scholarly journals Bat Severe Acute Respiratory Syndrome-Like Coronavirus WIV1 Encodes an Extra Accessory Protein, ORFX, Involved in Modulation of the Host Immune Response

2016 ◽  
Vol 90 (14) ◽  
pp. 6573-6582 ◽  
Author(s):  
Lei-Ping Zeng ◽  
Yu-Tao Gao ◽  
Xing-Yi Ge ◽  
Qian Zhang ◽  
Cheng Peng ◽  
...  
Keyword(s):  
Immune Response ◽  
Severe Acute Respiratory Syndrome ◽  
Viral Replication ◽  
Fluorescent Protein ◽  
Host Immune Response ◽  
Open Reading Frame ◽  
Functional Gene ◽  
Interspecies Transmission ◽  
Reading Frame

ABSTRACTBats harbor severe acute respiratory syndrome (SARS)-like coronaviruses (SL-CoVs) from which the causative agent of the 2002-2003 SARS pandemic is thought to have originated. However, despite the fact that a large number of genetically diverse SL-CoV sequences have been detected in bats, only two strains (named WIV1 and WIV16) have been successfully culturedin vitro. These two strains differ from SARS-CoV only in containing an extra open reading frame (ORF) (named ORFX), between ORF6 and ORF7, which has no homology to any known protein sequences. In this study, we constructed a full-length cDNA clone of SL-CoV WIV1 (rWIV1), an ORFX deletion mutant (rWIV1-ΔX), and a green fluorescent protein (GFP)-expressing mutant (rWIV1-GFP-ΔX). Northern blotting and fluorescence microscopy indicate that ORFX was expressed during WIV1 infection. A virus infection assay showed that rWIV1-ΔX replicated as efficiently as rWIV1 in Vero E6, Calu-3, and HeLa-hACE2 cells. Further study showed that ORFX could inhibit interferon production and activate NF-κB. Our results demonstrate for the first time that the unique ORFX in the WIV1 strain is a functional gene involving modulation of the host immune response but is not essential forin vitroviral replication.IMPORTANCEBats harbor genetically diverse SARS-like coronaviruses (SL-CoVs), and some of them have the potential for interspecies transmission. A unique open reading frame (ORFX) was identified in the genomes of two recently isolated bat SL-CoV strains (WIV1 and -16). It will therefore be critical to clarify whether and how this protein contributes to virulence during viral infection. Here we revealed that the unique ORFX is a functional gene that is involved in the modulation of the host immune response but is not essential forin vitroviral replication. Our results provide important information for further exploration of the ORFX function in the future. Moreover, the reverse genetics system we constructed will be helpful for study of the pathogenesis of this group of viruses and to develop therapeutics for future control of emerging SARS-like infections.

scholarly journals The p23 Protein of Hibiscus Chlorotic Ringspot Virus Is Indispensable for Host-Specific Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 12312-12319 ◽  
Author(s):  
Xiao-Zhen Liang ◽  
Andrew P. Lucy ◽  
Shou-Wei Ding ◽  
Sek-Man Wong
Keyword(s):  
Viral Replication ◽  
Fluorescent Protein ◽  
Chenopodium Quinoa ◽  
Hibiscus Cannabinus ◽  
Ringspot Virus ◽  
Site Directed Mutagenesis ◽  
Loss Of Function ◽  
Reading Frame

ABSTRACT Hibiscus chlorotic ringspot virus (HCRSV) possesses a novel open reading frame (ORF) which encodes a putative 23-kDa protein (p23). We report here the in vivo detection of p23 and demonstrate its essential role in viral replication. The expression of p23 could be detected in protein extracts from transfected kenaf (Hibiscus cannabinus L.) protoplasts and in HCRSV-infected leaves. Further, direct immunoblotting of infected kenaf leaves also showed the presence of p23, and transient expression in onion and kenaf cells demonstrated that the protein is distributed throughout the cell. Site-directed mutagenesis showed that mutations introduced into the ORF of p23 abolished viral replication in kenaf protoplasts and plants but not in Chenopodium quinoa L. The loss of function of the p23 mutant M23/S33-1 could be complemented in trans upon the induced expression of p23 from an infiltrated construct bearing the ORF (pCam23). Altogether, these results demonstrate that p23 is a bona fide HCRSV protein that is expressed in vivo and suggest that p23 is indispensable for the host-specific replication of HCRSV. In addition, we show that p23 does not bind nucleic acids in vitro and does not act as a suppressor of posttranscriptional gene silencing in transgenic tobacco carrying a green fluorescent protein.

