Characterization of the SARS-CoV-2 ExoN (nsp14ExoN-nsp10) complex: implications for its role in viral genome stability and inhibitor identification.

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14-nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14-nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3'-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14-nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12-nsp7-nsp8 (nsp12-7-8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14-nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.

Severe Acute Respiratory Syndrome Coronavirus-2 Inactivation Activity of the Polyphenol-Rich Tea Leaf Extract with Concentrated Theaflavins and Other Virucidal Catechins.

Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is producing a large number of infections and deaths globally, the development of supportive and auxiliary treatments is attracting increasing attention. Here, we evaluated SARS-CoV-2-inactivation activity of the polyphenol-rich tea leaf extract TY-1 containing concentrated theaflavins and other virucidal catechins. The TY-1 was mixed with SARS-CoV-2 solution, and its virucidal activity was evaluated. To evaluate the inhibition activity of TY-1 in SARS-CoV-2 infection, TY-1 was co-added with SARS-CoV-2 into cell culture media. After 1 h of incubation, the cell culture medium was replaced, and the cells were further incubated in the absence of TY-1. The viral titers were then evaluated. To evaluate the impacts of TY-1 on viral proteins and genome, TY-1-treated SARS-CoV-2 structural proteins and viral RNA were analyzed using western blotting and real-time RT-PCR, respectively. TY-1 showed time- and concentration-dependent virucidal activity. TY-1 inhibited SARS-CoV-2 infection of cells. The results of western blotting and real-time RT-PCR suggested that TY-1 induced structural change in the S2 subunit of the S protein and viral genome destruction, respectively. Our findings provided basic insights in vitro into the possible value of TY-1 as a virucidal agent, which could enhance the current SARS-CoV-2 control measures.

Unconventional viral gene expression mechanisms as therapeutic targets.

Unlike the human genome that comprises mostly noncoding and regulatory sequences, viruses have evolved under the constraints of maintaining a small genome size while expanding the efficiency of their coding and regulatory sequences. As a result, viruses use strategies of transcription and translation in which one or more of the steps in the conventional gene-protein production line are altered. These alternative strategies of viral gene expression (also known as gene recoding) can be uniquely brought about by dedicated viral enzymes or by co-opting host factors (known as host dependencies). Targeting these unique enzymatic activities and host factors exposes vulnerabilities of a virus and provides a paradigm for the design of novel antiviral therapies. In this Review, we describe the types and mechanisms of unconventional gene and protein expression in viruses, and provide a perspective on how future basic mechanistic work could inform translational efforts that are aimed at viral eradication.

The SARS-CoV-2-Inactivating Activity of Hydroxytyrosol-Rich Aqueous Olive Pulp Extract (HIDROX®) and Its Use as a Virucidal Cream for Topical Application.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally. Although measures to control SARS-CoV-2, namely, vaccination, medication, and chemical disinfectants are being investigated, there is an increase in the demand for auxiliary antiviral approaches using natural compounds. Here we have focused on hydroxytyrosol (HT)-rich aqueous olive pulp extract (HIDROX®) and evaluated its SARS-CoV-2-inactivating activity in vitro. We showed that the HIDROX solution exhibits time- and concentration-dependent SARS-CoV-2-inactivating activities, and that HIDROX has more potent virucidal activity than pure HT. The evaluation of the mechanism of action suggested that both HIDROX and HT induced structural changes in SARS-CoV-2, which changed the molecular weight of the spike proteins. Even though the spike protein is highly glycosylated, this change was induced regardless of the glycosylation status. In addition, HIDROX or HT treatment disrupted the viral genome. Moreover, the HIDROX-containing cream applied on film showed time- and concentration-dependent SARS-CoV-2-inactivating activities. Thus, the HIDROX-containing cream can be applied topically as an antiviral hand cream. Our findings suggest that HIDROX contributes to improving SARS-CoV-2 control measures.

