Overexpression of 7a, a Protein Specifically Encoded by the Severe Acute Respiratory Syndrome Coronavirus, Induces Apoptosis via a Caspase-Dependent Pathway

ABSTRACT Besides genes that are homologous to proteins found in other coronaviruses, the severe acute respiratory syndrome coronavirus genome also contains nine other potential open reading frames. Previously, we have characterized the expression and cellular localization of two of these “accessory” viral proteins, 3a (previously termed U274) and 7a (previously termed U122). In this study, we further examined whether they can induce apoptosis, which has been observed clinically. We showed that the overexpression of 7a, but not of 3a or the viral structural proteins, nucleocapsid, membrane, and envelope, induces apoptosis. 7a induces apoptosis via a caspase-dependent pathway and in cell lines derived from different organs, including lung, kidney, and liver.

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Mapping the Non-Structural Transmembrane Proteins of SARS-CoV-2

10.20944/preprints202012.0366.v1 ◽

2020 ◽

Author(s):

Sunil Thomas

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Drug Targets ◽

Transmembrane Proteins ◽

Host Immunity ◽

Open Reading Frames ◽

Structural Proteins ◽

Wild Animals ◽

Cell Organelles ◽

Enveloped Virus ◽

Reading Frames

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the disease COVID-19 has wreaked havoc on the health and economy of humanity. In addition, the disease is observed in domestic and wild animals. The disease has impacted directly and indirectly every corner of the planet. Currently, there are no vaccines and effective therapies for COVID-19. SARS-CoV-2 is an enveloped virus with a single-stranded RNA genome of 29.8 kb. More than two-thirds of the genome comprises Orf1ab encoding 16 non-structural proteins (nsps) followed by mRNAs encoding structural proteins, spike (S), envelop (E), membrane (M), and nucleocapsid (N). These genes are interspaced with several accessory genes (open reading frames [Orf] 3a, 3b, 6, 7a, 7b, 8, 9b, 9c and 10). The functions of these proteins are of particular interest for understanding the pathogenesis of SARS-CoV-2. Several of the nsps (nsp3, nsp4, nsp6) and Orf3a are transmembrane proteins involved in regulating the host immunity, modifying host cell organelles for viral replication and escape and hence considered drug targets. In this paper we report mapping the transmembrane structure of the non-structural proteins of SARS-CoV-2.

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A Novel P/V/C Gene in a New Member of theParamyxoviridae Family, Which Causes Lethal Infection in Humans, Horses, and Other Animals

Journal of Virology ◽

10.1128/jvi.72.2.1482-1490.1998 ◽

1998 ◽

Vol 72 (2) ◽

pp. 1482-1490 ◽

Cited By ~ 76

Author(s):

Lin-Fa Wang ◽

Wojtek P. Michalski ◽

Meng Yu ◽

L. Ian Pritchard ◽

Gary Crameri ◽

...

Keyword(s):

Basic Protein ◽

Viral Proteins ◽

Open Reading Frames ◽

Sequence Comparisons ◽

V Protein ◽

Reading Frame ◽

Lethal Infection ◽

The Family ◽

Low Levels ◽

Reading Frames

ABSTRACT In 1994, a new member of the family Paramyxoviridaeisolated from fatal cases of respiratory disease in horses and humans was shown to be distantly related to morbilliviruses and provisionally called equine morbillivirus (K. Murray et al., Science 268:94–97, 1995). To facilitate characterization and classification, the virus was purified, viral proteins were identified, and the P/V/C gene was cloned and sequenced. The coding strategy of the gene is similar to that of Sendai and measles viruses, members of the Paramyxovirusand Morbillivirus genera, respectively, in the subfamilyParamyxovirinae. The P/V/C gene contains four open reading frames, three of which, P, C, and V, have Paramyxovirinaecounterparts. The P and C proteins are larger and smaller, respectively, than are cognate proteins in members of the subfamily, and the V protein is made as a result of a single G insertion during transcription. The P/V/C gene has two unique features. (i) A fourth open reading frame is located between those of the C and V proteins and potentially encodes a small basic protein similar to those found in some members of the Rhabdoviridae andFiloviridae families. (ii) There is also a long untranslated 3′ sequence, a feature common in Filoviridaemembers. Sequence comparisons confirm that although the virus is a member of the Paramyxovirinae subfamily, it displays only low levels of homology with paramyxoviruses and morbilliviruses and negligible homologies with rubulaviruses.

