Comparative assessment of favipiravir and remdesivir against human coronavirus NL63 in molecular docking and cell culture models.

Human coronavirus NL63 (HCoV-NL63) mainly affects young children and immunocompromised patients, causing morbidity and mortality in a subset of patients. Since no specific treatment is available, this study aims to explore the anti-SARS-CoV-2 agents including favipiravir and remdesivir for treating HCoV-NL63 infection. We first successfully modelled the 3D structure of HCoV-NL63 RNA-dependent RNA polymerase (RdRp) based on the experimentally solved SARS-CoV-2 RdRp structure. Molecular docking indicated that favipiravir has similar binding affinities to SARS-CoV-2 and HCoV-NL63 RdRp with LibDock scores of 75 and 74, respectively. The LibDock scores of remdesivir to SARS-CoV-2 and HCoV-NL63 were 135 and 151, suggesting that remdesivir may have a higher affinity to HCoV-NL63 compared to SARS-CoV-2 RdRp. In cell culture models infected with HCoV-NL63, both favipiravir and remdesivir significantly inhibited viral replication and production of infectious viruses. Overall, remdesivir compared to favipiravir is more potent in inhibiting HCoV-NL63 in cell culture. Importantly, there is no evidence of resistance development upon long-term exposure to remdesivir. Furthermore, combining favipiravir or remdesivir with the clinically used antiviral cytokine interferon-alpha resulted in synergistic effects. These findings provided a proof-of-concept that anti-SARS-CoV-2 drugs, in particular remdesivir, have the potential to be repurposed for treating HCoV-NL63 infection.

Theoretical Analysis Reveals the Cost and Benefit of Proofreading in Coronavirus Genome Replication.

Severe acute respiratory syndrome coronaviruses have unusually large RNA genomes replicated by a multiprotein complex containing an RNA-dependent RNA polymerase (RdRp). Exonuclease activity enables the RdRp complex to remove wrongly incorporated bases via proofreading, a process not utilized by other RNA viruses. However, it is unclear why the RdRp complex needs proofreading and what the associated trade-offs are. Here we investigate the interplay among the accuracy, speed, and energetic cost of proofreading in the RdRp complex using a kinetic model and bioinformatics analysis. We find that proofreading nearly optimizes the rate of functional virus production. However, we find that further optimization would lead to a significant increase in the proofreading cost. Unexpected importance of the cost minimization is further supported by other global analyses. We speculate that cost optimization could help avoid cell defense responses. Thus, proofreading is essential for the production of functional viruses, but its rate is limited by energy costs.

Mapping major SARS-CoV-2 drug targets and assessment of druggability using computational fragment screening: Identification of an allosteric small-molecule binding site on the Nsp13 helicase.

The 2019 emergence of, SARS-CoV-2 has tragically taken an immense toll on human life and far reaching impacts on society. There is a need to identify effective antivirals with diverse mechanisms of action in order to accelerate preclinical development. This study focused on five of the most established drug target proteins for direct acting small molecule antivirals: Nsp5 Main Protease, Nsp12 RNA-dependent RNA polymerase, Nsp13 Helicase, Nsp16 2'-O methyltransferase and the S2 subunit of the Spike protein. A workflow of solvent mapping and free energy calculations was used to identify and characterize favorable small-molecule binding sites for an aromatic pharmacophore (benzene). After identifying the most favorable sites, calculated ligand efficiencies were compared utilizing computational fragment screening. The most favorable sites overall were located on Nsp12 and Nsp16, whereas the most favorable sites for Nsp13 and S2 Spike had comparatively lower ligand efficiencies relative to Nsp12 and Nsp16. Utilizing fragment screening on numerous possible sites on Nsp13 helicase, we identified a favorable allosteric site on the N-terminal zinc binding domain (ZBD) that may be amenable to virtual or biophysical fragment screening efforts. Recent structural studies of the Nsp12:Nsp13 replication-transcription complex experimentally corroborates ligand binding at this site, which is revealed to be a functional Nsp8:Nsp13 protein-protein interaction site in the complex. Detailed structural analysis of Nsp13 ZBD conformations show the role of induced-fit flexibility in this ligand binding site and identify which conformational states are associated with efficient ligand binding. We hope that this map of over 200 possible small-molecule binding sites for these drug targets may be of use for ongoing discovery, design, and drug repurposing efforts. This information may be used to prioritize screening efforts or aid in the process of deciphering how a screening hit may bind to a specific target protein.