Ritonavir may inhibit exoribonuclease activity of nsp14 from the SARS-CoV-2 virus and potentiate the activity of chain terminating drugs.

SARS-CoV-2is the causative agent for the ongoing COVID19 pandemic, and this virus belongs to the Coronaviridae family. The nsp14 protein of SARS-CoV-2 houses a 3' to 5' exoribonuclease activity responsible for removing mismatches that arise during genome duplication. A homology model of nsp10-nsp14 complex was used to carry out in silico screening to identify molecules among natural products, or FDA approved drugs that can potentially inhibit the activity of nsp14. This exercise showed that ritonavir might bind to the exoribonuclease active site of the nsp14 protein. A model of the SARS-CoV-2-nsp10-nsp14 complex bound to substrate RNA showed that the ritonavir binding site overlaps with that of the 3' nucleotide of substrate RNA. A comparison of the calculated energies of binding for RNA and ritonavir suggested that the drug may bind to the active site of nsp14 with significant affinity. It is, therefore, possible that ritonavir may prevent association with substrate RNA and thus inhibit the exoribonuclease activity of nsp14. Overall, our computational studies suggest that ritonavir may serve as an effective inhibitor of the nsp14 protein. nsp14 is known to attenuate the inhibitory effect of drugs that function through premature termination of viral genome replication. Hence, ritonavir may potentiate the therapeutic properties of drugs such as remdesivir, favipiravir and ribavirin.

Saxifraga spinulosa-Derived Components Rapidly Inactivate Multiple Viruses Including SARS-CoV-2.

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), and norovirus (NV) are highly contagious pathogens that threaten human health. Here we focused on the antiviral potential of the medicinal herb, Saxifraga spinulosa (SS). Water-soluble extracts of SS were prepared, and their virus-inactivating activity was evaluated against the human virus pathogens SARS-CoV-2 and IAV; we also examined virucidal activity against feline calicivirus and murine norovirus, which are surrogates for human NV. Among our findings, we found that SS-derived gallocatechin gallate compounds were capable of inactivating all viruses tested. Interestingly, a pyrogallol-enriched fraction (Fr 1C) inactivated all viruses more rapidly and effectively than did any of the component compounds used alone. We found that 25 µg/mL of Fr 1C inactivated >99.6% of SARS-CoV-2 within 10 s (reduction of ≥2.33 log10 TCID50/mL). Fr 1C resulted in the disruption of viral genomes and proteins as determined by gel electrophoresis, electron microscopy, and reverse transcription-PCR. Taken together, our results reveal the potential of Fr 1C for development as a novel antiviral disinfectant.

Potential Drug Targets Against Hepatitis B Virus Based on Both Virus and Host Factors.

BACKGROUND: Hepatitis B is a very harmful and epidemic disease caused by hepatitis B virus (HBV). Although an effective anti-HBV vaccine is available, chronic infection poses still a huge health burden in the whole world. The present anti-HBV drugs including nucleoside analogues and interferonalpha have their limitations without exception. There is no effective drug and therapeutic method that can really and truly cure hepatitis B so far. The variability of HBV genome results in that a significant number of patients develop drug resistance during the long-term use of anti-HBV drugs. Hence, it is urgently needed to discover novel targets and develop new drugs against hepatitis B. OBJECTIVE: The review aims to provide the theory support for designing of the anti-HBV innovative drugs by offering a summary of the current situation of antiviral potential targets. RESULTS AND CONCLUSION: Since HBV is obligate intracellular parasite, and as such it depends on host cellular components and functions to replicate itself. The targeting both virus and host might be a novel therapeutic option for hepatitis B. Accordingly, we analyse the advances in the study of the potential drug targets for anti-HBV infection, focusing on targeting virus genome, on targeting host cellular functions and on targeting virus-host proteins interactions, respectively. Meanwhile, the immune targets against chronic hepatitis B are also emphasized. In short, the review provides a summary of antiviral therapeutic strategies to target virus factors, host factors and immune factors for future designing of the innovative drug against HBV infection.