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Rearrangement of Influenza Virus Spliced Segments for the Development of Live-Attenuated Vaccines

Journal of Virology ◽

10.1128/jvi.00410-16 ◽

2016 ◽

Vol 90 (14) ◽

pp. 6291-6302 ◽

Cited By ~ 24

Author(s):

Aitor Nogales ◽

Marta L. DeDiego ◽

David J. Topham ◽

Luis Martínez-Sobrido

Keyword(s):

Influenza Virus ◽

Viral Infections ◽

Viral Proteins ◽

Influenza Viruses ◽

Open Reading Frames ◽

Wild Type Virus ◽

Wild Type ◽

Live Attenuated Vaccines ◽

Nonstructural Protein 1 ◽

Reading Frames

ABSTRACTInfluenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease which is most effectively prevented through vaccination. Segments 7 (M) and 8 (NS) of the influenza virus genome encode mRNA transcripts that are alternatively spliced to express two different viral proteins. This study describes the generation, using reverse genetics, of three different recombinant influenza A/Puerto Rico/8/1934 (PR8) H1N1 viruses containing M or NS viral segments individually or modified M or NS viral segments combined in which the overlapping open reading frames of matrix 1 (M1)/M2 for the modified M segment and the open reading frames of nonstructural protein 1 (NS1)/nuclear export protein (NEP) for the modified NS segment were split by using the porcine teschovirus 1 (PTV-1) 2A autoproteolytic cleavage site. Viruses with an M split segment were impaired in replication at nonpermissive high temperatures, whereas high viral titers could be obtained at permissive low temperatures (33°C). Furthermore, viruses containing the M split segment were highly attenuatedin vivo, while they retained their immunogenicity and provided protection against a lethal challenge with wild-type PR8. These results indicate that influenza viruses can be effectively attenuated by the rearrangement of spliced segments and that such attenuated viruses represent an excellent option as safe, immunogenic, and protective live-attenuated vaccines. Moreover, this is the first time in which an influenza virus containing a restructured M segment has been described. Reorganization of the M segment to encode M1 and M2 from two separate, nonoverlapping, independent open reading frames represents a useful tool to independently study mutations in the M1 and M2 viral proteins without affecting the other viral M product.IMPORTANCEVaccination represents our best therapeutic option against influenza viral infections. However, the efficacy of current influenza vaccines is suboptimal, and novel approaches are necessary for the prevention of disease caused by this important human respiratory pathogen. In this work, we describe a novel approach to generate safer and more efficient live-attenuated influenza virus vaccines (LAIVs) based on recombinant viruses whose genomes encode nonoverlapping and independent M1/M2 (split M segment [Ms]) or both M1/M2 and NS1/NEP (Ms and split NS segment [NSs]) open reading frames. Viruses containing a modified M segment were highly attenuated in mice but were able to confer, upon a single intranasal immunization, complete protection against a lethal homologous challenge with wild-type virus. Notably, the protection efficacy conferred by our viruses with split M segments was better than that conferred by the current temperature-sensitive LAIV. Altogether, these results open a new avenue for the development of safer and more protective LAIVs on the basis of the reorganization of spliced viral RNA segments in the genome.

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A Novel Severe Acute Respiratory Syndrome Coronavirus Protein, U274, Is Transported to the Cell Surface and Undergoes Endocytosis

Journal of Virology ◽

10.1128/jvi.78.13.6723-6734.2004 ◽

2004 ◽

Vol 78 (13) ◽

pp. 6723-6734 ◽

Cited By ~ 96

Author(s):

Yee-Joo Tan ◽

Eileen Teng ◽

Shuo Shen ◽

Timothy H. P. Tan ◽

Phuay-Yee Goh ◽

...

Keyword(s):

