , Karyopherins/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , SARS-CoV-2/pathogenicity , Active Transport, Cell Nucleus , Animals , COVID-19/immunology , COVID-19/metabolism , COVID-19/virology , Cell Nucleus/immunology , Cell Nucleus/metabolism , Cell Nucleus/virology , Host-Pathogen Interactions , Humans , Karyopherins/metabolism , Molecular Targeted Therapy , Receptors, Cytoplasmic and Nuclear/metabolism , SARS-CoV-2/immunology
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