UBR5 Acts as an Antiviral Host Factor against MERS-CoV via Promoting Ubiquitination and Degradation of ORF4b.

Within the past 2 decades, three highly pathogenic human coronaviruses have emerged, namely, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The health threats and economic burden posed by these tremendously severe coronaviruses have paved the way for research on their etiology, pathogenesis, and treatment. Compared to SARS-CoV and SARS-CoV-2, MERS-CoV genome encoded fewer accessory proteins, among which the ORF4b protein had anti-immunity ability in both the cytoplasm and nucleus. Our work for the first time revealed that ORF4b protein was unstable in the host cells and could be degraded by the ubiquitin proteasome system. After extensive screenings, it was found that UBR5 (ubiquitin protein ligase E3 component N-recognin 5), a member of the HECT E3 ubiquitin ligases, specifically regulated the ubiquitination and degradation of ORF4b. Similar to ORF4b, UBR5 can also translocate into the nucleus through its nuclear localization signal, enabling it to regulate ORF4b stability in both the cytoplasm and nucleus. Through further experiments, lysine 36 was identified as the ubiquitination site on the ORF4b protein, and this residue was highly conserved in various MERS-CoV strains isolated from different regions. When UBR5 was knocked down, the ability of ORF4b to suppress innate immunity was enhanced and MERS-CoV replication was stronger. As an anti-MERS-CoV host protein, UBR5 targets and degrades ORF4b protein through the ubiquitin proteasome system, thereby attenuating the anti-immunity ability of ORF4b and ultimately inhibiting MERS-CoV immune escape, which is a novel antagonistic mechanism of the host against MERS-CoV infection. IMPORTANCE ORF4b was an accessory protein unique to MERS-CoV and was not present in SARS-CoV and SARS-CoV-2 which can also cause severe respiratory disease. Moreover, ORF4b inhibited the production of antiviral cytokines in both the cytoplasm and the nucleus, which was likely to be associated with the high lethality of MERS-CoV. However, whether the host proteins regulate the function of ORF4b is unknown. Our study first determined that UBR5, a host E3 ligase, was a potential host anti-MERS-CoV protein that could reduce the protein level of ORF4b and diminish its anti-immunity ability by inducing ubiquitination and degradation. Based on the discovery of ORF4b-UBR5, a critical molecular target, further increasing the degradation of ORF4b caused by UBR5 could provide a new strategy for the clinical development of drugs for MERS-CoV.

A New Antiviral Regimen Against SARS-CoV-2 Based on Nanoviricide’s Biopolymer (NV-CoV-2)

NV-CoV-2, a nanoviricide composed of covalently attached polyethylene glycol and alkyl pendants that are designed to bind free virion particles of multiple strains of coronaviruses in a broad-spectrum manner at multiple points. The binding interaction is like a nano-velcro-tape and may cause a lipid–lipid fusion between nanoviricide micelle and the lipid envelope of the virus. A nanoviricide can encapsulate the virus and dismantle it without any involvement of the host immune system, ultimately disabling the infectibility of the host cells. Thus, it may be expected to count a stronger and synergistic antiviral effect by combining NV-CoV-2 with other anti-coronavirus regimens like remdesivir. Furthermore, some ligands similar to the SARS-CoV S-protein are designed by molecular modeling and attached to the nanoviricide at the same site as where the cognate cellular receptor, ACE2, binds. As a result, a competitive binding inhibition may occur. A nanoviricide can encapsulate other antiviral compounds and protect them from serum-mediated degradation in vivo. This makes the antiviral compounds available for a longer period of time to interact with RNA polymerase and inhibit it. Altogether, a multipoint antiviral efficacy can be achieved with our nanoviricide, NV-CoV-2. Copyright © 2022 Chakraborty, Diwan, Barton, Arora, Thakur, Chiniga, Tatake, Pandey, Holkar, Holkar and Pond.

Mechanism of Antiviral Activities of Nanoviricide's Platform Technology based Biopolymer (NV-CoV-2).

