The importance of accessory protein variants in the pathogenicity of SARS-CoV-2.
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ABSTRACT:
SUBMITTER: Hassan SS
PROVIDER: S-EPMC8785432 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A global pandemic is underway caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome, like its predecessor SARS-CoV, contains open reading frames that encode accessory proteins involved in virus-host interactions active during infection and which likely contribute to pathogenesis. One of these accessory proteins is 7b, with only 44 (SARS-CoV) and 43 (SARS-CoV-2) residues. It has one predicted transmembrane domain fully conserved, which suggests a functional role, whereas most variability is contained in the predicted cytoplasmic C-terminus. In SARS-CoV, 7b protein is expressed in infected cells, and the transmembrane domain was necessary and sufficient for Golgi localization. Also, anti-p7b antibodies have been found in the sera of SARS-CoV convalescent patients. In the present study, we have investigated the hypothesis that SARS-2 7b protein forms oligomers with ion channel activity. We show that in both SARS viruses 7b is almost completely α-helical and has a single transmembrane domain. In SDS, 7b forms various oligomers, from monomers to tetramers, but only monomers when exposed to reductants. Combination of SDS gel electrophoresis and analytical ultracentrifugation (AUC) in both equilibrium and velocity modes suggests a dimer-tetramer equilibrium, but a monomer-dimer-tetramer equilibrium in the presence of reductant. This data suggests that although disulfide-linked dimers may be present, they are not essential to form tetramers. Inclusion of pentamers or higher oligomers in the SARS-2 7b model were detrimental to fit quality. Preliminary models of this association was generated with AlphaFold2, and two alternative models were exposed to a molecular dynamics simulation in presence of a model lipid membrane. However, neither of the two models provided any evident pathway for ions. To confirm this, SARS-2 p7b was studied using Planar Bilayer Electrophysiology. Addition of p7b to model membranes produced occasional membrane permeabilization, but this was not consistent with bona fide ion channels made of a tetrameric assembly of α-helices.
| S-EPMC9680129 | biostudies-literature
Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. As of 19 May 2021, John Hopkins University's COVID-19 tracking platform reported 3.3 million deaths associated with SARS-CoV-2 infection. Currently, the World Health Organization has granted emergency use listing (EUL) to six COVID-19 vaccine candidates. However, much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse-genetics system approach to successfully engineer recombinant SARS-CoV-2 (rSARS-CoV-2) constructs; we removed individual viral ORF3a, -6, -7a, -7b, and -8 proteins from them, and we characterized the resulting recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF-deficient (ΔORF) viruses producing smaller plaques than those of the wild type (rSARS-CoV-2/WT). However, growth kinetics of ΔORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mice with the ΔORF rSARS-CoV-2s identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while ΔORF7a, ΔORF7b, and ΔORF8 rSARS-CoV-2s induced pathology comparable to that of rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse-genetics system to generate rSARS-CoV-2 constructs and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19. IMPORTANCE Despite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and the contribution of viral proteins to disease outcome remain elusive. Our study aims (i) to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins to viral pathogenesis and disease outcome and (ii) to develop a synergistic platform combining our robust reverse-genetics system to generate recombinant SARS-CoV-2 constructs with a validated rodent model of infection and disease. We demonstrate that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as a foundation for generating attenuated forms of the virus to develop live attenuated vaccines for the treatment of SARS-CoV-2.
