Membrane Atg8ylation, stress granule formation, and MTOR regulation during lysosomal damage.

The functions of mammalian Atg8 proteins (mATG8s) expand beyond canonical autophagy and include processes collectively referred to as Atg8ylation. Global modulation of protein synthesis under stress conditions is governed by MTOR and liquid-liquid phase separated condensates containing ribonucleoprotein particles known as stress granules (SGs). We report that lysosomal damage induces SGs acting as a hitherto unappreciated inhibitor of protein translation via EIF2A/eIF2α phosphorylation while favoring an ATF4-dependent integrated stress response. SGs are induced by lysosome-damaging agents, SARS-CoV-2 open reading frame 3a protein (ORF3a) expression, Mycobacterium tuberculosis infection, and exposure to proteopathic MAPT/tau. Proteomic studies revealed recruitment to damaged lysosomes of the core SG proteins NUFIP2 and G3BP1 along with the GABARAPs of the mATG8 family. The recruitment of these proteins is independent of SG condensates or canonical autophagy. GABARAPs interact directly with NUFIP2 and G3BP1 whereas Atg8ylation is needed for their recruitment to damaged lysosomes. At the lysosome, NUFIP2 contributes to MTOR inactivation together with LGALS8 (galectin 8) via the Ragulator-RRAGA-RRAGB complex. The separable functions of NUFIP2 and G3BP1 in SG formation vis-a-vis their role in MTOR inactivation are governed by GABARAP and Atg8ylation. Thus, cells employ membrane Atg8ylation to control and coordinate SG and MTOR responses to lysosomal damage.Abbreviations: Atg8: autophagy related 8; ATG: autophagy related; ATF4: activating transcription factor 4; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; GABARAP: GABA type A receptor-associated protein; G3BP1: G3BP stress granule assembly factor 1; LLOMe: L-leucyl-L-leucine methyl ester; LysoIP: lysosome immunopurification; mRNA: messenger ribonucleic acid; MTOR: mechanistic target of rapamycin kinase; NUFIP2: nuclear FMR1 interacting protein 2; ORF3a: open reading frame 3a protein; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SG: stress granule; TIA1: TIA1 cytotoxic granule associated RNA binding protein.

Structural biology of SARS-CoV-2 accessory proteins

The coronavirus SARS-CoV-2 is the causative agent for the COVID-19 pandemic. Its proteome is typically separated into three classes of proteins: (1) Structural proteins which facilitate the transport and host cell infiltration of the viral RNA, (2) non-structural proteins which are thought to be essential for the viral life cycle and are all produced from open reading frame 1ab (ORF1ab) on the RNA, and (3) everything else, called accessory proteins. Although it was originally thought that these accessory proteins are non-essential for viral replication, a growing body of evidence suggests that these diverse proteins have crucial roles in virus-host interactions, in particular in the way they interfere with the signalling pathways that modulate the host cell's response to infection and viral pathogenicity. Here, we summarize efforts to structurally characterize the accessory proteins from SARS-CoV-2.

SARS-CoV-2 ORF3a sensitizes cells to ferroptosis via Keap1-NRF2 axis.

Viral infection-induced cell death has long been considered as a double-edged sword in the inhibition or exacerbation of viral infections. Patients with severe Coronavirus Disease 2019 (COVID-19) are characterized by multiple organ dysfunction syndrome and cytokine storm, which may result from SARS-CoV-2-induced cell death. Previous studies have observed enhanced ROS level and signs of ferroptosis in SARS-CoV-2 infected cells or specimens of patients with COVID-19, but the exact mechanism is not clear yet. Here, we find SARS-CoV-2 ORF3a sensitizes cells to ferroptosis via Keap1-NRF2 axis. SARS-CoV-2 ORF3a promotes the degradation of NRF2 through recruiting Keap1, thereby attenuating cellular resistance to oxidative stress and facilitated cells to ferroptotic cell death. Our study uncovers that SARS-CoV-2 ORF3a functions as a positive regulator of ferroptosis, which might explain SARS-CoV-2-induced damage in multiple organs in COVID-19 patients and imply the potential of ferroptosis inhibition in COVID-19 treatment.

