scholarly journals Localization of SARS-CoV-2 Capping Enzymes Revealed by an Antibody against the nsp10 Subunit

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1487
Author(s):  
Vladimira Horova ◽  
Barbora Landova ◽  
Jan Hodek ◽  
Karel Chalupsky ◽  
Petra Krafcikova ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Severe Acute Respiratory Syndrome ◽  
Subcellular Localization ◽  
Protein Complexes ◽  
Structural Protein ◽  
Biological Characterization ◽  
Replication Complex ◽  
A Subunit ◽  
Infected Cells ◽  
Capping Enzymes

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2’-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2’-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.

scholarly journals Severe Acute Respiratory Syndrome Coronavirus 3a Protein Is Released in Membranous Structures from 3a Protein-Expressing Cells and Infected Cells

2006 ◽  
Vol 80 (1) ◽  
pp. 210-217 ◽  
Author(s):  
Cheng Huang ◽  
Krishna Narayanan ◽  
Naoto Ito ◽  
C. J. Peters ◽  
Shinji Makino
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Buoyant Density ◽  
Protein A ◽  
Biological Properties ◽  
Structural Protein ◽  
Membrane Structures ◽  
Capture Assay ◽  
Release Analysis ◽  
Infected Cells ◽  
Detergent Resistant Membranes

ABSTRACT Severe acute respiratory syndrome coronavirus (SCoV) accessory protein 3a is a virus structural protein. We demonstrate here that 3a protein was released efficiently in membranous structures from various cell lines expressing 3a protein. A subpopulation of the released 3a protein is associated with detergent-resistant membranes. The presence of the YxxΦ and diacidic motifs, located within the cytoplasmic tail of the 3a protein, was not required for its efficient release. Analysis of supernatant from SCoV-infected cells with sucrose gradient sedimentation and virus capture assay indicated that the 3a protein was released from infected cells in two distinct populations, as a component of SCoV particles, and in membrane structures with a lower buoyant density. These data provide new insights into the biological properties of SCoV 3a protein.

scholarly journals Non-structural protein 4A of Hepatitis C virus accumulates on mitochondria and renders the cells prone to undergoing mitochondria-mediated apoptosis

2006 ◽  
Vol 87 (7) ◽  
pp. 1935-1945 ◽  
Author(s):  
Yuki Nomura-Takigawa ◽  
Motoko Nagano-Fujii ◽  
Lin Deng ◽  
Sohei Kitazawa ◽  
Satoshi Ishido ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Replication ◽  
Hcv Rna ◽  
Structural Protein ◽  
Cellular Functions ◽  
Replication Complex ◽  
Huh7 Cells ◽  
Infected Cells ◽  
Rna Replicon

Non-structural protein 4A (NS4A) of Hepatitis C virus (HCV) functions as a cofactor for NS3 by forming a complex with it to augment its enzymic activities. NS4A also forms a complex with other HCV proteins, such as NS4B/NS5A, to facilitate the formation of the viral RNA replication complex on the endoplasmic reticulum (ER) membrane. In addition to its essential role in HCV replication, NS4A is thought to be involved in viral pathogenesis by affecting cellular functions. In this study, it was demonstrated that NS4A was localized not only on the ER, but also on mitochondria when expressed either alone or together with NS3 in the form of the NS3/4A polyprotein and in the context of HCV RNA replication in Huh7 cells harbouring an HCV RNA replicon. Moreover, NS4A expression altered the intracellular distribution of mitochondria significantly and caused mitochondrial damage, as evidenced by the collapsed mitochondrial transmembrane potential and release of cytochrome c into the cytoplasm, which led ultimately to induction of apoptosis through activation of caspase-3, but not caspase-8. Consistently, Huh7 cells expressing NS3/4A and those harbouring an HCV RNA replicon were shown to be more prone to undergoing actinomycin D-induced, mitochondria-mediated apoptosis, compared with the control Huh7 cells. Taken together, these results suggest the possibility that HCV exerts cytopathic effect (CPE) on the infected cells under certain conditions and that NS4A is responsible, at least in part, for the conditional CPE in HCV-infected cells.

scholarly journals Severe Acute Respiratory Syndrome Coronavirus 3a Protein Is a Viral Structural Protein

2005 ◽  
Vol 79 (5) ◽  
pp. 3182-3186 ◽  
Author(s):  
Naoto Ito ◽  
Eric C. Mossel ◽  
Krishna Narayanan ◽  
Vsevolod L. Popov ◽  
Cheng Huang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Severe Acute Respiratory Syndrome ◽  
Posttranslational Modifications ◽  
Accessory Protein ◽  
Structural Protein ◽  
Viral Structural Protein ◽  
Infected Cells

ABSTRACT The present study showed the association of a severe acute respiratory syndrome coronavirus (SCoV) accessory protein, 3a, with plasma membrane and intracellular SCoV particles in infected cells. 3a protein appeared to undergo posttranslational modifications in infected cells and was incorporated into SCoV particles, establishing that 3a protein was a SCoV structural protein.