NV-CoV-2 is a nanoviricide that is covalently bonded with polyethylene glycol (PEG) and alkyl pendants. This molecular design is used to attack many strains of coronaviruses in a broad-spectrum manner. The ligand works by competitive inhibition and binds to the same site on the S-protein of SARS-CoV that attaches to the cognate cellular receptor, ACE2. This prevents SARS-CoV from binding and infecting the cell. NV-CoV-2 is designed to bind to the free virion particles at multiple points encapsulate the virus and disable its ability to infect the cells. The multi-point binding interaction, like a nano-velcro-tape, may lead to lipid-lipid fusion of the alkyl chains in the nanoviricide micelle with the lipid envelope of the virus. The virus becomes dismantled to a capsid form before the host immune system becomes involved. This putative mechanism is orthogonal to many other anti-coronavirus agents in development. Thus, it maybe possible to produce a stronger antiviral effect when combining NV-CoV-2 therapy with other anti-coronavirus therapies such as Remdesivir (RDV). NV-CoV-2 can encapsulate other antiviral compounds as well. In this study, RDV was encapsulated and protected from serum-mediated degradation in vivo. As a result, RDV was available for a longer period of time to interact with RNA polymerase and inhibit.

Studies in the antiviral molecular mechanisms of 25-hydroxycholesterol: Disturbing cholesterol homeostasis and post-translational modification of proteins.

Efficient antiviral drug discovery has been a pressing issue of global public health concern since the outbreak of coronavirus disease 2019. In recent years, numerous in vitro and in vivo studies have shown that 25-hydroxycholesterol (25HC), a reactive oxysterol catalyzed by cholesterol-25-hydroxylase, exerts broad-spectrum antiviral activity with high efficiency and low toxicity. 25HC restricts viral internalization and disturbs the maturity of viral proteins using multiple mechanisms. First, 25HC reduces lipid rafts and cholesterol in the cytomembrane by inhibiting sterol-regulatory element binding proteins-2, stimulating liver X receptor, and activating Acyl-coenzyme A: cholesterol acyl-transferase. Second, 25HC impairs endosomal pathways by restricting the function of oxysterol-binding protein or Niemann-pick protein C1, causing the virus to fail to release nucleic acid. Third, 25HC disturbs the prenylation of viral proteins by suppressing the sterol-regulatory element binding protein pathway and glycosylation by increasing the sensitivity of glycans to endoglycosidase. This paper reviews previous studies on the antiviral activity of 25HC in order to fully understand its role in innate immunity and how it may contribute to the development of urgently needed broad-spectrum antiviral drugs.

Unique Mode of Antiviral Action of a Marine Alkaloid against Ebola Virus and SARS-CoV-2.

Lamellarin α 20-sulfate is a cell-impenetrable marine alkaloid that can suppress infection that is mediated by the envelope glycoprotein of human immunodeficiency virus type 1. We explored the antiviral action and mechanisms of this alkaloid against emerging enveloped RNA viruses that use endocytosis for infection. The alkaloid inhibited the infection of retroviral vectors that had been pseudotyped with the envelope glycoprotein of Ebola virus and SARS-CoV-2. The antiviral effects of lamellarin were independent of the retrovirus Gag-Pol proteins. Interestingly, although heparin and dextran sulfate suppressed the cell attachment of vector particles, lamellarin did not. In silico structural analyses of the trimeric glycoprotein of the Ebola virus disclosed that the principal lamellarin-binding site is confined to a previously unappreciated cavity near the NPC1-binding site and fusion loop, whereas those for heparin and dextran sulfate were dispersed across the attachment and fusion subunits of the glycoproteins. Notably, lamellarin binding to this cavity was augmented under conditions where the pH was 5.0. These results suggest that the final action of the alkaloid against Ebola virus is specific to events following endocytosis, possibly during conformational glycoprotein changes in the acidic environment of endosomes. Our findings highlight the unique biological and physicochemical features of lamellarin α 20-sulfate and should lead to the further use of broadly reactive antivirals to explore the structural mechanisms of virus replication.

