Amantadine has potential for the treatment of COVID-19 because it inhibits known and novel ion channels encoded by SARS-CoV-2.

The dire need for COVID-19 treatments has inspired strategies of repurposing approved drugs. Amantadine has been suggested as a candidate, and cellular as well as clinical studies have indicated beneficial effects of this drug. We demonstrate that amantadine and hexamethylene-amiloride (HMA), but not rimantadine, block the ion channel activity of Protein E from SARS-CoV-2, a conserved viroporin among coronaviruses. These findings agree with their binding to Protein E as evaluated by solution NMR and molecular dynamics simulations. Moreover, we identify two novel viroporins of SARS-CoV-2; ORF7b and ORF10, by showing ion channel activity in a X. laevis oocyte expression system. Notably, amantadine also blocks the ion channel activity of ORF10, thereby providing two ion channel targets in SARS-CoV-2 for amantadine treatment in COVID-19 patients. A screen of known viroporin inhibitors on Protein E, ORF7b, ORF10 and Protein 3a from SARS-CoV-2 revealed inhibition of Protein E and ORF7b by emodin and xanthene, the latter also blocking Protein 3a. This illustrates a general potential of well-known ion channel blockers against SARS-CoV-2 and specifically a dual molecular basis for the promising effects of amantadine in COVID-19 treatment. We therefore propose amantadine as a novel, cheap, readily available and effective way to treat COVID-19.

Non-Canonical Translation Initiation Mechanisms Employed by Eukaryotic Viral mRNAs.

Viruses exploit the translation machinery of an infected cell to synthesize their proteins. Therefore, viral mRNAs have to compete for ribosomes and translation factors with cellular mRNAs. To succeed, eukaryotic viruses adopt multiple strategies. One is to circumvent the need for m7G-cap through alternative instruments for ribosome recruitment. These include internal ribosome entry sites (IRESs), which make translation independent of the free 5' end, or cap-independent translational enhancers (CITEs), which promote initiation at the uncapped 5' end, even if located in 3' untranslated regions (3' UTRs). Even if a virus uses the canonical cap-dependent ribosome recruitment, it can still perturb conventional ribosomal scanning and start codon selection. The pressure for genome compression often gives rise to internal and overlapping open reading frames. Their translation is initiated through specific mechanisms, such as leaky scanning, 43S sliding, shunting, or coupled termination-reinitiation. Deviations from the canonical initiation reduce the dependence of viral mRNAs on translation initiation factors, thereby providing resistance to antiviral mechanisms and cellular stress responses. Moreover, viruses can gain advantage in a competition for the translational machinery by inactivating individual translational factors and/or replacing them with viral counterparts. Certain viruses even create specialized intracellular "translation factories", which spatially isolate the sites of their protein synthesis from cellular antiviral systems, and increase availability of translational components. However, these virus-specific mechanisms may become the Achilles' heel of a viral life cycle. Thus, better understanding of the unconventional mechanisms of viral mRNA translation initiation provides valuable insight for developing new approaches to antiviral therapy.

Binding of boswellic acids to functional proteins of the SARS-CoV-2 virus: Bioinformatic studies.

Boswellic acids (BAs) have been shown to possess antiviral activity. Using bioinformatic methods, it was tested whether or not acetyl-11-keto-ß-boswellic acid (AKBA), 11-keto-ß-boswellic acid (KBA), ß-boswellic acid (BBA), and the phosphorylated active metabolite of Remdesivir® (RGS-P3) bind to functional proteins of SARS-CoV-2, that is, the replicase polyprotein P0DTD1, the spike glycoprotein P0DTC2, and the nucleoprotein P0DTC9. Using P0DTD1, AKBA and KBA showed micromolar binding affinity to the RNA-dependent RNA polymerase (RdRp) and to the main proteinase complex Mpro . Phosphorylated BAs even bond in the nanomolar range. Due to their positive and negative charges, BAs and RGS-P3 bond to corresponding negative and positive areas of the protein. BAs and RGS-P3 docked in the tunnel-like cavity of RdRp. BAs also docked into the elongated surface rim of viral Mpro . In both cases, binding occurred with active site amino acids in the lower micromolecular to upper nanomolar range. KBA, BBA, and RGS-P3 also bond to P0DTC2 and P0DTC9. The binding energies for BAs were in the range of -5.8 to -6.3 kcal/mol. RGS-P3 and BAs occluded the centrally located pore of the donut-like protein structure of P0DTC9 and, in the case of P0DTC2, RGS-P3 and BAs impacted the double-wing-like protein structure. The data of this bioinformatics study clearly show that BAs bind to three functional proteins of the SARS-CoV-2 virus responsible for adhesion and replication, as does RGS-P3, a drug on the market to treat this disease. The binding effectiveness of BAs can be increased through phosphate esterification. Whether or not BAs are druggable against the SARS-CoV-2 disease remains to be established.