Autophagy Receptor p62 Regulates SARS-CoV-2-Induced Inflammation in COVID-19.

As autophagy can promote or inhibit inflammation, we examined autophagy-inflammation interplay in COVID-19. Autophagy markers in the blood of 19 control subjects and 26 COVID-19 patients at hospital admission and one week later were measured by ELISA, while cytokine levels were examined by flow cytometric bead immunoassay. The antiviral IFN-α and proinflammatory TNF, IL-6, IL-8, IL-17, IL-33, and IFN-γ were elevated in COVID-19 patients at both time points, while IL-10 and IL-1ß were increased at admission and one week later, respectively. Autophagy markers LC3 and ATG5 were unaltered in COVID-19. In contrast, the concentration of autophagic cargo receptor p62 was significantly lower and positively correlated with TNF, IL-10, IL-17, and IL-33 at hospital admission, returning to normal levels after one week. The expression of SARS-CoV-2 proteins NSP5 or ORF3a in THP-1 monocytes caused an autophagy-independent decrease or autophagy-inhibition-dependent increase, respectively, of intracellular/secreted p62, as confirmed by immunoblot/ELISA. This was associated with an NSP5-mediated decrease in TNF/IL-10 mRNA and an ORF3a-mediated increase in TNF/IL-1ß/IL-6/IL-10/IL-33 mRNA levels. A genetic knockdown of p62 mimicked the immunosuppressive effect of NSP5, and a p62 increase in autophagy-deficient cells mirrored the immunostimulatory action of ORF3a. In conclusion, the proinflammatory autophagy receptor p62 is reduced inacute COVID-19, and the balance between autophagy-independent decrease and autophagy blockade-dependent increase of p62 levels could affect SARS-CoV-induced inflammation.

Severe acute respiratory syndrome coronavirus-2 accessory proteins ORF3a and ORF7a modulate autophagic flux and Ca2+ homeostasis in yeast.

Virus infection involves the manipulation of key host cell functions by specialized virulence proteins. The Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) small accessory proteins ORF3a and ORF7a have been implicated in favoring virus replication and spreading by inhibiting the autophagic flux within the host cell. Here, we apply yeast models to gain insights into the physiological functions of both SARS-CoV-2 small open reading frames (ORFs). ORF3a and ORF7a can be stably overexpressed in yeast cells, producing a decrease in cellular fitness. Both proteins show a distinguishable intracellular localization. ORF3a localizes to the vacuolar membrane, whereas ORF7a targets the endoplasmic reticulum. Overexpression of ORF3a and ORF7a leads to the accumulation of Atg8 specific autophagosomes. However, the underlying mechanism is different for each viral protein as assessed by the quantification of the autophagic degradation of Atg8-GFP fusion proteins, which is inhibited by ORF3a and stimulated by ORF7a. Overexpression of both SARS-CoV-2 ORFs decreases cellular fitness upon starvation conditions, where autophagic processes become essential. These data confirm previous findings on SARS-CoV-2 ORF3a and ORF7a manipulating autophagic flux in mammalian cell models and are in agreement with a model where both small ORFs have synergistic functions in stimulating intracellular autophagosome accumulation, ORF3a by inhibiting autophagosome processing at the vacuole and ORF7a by promoting autophagosome formation at the ER. ORF3a has an additional function in Ca2+ homeostasis. The overexpression of ORF3a confers calcineurin-dependent Ca2+ tolerance and activates a Ca2+ sensitive FKS2-luciferase reporter, suggesting a possible ORF3a-mediated Ca2+ efflux from the vacuole. Taken together, we show that viral accessory proteins can be functionally investigated in yeast cells and that SARS-CoV-2 ORF3a and ORF7a proteins interfere with autophagosome formation and processing as well as with Ca2+ homeostasis from distinct cellular targets.

Exploring polyamine interactions and binding pockets in SARS-CoV-2 ORF3a.

