In vivo functional characterization of the SARS-Coronavirus 3a protein in Drosophila.
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ABSTRACT:
SUBMITTER: Wong SL
PROVIDER: S-EPMC7117541 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Proteomics was used to identify a protein encoded by ORF 3a in a SARS-associated coronavirus (SARS-CoV). Immuno-blotting revealed that interchain disulfide bonds might be formed between this protein and the spike protein. ELISA indicated that sera from SARS patients have significant positive reactions with synthesized peptides derived from the 3a protein. These results are concordant with that of a spike protein-derived peptide. A tendency exists for co-mutation between the 3a protein and the spike protein of SARS-CoV isolates, suggesting that the function of the 3a protein correlates with the spike protein. Taken together, the 3a protein might be tightly correlated to the spike protein in the SARS-CoV functions. The 3a protein may serve as a new clinical marker or drug target for SARS treatment.
| S-EPMC7127270 | biostudies-literature
Project description:The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a positive stranded RNA virus with ?30kb genome. Among all open reading frames (orfs) of this virus, the orf3a is the largest, and encodes a protein of 274 amino acids, named as 3a protein. Sequence analysis suggests that the orf3a aligned to one calcium pump present in Plasmodium falciparum and the enzyme glutamine synthetase found in Leptospira interrogans. This sequence similarity was found to be limited only to amino acid residues 209-264 which form the cytoplasmic domain of the orf3a. Furthermore, this region was predicted to be involved in the calcium binding. Owing to this hypothesis, we were driven to establish its calcium binding property in vitro. Here, we expressed and purified the cytoplasmic domain of the 3a protein, called Cyto3a, as a recombinant His-tagged protein in the E. coli. The calcium binding nature was established by performing various staining methods such as ruthenium red and stains-all. (45)Ca overlay method was also done to further support the data. Since the 3a protein forms ion channels, we were interested to see any conformational changes occurring in the Cyot3a upon calcium binding, using fluorescence spectroscopy and circular dichroism. These studies clearly indicate a significant change in the conformation of the Cyto3a protein after binding with calcium. Our results strongly suggest that the cytoplasmic domain of the 3a protein of SARS-CoV binds calcium in vitro, causing a change in protein conformation.
| S-EPMC7114474 | biostudies-literature
Project description:Set of microarray experiments used to identify an unknown coronavirus in a viral culture derived from a patient with SARS. March 2003. Keywords = SARS Keywords = coronavirus Keywords = viral discovery Keywords = viruses Keywords = respiratory infection
2010-06-05 | E-GEOD-546 | biostudies-arrayexpress
Project description:The molecular mechanisms underlying the severe lung pathology that occurs during SARS-CoV infections remain incompletely understood. The largest of the SARS-CoV accessory protein open reading frames (SARS 3a) oligomerizes, dynamically inserting into late endosomal, lysosomal, and trans-Golgi-network membranes. While previously implicated in a non-inflammatory apoptotic cell death pathway, here we extend the range of SARS 3a pathophysiologic targets by examining its effects on necrotic cell death pathways. We show that SARS 3a interacts with Receptor Interacting Protein 3 (Rip3), which augments the oligomerization of SARS 3a helping drive necrotic cell death. In addition, by inserting into lysosomal membranes SARS 3a triggers lysosomal damage and dysfunction. Consequently, Transcription Factor EB (TFEB) translocates to the nucleus increasing the transcription of autophagy- and lysosome-related genes. Finally, SARS 3a activates caspase-1 either directly or via an enhanced potassium efflux, which triggers NLRP3 inflammasome assembly. In summary, Rip3-mediated oligomerization of SARS 3a causes necrotic cell death, lysosomal damage, and caspase-1 activation-all likely contributing to the clinical manifestations of SARS-CoV infection.
| S-EPMC6125346 | biostudies-literature
Project description:Animal microRNAs (miRNA) are implicated in the control of nearly all cellular functions. Due to high sequence redundancy within the miRNA gene pool, loss of most of these 21- to 24-bp long RNAs individually does not cause a phenotype. Thus, only very few miRNAs have been associated with clear functional roles. We constructed a transgenic UAS-miRNA library in Drosophila melanogaster that contains 180 fly miRNAs. This library circumvents the redundancy issues by facilitating the controlled misexpression of individual miRNAs and is a useful tool to complement loss-of-function approaches. Demonstrating the effectiveness of our library, 78 miRNAs induced clear phenotypes. Most of these miRNAs were previously unstudied. Furthermore, we present a simple system to create GFP sensors to monitor miRNA expression and test direct functional interactions in vivo. Finally, we focus on the miR-92 family and identify a direct target gene that is responsible for the specific wing phenotype induced by the misexpression of miR-92 family members.
| S-EPMC3512159 | biostudies-literature
Project description:Background The novel coronavirus SARS-CoV-2 is currently a global threat to health and economies. Therapeutics and vaccines are in rapid development; however, none of these therapeutics are considered as absolute cure, and the potential to mutate makes it necessary to find therapeutics that target a highly conserved regions of the viral structure. Results In this study, we characterized an essential but poorly understood coronavirus accessory X4 protein, a core and stable component of the SARS-CoV family. Sequence analysis shows a conserved ~ 90% identity between the SARS-CoV-2 and previously characterized X4 protein in the database. QMEAN Z score of the model protein shows a value of around 0.5, within the acceptable range 0–1. A MolProbity score of 2.96 was obtained for the model protein and indicates a good quality model. The model has Ramachandran values of φ = − 57o and ψ = − 47o for α-helices and values of φ = − 130o and ψ = + 140o for twisted sheets. Conclusions The protein data obtained from this study provides robust information for further in vitro and in vivo experiment, targeted at devising therapeutics against the virus. Phylogenetic analysis further supports previous evidence that the SARS-CoV-2 is positioned with the SL-CoVZC45, BtRs-BetaCoV/YN2018B and the RS4231 Bat SARS-like corona viruses.