The interplay of SARS-CoV-2 evolution and constraints imposed by the structure and functionality of its proteins.

The unprecedented pace of the sequencing of the SARS-CoV-2 virus genomes provides us with unique information about the genetic changes in a single pathogen during ongoing pandemic. By the analysis of close to 200,000 genomes we show that the patterns of the SARS-CoV-2 virus mutations along its genome are closely correlated with the structural and functional features of the encoded proteins. Requirements of foldability of proteins' 3D structures and the conservation of their key functional regions, such as protein-protein interaction interfaces, are the dominant factors driving evolutionary selection in protein-coding genes. At the same time, avoidance of the host immunity leads to the abundance of mutations in other regions, resulting in high variability of the missense mutation rate along the genome. "Unexplained" peaks and valleys in the mutation rate provide hints on function for yet uncharacterized genomic regions and specific protein structural and functional features they code for. Some of these observations have immediate practical implications for the selection of target regions for PCR-based COVID-19 tests and for evaluating the risk of mutations in epitopes targeted by specific antibodies and vaccine design strategies.

Complement Decay-Accelerating Factor is a modulator of influenza A virus lung immunopathology.

Clearance of viral infections, such as SARS-CoV-2 and influenza A virus (IAV), must be fine-tuned to eliminate the pathogen without causing immunopathology. As such, an aggressive initial innate immune response favors the host in contrast to a detrimental prolonged inflammation. The complement pathway bridges innate and adaptive immune system and contributes to the response by directly clearing pathogens or infected cells, as well as recruiting proinflammatory immune cells and regulating inflammation. However, the impact of modulating complement activation in viral infections is still unclear. In this work, we targeted the complement decay-accelerating factor (DAF/CD55), a surface protein that protects cells from non-specific complement attack, and analyzed its role in IAV infections. We found that DAF modulates IAV infection in vivo, via an interplay with the antigenic viral proteins hemagglutinin (HA) and neuraminidase (NA), in a strain specific manner. Our results reveal that, contrary to what could be expected, DAF potentiates complement activation, increasing the recruitment of neutrophils, monocytes and T cells. We also show that viral NA acts on the heavily sialylated DAF and propose that the NA-dependent DAF removal of sialic acids exacerbates complement activation, leading to lung immunopathology. Remarkably, this mechanism has no impact on viral loads, but rather on the host resilience to infection, and may have direct implications in zoonotic influenza transmissions.

Neurological Implications of COVID-19: Role of Redox Imbalance and Mitochondrial Dysfunction.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 or COVID-19 has been declared as a pandemic disease by the World Health Organization (WHO). Globally, this disease affected 159 million of the population and reported ~ 3.3 million deaths to the current date (May 2021). There is no definitive treatment strategy that has been identified, although this disease has prevailed in its current form for the past 18 months. The main challenges in the (SARS-CoV)-2 infections are in identifying the heterogeneity in viral strains and the plausible mechanisms of viral infection to human tissues. In parallel to the investigations into the patho-mechanism of SARS-CoV-2 infection, understanding the fundamental processes underlying the clinical manifestations of COVID-19 is very crucial for designing effective therapies. Since neurological symptoms are very apparent in COVID-19 infected patients, here, we tried to emphasize the involvement of redox imbalance and subsequent mitochondrial dysfunction in the progression of the COVID-19 infection. It has been articulated that mitochondrial dysfunction is very apparent and also interlinked to neurological symptoms in COVID-19 infection. Overall, this article provides an in-depth overview of redox imbalance and mitochondrial dysfunction involvement in aggravating COVID-19 infection and its probable contribution to the neurological manifestation of the disease.

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.

The ORF8 protein of SARS-CoV-2 induced endoplasmic reticulum stress and mediated immune evasion by antagonizing production of interferon beta.