Ongoing global pandemic caused by coronavirus (COVID-19) requires urgent development of vaccines, treatments, and diagnostic tools. Open reading frame 3a (ORF3a) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is considered to be a potential drug target for COVID-19 treatment. ORF3a is an accessory protein that plays a significant role in virus-host interactions and in facilitating host immune responses. Using putrescine, spermidine and spermine, an aliphatic polyamine for the activity suppression of ORF3a appears to be a promising approach in finding new targets for drug design. In this study, we explored the possible binding poses of polyamines to the ORF3a protein using a combination of various computational approaches i.e. pocket prediction, blind and site-specific molecular docking, molecular dynamics and ligand flooding simulations. The results showed that the tip of cytoplasmic domain and the upper tunnel of transmembrane domain of ORF3a provide a suitable binding site specific for the polyamines. MD simulations revealed the stability of spermidine binding in the upper tunnel pocket of ORF3a through salt bridge and hydrogen bond interactions between the amine groups of the ligand and negatively charged residues of ORF3a. These findings can be helpful in designing new therapeutic drugs.

Targeting viral ORF3a protein: A new approach to mitigate COVID- 19 induced Immune cell apoptosis and associated respiratory complications

Infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a growing concern to the global well-being of the public at present. Different amino acid mutations alter the biological and epidemiological characteristics, as well as immune resistance of SARS-CoV-2. The virus-induced pulmonary impairment and inflammatory cytokine storm are directly related to its clinical manifestations. But, the fundamental mechanisms of inflammatory responses are found to be the reason for the death of immune cells which render the host immune system failure. Apoptosis of immune cells is one of the most common forms of programmed cell death induced by the virus for its survival and virulence property. ORF3a, a SARS-CoV-2 accessory viral protein, induces apoptosis in host cells and suppress the defense mechanism. This suggests, inhibiting SARS-CoV-2 ORF3a protein is a good therapeutic strategy for the treatment in COVID-19 infection by promoting the host immune defense mechanism.Copyright © 2023 Tabriz University of Medical Sciences. All rights reserved.

Polypharmacology of some medicinal plant metabolites against SARS-CoV-2 and host targets: Molecular dynamics evaluation of NSP9 RNA binding protein.

Medicinal plants as rich sources of bioactive compounds are now being explored for drug development against COVID-19. 19 medicinal plants known to exhibit antiviral and anti-inflammatory effects were manually curated, procuring a library of 521 metabolites; this was virtually screened against NSP9, including some other viral and host targets and were evaluated for polypharmacological indications. Leads were identified via rigorous scoring thresholds and ADMET filtering. MM-GBSA calculation was deployed to select NSP9-Lead complexes and the complexes were evaluated for their stability and protein-ligand communication via MD simulation. We identified 5 phytochemical leads for NSP9, 23 for Furin, 18 for ORF3a, and 19 for IL-6. Ochnaflavone and Licoflavone B, obtained from Lonicera japonica (Japanese Honeysuckle) and Glycyrrhiza glabra (Licorice), respectively, were identified to have the highest potential polypharmacological properties for the aforementioned targets and may act on multiple pathways simultaneously to inhibit viral entry, replication, and disease progression. Additionally, MD simulation supports the robust stability of Ochnaflavone and Licoflavone B against NSP9 at the active sites via hydrophobic interactions, H-bonding, and H-bonding facilitated by water. This study promotes the initiation of further experimental analysis of natural product-based anti-COVID-19 therapeutics. Communicated by Ramaswamy H. Sarma.

Genetic variety of ORF3a shapes SARS-CoV-2 fitness through modulation of lipid droplet.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection leads to the accumulation of lipid droplets (LD), the central hubs of the lipid metabolism, in vitro or in type II pneumocytes and monocytes from coronavirus disease 19 (COVID-19) patients and blockage of LD formation by specific inhibitors impedes SARS-CoV-2 replication. Here, we showed that ORF3a is necessary and sufficient to trigger LD accumulation during SARS-CoV-2 infection, leading to efficient virus replication. Although highly mutated during evolution, ORF3a-mediated LD modulation is conserved in most SARS-CoV-2 variants except the Beta strain and is a major difference between SARS-CoV and SARS-CoV-2 that depends on the genetic variations on the amino acid position 171, 193, and 219 of ORF3a. Importantly, T223I substitution in recent Omicron strains (BA.2-BF.8) impairs ORF3a-Vps39 association and LD accumulation, leading to less efficient replication and potentially contributing to lower pathogenesis of the Omicron strains. Our work characterized how SARS-CoV-2 modulates cellular lipid homeostasis to benefit its replication during virus evolution, making ORF3a-LD axis a promising drug target for the treatment of COVID-19.