Viral macrodomains: a structural and evolutionary assessment of the pharmacological potential.

Viral macrodomains possess the ability to counteract host ADP-ribosylation, a post-translational modification implicated in the creation of an antiviral environment via immune response regulation. This brought them into focus as promising therapeutic targets, albeit the close homology to some of the human macrodomains raised concerns regarding potential cross-reactivity and adverse effects for the host. Here, we evaluate the structure and function of the macrodomain of SARS-CoV-2, the causative agent of COVID-19. We show that it can antagonize ADP-ribosylation by PARP14, a cellular (ADP-ribosyl)transferase necessary for the restriction of coronaviral infections. Furthermore, our structural studies together with ligand modelling revealed the structural basis for poly(ADP-ribose) binding and hydrolysis, an emerging new aspect of viral macrodomain biology. These new insights were used in an extensive evolutionary analysis aimed at evaluating the druggability of viral macrodomains not only from the Coronaviridae but also Togaviridae and Iridoviridae genera (causing diseases such as Chikungunya and infectious spleen and kidney necrosis virus disease, respectively). We found that they contain conserved features, distinct from their human counterparts, which may be exploited during drug design.

Is oseltamivir suitable for fighting against COVID-19: In silico assessment, in vitro and retrospective study.

BACKGROUND: Oseltamivir is a first-line antiviral drug, especially in primary hospitals. During the ongoing outbreak of coronavirus disease 2019 (COVID-19), most patients with COVID-19 who are symptomatic have used oseltamivir. Considering its popular and important role as an antiviral drug, it is necessary to evaluate oseltamivir in the treatment of COVID-19. OBJECTIVE: To evaluate the effect of oseltamivir against COVID-19. METHODS: Swiss-model was used to construct the structure of the N-terminal RNA-binding domain (NRBD) of the nucleoprotein (NC), papain-like protease (PLpro), and RNA-directed RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). TM-align program was performed to compare the structure of the viral proteins with the structure of the neuraminidase of influenza A. Molecular docking was used to analyze the theoretical possibility of effective binding of oseltamivir with the active centers of the viral proteins. In vitro study was used to evaluate the antiviral efficiency of oseltamivir against SARS-CoV-2. By clinical case analysis, we statistically evaluated whether the history of oseltamivir use influenced the progression of the disease. RESULTS: The structures of NRBD, PLpro, and RdRp were built successfully. The results from TM-align suggested that the S protein, NRBD, 3C-like protease (3CLpro), PLPrO, and RdRp were structurally similar to the influenza A neuraminidase, with TM-scores of 0.30077, 0.19254, 0.28766, 0.30666, and 0.34047, respectively. Interestingly, the active center of 3CL pro was found to be similar to the active center from the neuraminidase of influenza A. Through an analysis of molecular docking, we discovered that oseltamivir carboxylic acid was more favorable to bind to the active site of 3CLpro effectively, but its inhibitory effect was not strong compared with the positive group. Finally, we used in vitro study and retrospective case analysis to verify our speculations. We found that oseltamivir is ineffective against SARS-CoV-2 in vitro study and the clinical use of oseltamivir did not improve the patients' symptoms and signs and did not slow the disease progression. CONCLUSIONS: We consider that oseltamivir isn't suitable for the treatment of COVID-19. During the outbreak of novel coronavirus, when oseltamivir is not effective for the patients after they take it, health workers should be highly vigilant about the possibility of COVID-19.

Tag mechanism as a strategy for the RNA replicase to resist parasites in the RNA world.

