DSC Study of SARS-CoV-2 ORF10 Protein and Its Complexes with Water-Soluble Metal Phthalocyanines

A theoretical and experimental study of the interaction of the SARS-CoV-2 ORF10 protein with sulfosubstituted cobalt(II) and copper(II) phthalocyanines was carried out. The structures of the most probable complexes of metal phthalocyanines with the ORF10 protein were obtained by molecular docking methods. Cobalt(II) tetrasulfophthal ocyanine binds to the protein in the monomeric state, while the interaction ofORF1 0 with copper(II) tetrasulfophthalocyanine causes aggregation of the formed protein complexes, which was shown by the UV-Vis spectroscopy. Thermal denaturation of the ORF10 protein and its complexes with metal phthalocyanines was studied by differential scanning calorimetry. A joint analysis of the spectral and thermochemical data made it possible to propose a description of the mechanism of thermal denaturation ofthe ORF10 protein.

Effect of cannabidiol on apoptosis and cellular interferon and interferon-stimulated gene responses to the SARS-CoV-2 genes ORF8, ORF10 and M protein.

AIMS: To study effects on cellular innate immune responses to ORF8, ORF10, and Membrane protein (M protein) from the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, in combination with cannabidiol (CBD). MAIN METHODS: HEK293 cells transfected with plasmids expressing control vector, ORF8, ORF10, or M protein were assayed for cell number and markers of apoptosis at 24 h, and interferon and interferon-stimulated gene expression at 14 h, with or without CBD. Cells transfected with polyinosinic:polycytidylic acid (Poly (I:C)) were also studied as a general model of RNA-type viral infection. KEY FINDINGS: Reduced cell number and increased early and late apoptosis were found when expression of viral genes was combined with 1-2 µM CBD treatment, but not in control-transfected cells treated with CBD, or in cells expressing viral genes but treated only with vehicle. In cells expressing viral genes, CBD augmented expression of IFNγ, IFNλ1 and IFNλ2/3, as well as the 2'-5'-oligoadenylate synthetase (OAS) family members OAS1, OAS2, OAS3, and OASL. CBD also augmented expression of these genes in control cells not expressing viral genes, but without enhancing apoptosis. CBD similarly enhanced the cellular anti-viral response to Poly (I:C). SIGNIFICANCE: Our results demonstrate a poor ability of HEK293 cells to respond to SARS-CoV-2 genes alone, but an augmented innate anti-viral response to these genes in the presence of CBD. Thus, CBD may prime components of the innate immune system, increasing readiness to respond to RNA-type viral infection without activating apoptosis, and could be studied for potential in prophylaxis.

Computational Structural and Functional Analyses of ORF10 in Novel Coronavirus SARS-CoV-2 Variants to Understand Evolutionary Dynamics.

Introduction: In an effort to combat SARS-CoV-2 through multi-subunit vaccine design, during studies using whole genome and immunome, ORF10, located at the 3' end of the genome, displayed unique features. It showed no homology to any known protein in other organisms, including SARS-CoV. It was observed that its nucleotide sequence is 100% identical in the SARS-CoV-2 genomes sourced worldwide, even in the recent-most VoCs and VoIs of B.1.1.529 (Omicron), B.1.617 (Delta), B.1.1.7 (Alpha), B.1.351 (Beta), and P.1 (Gamma) lineages, implicating its constant nature throughout the evolution of deadly variants. Aim: The structure and function of SARS-CoV-2 ORF10 and the role it may play in the viral evolution is yet to be understood clearly. The aim of this study is to predict its structure, function, and understand evolutionary dynamics on the basis of mutations and likely heightened immune responses in the immunopathogenesis of this deadly virus. Methods: Sequence analysis, ab-initio structure modeling and an understanding of the impact of likely substitutions in key regions of protein was carried out. Analyses of viral T cell epitopes and primary anchor residue mutations was done to understand the role it may play in the evolution as a molecule with likely enhanced immune response and consequent immunopathogenesis. Results: Few amino acid substitution mutations are observed, most probably due to the ribosomal frameshifting, and these mutations may not be detrimental to its functioning. As ORF10 is observed to be an expressed protein, ab-initio structure modeling shows that it comprises mainly an α-helical region and maybe an ER-targeted membrane mini-protein. Analyzing the whole proteome, it is observed that ORF10 presents amongst the highest number of likely promiscuous and immunogenic CTL epitopes, specifically 11 out of 30 promiscuous ones and 9 out of these 11, immunogenic CTL epitopes. Reactive T cells to these epitopes have been uncovered in independent studies. Majority of these epitopes are located on the α-helix region of its structure, and the substitution mutations of primary anchor residues in these epitopes do not affect immunogenicity. Its conserved nucleotide sequence throughout the evolution and diversification of virus into several variants is a puzzle yet to be solved. Conclusions: On the basis of its sequence, structure, and epitope mapping, it is concluded that it may function like those mini-proteins used to boost immune responses in medical applications. Due to the complete nucleotide sequence conservation even a few years after SARS-CoV-2 genome was first sequenced, it poses a unique puzzle to be solved, in view of the evolutionary dynamics of variants emerging in the populations worldwide.