A novel diG motif in ORF3a protein of SARS-Cov-2 for intracellular transport.

The ongoing SARS-CoV-2/COVID-19 pandemic caused a global public health crisis. Yet, everyone's response to SARS-CoV-2 infection varies, and different viral variants confer diverse pathogenicity. Thus, it is imperative to understand how viral determinants contribute to COVID-19. Viral ORF3a protein is one of those viral determinants, as its functions are linked to induction of cell and tissues damages, disease severity and cytokine storm that is a major cause of COVID-19-related death. ORF3a is a membrane-associated protein. Upon synthesis, it is transported from endoplasmic reticulum, Golgi apparatus to plasma membrane and subcellular endomembranes including endosomes and lysosomes. However, how ORF3a is transported intracellularly remains elusive. The goal of this study was to carry out a systematic mutagenesis study to determine the structural relationship of ORF3a protein with its subcellular locations. Single amino acid (aa) and deletion mutations were generated in the putative function-relevant motifs and other regions of interest. Immunofluorescence and ImageJ analyses were used to determine and quantitate subcellular locations of ORF3a mutants in comparison with wildtype ORF3a. The wildtype ORF3a localizes predominantly (Pearson's coefficients about 0.8) on the membranes of endosomes and lysosomes. Consistent with earlier findings, deletion of the YXXΦ motif, which is required for protein export, retained ORF3a in the Golgi apparatus. Interestingly, mutations in a double glycine (diG) region (aa 187-188) displayed a similar phenotype to the YXXΦ deletion, implicating a similar role of the diG motif in intracellular transport. Indeed, interrupting any one of the two glycine residues such as deletion of a single (dG188), both (dG187/dG188) or substitution (G188Y) of these residues led to ORF3a retention in the Golgi apparatus (Pearson's coefficients ≥0.8). Structural analyses further suggest that the diG motif supports a type-II ß-turn between the anti-parallel ß4 and ß5 sheets and connects to the YXXΦ motif via hydrogen bonds between two monomers. The diG- YXXΦ interaction forms a hand-in-hand configuration that could facilitate dimerization. Together, these observations suggest a functional role of the diG motif in intracellular transport of ORF3a.

SARS-CoV-2 ORF3a positively regulates NF-κB activity by enhancing IKKß-NEMO interaction.

Coronavirus disease 2019 (COVID-19) is a global pandemic caused by SARS-CoV-2 infection. Patients with severe COVID-19 exhibit robust induction of proinflammatory cytokines, which are closely associated with the development of acute respiratory distress syndrome. However, the underlying mechanisms of the NF-κB activation mediated by SARS-CoV-2 infection remain poorly understood. Here, we screened SARS-CoV-2 genes and found that ORF3a induces proinflammatory cytokines by activating the NF-κB pathway. Moreover, we found that ORF3a interacts with IKKß and NEMO and enhances the interaction of IKKß-NEMO, thereby positively regulating NF-κB activity. Together, these results suggest ORF3a may play pivotal roles in the pathogenesis of SARS-CoV-2 and provide novel insights into the interaction between host immune responses and SARS-CoV-2 infection.

Probing the mechanical properties of ORF3a protein, a transmembrane channel of SARS-CoV-2 virus: Molecular dynamics study.