scholarly journals Subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein

2005 ◽  
Vol 86 (12) ◽  
pp. 3303-3310 ◽  
Author(s):  
Jaehwan You ◽  
Brian K. Dove ◽  
Luis Enjuanes ◽  
Marta L. DeDiego ◽  
Enrique Alvarez ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Subcellular Localization ◽  
Rna Binding ◽  
Cell Signalling ◽  
Bioinformatic Analysis ◽  
Binding Motif ◽  
Nuclear Localization Signals ◽  
N Protein ◽  
Localization Signal ◽  
Infected Cells

The coronavirus nucleocapsid (N) protein is a viral RNA-binding protein with multiple functions in terms of virus replication and modulating cell signalling pathways. N protein is composed of three distinct regions containing RNA-binding motif(s), and appropriate signals for modulating cell signalling. The subcellular localization of severe acute respiratory syndrome coronavirus (SARS-CoV) N protein was studied. In infected cells, SARS-CoV N protein localized exclusively to the cytoplasm. In contrast to the avian coronavirus N protein, overexpressed SARS-CoV N protein remained principally localized to the cytoplasm, with very few cells exhibiting nucleolar localization. Bioinformatic analysis and deletion mutagenesis coupled to confocal microscopy and live-cell imaging, revealed that SARS-CoV N protein regions I and III contained nuclear localization signals and region II contained a nucleolar retention signal. However, cytoplasmic localization was directed by region III and was the dominant localization signal in the protein.

scholarly journals Development of Bacteriophage Virus-Like Particle Vaccines Displaying Conserved Epitopes of Dengue Virus Non-Structural Protein 1

Vaccines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 726
Author(s):  
Nikole L. Warner ◽  
Kathryn M. Frietze
Keyword(s):  
Dengue Virus ◽  
Population Based ◽  
Ns1 Protein ◽  
Structural Protein ◽  
Denv Serotypes ◽  
Conserved Regions ◽  
Virus Like Particle ◽  
Dependent Cell ◽  
Infected Cells ◽  
Plasma Leakage

Dengue virus (DENV) is a major global health problem, with over half of the world’s population at risk of infection. Despite over 60 years of efforts, no licensed vaccine suitable for population-based immunization against DENV is available. Here, we describe efforts to engineer epitope-based vaccines against DENV non-structural protein 1 (NS1). NS1 is present in DENV-infected cells as well as secreted into the blood of infected individuals. NS1 causes disruption of endothelial cell barriers, resulting in plasma leakage and hemorrhage. Immunizing against NS1 could elicit antibodies that block NS1 function and also target NS1-infected cells for antibody-dependent cell cytotoxicity. We identified highly conserved regions of NS1 from all four DENV serotypes. We generated synthetic peptides to these regions and chemically conjugated them to bacteriophage Qβ virus-like particles (VLPs). Mice were immunized two times with the candidate vaccines and sera were tested for the presence of antibodies that bound to the cognate peptide, recombinant NS1 from all four DENV serotypes, and DENV-2-infected cells. We found that two of the candidate vaccines elicited antibodies that bound to recombinant NS1, and one candidate vaccine elicited antibodies that bound to DENV-infected cells. These results show that an epitope-specific vaccine against conserved regions of NS1 could be a promising approach for DENV vaccines or therapeutics to bind circulating NS1 protein.

scholarly journals A GFP Reporter MR766-Based Flow Cytometry Neutralization Test for Rapid Detection of Zika Virus-Neutralizing Antibodies in Serum Specimens

Vaccines ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 66 ◽  
Author(s):  
Frumence ◽  
Viranaicken ◽  
Gadea ◽  
Desprès
Keyword(s):  
Flow Cytometry ◽  
Zika Virus ◽  
Neutralizing Antibodies ◽  
Neutralization Test ◽  
Health Concern ◽  
Structural Protein ◽  
Adult Mice ◽  
Gfp Reporter ◽  
Infected Cells ◽  
High Level

Zika virus (ZIKV) is an emerging arthropod-borne virus of major public health concern. ZIKV infection is responsible for congenital Zika disease and other neurological defects. Antibody-mediated virus neutralization is an essential component of protective antiviral immunity against ZIKV. In the present study, we assessed whether our GFP reporter ZIKV derived from African viral strain MR766 could be useful for the development of a flow cytometry neutralization test (FNT), as an alternative to the conventional plaque-reduction neutralization test (PRNT). To improve the efficacy of GFP-expressing MR766, we selected virus variant MR766GFP showing a high level of GFP signal in infected cells. A MR766GFP-based FNT was assayed with immune sera from adult mice that received ZIKBeHMR-2. The chimeric ZIKV clone ZIKBeHMR-2 comprises the structural protein region of epidemic strain BeH819015 into MR766 backbone. We reported that adult mice inoculated with ZIKBeHMR-2 developed high levels of neutralizing anti-ZIKV antibodies. Comparative analysis between MR766GFP-based FNT and conventional PRNT was performed using mouse anti-ZIKBeHMR-2 immune sera. Indistinguishable neutralization patterns were observed when compared with PRNT50 and FNT50. We consider that the newly developed MR766GFP-based FNT is a valid format for measuring ZIKV-neutralizing antibodies in serum specimens.

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