Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
1.
Chem Commun (Camb) ; 57(93): 12476-12479, 2021 Nov 23.
Article in English | MEDLINE | ID: covidwho-1500757

ABSTRACT

We identified small-molecule enhancers of cellular stress granules by observing molecular crowding of proteins and RNAs in a time-dependent manner. Hit molecules sensitized the IRF3-mediated antiviral mechanism in the presence of poly(I:C) and inhibited the replication of SARS-CoV-2 by inducing stress granule formation. Thus, modulating multimolecular crowding can be a promising strategy against SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Benzopyrans/pharmacology , Cytoplasmic Granules/drug effects , Pyrazoles/pharmacology , SARS-CoV-2/drug effects , Virus Replication/drug effects , Animals , Antiviral Agents/chemistry , Benzopyrans/chemistry , Cell Line, Tumor , Chlorocebus aethiops , Cytoplasmic Granules/metabolism , Dose-Response Relationship, Drug , Drug Combinations , Humans , Interferon Regulatory Factor-3/metabolism , Lopinavir/pharmacology , Microbial Sensitivity Tests , Molecular Structure , Poly I-C/pharmacology , Pyrazoles/chemistry , Structure-Activity Relationship , Vero Cells
2.
Viruses ; 13(6)2021 05 21.
Article in English | MEDLINE | ID: covidwho-1359299

ABSTRACT

Viral RNAs contain the information needed to synthesize their own proteins, to replicate, and to spread to susceptible cells. However, due to their reduced coding capacity RNA viruses rely on host cells to complete their multiplication cycle. This is largely achieved by the concerted action of regulatory structural elements on viral RNAs and a subset of host proteins, whose dedicated function across all stages of the infection steps is critical to complete the viral cycle. Importantly, not only the RNA sequence but also the RNA architecture imposed by the presence of specific structural domains mediates the interaction with host RNA-binding proteins (RBPs), ultimately affecting virus multiplication and spreading. In marked difference with other biological systems, the genome of positive strand RNA viruses is also the mRNA. Here we focus on distinct types of positive strand RNA viruses that differ in the regulatory elements used to promote translation of the viral RNA, as well as in the mechanisms used to evade the series of events connected to antiviral response, including translation shutoff induced in infected cells, assembly of stress granules, and trafficking stress.


Subject(s)
Host-Pathogen Interactions , RNA Viruses/physiology , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , Response Elements , Biological Transport , Cytoplasmic Granules/metabolism , Gene Expression Regulation, Viral , Humans , Protein Biosynthesis , RNA Virus Infections/metabolism , RNA Virus Infections/virology , RNA, Viral/chemistry , Stress, Physiological , Transport Vesicles/metabolism , Virus Replication
3.
J Biol Chem ; 297(1): 100821, 2021 07.
Article in English | MEDLINE | ID: covidwho-1240418

ABSTRACT

Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) necessary for the viral life cycle, but it remains unknown whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins are methylated. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG motifs, preferred PRMT target sequences. We confirmed arginine methylation of N protein by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated from cell culture. Type I PRMT inhibitor (MS023) or substitution of R95 or R177 with lysine inhibited interaction of N protein with the 5'-UTR of SARS-CoV-2 genomic RNA, a property required for viral packaging. We also defined the N protein interactome in HEK293 cells, which identified PRMT1 and many of its RGG/RG substrates, including the known interacting protein G3BP1 as well as other components of stress granules (SGs), which are part of the host antiviral response. Methylation of R95 regulated the ability of N protein to suppress the formation of SGs, as R95K substitution or MS023 treatment blocked N-mediated suppression of SGs. Also, the coexpression of methylarginine reader Tudor domain-containing protein 3 quenched N protein-mediated suppression of SGs in a dose-dependent manner. Finally, pretreatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. Because type I PRMT inhibitors are already undergoing clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may represent an additional therapeutic application of these drugs.


Subject(s)
Arginine/metabolism , COVID-19/metabolism , COVID-19/virology , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/metabolism , RNA, Viral/metabolism , SARS-CoV-2/physiology , Amino Acid Motifs , COVID-19/genetics , Cytoplasmic Granules/genetics , Cytoplasmic Granules/metabolism , HEK293 Cells , Humans , Methylation , Nucleocapsid Proteins/genetics , RNA Stability , RNA, Viral/chemistry , RNA, Viral/genetics , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Virus Replication
4.
J Virol ; 95(9)2021 04 12.
Article in English | MEDLINE | ID: covidwho-1180915