SARS-CoV-2-encoded accessory protein ORF3a was found to be a conserved coronavirus protein that shows crucial roles in apoptosis in cells as well as in virus release and replications. To complete the knowledge and identify the unknown of this protein, further comprehensive research is needed to clarify the leading role of ORF3a in the functioning of the coronavirus. One of the efficient approaches to determining the functionality of this protein is to investigate the mechanical properties and study its structural dynamics in the presence of physical stimuli. Herein, performing all-atom steered molecular dynamics (SMD) simulations, the mechanical properties of the force-bearing components of the ORF3a channel are calculated in different physiological conditions. As variations occurring in ORF3a may lead to alteration in protein structure and function, the G49V mutation was also simulated to clarify the relationship between the mechanical properties and chemical stability of the protein by comparing the behavior of the wild-type and mutant Orf3a. From a physiological conditions point of view, it was observed that in the solvated system, the presence of water molecules reduces Young's modulus of TM1 by ∼30 %. Our results also show that by substitution of Gly49 with valine, Young's modulus of the whole helix increases from 1.61 ± 0.20 to 2.08 ± 0.15 GPa, which is consistent with the calculated difference in free energy of wild-type and mutant helices. In addition to finding a way to fight against Covid-19 disease, understanding the mechanical behavior of these biological nanochannels can lead to the development of the potential applications of the ORF3a protein channel, such as tunable nanovalves in smart drug delivery systems, nanofilters in the new generation of desalination systems, and promising applications in DNA sequencing.

SARS-CoV-2 viral protein ORF3A injures renal tubules by interacting with TRIM59 to induce STAT3 activation.

Acute kidney injury occurs frequently in COVID-19 patients infected by the coronavirus SARS-CoV-2, and infection of kidney cells by this virus has been reported. However, little is known about the direct impact of the SARS-CoV-2 infection upon the renal tubular cells. We report that SARS-CoV-2 activated signal transducer and activator of transcription 3 (STAT3) signaling and caused cellular injury in the human renal tubular cell line. Mechanistically, the viral protein ORF3A of SARS-CoV-2 augmented both NF-κB and STAT3 signaling and increased the expression of kidney injury molecule 1. SARS-CoV-2 infection or expression of ORF3A alone elevated the protein level of tripartite motif-containing protein 59 (TRIM59), an E3 ubiquitin ligase, which interacts with both ORF3A and STAT3. The excessive TRIM59 in turn dissociated the phosphatase TCPTP from binding to STAT3 and hence inhibited the dephosphorylation of STAT3, leading to persistent STAT3 activation. Consistently, ORF3A induced renal injury in zebrafish and mice. In addition, expression of TRIM59 was elevated in the kidney autopsies of COVID-19 patients with acute kidney injury. Thus, the aberrant activation of STAT3 signaling by TRIM59 plays a significant role in the renal tubular cell injury caused by SARS-CoV-2, which suggests a potential targeted therapy for the renal complications of COVID-19.

Genome-Wide Characterization of SARS-CoV-2 Cytopathogenic Proteins in the Search of Antiviral Targets.

Therapeutic inhibition of critical viral functions is important for curtailing coronavirus disease 2019 (COVID-19). We sought to identify antiviral targets through the genome-wide characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins that are crucial for viral pathogenesis and that cause harmful cytopathogenic effects. All 29 viral proteins were tested in a fission yeast cell-based system using inducible gene expression. Twelve proteins, including eight nonstructural proteins (NSP1, NSP3, NSP4, NSP5, NSP6, NSP13, NSP14, and NSP15) and four accessory proteins (ORF3a, ORF6, ORF7a, and ORF7b), were identified that altered cellular proliferation and integrity and induced cell death. Cell death correlated with the activation of cellular oxidative stress. Of the 12 proteins, ORF3a was chosen for further study in mammalian cells because it plays an important role in viral pathogenesis and its activities are linked to lung tissue damage and a cytokine storm. In human pulmonary and kidney epithelial cells, ORF3a induced cellular oxidative stress associated with apoptosis and necrosis and caused activation of proinflammatory response with production of the cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IFN-ß1, possibly through the activation of nuclear factor kappa B (NF-κB). To further characterize the mechanism, we tested a natural ORF3a Beta variant, Q57H, and a mutant with deletion of the highly conserved residue, ΔG188. Compared with wild-type ORF3a, the ΔG188 variant yielded more robust activation of cellular oxidative stress, cell death, and innate immune response. Since cellular oxidative stress and inflammation contribute to cell death and tissue damage linked to the severity of COVID-19, our findings suggest that ORF3a is a promising, novel therapeutic target against COVID-19. IMPORTANCE The ongoing COVID-19 pandemic caused by SARS-CoV-2 has claimed over 5.5 million lives with more than 300 million people infected worldwide. While vaccines are effective, the emergence of new viral variants could jeopardize vaccine protection. Treatment of COVID-19 by antiviral drugs provides an alternative to battle against the disease. The goal of this study was to identify viral therapeutic targets that can be used in antiviral drug discovery. Utilizing a genome-wide functional analysis in a fission yeast cell-based system, we identified 12 viral candidates, including ORF3a, which cause cellular oxidative stress, inflammation, apoptosis, and necrosis that contribute to cytopathogenicity and COVID-19. Our findings indicate that antiviral agents targeting ORF3a could have a great impact on COVID-19.