| S-EPMC8354228 | biostudies-literature
Project description:As the world continues to fight the coronavirus pandemic caused by SARS‐CoV‐2, we are gaining valuable insights by comparing this virus to other human coronaviruses such as SARS‐CoV and MERS‐CoV that have also caused outbreaks. Coronaviruses infect many animal species, but disease severity varies between strains. Viral accessory proteins are generally implicated in increased infectivity, pathogenicity, and virulence. The accessory protein open reading frame 8 (ORF8), although not essential for viral replication, appears to play a critical role in disease severity by inhibiting multiple pathways of the immune response. Furthermore, the ORF8 gene is found within a highly variable portion of the genome, increasing the possibility of dangerous mutant forms arising. Structure‐based design of therapeutics holds great promise for effective targeting of such rapidly evolving threats. Thus, we designed physical models and computer representations of ORF8, based on the published structure 7JTL, to better evaluate the structural features responsible for viral pathogenicity. ORF8 exists as a homodimer held together by amino acid residues that interact through hydrophobic interactions, hydrogen bonding, a salt bridge, and a disulfide bond. These interactions occur in a region referred to as the covalent interface. Another interface, referred to as the noncovalent interface, exists between separate homodimers and results in oligomerization. The homodimers in the oligomer are held together by an intermolecular beta sheet that is stabilized by an extensive hydrophobic surface. These two dimerization interfaces are unique to SARS‐CoV‐2 and have been proposed to be involved in the protein's ability to evade and suppress the host immune response. The computer and physical representations allow for better visualization of the ORF8 structure to evaluate the role of the multimeric structure. ORF8 has been found to interact with various host proteins, including Interleukin‐17 Receptor A (IL17RA), a pro‐inflammatory cytokine. To further corroborate the existence of this interaction and to better understand the role oligomerization might play in pathogenicity, we assessed the possible interactions of ORF8 (7JTL) and IL17RA (5N9B) through theoretical modeling using available tools such as HDOCK. A better understanding of these inter‐subunit interactions may improve our understanding of the unique mechanism the virus uses to evade the immune system and may be instrumental in the development of effective therapeutics.
| S-EPMC8239924 | biostudies-literature
Project description:The evolution of SARS-CoV-2 variants with increased fitness has been accompanied by structural changes in the spike (S) proteins, which are the major target for the adaptive immune response. Single-particle cryo-EM analysis of soluble S protein from SARS-CoV-2 variants has revealed this structural adaptation at high resolution. The analysis of S trimers in situ on intact virions has the potential to provide more functionally relevant insights into S structure and virion morphology. Here, we characterized B.1, Alpha, Beta, Gamma, Delta, Kappa, and Mu variants by cryo-electron microscopy and tomography, assessing S cleavage, virion morphology, S incorporation, "in-situ" high-resolution S structures and the range of S conformational states. We found no evidence for adaptive changes in virion morphology, but describe multiple different positions in the S protein where amino acid changes alter local protein structure. Taken together, our data is consistent with a model where amino acid changes at multiple positions from the top to the base of the spike cause structural changes that can modulate the conformational dynamics of the S protein.
| S-SCDT-10_1038-S44318-024-00303-1 | biostudies-other
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:Viruses use many different strategies to evade host immune responses. In the case of SARS-CoV-2, its Spike mutates rapidly to escape from neutralizing antibodies. In addition to this strategy, ORF8, a small accessory protein encoded by SARS-CoV-2, helps immune evasion by reducing the susceptibility of SARS-CoV-2-infected cells to the cytotoxic CD8+ T cell response. Interestingly, among all accessory proteins, ORF8 is rapidly evolving and a deletion in this protein has been linked to milder disease. Here, we studied the effect of ORF8 on peripheral blood mononuclear cells (PBMC). Specifically, we found that ORF8 can bind monocytes as well as NK cells. Strikingly, ORF8 binds CD16a (FcγRIIIA) with nanomolar affinity and decreases the overall level of CD16 at the surface of monocytes and, to a lesser extent, NK cells. This decrease significantly reduces the capacity of PBMCs and particularly monocytes to mediate antibody-dependent cellular cytotoxicity (ADCC). Overall, our data identifies a new immune-evasion activity used by SARS-CoV-2 to escape humoral responses.