SARS-CoV-2 ORF10 antagonizes STING-dependent interferon activation and autophagy.

A characteristic feature of COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is the dysregulated immune response with impaired type I and III interferon (IFN) expression and an overwhelming inflammatory cytokine storm. RIG-I-like receptors (RLRs) and cGAS-STING signaling pathways are responsible for sensing viral infection and inducing IFN production to combat invading viruses. Multiple proteins of SARS-CoV-2 have been reported to modulate the RLR signaling pathways to achieve immune evasion. Although SARS-CoV-2 infection also activates the cGAS-STING signaling by stimulating micronuclei formation during the process of syncytia, whether SARS-CoV-2 modulates the cGAS-STING pathway requires further investigation. Here, we screened 29 SARS-CoV-2-encoded viral proteins to explore the viral proteins that affect the cGAS-STING signaling pathway and found that SARS-CoV-2 open reading frame 10 (ORF10) targets STING to antagonize IFN activation. Overexpression of ORF10 inhibits cGAS-STING-induced interferon regulatory factor 3 phosphorylation, translocation, and subsequent IFN induction. Mechanistically, ORF10 interacts with STING, attenuates the STING-TBK1 association, and impairs STING oligomerization and aggregation and STING-mediated autophagy; ORF10 also prevents the endoplasmic reticulum (ER)-to-Golgi trafficking of STING by anchoring STING in the ER. Taken together, these findings suggest that SARS-CoV-2 ORF10 impairs the cGAS-STING signaling by blocking the translocation of STING and the interaction between STING and TBK1 to antagonize innate antiviral immunity.

Spectral and theoretical study of SARS-CoV-2 ORF10 protein interaction with endogenous and exogenous macroheterocyclic compounds.

The coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 coronavirus has spread rapidly around the world in a matter of weeks. Most of the current recommendations developed for the use of antivirals in COVID-19 were developed during the initial waves of the pandemic, when resources were limited and administrative or pragmatic criteria took precedence. The choice of drugs for the treatment of COVID-19 was carried out from drugs approved for medical use. COVID-19 is a serious public health problem and the search for drugs that can relieve the disease in infected patients at various stages is still necessary. Therefore, the search for effective drugs with inhibitory and/or virucidal activity is a paramount task. Accessory proteins of the virus play a significant role in the pathogenesis of the disease, as they modulate the host's immune response. This paper studied the interaction of one of the SARS-CoV-2 accessory proteins ORF10 with macroheterocyclic compounds - protoporphyrin IX d.m.e., Fe(III)protoporphyrin d.m.e. and 5,10,15,20-tetrakis(3'-pyridyl)chlorin tetraiodide, which are potential inhibitors and virucidal agents. The SARS-CoV-2 ORF10 protein shows the highest affinity for Chlorin, which binds hydrophobically to the alpha structured region of the protein. Protoporphyrin is able to form several complexes with ORF10 close in energy, with alpha- and beta-molecular recognition features, while Fe(III)protoporphyrin forms complexes with the orientation of the porphyrin macrocycle parallel to the ORF10 alpha-helix. Taking into account the nature of the interaction with ORF10, it has been suggested that Chlorin may have virucidal activity upon photoexposure. The SARS-CoV-2 ORF10 protein was expressed in Escherichia coli cells, macroheterocyclic compounds were synthesized, and the structure was confirmed. The interaction between macrocycles with ORF10 was studied by spectral methods. The results of in silico studies were confirmed by experimental data.