Glycyrrhizin inhibits human parainfluenza virus type 2 replication by the inhibition of genome RNA, mRNA and protein syntheses.

The effect of glycyrrhizin on the replication of human parainfluenza virus type 2 (hPIV-2) was examined. Cell fusion induced by hPIV-2 was inhibited by glycyrrhizin, and glycyrrhizin reduced the number of viruses released from the cells. Glycyrrhizin did not change cell morphology at 1 day of culture, but caused some damage at 4 days, as determined by the effect on actin microfilaments. However, it affected the cell viability at 1 day: about 20% of the cells were not alive by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 1 day of culture. Real-time polymerase chain reaction (PCR) and PCR showed that virus genome synthesis was largely inhibited. mRNA synthesis was also inhibited by glycyrrhizin. Viral protein synthesis was largely inhibited as observed by an indirect immunofluorescence study. Multinucleated giant cell formation was studied using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2ΔMGFP). A few single cells with fluorescence were observed, but the formation of giant cells was completely blocked. Taken together, it was shown that viral genome, mRNA and protein syntheses, including F and HN proteins, were inhibited by glycyrrhizin, and consequently multinucleated giant cell formation was not observed and the infectious virus was not detected in the culture medium.

The protective effect of 3-deoxysappanchalcone on in vitro influenza virus-induced apoptosis and inflammation.

Influenza virus is one of the most important causes of acute respiratory disease. Viral infection and viral replication activate multiple cell signalling pathways. Apoptosis of infected cells and immune response against viral replication, which are generally considered to be protective mechanisms, are also probably mediated by viruses, which lead to severe health problems. We previously reported that 3-deoxysappanchalcone (3-DSC), a compound that is isolated from Caesalpinia sappan, exhibited in vitro anti-influenza activity. In the present study, we further identified that 3-DSC inhibited viral genomic replication and transcription only at a relatively high concentration. We then evaluated the effect of 3-DSC on the regulation of virus-induced cellular apoptosis. 3-DSC ameliorated virus-induced DNA fragmentation in a concentration-dependent manner, which tends to be a consequence of its inhibition of upstream caspase activation. 3-DSC also protected host cells against influenza-induced inflammation by suppressing CCL5 and CXCL10 secretions in endothelial cells and reducing the production of IL-6 and IL-1ß in monocytes/macrophages. In conclusion, our results demonstrate that anti-influenza virus mechanisms of 3-DSC involved anti-apoptosis and anti-inflammation activities in vitro. Moreover, 3-DSC could be a promising drug candidate for influenza treatment.

Antiviral activity of gliotoxin, gentian violet and brilliant green against Nipah and Hendra virus in vitro.

BACKGROUND: Using a recently described monolayer assay amenable to high throughput screening format for the identification of potential Nipah virus and Hendra virus antivirals, we have partially screened a low molecular weight compound library (>8,000 compounds) directly against live virus infection and identified twenty eight promising lead molecules. Initial single blind screens were conducted with 10 microM compound in triplicate with a minimum efficacy of 90% required for lead selection. Lead compounds were then further characterised to determine the median efficacy (IC50), cytotoxicity (CC50) and the in vitro therapeutic index in live virus and pseudotype assay formats. RESULTS: While a number of leads were identified, the current work describes three commercially available compounds: brilliant green, gentian violet and gliotoxin, identified as having potent antiviral activity against Nipah and Hendra virus. Similar efficacy was observed against pseudotyped Nipah and Hendra virus, vesicular stomatitis virus and human parainfluenza virus type 3 while only gliotoxin inhibited an influenza A virus suggesting a non-specific, broad spectrum activity for this compound. CONCLUSION: All three of these compounds have been used previously for various aspects of anti-bacterial and anti-fungal therapy and the current results suggest that while unsuitable for internal administration, they may be amenable to topical antiviral applications, or as disinfectants and provide excellent positive controls for future studies.