Molecular docking and dynamics studies of curcumin with COVID-19 proteins.

Coronavirus disease 2019 (COVID-19) is caused by a Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2), which is a positive-strand RNA virus. The SARS-CoV-2 genome and its association to SAR-CoV-1 vary from ca. 66 to 96% depending on the type of betacoronavirideae family members. With several drugs, viz. chloroquine, hydroxychloroquine, ivermectin, artemisinin, remdesivir, azithromycin considered for clinical trials, there has been an inherent need to find distinctive antiviral mechanisms of these drugs. Curcumin, a natural bioactive molecule has been shown to have therapeutic potential for various diseases, and its effect on COVID-19 is also currently being explored. In this study, we show the binding potential of curcumin targeted to a variety of SARS-CoV-2 proteins, viz. spike glycoproteins (PDB ID: 6VYB), nucleocapsid phosphoprotein (PDB ID: 6VYO), spike protein-ACE2 (PDB ID: 6M17) along with nsp10 (PDB ID: 6W4H) and RNA dependent RNA polymerase (PDB ID: 6M71) structures. Furthermore, representative docking complexes were validated using molecular dynamics simulations and mechanistic studies at 100 ns was carried on nucleocapsid and nsp10 proteins with curcumin complexes which resulted in stable and efficient binding energies and correlated with that of docked binding energies of the complexes. Both the docking and simulation studies indicate that curcumin has the potential as an antiviral against COVID-19.

Repurposing Chloroquine Against Multiple Diseases With Special Attention to SARS-CoV-2 and Associated Toxicity.

Chloroquine and its derivatives have been used since ages to treat malaria and have also been approved by the FDA to treat autoimmune diseases. The drug employs pH-dependent inhibition of functioning and signalling of the endosome, lysosome and trans-Golgi network, immunomodulatory actions, inhibition of autophagy and interference with receptor binding to treat cancer and many viral diseases. The ongoing pandemic of COVID-19 has brought the whole world on the knees, seeking an urgent hunt for an anti-SARS-CoV-2 drug. Chloroquine has shown to inhibit receptor binding of the viral particles, interferes with their replication and inhibits "cytokine storm". Though multiple modes of actions have been employed by chloroquine against multiple diseases, viral diseases can provide an added advantage to establish the anti-SARS-CoV-2 mechanism, the in vitro and in vivo trials against SARS-CoV-2 have yielded mixed results. The toxicological effects and dosage optimization of chloroquine have been studied for many diseases, though it needs a proper evaluation again as chloroquine is also associated with several toxicities. Moreover, the drug is inexpensive and is readily available in many countries. Though much of the hope has been created by chloroquine and its derivatives against multiple diseases, repurposing it against SARS-CoV-2 requires large scale, collaborative, randomized and unbiased clinical trials to avoid false promises. This review summarizes the use and the mechanism of chloroquine against multiple diseases, its side-effects, mechanisms and the different clinical trials ongoing against "COVID-19".

MiR-10a-5p-Mediated Syndecan 1 Suppression Restricts Porcine Hemagglutinating Encephalomyelitis Virus Replication.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a single-stranded RNA coronavirus that causes nervous dysfunction in the infected hosts and leads to widespread alterations in the host transcriptome by modulating specific microRNA (miRNA) levels. MiRNAs contribute to RNA virus pathogenesis by promoting antiviral immune response, enhancing viral replication, or altering miRNA-mediated host gene regulation. Thus, exploration of the virus-miRNA interactions occurring in PHEV-infected host may lead to the identification of novel mechanisms combating the virus life cycle or pathogenesis. Here, we discovered that the expression of miR-10a-5p was constitutively up-regulated by PHEV in both the N2a cells in vitro and mice brain in vivo. Treatment with miR-10a-5p mimics allowed miR-10a-5p enrichment and resulted in a significant restriction in PHEV replication, suggesting widespread negative regulation of the RNA virus infection by miR-10a-5p. The outcomes were also evidenced by miR-10a-5p inhibitor over-expression. Luciferase reporter, quantitative real-time PCR (qRT-PCR), and western blotting analysis further showed that Syndecan 1 (SDC1), a cell surface proteoglycan associated with host defense mechanisms, acts as a target gene of miR-10a-5p during PHEV infection. Naturally, siRNA-mediated knockdown of SDC1 leads to a reduction in viral replication, implying that SDC1 expression is likely a favorable condition for viral replication. Together, the findings demonstrated that the abundant miR-10a-5p leads to downstream suppression of SDC1, and it functions as an antiviral mechanism in the PHEV-induced disease, providing a potential strategy for the prevention and treatment of PHEV infection in the future work.