scholarly journals Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’

2001 ◽  
Vol 82 (5) ◽  
pp. 1013-1025 ◽  
Author(s):  
Michelle L. L. Donnelly ◽  
Garry Luke ◽  
Amit Mehrotra ◽  
Xuejun Li ◽  
Lorraine E. Hughes ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Foot And Mouth Disease ◽  
Peptide Bond ◽  
Open Reading Frame ◽  
Translation Product ◽  
Reading Frame ◽  
C Terminus ◽  
Mouth Disease Virus ◽  
Green Fluorescent

The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa long. A ‘primary’ intramolecular polyprotein processing event mediated by 2A occurs at its own C terminus. FMDV 2A activity was studied in artificial polyproteins in which sequences encoding reporter proteins flanked the 2A sequence such that a single, long, open reading frame was created. The self-processing properties of these artificial polyproteins were investigated and the co-translational ‘cleavage’ products quantified. The processing products from our artificial polyprotein systems showed a molar excess of ‘cleavage’ product N-terminal of 2A over the product C-terminal of 2A. A series of experiments was performed to characterize our in vitro translation systems. These experiments eliminated the translational or transcriptional properties of the in vitro systems as an explanation for this imbalance. In addition, the processing products derived from a control construct encoding the P1P2 region of the human rhinovirus polyprotein, known to be proteolytically processed, were quantified and found to be equimolar. Translation of a construct encoding green fluorescent protein (GFP), FMDV 2A and β-glucuronidase, also in a single open reading frame, in the presence of puromycin, showed this antibiotic to be preferentially incorporated into the [GFP2A] translation product. We conclude that the discrete translation products from our artificial polyproteins are not produced by proteolysis. We propose that the FMDV 2A sequence, rather than representing a proteolytic element, modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNAGly ester linkage, thereby releasing the polypeptide from the translational complex, in a manner that allows the synthesis of a discrete downstream translation product to proceed. This process produces a ribosomal ‘skip’ from one codon to the next without the formation of a peptide bond.

scholarly journals Restriction of SARS-CoV-2 Replication by Targeting Programmed −1 Ribosomal Frameshifting In Vitro

2020 ◽  
Author(s):  
Yu Sun ◽  
Laura Abriola ◽  
Yulia V. Surovtseva ◽  
Brett D. Lindenbach ◽  
Junjie U. Guo
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
High Throughput ◽  
Open Reading Frame ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Rna Pseudoknot ◽  
Proof Of Principle

SUMMARYTranslation of open reading frame 1b (ORF1b) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires programmed −1 ribosomal frameshifting (−1 PRF) promoted by an RNA pseudoknot. The extent to which SARS-CoV-2 replication may be sensitive to changes in −1 PRF efficiency is currently unknown. Through an unbiased, reporter-based high-throughput compound screen, we identified merafloxacin, a fluoroquinolone antibacterial, as a −1 PRF inhibitor of SARS-CoV-2. Frameshift inhibition by merafloxacin is robust to mutations within the pseudoknot region and is similarly effective on −1 PRF of other beta coronaviruses. Importantly, frameshift inhibition by merafloxacin substantially impedes SARS-CoV-2 replication in Vero E6 cells, thereby providing the proof of principle of targeting −1 PRF as an effective antiviral strategy for SARS-CoV-2.

scholarly journals Severe Acute Respiratory Syndrome Coronavirus Open Reading Frame (ORF) 3b, ORF 6, and Nucleocapsid Proteins Function as Interferon Antagonists

2006 ◽  
Vol 81 (2) ◽  
pp. 548-557 ◽  
Author(s):  
Sarah A. Kopecky-Bromberg ◽  
Luis Martínez-Sobrido ◽  
Matthew Frieman ◽  
Ralph A. Baric ◽  
Peter Palese
Keyword(s):  
Immune Response ◽  
Severe Acute Respiratory Syndrome ◽  
Innate Immune Response ◽  
Nuclear Translocation ◽  
Innate Immune ◽  
Open Reading Frame ◽  
Interferon Response ◽  
Reading Frame ◽  
N Protein ◽  
Interferon Antagonists