| S-EPMC8105150 | biostudies-literature
Project description:The spike (S) glycoprotein of coronaviruses mediates viral entry into host cells. It is a type 1 viral fusion protein that characteristically contains two heptad repeat regions, denoted HR-N and HR-C, that form coiled-coil structures within the ectodomain of the protein. Previous studies have shown that the two heptad repeat regions can undergo a conformational change from their native state to a 6-helix bundle (trimer of dimers), which mediates fusion of viral and host cell membranes. Here we describe the biophysical analysis of the two predicted heptad repeat regions within the severe acute respiratory syndrome coronavirus S protein. Our results show that in isolation the HR-N region forms a stable alpha-helical coiled coil that associates in a tetrameric state. The HR-C region in isolation formed a weakly stable trimeric coiled coil. When mixed together, the two peptide regions (HR-N and HR-C) associated to form a very stable alpha-helical 6-stranded structure (trimer of heterodimers). Systematic peptide mapping showed that the site of interaction between the HR-N and HR-C regions is between residues 916-950 of HR-N and residues 1151-1185 of HR-C. Additionally, interchain disulfide bridge experiments showed that the relative orientation of the HR-N and HR-C helices in the complex was antiparallel. Overall, the structure of the hetero-stranded complex is consistent with the structures observed for other type 1 viral fusion proteins in their fusion-competent state.
| S-EPMC8060857 | biostudies-literature
Project description:Set of microarray experiments used to identify an unknown coronavirus in a viral culture derived from a patient with SARS. March 2003. Keywords = SARS Keywords = coronavirus Keywords = viral discovery Keywords = viruses Keywords = respiratory infection Keywords: repeat sample
2003-10-19 | GSE546 | GEO
Project description:BackgroundA recent publication reported that a tyrosine-dependent sorting signal, present in cytoplasmic tail of the spike protein of most coronaviruses, mediates the intracellular retention of the spike protein. This motif is missing from the spike protein of the severe acute respiratory syndrome-coronavirus (SARS-CoV), resulting in high level of surface expression of the spike protein when it is expressed on its own in vitro.Presentation of the hypothesisIt has been shown that the severe acute respiratory syndrome-coronavirus genome contains open reading frames that encode for proteins with no homologue in other coronaviruses. One of them is the 3a protein, which is expressed during infection in vitro and in vivo. The 3a protein, which contains a tyrosine-dependent sorting signal in its cytoplasmic domain, is expressed on the cell surface and can undergo internalization. In addition, 3a can bind to the spike protein and through this interaction, it may be able to cause the spike protein to become internalized, resulting in a decrease in its surface expression.Testing the hypothesisThe effects of 3a on the internalization of cell surface spike protein can be examined biochemically and the significance of the interplay between these two viral proteins during viral infection can be studied using reverse genetics methodology.Implication of the hypothesisIf this hypothesis is proven, it will indicate that the severe acute respiratory syndrome-coronavirus modulates the surface expression of the spike protein via a different mechanism from other coronaviruses. The interaction between 3a and S, which are expressed from separate subgenomic RNA, would be important for controlling the trafficking properties of S. The cell surface expression of S in infected cells significantly impacts viral assembly, viral spread and viral pathogenesis. Modulation by this unique pathway could confer certain advantages during the replication of the severe acute respiratory syndrome-coronavirus.
| S-EPMC549520 | biostudies-literature
Project description:The entry of the SARS coronavirus (SCV) into cells is initiated by binding of its spike envelope glycoprotein (S) to a receptor, ACE2. We and others identified the receptor-binding domain (RBD) by using S fragments of various lengths but all including the amino acid residue 318 and two other potential glycosylation sites. To further characterize the role of glycosylation and identify residues important for its function as an interacting partner of ACE2, we have cloned, expressed and characterized various soluble fragments of S containing RBD, and mutated all potential glycosylation sites and 32 other residues. The shortest of these fragments still able to bind the receptor ACE2 did not include residue 318 (which is a potential glycosylation site), but started at residue 319, and has only two potential glycosylation sites (residues 330 and 357). Mutation of each of these sites to either alanine or glutamine, as well as mutation of residue 318 to alanine in longer fragments resulted in the same decrease of molecular weight (by approximately 3 kDa) suggesting that all glycosylation sites are functional. Simultaneous mutation of all glycosylation sites resulted in lack of expression suggesting that at least one glycosylation site (any of the three) is required for expression. Glycosylation did not affect binding to ACE2. Alanine scanning mutagenesis of the fragment S319-518 resulted in the identification of ten residues (K390, R426, D429, T431, I455, N473, F483, Q492, Y494, R495) that significantly reduced binding to ACE2, and one residue (D393) that appears to increase binding. Mutation of residue T431 reduced binding by about 2-fold, and mutation of the other eight residues--by more than 10-fold. Analysis of these data and the mapping of these mutations on the recently determined crystal structure of a fragment containing the RBD complexed to ACE2 (Li, F, Li, W, Farzan, M, and Harrison, S. C., submitted) suggested the existence of two hot spots on the S RBD surface, R426 and N473, which are likely to contribute significant portion of the binding energy. The finding that most of the mutations (23 out of 34 including glycosylation sites) do not affect the RBD binding function indicates possible mechanisms for evasion of immune responses.
| S-EPMC1236967 | biostudies-literature