Insights into antiviral mechanisms of remdesivir, lopinavir/ritonavir and chloroquine/hydroxychloroquine affecting the new SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is a kind of viral pneumonia with an unusual outbreak in Wuhan, China, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). There is currently no licensed antiviral treatment available to prevent human CoV infection. The widespread clinical use and existing knowledge on antiviral mechanisms of remdesivir, lopinavir/ritonavir and chloroquine/hydroxychloroquine in the treatment of previous epidemic diseases, namely, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), may be helpful in the combat with novel SARS-CoV-2 infection. Recent clinical evidence didn't confirm the beneficial role of lopinavir/ritonavir and chloroquine/hydroxychloroquine for COVID-19 patients and their use was reassessed. We provide an overview of the current evidence into the mechanisms of action of these available drugs which are repurposed for treatment of the new virus. Available data identifies remdesivir as an adenosine analogue that can target the RNA-dependent RNA polymerase and block viral RNA synthesis. It has been a promising antiviral drug against a wide array of RNA viruses. 3CLpro is a major CoV protease that cleaves the large replicase polyproteins during viral replication and can be targeted by the protease inhibitor lopinavir/ritonavir but the clinical effects are controversial. Chloroquine/Hydroxychloroquine could impair the replication of SARSCoV-2 by multiple mechanisms and their immunomodulatory properties could ameliorate clinical manifestations that are mediated by immune reactions of the host although its beneficial effects are under question and need to be proven at the clinical level. Existing in vitro and in vivo evidence delineate the molecular mechanisms of these drugs in CoV-infected cells. Numerous studies demonstrated the ability of remdesivir to inhibit SARS-CoV-2 replication but future research would be needed to understand the exact mode of action of lopinavir/ritonavir and chloroquine/hydroxychloroquine in SARS-CoV-2 infected cells and to use this knowledge in the treatment of the current COVID-19.

Antiviral Potential of Nanoparticles-Can Nanoparticles Fight Against Coronaviruses?

Infectious diseases account for more than 20% of global mortality and viruses are responsible for about one-third of these deaths. Highly infectious viral diseases such as severe acute respiratory (SARS), Middle East respiratory syndrome (MERS) and coronavirus disease (COVID-19) are emerging more frequently and their worldwide spread poses a serious threat to human health and the global economy. The current COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of 27 July 2020, SARS-CoV-2 has infected over 16 million people and led to the death of more than 652,434 individuals as on 27 July 2020 while also causing significant economic losses. To date, there are no vaccines or specific antiviral drugs to prevent or treat COVID-19. Hence, it is necessary to accelerate the development of antiviral drugs and vaccines to help mitigate this pandemic. Non-Conventional antiviral agents must also be considered and exploited. In this regard, nanoparticles can be used as antiviral agents for the treatment of various viral infections. The use of nanoparticles provides an interesting opportunity for the development of novel antiviral therapies with a low probability of developing drug resistance compared to conventional chemical-based antiviral therapies. In this review, we first discuss viral mechanisms of entry into host cells and then we detail the major and important types of nanomaterials that could be used as antiviral agents. These nanomaterials include silver, gold, quantum dots, organic nanoparticles, liposomes, dendrimers and polymers. Further, we consider antiviral mechanisms, the effects of nanoparticles on coronaviruses and therapeutic approaches of nanoparticles. Finally, we provide our perspective on the future of nanoparticles in the fight against viral infections.