Amino Acids ◽

Severe Acute Respiratory Syndrome ◽

Cell Surface ◽

Open Reading Frames ◽

Structural Proteins ◽

Replicase Gene ◽

Subgenomic Rna ◽

C Terminus ◽

Infected Cells ◽

Potential Orfs

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) genome contains open reading frames (ORFs) that encode for several genes that are homologous to proteins found in all known coronaviruses. These are the replicase gene 1a/1b and the four structural proteins, nucleocapsid (N), spike (S), membrane (M), and envelope (E), and these proteins are expected to be essential for the replication of the virus. In addition, this genome also contains nine other potential ORFs varying in length from 39 to 274 amino acids. The largest among these is the first ORF of the second longest subgenomic RNA, and this protein (termed U274 in the present study) consists of 274 amino acids and contains three putative transmembrane domains. Using antibody specific for the C terminus of U274, we show U274 to be expressed in SARS-CoV-infected Vero E6 cells and, in addition to the full-length protein, two other processed forms were also detected. By indirect immunofluorescence, U274 was localized to the perinuclear region, as well as to the plasma membrane, in both transfected and infected cells. Using an N terminus myc-tagged U274, the topology of U274 and its expression on the cell surface were confirmed. Deletion of a cytoplasmic domain of U274, which contains Yxxφ and diacidic motifs, abolished its transport to the cell surface. In addition, U274 expressed on the cell surface can internalize antibodies from the culture medium into the cells. Coimmunoprecipitation experiments also showed that U274 could interact specifically with the M, E, and S structural proteins, as well as with U122, another protein that is unique to SARS-CoV.

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Identification of Trichoplusia ni ascovirus 2c virion structural proteins

Journal of General Virology ◽

10.1099/vir.0.82951-0 ◽

2007 ◽

Vol 88 (8) ◽

pp. 2194-2197 ◽

Cited By ~ 11

Author(s):

Liwang Cui ◽

Xiaowen Cheng ◽

Lianchao Li ◽

Jianyong Li

Keyword(s):

Mass Spectrometry ◽

Trichoplusia Ni ◽

Major Capsid Protein ◽

Protein Sequences ◽

Open Reading Frames ◽

Structural Proteins ◽

Tandem Mass ◽

Double Stranded Dna ◽

Coomassie Blue ◽

Reading Frames

Ascoviruses are a family of insect viruses with circular, double-stranded DNA genomes. With the sequencing of the Trichoplusia ni ascovirus 2c (TnAV-2c) genome, the virion structural proteins were identified by using tandem mass spectrometry. From at least eight protein bands visible on a Coomassie blue-stained gel of TnAV-2c virion proteins, seven bands generated protein sequences that matched predicted open reading frames (ORFs) in the genome, i.e. ORFs 2, 43, 115, 141, 142, 147 and 153. Among these ORFs, only ORF153, encoding the major capsid protein, has been characterized previously.

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TBIO-26. NON-CANONICAL OPEN READING FRAMES ENCODE FUNCTIONAL PROTEINS ESSENTIAL FOR CANCER CELL SURVIVAL

Neuro-Oncology ◽

10.1093/neuonc/noaa222.849 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii471-iii471

Author(s):

John Prensner ◽

Oana Enache ◽

Victor Luria ◽

Karsten Krug ◽

Karl Clauser ◽

...

Keyword(s):

Cell Lines ◽

Cancer Cell ◽

Protein Translation ◽

Cancer Cell Lines ◽

Open Reading Frames ◽

Sequencing Analysis ◽

Multiple Cancer ◽

Cancer Types ◽

Non Coding Rnas ◽

Reading Frames

Abstract The brain is the foremost non-gonadal tissue for expression of non-coding RNAs of unclear function. Yet, whether such transcripts are truly non-coding or rather the source of non-canonical protein translation is unknown. Here, we used functional genomic screens to establish the cellular bioactivity of non-canonical proteins located in putative non-coding RNAs or untranslated regions of protein-coding genes. We experimentally interrogated 553 open reading frames (ORFs) identified by ribosome profiling for three major phenotypes: 257 (46%) demonstrated protein translation when ectopically expressed in HEK293T cells, 401 (73%) induced gene expression changes following ectopic expression across 4 cancer cell types, and 57 (10%) induced a viability defect when the endogenous ORF was knocked out using CRISPR/Cas9 in 8 human cancer cell lines. CRISPR tiling and start codon mutagenesis indicated that the biological impact of these non-canonical ORFs required their translation as opposed to RNA-mediated effects. We functionally characterized one of these ORFs, G029442—renamed GREP1 (Glycine-Rich Extracellular Protein-1)—as a cancer-implicated gene with high expression in multiple cancer types, such as gliomas. GREP1 knockout in >200 cancer cell lines reduced cell viability in multiple cancer types, including glioblastoma, in a cell-autonomous manner and produced cell cycle arrest via single-cell RNA sequencing. Analysis of the secretome of GREP1-expressing cells showed increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth inhibitory effect of GREP1 knock-out. Taken together, these experiments suggest that the non-canonical ORFeome is surprisingly rich in biologically active proteins and potential cancer therapeutic targets deserving of further study.