Structural insights into ORF10 recognition by ZYG11B.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a major threat to human health. As a unique putative protein of SARS-CoV-2, the N-terminus of ORF10 can be recognized by ZYG11B, a substrate receptor of the Cullin 2-RING E3 ubiquitin ligase (CRL2). Here we elucidated recognition mechanism of ORF10 N-terminus by ZYG11B through presenting the crystal structure of ZYG11B bound to ORF10 N-terminal peptide. Our work expands the current understanding of ORF10 interaction with ZYG11B, and may also inspire the development of novel therapies for COVID-19.

Emergence of unique SARS-CoV-2 ORF10 variants and their impact on protein structure and function.

The devastating impact of the ongoing coronavirus disease 2019 (COVID-19) on public health, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has made targeting the COVID-19 pandemic a top priority in medical research and pharmaceutical development. Surveillance of SARS-CoV-2 mutations is essential for the comprehension of SARS-CoV-2 variant diversity and their impact on virulence and pathogenicity. The SARS-CoV-2 open reading frame 10 (ORF10) protein interacts with multiple human proteins CUL2, ELOB, ELOC, MAP7D1, PPT1, RBX1, THTPA, TIMM8B, and ZYG11B expressed in lung tissue. Mutations and co-occurring mutations in the emerging SARS-CoV-2 ORF10 variants are expected to impact the severity of the virus and its associated consequences. In this article, we highlight 128 single mutations and 35 co-occurring mutations in the unique SARS-CoV-2 ORF10 variants. The possible predicted effects of these mutations and co-occurring mutations on the secondary structure of ORF10 variants and host protein interactomes are presented. The findings highlight the possible effects of mutations and co-occurring mutations on the emerging 140 ORF10 unique variants from secondary structure and intrinsic protein disorder perspectives.

SARS-CoV-2 ORF10 suppresses the antiviral innate immune response by degrading MAVS through mitophagy.

The global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused severe morbidity and mortality in humans. It is urgent to understand the function of viral genes. However, the function of open reading frame 10 (ORF10), which is uniquely expressed by SARS-CoV-2, remains unclear. In this study, we showed that overexpression of ORF10 markedly suppressed the expression of type I interferon (IFN-I) genes and IFN-stimulated genes. Then, mitochondrial antiviral signaling protein (MAVS) was identified as the target via which ORF10 suppresses the IFN-I signaling pathway, and MAVS was found to be degraded through the ORF10-induced autophagy pathway. Furthermore, overexpression of ORF10 promoted the accumulation of LC3 in mitochondria and induced mitophagy. Mechanistically, ORF10 was translocated to mitochondria by interacting with the mitophagy receptor Nip3-like protein X (NIX) and induced mitophagy through its interaction with both NIX and LC3B. Moreover, knockdown of NIX expression blocked mitophagy activation, MAVS degradation, and IFN-I signaling pathway inhibition by ORF10. Consistent with our observations, in the context of SARS-CoV-2 infection, ORF10 inhibited MAVS expression and facilitated viral replication. In brief, our results reveal a novel mechanism by which SARS-CoV-2 inhibits the innate immune response; that is, ORF10 induces mitophagy-mediated MAVS degradation by binding to NIX.

Rare mutations in the accessory proteins ORF6, ORF7b, and ORF10 of the SARS-CoV-2 genomes.