D155Y substitution of SARS-CoV-2 ORF3a weakens binding with Caveolin-1.

The clinical manifestation of the recent pandemic COVID-19, caused by the novel SARS-CoV-2 virus, varies from mild to severe respiratory illness. Although environmental, demographic and co-morbidity factors have an impact on the severity of the disease, contribution of the mutations in each of the viral genes towards the degree of severity needs a deeper understanding for designing a better therapeutic approach against COVID-19. Open Reading Frame-3a (ORF3a) protein has been found to be mutated at several positions. In this work, we have studied the effect of one of the most frequently occurring mutants, D155Y of ORF3a protein, found in Indian COVID-19 patients. Using computational simulations we demonstrated that the substitution at 155th changed the amino acids involved in salt bridge formation, hydrogen-bond occupancy, interactome clusters, and the stability of the protein compared with the other substitutions found in Indian patients. Protein-protein docking using HADDOCK analysis revealed that substitution D155Y weakened the binding affinity of ORF3a with caveolin-1 compared with the other substitutions, suggesting its importance in the overall stability of ORF3a-caveolin-1 complex, which may modulate the virulence property of SARS-CoV-2.

Safety and immunogenicity of the bi-cistronic GLS-5310 COVID-19 DNA vaccine delivered with the GeneDerm suction device.

OBJECTIVES: The CoV2-001 phase I randomized trial evaluated the safety and immunogenicity of the GLS-5310 bi-cistronic DNA vaccine through 48 weeks of follow-up. DESIGN: A total of 45 vaccine-naïve participants were recruited between December 31, 2020, and March 30, 2021. GLS-5310, encoding for the SARS-CoV-2 spike and open reading frame 3a (ORF3a) proteins, was administered intradermally at 0.6 mg or 1.2 mg per dose, followed by application of the GeneDerm suction device as part of a two-dose regimen spaced either 8 or 12 weeks between vaccinations. RESULTS: GLS-5310 was well tolerated with no serious adverse events reported. Antibody and T cell responses were dose-independent. Anti-spike antibodies were induced in 95.5% of participants with an average geometric mean titer of ∼480 four weeks after vaccination and declined minimally through 48 weeks. Neutralizing antibodies were induced in 55.5% of participants with post-vaccination geometric mean titer of 28.4. T cell responses were induced in 97.8% of participants, averaging 716 site forming units/106 cells four weeks after vaccination, increasing to 1248 at week 24, and remaining greater than 1000 through 48 weeks. CONCLUSION: GLS-5310 administered with the GeneDerm suction device was well tolerated and induced high levels of binding antibodies and T-cell responses. Antibody responses were similar to other DNA vaccines, whereas T cell responses were many-fold greater than DNA and non-DNA vaccines.

The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins.

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a positively charged aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a's ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

Inhibition of SARS-CoV-2 Viral Channel Activity Using FDA-Approved Channel Modulators Independent of Variants.