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Assembly of the K40 Antigen in Escherichia coli: Identification of a Novel Enzyme Responsible for Addition ofl-Serine Residues to the Glycan Backbone and Its Requirement for K40 Polymerization

Journal of Bacteriology ◽

10.1128/jb.181.3.772-780.1999 ◽

1999 ◽

Vol 181 (3) ◽

pp. 772-780 ◽

Cited By ~ 10

Author(s):

Paul A. Amor ◽

Jeremy A. Yethon ◽

Mario A. Monteiro ◽

Chris Whitfield

Keyword(s):

Escherichia Coli ◽

Lipid A ◽

Glucuronic Acid ◽

Repeat Unit ◽

Compositional Analysis ◽

Open Reading Frames ◽

Insertional Inactivation ◽

Dependent Pathway ◽

Reading Frames ◽

Polyclonal Serum

ABSTRACT Escherichia coli O8:K40 coexpresses two distinct lipopolysaccharide (LPS) structures on its surface. The O8 polysaccharide is a mannose homopolymer with a trisaccharide repeat unit and is synthesized by an ABC-2 transport-dependent pathway. The K40LPS backbone structure is composed of a trisaccharide repeating unit of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) and has an uncommon substitution, anl-serine moiety attached to glucuronic acid. The gene cluster responsible for synthesis of the K40 polysaccharide has previously been cloned and sequenced and was found to contain six open reading frames (ORFs) (P. A. Amor and C. Whitfield, Mol. Microbiol. 26:145–161, 1997). Here, we demonstrate that insertional inactivation of orf1 results in the accumulation of a semirough (SR)-K40LPS form which retains reactivity with specific polyclonal serum in Western immunoblots. Structural and compositional analysis of the SR-K40LPS reveals that it comprises a single K40 repeat unit attached to lipid A core. The lack of polymerization of the K40 polysaccharide indicates thatorf1 encodes the K40 polymerase (Wzy) and that assembly of the K40 polysaccharide occurs via a Wzy-dependent pathway (in contrast to that of the O8 polysaccharide). Inactivation of orf3also results in the accumulation of an SR-LPS form which fails to react with specific polyclonal K40 serum in Western immunoblots. Methylation linkage analysis and fast atom bombardment-mass spectrometry of this SR-LPS reveals that the biological repeat unit of the K40 polysaccharide is GlcNAc-GlcA-GlcNAc. Additionally, this structure lacks the l-serine substitution of GlcA. These results show that (i) orf3 encodes the enzyme responsible for the addition of the l-serine residue to the K40 backbone and (ii) substitution of individual K40 repeats withl-serine is essential for their recognition and polymerization into the K40 polysaccharide by Wzy.

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The SARS-CoV-2 antibody landscape is lower in magnitude for structural proteins, diversified for accessory proteins and stable long-term in children

10.1101/2021.01.03.21249180 ◽

2021 ◽

Author(s):

Asmaa Hachim ◽

Haogao Gu ◽

Otared Kavian ◽

Mike YW Kwan ◽

Wai-hung Chan ◽

...

Keyword(s):

Antibody Response ◽

Principal Component ◽

Humoral Response ◽

Open Reading Frames ◽

Clinical Severity ◽

Structural Proteins ◽

Accessory Proteins ◽

Longitudinal Sampling ◽

Reading Frames

AbstractBackgroundChildren are less clinically affected by SARS-CoV-2 infection than adults with the majority of cases being mild or asymptomatic and the differences in infection outcomes are poorly understood. The kinetics, magnitude and landscape of the antibody response may impact the clinical severity and serological diagnosis of COVID-19. Thus, a comprehensive investigation of the antibody landscape in children and adults is needed.MethodsWe tested 254 plasma from 122 children with symptomatic and asymptomatic SARS-CoV-2 infections in Hong Kong up to 206 days post symptom onset, including 146 longitudinal samples from 58 children. Adult COVID-19 patients and pre-pandemic controls were included for comparison. We assessed antibodies to a 14-wide panel of SARS-CoV-2 structural and accessory proteins by Luciferase Immunoprecipitation System (LIPS).FindingsChildren have lower levels of Spike and Nucleocapsid antibodies than adults, and their cumulative humoral response is more expanded to accessory proteins (NSP1 and Open Reading Frames (ORFs)). Sensitive serology using the three N, ORF3b, ORF8 antibodies can discriminate COVID-19 in children. Principal component analysis revealed distinct serological signatures in children and the highest contribution to variance were responses to non-structural proteins ORF3b, NSP1, ORF7a and ORF8. Longitudinal sampling revealed maintenance or increase of antibodies for at least 6 months, except for ORF7b antibodies which showed decline. It was interesting to note that children have higher antibody responses towards known IFN antagonists: ORF3b, ORF6 and ORF7a. The diversified SARS-CoV-2 antibody response in children may be an important factor in driving control of SARS-CoV-2 infection.