A total number of 3080 SARS-CoV-2 genomes from all continents are considered from the NCBI database. Every accessory protein ORF6, ORF7b, and ORF10 of SARS-CoV-2 possess a single missense mutation in less than 1.5% of the 3080 genomes. It has now been observed that different non-synonymous mutations occurred in these three accessory proteins. Most of these rare mutations are changing the amino acids such as hydrophilic to hydrophobic, acidic or basic to hydrophobic, and vice versa etc. So these highly conserved proteins might play an essential role in virus pathogenicity. This study opens a question whether it carries some messages about the virus rapid replications, and virulence.

Notable sequence homology of the ORF10 protein introspects the architecture of SARS-CoV-2.

The current Coronavirus Disease 19 (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) shows similar pathology to MERS and SARS-CoV, with a current estimated fatality rate of 1.4%. Open reading frame 10 (ORF10) is a unique SARS-CoV-2 accessory protein, which contains eleven cytotoxic T lymphocyte (CTL) epitopes each of nine amino acids in length. Twenty-two unique SARS-CoV-2 ORF10 variants have been identified based on missense mutations found in sequence databases. Some of these mutations are predicted to decrease the stability of ORF10 in silico physicochemical and structural comparative analyses were carried out on SARS-CoV-2 and Pangolin-CoV ORF10 proteins, which share 97.37% amino acid (aa) homology. Though there is a high degree of ORF10 protein similarity of SARS-CoV-2 and Pangolin-CoV, there are differences of these two ORF10 proteins related to their sub-structure (loop/coil region), solubility, antigenicity and shift from strand to coil at aa position 26 (tyrosine). SARS-CoV-2 ORF10, which is apparently expressed in vivo since reactive T cell clones are found in convalescent patients should be monitored for changes which could correlate with the pathogenesis of COVID-19.

The percentages of SARS-CoV-2 protein similarity and identity with SARS-CoV and BatCoV RaTG13 proteins can be used as indicators of virus origin.

There are three types of proteins in coronaviruses: nonstructural, structural, and accessory proteins. Coronavirus proteins are essential for viral replication and for the binding and invasion of hosts and the regulation of host cell metabolism and immunity. This study investigated the amino acid sequence similarity and identity percentages of 10 proteins in SARS-CoV-2, SARS-CoV and the Rhinolophus affinis bat coronavirus (BatCoV RaTG13). The investigated proteins were the 1ab polyprotein, spike protein, orf3a, the envelope protein, the membrane protein, orf6, orf7a, orf7b, orf8, and the nucleocapsid protein. The online sequence alignment service of The European Molecular Biology Open Software Suite (EMBOSS) was used to determine the percentages of protein similarity and identity in the three viruses. The results showed that the similarity and identity percentages of the SARS-CoV-2 and BatCoV RaTG13 proteins were both greater than 95%, while the identity and similarity percentages of SARS-CoV-2 and SARS-CoV were both greater than 38%. The proteins of SARS-CoV-2 and BatCoV RaTG13 have high identity and similarity compared to those of SARS-CoV-2 and SARS-CoV. GRAPHIC ABSTRACT: The proteins of the SARS-CoV-2 are most identical and similar to those of BatCoV RaTG13 than to the proteins of SARS-CoV. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42485-021-00060-3.

ORF10-Cullin-2-ZYG11B complex is not required for SARS-CoV-2 infection.

In order to understand the transmission and virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to understand the functions of each of the gene products encoded in the viral genome. One feature of the SARS-CoV-2 genome that is not present in related, common coronaviruses is ORF10, a putative 38-amino acid protein-coding gene. Proteomic studies found that ORF10 binds to an E3 ubiquitin ligase containing Cullin-2, Rbx1, Elongin B, Elongin C, and ZYG11B (CRL2ZYG11B). Since CRL2ZYG11B mediates protein degradation, one possible role for ORF10 is to "hijack" CRL2ZYG11B in order to target cellular, antiviral proteins for ubiquitylation and subsequent proteasomal degradation. Here, we investigated whether ORF10 hijacks CRL2ZYG11B or functions in other ways, for example, as an inhibitor or substrate of CRL2ZYG11B While we confirm the ORF10-ZYG11B interaction and show that the N terminus of ORF10 is critical for it, we find no evidence that ORF10 is functioning to inhibit or hijack CRL2ZYG11B Furthermore, ZYG11B and its paralog ZER1 are dispensable for SARS-CoV-2 infection in cultured cells. We conclude that the interaction between ORF10 and CRL2ZYG11B is not relevant for SARS-CoV-2 infection in vitro.