The open reading frame 8 (orf8) is an accessory protein of SARS-CoV-2. It has 121 amino acids with two genotypes, orf8L and orf8S. In this study, we overexpressed the orf8L and orf8S of SARS-CoV-2 as well as the orf8b of SARS-CoV to investigate their roles in the regulation of endoplasmic reticulum (ER) stress and the inhibition of interferon beta (IFNß) production. We found that the two genotypes of SARS-CoV-2 orf8 are capable of inducing ER stress without significant difference by triggering the activating transcription factor 6 (ATF6) and inositol-requiring enzymes 1 (IRE1) branches of the ER stress pathway. However, the third branch of ER stress pathway, i.e. the protein kinase-like ER kinase (PERK), was unaffected by the overexpression of SARS-CoV-2 orf8L or orf8S. Moreover, both orf8L and orf8S of SARS-CoV-2 are capable of down regulating the production of IFNß and interferon-stimulated genes (ISG), ISG15 and ISG56 induced by polyinosinic-polycytidylic acid (poly (I:C)). Moreover, we also found decreased nuclear translocation of Interferon regulatory factor 3 (IRF3), after overexpressing orf8L and orf8S induced by poly (I:C). Our data demonstrated that SARS-CoV-2 orf8 protein could induce ER stress by activating the ATF6 and IRE1 pathways, but not the PERK pathway, and functions as an interferon antagonist to inhibit the production of IFNß. However, these functions appeared not to be affected by the genotypes of SARS-CoV-2 orf8L and orf8S.

Open Science Resources for the Mass Spectrometry-Based Analysis of SARS-CoV-2.

The SARS-CoV-2 virus is the causative agent of the 2020 pandemic leading to the COVID-19 respiratory disease. With many scientific and humanitarian efforts ongoing to develop diagnostic tests, vaccines, and treatments for COVID-19, and to prevent the spread of SARS-CoV-2, mass spectrometry research, including proteomics, is playing a role in determining the biology of this viral infection. Proteomics studies are starting to lead to an understanding of the roles of viral and host proteins during SARS-CoV-2 infection, their protein-protein interactions, and post-translational modifications. This is beginning to provide insights into potential therapeutic targets or diagnostic strategies that can be used to reduce the long-term burden of the pandemic. However, the extraordinary situation caused by the global pandemic is also highlighting the need to improve mass spectrometry data and workflow sharing. We therefore describe freely available data and computational resources that can facilitate and assist the mass spectrometry-based analysis of SARS-CoV-2. We exemplify this by reanalyzing a virus-host interactome data set to detect protein-protein interactions and identify host proteins that could potentially be used as targets for drug repurposing.

Type I and III IFN-mediated antiviral actions counteracted by SARS-CoV-2 proteins and host inherited factors.

SARS-CoV-2 is a recently identified coronavirus accountable for the current pandemic disease known as COVID-19. Different patterns of disease progression infer a diverse host immune response, with interferon (IFN) being pivotal. IFN-I and III are produced and released by virus-infected cells during the interplay with SARS-CoV-2, thus establishing an antiviral state in target cells. However, the efficacy of IFN and its role in the possible outcomes of the disease are not yet defined, as it is influenced both by factors inherent to the virus and to the host. The virus exhibits multiple strategies to counteract the innate immune response, including those shared by SARS-CoV and MERS-CoV and other novel ones. Inborn errors in the host may affect IFN-related effector proteins or decrease its levels in plasma upon neutralization by preexistent autoantibodies. This battle between the IFN response triggered upon SARS-CoV-2 infection, its magnitude and timing, and the efficacy of its antiviral tools in dispute against the viral evasion strategies together with the genetic factors of the host, generate a scenario whose fate contributes to defining the severity of COVID-19.

Why COVID-19 Transmission Is More Efficient and Aggressive Than Viral Transmission in Previous Coronavirus Epidemics?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing a pandemic of coronavirus disease 2019 (COVID-19). The worldwide transmission of COVID-19 from human to human is spreading like wildfire, affecting almost every country in the world. In the past 100 years, the globe did not face a microbial pandemic similar in scale to COVID-19. Taken together, both previous outbreaks of other members of the coronavirus family (severe acute respiratory syndrome (SARS-CoV) and middle east respiratory syndrome (MERS-CoV)) did not produce even 1% of the global harm already inflicted by COVID-19. There are also four other CoVs capable of infecting humans (HCoVs), which circulate continuously in the human population, but their phenotypes are generally mild, and these HCoVs received relatively little attention. These dramatic differences between infection with HCoVs, SARS-CoV, MERS-CoV, and SARS-CoV-2 raise many questions, such as: Why is COVID-19 transmitted so quickly? Is it due to some specific features of the viral structure? Are there some specific human (host) factors? Are there some environmental factors? The aim of this review is to collect and concisely summarize the possible and logical answers to these questions.

IL6-mediated HCoV-host interactome regulatory network and GO/Pathway enrichment analysis.