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Think Different with RNA Therapy: Can Antisense Oligonucleotides Be Used to Inhibit Replication and Transcription of SARS-Cov-2?

10.20944/preprints202004.0412.v1 ◽

2020 ◽

Cited By ~ 2

Author(s):

Eric Barrey ◽

Veronica Burzio ◽

Sophie Dhorne-Pollet ◽

Jean-François Eléouët ◽

Bernard Delmas

Keyword(s):

Antisense Oligonucleotide ◽

Antisense Oligonucleotides ◽

Transmission Rate ◽

Drug Repurposing ◽

Viral Proteins ◽

Open Reading Frames ◽

Viral Rna ◽

Medical Treatments ◽

Locked Nucleic Acids ◽

Reading Frames

The severity of the global COVID-19 pandemic, with a high transmission rate, 2.6-4.7% lethality and a huge economic impact, poses an urgent need for efficient medical treatments and vaccines. Currently, there are only non-specific treatments to assist the patients in acute respiratory distress during the inflammatory step following the preliminary infection by SARS-CoV-2. Clinical trials of drug repurposing were quickly launched at the international level. Specific treatments such as the transfusion of plasma from patients who have recovered into infected patients or the use of specific inhibitors of the viral RNA-polymerase complex are promising strategies to block infection. To complete the therapeutic arsenal, we believe that the opportunity of targeting the SARS-CoV-2 genome by RNA therapy should be deeply investigated. In the present paper, we propose to design specific antisense oligonucleotides targeting transcripts encoding viral proteins associated to replication and transcription of SARS-CoV-2, aiming to block infection. We designed antisense oligonucleotides targeting the genomic 5’ untranslated region (5’-UTR), open reading frames 1a and 1b (ORF1a and ORF1b) governing expression of the replicase/transcriptase complex, and the gene N encoding the nucleoprotein that is genome-associated. To maximize the probability of efficiency, we predicted the antisense oligonucleotides by using two design methods: i) conventional antisense oligonucleotides with 100% phosphorothioate modifications (ASO); ii) antisense locked nucleic acids GapmeR. After binding the viral RNA target, the hetero-duplexes antisense oligonucleotide-RNA are cleaved by RNAse H1. Nine potent ASO candidates were found and we selected five of them targeting ORF1a (3), ORF1b (1) and N (1). Nine GapmeR candidates were predicted with excellent properties and we retained four of them targeting 5’-UTR (1), ORF1a (3), ORF1b (1) and N (1). The most potent GapmeR candidate targets the 5’-UTR, a key genomic domain with multiple functions in the viral cycle. By this open publication, we are pleased to share these in silico results with the scientific community in hopes of stimulating innovation in translational research in order to fight the unprecedented COVID-19 pandemic. These antisense oligonucleotide candidates should be now experimentally evaluated.

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Genome Sequences of Two GH Clade SARS-CoV-2 Strains Isolated from Patients with COVID-19 in South Korea

Microbiology Resource Announcements ◽

10.1128/mra.01384-20 ◽

2021 ◽

Vol 10 (1) ◽

Author(s):

Minwoo Kim ◽

Youn-Jung Lee ◽

Jae Sun Yoon ◽

Jin Young Ahn ◽

Jung Ho Kim ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

South Korea ◽

Open Reading Frames ◽

Untranslated Regions ◽

Genome Sequences ◽

Nonsynonymous Substitutions ◽

Sequence Identity ◽

Reading Frames

ABSTRACT We report the genome sequences of two GH clade severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains isolated from nasopharyngeal swabs from patients with coronavirus disease 2019 (COVID-19) in South Korea. These strains had two mutations in the untranslated regions and seven nonsynonymous substitutions in open reading frames, compared with Wuhan/Hu-1/2019, showing 99.96% sequence identity.

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