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) is an α-coronavirus causing severe diarrhea and high mortality rates in suckling piglets and posing significant economic impact. PEDV replication is completed and results in a large amount of RNA in the cytoplasm. Stress granules (SGs) are dynamic cytosolic RNA granules formed under various stress conditions, including viral infections. Several previous studies suggested that SGs were involved in the antiviral activity of host cells to limit viral propagation. However, the underlying mechanisms are poorly understood. This study aimed to delineate the molecular mechanisms regulating the SG response to PEDV infection. SG formation is induced early during PEDV infection, but as infection proceeds, this ability is lost and SGs disappear at late stages of infection (>18 h postinfection). PEDV infection resulted in the cleavage of Ras-GTPase-activating protein-binding protein 1 (G3BP1) mediated by caspase-8. Using mutational analysis, the PEDV-induced cleavage site within G3BP1 was identified, which differed from the 3C protease cleavage site previously identified. Furthermore, G3BP1 cleavage by caspase-8 at D168 and D169 was confirmed in vitro as well as in vivo The overexpression of cleavage-resistant G3BP1 conferred persistent SG formation and suppression of viral replication. Additionally, the knockdown of endogenous G3BP1 abolished SG formation and potentiated viral replication. Taken together, these data provide new insights into novel strategies in which PEDV limits the host stress response and antiviral responses and indicate that caspase-8-mediated G3BP1 cleavage is important in the failure of host defense against PEDV infection.IMPORTANCE Coronaviruses (CoVs) are drawing extensive attention again since the outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019. CoVs are prone to variation and own the transmission capability by crossing the species barrier resulting in reemergence. How CoVs manipulate the antiviral responses of their hosts needs to be explored. Overall, the study provides new insight into how porcine epidemic diarrhea virus (PEDV) impaired SG assembly by targeting G3BP1 via the host proteinase caspase-8. These findings enhanced the understanding of PEDV infection and might help identify new antiviral targets that could inhibit viral replication and limit the pathogenesis of PEDV.


Subject(s)
Caspase 8/metabolism , Coronavirus Infections/metabolism , Cytoplasmic Granules/metabolism , Porcine epidemic diarrhea virus/physiology , Proteolysis , RNA Recognition Motif Proteins/metabolism , Virus Replication , Animals , Caspase 8/genetics , Chlorocebus aethiops , Coronavirus Infections/genetics , Coronavirus Infections/pathology , Cytoplasmic Granules/genetics , Cytoplasmic Granules/virology , HEK293 Cells , Humans , RNA Recognition Motif Proteins/genetics , Swine , Vero Cells
5.
PLoS Pathog ; 17(2): e1008690, 2021 02.
Article in English | MEDLINE | ID: covidwho-1105832

ABSTRACT

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-ß which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.


Subject(s)
COVID-19/virology , Cytoplasmic Granules/metabolism , Endoribonucleases/immunology , Endoribonucleases/metabolism , SARS-CoV-2/physiology , Viral Nonstructural Proteins/immunology , Viral Nonstructural Proteins/metabolism , COVID-19/metabolism , Cell Line , Coronavirus/immunology , Cytoplasmic Granules/immunology , Cytoplasmic Granules/virology , Humans , Interferon-beta/immunology , Interferon-beta/metabolism , SARS-CoV-2/metabolism , Signal Transduction , Virus Replication/physiology
6.
Arch Med Res ; 52(1): 48-57, 2021 01.
Article in English | MEDLINE | ID: covidwho-893598

ABSTRACT

BACKGROUND: Ras-GTPase activating protein SH3-domain-binding proteins (G3BP) are a small family of RNA-binding proteins implicated in regulating gene expression. Changes in expression of G3BPs are correlated to several cancers including thyroid, colon, pancreatic and breast cancer. G3BPs are important regulators of stress granule (SG) formation and function. SG are ribonucleoprotein (RNP) particles that respond to cellular stresses to triage mRNA resulting in transcripts being selectively degraded, stored or translated resulting in a change of gene expression which confers a survival response to the cell. These changes in gene expression contribute to the development of drug resistance. Many RNA viruses, including Chikungunya (and potentially Coronavirus), dismantle SG so that the cell cannot respond to the viral infection. Non-structural protein 3 (nsP3), from the Chikungunya virus, has been shown to translocate G3BP away from SG. Interestingly in cancer cells, the formation of SG is correlated to drug-resistance and blocking SG formation has been shown to reestablish the efficacy of the anticancer drug bortezomib. METHODS: Chikungunya nsP3 was transfected into breast cancer cell lines T47D and MCF7 to disrupt SG formation. Changes in the cytotoxicity of bortezomib were measured. RESULTS: Bortezomib cytotoxicity in breast cancer cell lines changed with a 22 fold decrease in its IC50 for T47D and a 7 fold decrease for MCF7 cells. CONCLUSIONS: Chikungunya nsP3 disrupts SG formation. As a result, it increases the cytotoxicity of the FDA approved drug, bortezomib. In addition, the increased cytotoxicity appears to correlate to improved bortezomib selectivity when compared to control cell lines.


Subject(s)
Bortezomib/pharmacology , Chikungunya Fever/drug therapy , Chikungunya virus/genetics , Cytoplasmic Granules/metabolism , DNA Helicases/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , RNA Helicases/metabolism , RNA Recognition Motif Proteins/metabolism , Viral Nonstructural Proteins/metabolism , Animals , Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/therapy , Chikungunya Fever/metabolism , Chikungunya Fever/pathology , Chikungunya virus/metabolism , Chlorocebus aethiops , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/pathology , DNA Helicases/genetics , Down-Regulation , Drug Resistance, Neoplasm , Female , HEK293 Cells , HeLa Cells , Humans , MCF-7 Cells , Poly-ADP-Ribose Binding Proteins/genetics , RNA Helicases/genetics , RNA Recognition Motif Proteins/genetics , Transfection , Vero Cells , Viral Nonstructural Proteins/administration & dosage , Viral Nonstructural Proteins/genetics
SELECTION OF CITATIONS
SEARCH DETAIL