| S-EPMC9230529 | biostudies-literature
Project description:Emerging variants enable the continuous spread of SARS-CoV-2 in humans. The factors contributing to behavioral differences in variants remain elusive despite associations with several Spike protein mutations. Exploring accessory proteins may provide a wider understanding of these differences since these proteins may affect viral processes that occur beyond infection. Various bioinformatics tools were utilized to identify significant accessory protein mutations and determine their structural and functional effects over time. The ViruClust web application was used to retrieve accessory protein amino acid sequences and determine mutation frequencies in these sequences across time. The structural and functional effects of the mutations were determined using Missense3D and PROVEAN, respectively. The accessory and Spike protein mutations were compared using mutation densities. Q57H and T151I of ORF3a; T21I and W27L of ORF6; G38V, V82A, and T120I of ORF7a; S31P and T40I of ORF7b; and R52I, C61F, and I121L of ORF8 were highly frequent in most variants of concern and were within known functional domains. Thus, these are good candidates for further experimental evaluation. Among the accessory proteins, ORF6 and ORF8 were highlighted because of their strong and weak correlation with Spike protein mutations, respectively.
| S-EPMC9349927 | biostudies-literature
Project description:We report a live-attenuated SARS-CoV-2 vaccine candidate with (i) re-engineered viral transcription regulator sequences and (ii) deleted open-reading-frames (ORF) 3, 6, 7, and 8 (∆3678). The ∆3678 virus replicates about 7,500-fold lower than wild-type SARS-CoV-2 on primary human airway cultures, but restores its replication on interferon-deficient Vero-E6 cells that are approved for vaccine production. The ∆3678 virus is highly attenuated in both hamster and K18-hACE2 mouse models. A single-dose immunization of the ∆3678 virus protects hamsters from wild-type virus challenge and transmission. Among the deleted ORFs in the ∆3678 virus, ORF3a accounts for the most attenuation through antagonizing STAT1 phosphorylation during type-I interferon signaling. We also developed an mNeonGreen reporter ∆3678 virus for high-throughput neutralization and antiviral testing. Altogether, the results suggest that ∆3678 SARS-CoV-2 may serve as a live-attenuated vaccine candidate and a research tool for potential biosafety level-2 use.
| S-EPMC9326133 | biostudies-literature
Project description:The interactions between Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and human host factors enable the virus to propagate infections that lead to Coronavirus Disease 2019 (COVID-19). The spike protein is the largest structural component of the virus and mediates interactions essential for infection, including with the primary angiotensin-converting enzyme 2 (ACE2) receptor. We performed two independent cell-based systematic screens to determine whether there are additional proteins by which the spike protein of SARS-CoV-2 can interact with human cells. We discovered that in addition to ACE2, expression of LRRC15 also causes spike protein binding. This interaction is distinct from other known spike attachment mechanisms such as heparan sulfates or lectin receptors. Measurements of orthologous coronavirus spike proteins implied the interaction was functionally restricted to SARS-CoV-2 by accessibility. We localized the interaction to the C-terminus of the S1 domain and showed that LRRC15 shares recognition of the ACE2 receptor binding domain. From analyzing proteomics and single-cell transcriptomics, we identify LRRC15 expression as being common in human lung vasculature cells and fibroblasts. Levels of LRRC15 were greatly elevated by inflammatory signals in the lungs of COVID-19 patients. Although infection assays demonstrated that LRRC15 alone is not sufficient to permit viral entry, we present evidence that it can modulate infection of human cells. This unexpected interaction merits further investigation to determine how SARS-CoV-2 exploits host LRRC15 and whether it could account for any of the distinctive features of COVID-19.
| S-EPMC9897555 | biostudies-literature
Project description:hACE2 transgenic mice were infected with the original SARS-CoV-2 strain (B.1) and the Beta (B.1.351) variant. Lung and spleen samples were collected 1 day post infection (DPI), 3 DPI and 5 DPI, and mRNA was sequenced.
2023-09-22 | E-MTAB-13308 | biostudies-arrayexpress