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) is highly pathogenic in humans, with a death rate near 10%. This high pathogenicity suggests that SARS-CoV has developed mechanisms to overcome the host innate immune response. It has now been determined that SARS-CoV open reading frame (ORF) 3b, ORF 6, and N proteins antagonize interferon, a key component of the innate immune response. All three proteins inhibit the expression of beta interferon (IFN-β), and further examination revealed that these SARS-CoV proteins inhibit a key protein necessary for the expression of IFN-β, IRF-3. N protein dramatically inhibited expression from an NF-κB-responsive promoter. All three proteins were able to inhibit expression from an interferon-stimulated response element (ISRE) promoter after infection with Sendai virus, while only ORF 3b and ORF 6 proteins were able to inhibit expression from the ISRE promoter after treatment with interferon. This indicates that N protein inhibits only the synthesis of interferon, while ORF 3b and ORF 6 proteins inhibit both interferon synthesis and signaling. ORF 6 protein, but not ORF 3b or N protein, inhibited nuclear translocation but not phosphorylation of STAT1. Thus, it appears that these three interferon antagonists of SARS-CoV inhibit the interferon response by different mechanisms.

scholarly journals Open Reading Frame 8a of the Human Severe Acute Respiratory Syndrome Coronavirus Not Only Promotes Viral Replication but Also Induces Apoptosis

2007 ◽  
Vol 196 (3) ◽  
pp. 405-415 ◽  
Author(s):  
Chia-Yen Chen ◽  
Yueh-Hsin Ping ◽  
Hsin-Chen Lee ◽  
Kuan-Hsuan Chen ◽  
Yuan-Ming Lee ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Viral Replication ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Cytopathic Effects ◽  
Antibody Assays ◽  
Cellular Apoptosis ◽  
Species Production ◽  
Interfering Rna ◽  
Dependent Pathway

Abstract Background A unique genomic difference between human and civet severe acute respiratory syndrome coronaviruses (SARS-CoVs) is that the former has a deletion of 29 nucleotides from open reading frame (orf) 8d that results in the generation of orf8a and orf8b. The objectives of the present study were to analyze antibody reactivity to ORF8a in patients with SARS and to elucidate the function of ORF8a. Methods Western-blot and immunofluorescent antibody assays were used to detect anti-ORF8a antibody. SARS-CoV HKU39849 was used to infect stable clones expressing ORF8a and cells transfected with small interfering RNA (siRNA). The virus loads (VLs) and cytopathic effects (CPEs) were recorded. Confocal microscopy and several mitochondria-related tests were used to study the function of ORF8a. Results Two (5.4%) of 37 patients with SARS had anti-ORF8a antibodies. The VLs in the stable clones expressing ORF8a were significantly higher than those in control subjects 5 days after infection. siRNA against orf8a significantly reduced VLs and interrupted the CPE. ORF8a was found to be localized in mitochondria, and overexpression resulted in increases in mitochondrial transmembrane potential, reactive oxygen species production, caspase 3 activity, and cellular apoptosis. Conclusions ORF8a not only enhances viral replication but also induces apoptosis through a mitochondria-dependent pathway.

scholarly journals Identification of Anti-Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV-2) Oxysterol Derivatives In Vitro

2021 ◽  
Vol 22 (6) ◽  
pp. 3163
Author(s):  
Hirofumi Ohashi ◽  
Feng Wang ◽  
Frank Stappenbeck ◽  
Kana Tsuchimoto ◽  
Chisa Kobayashi ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Viral Replication ◽  
Cultured Cells ◽  
Peak Plasma ◽  
Plasma Concentrations ◽  
Membrane Vesicles ◽  
Drug Candidates ◽  
Increased Risk ◽  
Derivatives Of

The development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is urgently needed to combat the coronavirus disease 2019 (COVID-19). We have previously studied the use of semi-synthetic derivatives of oxysterols, oxidized derivatives of cholesterol as drug candidates for the inhibition of cancer, fibrosis, and bone regeneration. In this study, we screened a panel of naturally occurring and semi-synthetic oxysterols for anti-SARS-CoV-2 activity using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 μM and 99% at 15 μM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fell into a therapeutically relevant range (19 μM), based on the dose-dependent curve for antiviral activity in our cell-based assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 by disrupting the formation of double-membrane vesicles (DMVs); intracellular membrane compartments associated with viral replication. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk of developing COVID-19.