BACKGROUND: SARS-CoV-2 has undergone mutations, yielding clinically relevant variants. HYPOTHESIS: We hypothesized that in SARS-CoV-2, two highly conserved Orf3a and E channels directly related to the virus replication were a target for the detection and inhibition of the viral replication, independent of the variant, using FDA-approved ion channel modulators. METHODS: A combination of a fluorescence potassium ion assay with channel modulators was developed to detect SARS-CoV-2 Orf3a/E channel activity. Two FDA-approved drugs, amantadine (an antiviral) and amitriptyline (an antidepressant), which are ion channel blockers, were tested as to whether they inhibited Orf3a/E channel activity in isolated virus variants and in nasal swab samples from COVID-19 patients. The variants were confirmed by PCR sequencing. RESULTS: In isolated SARS-CoV-2 Alpha, Beta, and Delta variants, the channel activity of Orf3a/E was detected and inhibited by emodin and gliclazide (IC50 = 0.42 mM). In the Delta swab samples, amitriptyline and amantadine inhibited the channel activity of viral proteins, with IC50 values of 0.73 mM and 1.11 mM, respectively. In the Omicron swab samples, amitriptyline inhibited the channel activity, with an IC50 of 0.76 mM. CONCLUSIONS: We developed an efficient method to screen FDA-approved ion channel modulators that could be repurposed to detect and inhibit SARS-CoV-2 viral replication, independent of variants.

Using Alphafold2 to Predict the Structure of the Gp5/M Dimer of Porcine Respiratory and Reproductive Syndrome Virus.

Porcine reproductive and respiratory syndrome virus is a positive-stranded RNA virus of the family Arteriviridae. The Gp5/M dimer, the major component of the viral envelope, is required for virus budding and is an antibody target. We used alphafold2, an artificial-intelligence-based system, to predict a credible structure of Gp5/M. The short disulfide-linked ectodomains lie flat on the membrane, with the exception of the erected N-terminal helix of Gp5, which contains the antibody epitopes and a hypervariable region with a changing number of carbohydrates. The core of the dimer consists of six curved and tilted transmembrane helices, and three are from each protein. The third transmembrane regions extend into the cytoplasm as amphiphilic helices containing the acylation sites. The endodomains of Gp5 and M are composed of seven ß-strands from each protein, which interact via ß-strand seven. The area under the membrane forms an open cavity with a positive surface charge. The M and Orf3a proteins of coronaviruses have a similar structure, suggesting that all four proteins are derived from the same ancestral gene. Orf3a, like Gp5/M, is acylated at membrane-proximal cysteines. The role of Gp5/M during virus replication is discussed, in particular the mechanisms of virus budding and models of antibody-dependent virus neutralization.

Targeting the YXXΦ Motifs of the SARS Coronaviruses 1 and 2 ORF3a Peptides by In Silico Analysis to Predict Novel Virus-Host Interactions.

The emerging SARS-CoV and SARS-CoV-2 belong to the family of "common cold" RNA coronaviruses, and they are responsible for the 2003 epidemic and the current pandemic with over 6.3 M deaths worldwide. The ORF3a gene is conserved in both viruses and codes for the accessory protein ORF3a, with unclear functions, possibly related to viral virulence and pathogenesis. The tyrosine-based YXXΦ motif (Φ: bulky hydrophobic residue-L/I/M/V/F) was originally discovered to mediate clathrin-dependent endocytosis of membrane-spanning proteins. Many viruses employ the YXXΦ motif to achieve efficient receptor-guided internalisation in host cells, maintain the structural integrity of their capsids and enhance viral replication. Importantly, this motif has been recently identified on the ORF3a proteins of SARS-CoV and SARS-CoV-2. Given that the ORF3a aa sequence is not fully conserved between the two SARS viruses, we aimed to map in silico structural differences and putative sequence-driven alterations of regulatory elements within and adjacently to the YXXΦ motifs that could predict variations in ORF3a functions. Using robust bioinformatics tools, we investigated the presence of relevant post-translational modifications and the YXXΦ motif involvement in protein-protein interactions. Our study suggests that the predicted YXXΦ-related features may confer specific-yet to be discovered-functions to ORF3a proteins, significant to the new virus and related to enhanced propagation, host immune regulation and virulence.