During these days of global emergency for the COVID-19 disease outbreak, there is an urgency to share reliable information able to help worldwide life scientists to get better insights and make sense of the large amount of data currently available. In this study we used the results presented in [1] to perform two different Systems Biology analyses on the HCoV-host interactome. In the first one, we reconstructed the interactome of the HCoV-host proteins, integrating it with highly reliable miRNA and drug interactions information. We then added the IL-6 gene, identified in recent publications [2] as heavily involved in the COVID-19 progression and, interestingly, we identified several interactions with the reconstructed interactome. In the second analysis, we performed a Gene Ontology and a Pathways enrichment analysis on the full set of the HCoV-host interactome proteins and on the ones belonging to a significantly dense cluster of interacting proteins identified in the first analysis. Results of the two analyses provide a compact but comprehensive glance on some of the current state-of-the-art regulations, GO, and pathways involved in the HCoV-host interactome, and that could support all scientists currently focusing on SARS-CoV-2 research.

A persistently replicating SARS-CoV-2 variant derived from an asymptomatic individual.

BACKGROUND: Since the first outbreak of SARS-CoV-2, the clinical characteristics of the Coronavirus Disease 2019 (COVID-19) have been progressively changed. Data reporting a viral intra-host and inter-host evolution favouring the appearance of mild SARS-CoV-2 strains are since being accumulating. To better understand the evolution of SARS-CoV-2 pathogenicity and its adaptation to the host, it is therefore crucial to investigate the genetic and phenotypic characteristics of SARS-CoV-2 strains circulating lately in the epidemic. METHODS: Nasopharyngeal swabs have been analyzed for viral load in the early (March 2020) and late (May 2020) phases of epidemic in Brescia, Italy. Isolation of SARS-CoV-2 from 2 high viral load specimens identified on March 9 (AP66) and on May 8 (GZ69) was performed on Vero E6 cells. Amount of virus released was assessed by quantitative PCR. Genotypic characterization of AP66 and GZ69 was performed by next generation sequencing followed by an in-depth in silico analysis of nucleotide mutations. RESULTS: The SARS-CoV-2 GZ69 strain, isolated in May from an asymptomatic healthcare worker, showed an unprecedented capability of replication in Vero E6 cells in the absence of any evident cytopathic effect. Vero E6 subculturing, up to passage 4, showed that SARS-CoV-2 GZ69 infection was as productive as the one sustained by the cytopathic strain AP66. Whole genome sequencing of the persistently replicating SARS-CoV-2 GZ69 has shown that this strain differs from the early AP66 variant in 9 nucleotide positions (C2939T; C3828T; G21784T; T21846C; T24631C; G28881A; G28882A; G28883C; G29810T) which lead to 6 non-synonymous substitutions spanning on ORF1ab (P892S; S1188L), S (K74N; I95T) and N (R203K, G204R) proteins. CONCLUSIONS: Identification of the peculiar SARS-CoV-2 GZ69 strain in the late Italian epidemic highlights the need to better characterize viral variants circulating among asymptomatic or paucisymptomatic individuals. The current approach could unravel the ways for future studies aimed at analyzing the selection process which favours viral mutations in the human host.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): An overview of viral structure and host response.

BACKGROUND AND AIM: As a result of its rapid spread in various countries around the world, on March 11, 2020, WHO issued an announcement of the change in coronavirus disease 2019 status from epidemic to pandemic disease. The virus that causes this disease is indicated originating from animals traded in a live animal market in Wuhan, China. Severe Acute Respiratory Syndrome Coronavirus 2 can attack lung cells because there are many conserved receptor entries, namely Angiotensin Converting Enzyme-2. The presence of this virus in host cells will initiate various protective responses leading to pneumonia and Acute Respiratory Distress Syndrome. This review aimed to provide an overview related to this virus and examine the body's responses and possible therapies. METHOD: We searched PubMed databases for Severe Acute Respiratory Syndrome Coronavirus-2, Middle East respiratory syndrome-related coronavirus and Severe Acute Respiratory Syndrome Coronavirus. Full texts were retrieved, analyzed and developed into an easy-to-understand review. RESULTS: We provide a complete review related to structure, origin, and how the body responds to this virus infection and explain the possibility of an immune system over-reaction or cytokine storm. We also include an explanation of how this virus creates modes of avoidance to evade immune system attacks. We further explain the therapeutic approaches that can be taken in the treatment and prevention of this viral infection. CONCLUSION: In summary, based on the structural and immune-evasion system of coronavirus, we suggest several approaches to treat the disease.

Forty years with coronaviruses.

I have been researching coronaviruses for more than forty years. This viewpoint summarizes some of the major findings in coronavirus research made before the SARS epidemic and how they inform current research on the newly emerged SARS-CoV-2.