SARS-CoV-2 Proteins: Are They Useful as Targets for COVID-19 Drugs and Vaccines?

The proteins of coronavirus are classified as non-structural, structural, and accessory. There are 16 non-structural viral proteins besides their precursors (1a and 1ab polyproteins). The non-structural proteins are named nsp1 to nsp16, and they act as enzymes, coenzymes, and binding proteins to facilitate the replication, transcription, and translation of the virus. The structural proteins are bound to the RNA in the nucleocapsid (N- protein) or to the lipid bilayer membrane of the viral envelope. The lipid bilayer proteins include the membrane protein (M), an envelope protein (E), and spike protein (S). Besides their role as structural proteins, they are essential for the host cells' binding and invasion. The SARS-CoV-2 contains six accessory proteins which participate in the viral replication, assembly and virus-host interactions. The SARS-CoV-2 accessory proteins are orf3a, orf6, orf7a, orf7b, orf8, and orf10. The functions of the SARS-CoV-2 are not well known, while the functions of their corresponding proteins in SARS-CoV are either well known or poorly studied. Recently, the Oxford University and Astrazeneca, Pfizer and BioNTech have made SARS-CoV-2 vaccines by targeting the spike protein gene. The US Food and Drug Administration (FDA) and the health authorities of the United Kingdom have approved and started conducting vaccinations using the Pfizer and BioNTech mRNA vaccine. Also, The FDA of the USA has approved the use of two monoclonal antibodies produced by Regeneron pharmaceuticals to target the spike protein for treating COVID-19. The SARS-CoV-2 proteins can be used for the diagnosis, as drug targets and in vaccination trials for COVID-19. In future COVID-19 research, more efforts should be made to elaborate the functions and structure of the SARS-CoV- 2 proteins so as to use them as targets for COVID-19 drugs and vaccines. Special attention should be paid to extensive research on the SARS-CoV-2 nsp3, orf8, and orf10.

Differential Expression of Viral Transcripts From Single-Cell RNA Sequencing of Moderate and Severe COVID-19 Patients and Its Implications for Case Severity.

With steady increase of new COVID-19 cases around the world, especially in the United States, health care resources in areas with the disease outbreak are quickly exhausted by overwhelming numbers of COVID-19 patients. Therefore, strategies that can effectively and quickly predict the disease progression and stratify patients for appropriate health care arrangements are urgently needed. We explored the features and evolutionary difference of viral gene expression in the SARS-CoV-2 infected cells from the bronchoalveolar lavage fluids of patients with moderate and severe COVID-19 using both single cell and bulk tissue transcriptome data. We found SARS-CoV-2 sequences were detectable in 8 types of immune related cells, including macrophages, T cells, and NK cells. We first reported that the SARS-CoV-2 ORF10 gene was differentially expressed in the severe vs. moderate samples. Specifically, ORF10 was abundantly expressed in infected cells of severe cases, while it was barely detectable in the infected cells of moderate cases. Consequently, the expression ratio of ORF10 to nucleocapsid (N) was significantly higher in severe than moderate cases (p = 0.0062). Moreover, we found transcription regulatory sequences (TRSs) of the viral leader sequence-independent fusions with a 5' joint point at position 1073 of SARS-CoV-2 genome were detected mainly in the patients with death outcome, suggesting its potential indication of clinical outcome. Finally, we identified the motifs in TRS of the viral leader sequence-dependent fusion events of SARS-CoV-2 and compared with that in SARS-CoV, suggesting its evolutionary trajectory. These results implicated potential roles and predictive features of viral transcripts in the pathogenesis of COVID-19 moderate and severe patients. Such features and evolutionary patterns require more data to validate in future.