The Drosophila suppressor of sable gene encodes a polypeptide with regions similar to those of RNA-binding proteins

1991 ◽  
Vol 11 (2) ◽  
pp. 894-905
Author(s):  
R A Voelker ◽  
W Gibson ◽  
J P Graves ◽  
J F Sterling ◽  
M T Eisenberg
Keyword(s):  
Rna Processing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Open Reading Frame ◽  
In Vitro Translation ◽  
Rna Recognition Motif ◽  
Reading Frame ◽  
Cdna Sequences ◽  
Suppressor Of Sable

The nucleotide sequence of the Drosophila melanogaster suppressor of sable [su(s)] gene has been determined. Comparison of genomic and cDNA sequences indicates that an approximately 7,860-nucleotide primary transcript is processed into an approximately 5-kb message, expressed during all stages of the life cycle, that contains an open reading frame capable of encoding a 1,322-amino-acid protein of approximately 150 kDa. The putative protein contains an RNA recognition motif-like region and a highly charged arginine-, lysine-, serine-, aspartic or glutamic acid-rich region that is similar to a region contained in several RNA-processing proteins. In vitro translation of in vitro-transcribed RNA from a complete cDNA yields a product whose size agrees with the size predicted by the open reading frame. Antisera against su(s) fusion proteins recognize the in vitro-translated protein and detect a protein of identical size in the nuclear fractions from tissue culture cells and embryos. The protein is also present in smaller amounts in cytoplasmic fractions of embryos. That the su(s) protein has regions similar in structure to RNA-processing protein is consistent with its known role in affecting the transcript levels of those alleles that it suppresses.

scholarly journals Ovine herpesvirus 2 replicates initially in the lung of experimentally infected sheep

2008 ◽  
Vol 89 (7) ◽  
pp. 1699-1708 ◽  
Author(s):  
Hong Li ◽  
Cristina W. Cunha ◽  
Christopher J. Davies ◽  
Katherine L. Gailbreath ◽  
Donald P. Knowles ◽  
...  
Keyword(s):  
Lymphoproliferative Disease ◽  
Infected Sheep ◽  
Open Reading Frame ◽  
Malignant Catarrhal Fever ◽  
Defence Mechanism ◽  
Reading Frame ◽  
Initial Infection ◽  
Nasal Secretions ◽  
Replication Site

Ovine herpesvirus 2 (OvHV-2), a rhadinovirus in the subfamily Gammaherpesvirinae, is the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal lymphoproliferative disease primarily of ruminants worldwide. Inability to propagate the virus in vitro has made it difficult to study OvHV-2 replication. Aerosol inoculation of sheep with OvHV-2 from nasal secretions collected from naturally infected sheep during shedding episodes results in infection of naive sheep, providing an excellent system to study OvHV-2 initial replication in the natural host. In this study, we showed that OvHV-2 delivered through the nasal route by nebulization resulted in infection in all lambs, but no infection was established in any lambs after intravenous or intraperitoneal injection. In nebulized lambs, while it was not detected initially in any other tissues, OvHV-2 DNA became detectable in the lung at 3 days post-infection (p.i.), increased to about 900 copies per 50 ng DNA at 5 days p.i., reached peak levels (∼7500 copies) at 7 days p.i., and then declined to an average of 800 copies at 9 days p.i. Transcripts of OvHV-2 open reading frame 25 (coding for the capsid protein), an indicator of virus replication, were only detected in lung tissues, and were positively correlated with OvHV-2 DNA levels in the lungs. In addition, selected immune response genes were also highly expressed in the lung at 5 and 7 days p.i. The data indicate that lung is the primary replication site for OvHV-2 during initial infection in sheep and suggest that viral replication is promptly controlled by a host defence mechanism.

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