The idea that life may have started with an "RNA world" is attractive. Wherein, a crucial event (perhaps at the very beginning of the scenario) should have been the emergence of a ribozyme that catalyzes its own replication, i.e., an RNA replicase. Although now there is experimental evidence supporting the chemical feasibility of such a ribozyme, the evolutionary dynamics of how the replicase could overcome the "parasite" problem (because other RNAs may also exploit this ribozyme) and thrive, as described in the scenario, remains unclear. It has been suggested that spatial limitation may have been important for the replicase to confront parasites. However, more studies showed that such a mechanism is not sufficient when this ribozyme's altruistic trait is taken into full consideration. "Tag mechanism", which means labeling the replicase with a short subsequence for recognition in replication, may be a further mechanism supporting the thriving of the replicase. However, because parasites may also "equip" themselves with the tag, it is far from clear whether the tag mechanism could take effect. Here, we conducted a computer simulation using a Monte-Carlo model to study the evolutionary dynamics surrounding the development of a tag-driven (polymerase-type) RNA replicase in the RNA world. We concluded that (1) with the tag mechanism the replicase could resist the parasites and become prosperous, (2) the main underlying reason should be that the parasitic molecules, especially those strong parasites, are more difficult to appear in the tag-driven system, and (3) the tag mechanism has a synergic effect with the spatial limitation mechanism-while the former provides "time" for the replicase to escape from parasites, the latter provides "space" for the replicase to escape. Notably, tags may readily serve as "control handles", and once the tag mechanism was exploited, the evolution towards complex life may have been much easier.

Viral factors in influenza pandemic risk assessment.

The threat of an influenza A virus pandemic stems from continual virus spillovers from reservoir species, a tiny fraction of which spark sustained transmission in humans. To date, no pandemic emergence of a new influenza strain has been preceded by detection of a closely related precursor in an animal or human. Nonetheless, influenza surveillance efforts are expanding, prompting a need for tools to assess the pandemic risk posed by a detected virus. The goal would be to use genetic sequence and/or biological assays of viral traits to identify those non-human influenza viruses with the greatest risk of evolving into pandemic threats, and/or to understand drivers of such evolution, to prioritize pandemic prevention or response measures. We describe such efforts, identify progress and ongoing challenges, and discuss three specific traits of influenza viruses (hemagglutinin receptor binding specificity, hemagglutinin pH of activation, and polymerase complex efficiency) that contribute to pandemic risk.

Assessment of the internal genes of influenza A (H7N9) virus contributing to high pathogenicity in mice.

UNLABELLED: The recently identified H7N9 influenza A virus has caused severe economic losses and worldwide public concern. Genetic analysis indicates that its six internal genes all originated from H9N2 viruses. However, the H7N9 virus is more highly pathogenic in humans than H9N2, which suggests that the internal genes of H7N9 have mutated. To analyze which H7N9 virus internal genes contribute to its high pathogenicity, a series of reassortants was generated by reverse genetics, with each virus containing a single internal gene of the typical A/Anhui/1/2013 (H7N9) (AH-H7N9) virus in the genetic background of the A/chicken/Shandong/lx1023/2007 (H9N2) virus. The replication ability, polymerase activity, and pathogenicity of these viruses were then evaluated in vitro and in vivo. These recombinants displayed high genetic compatibility, and the H7N9-derived PB2, M, and NP genes were identified as the virulence genes for the reassortants in mice. Further investigation confirmed that the PB2 K627 residue is critical for the high pathogenicity of the H7N9 virus and the reassortant containing the H7N9-derived PB2 segment (H9N2-AH/PB2). Notably, the H7N9-derived PB2 gene displayed greater compatibility with the H9N2 genome than that of H7N9, endowing the H9N2-AH/PB2 reassortant with greater viability and virulence than the parental H7N9 virus. In addition, the H7N9 virus, with the exception of the H9N2 reassortants, could effectively replicate in human A549 cells. Our results indicate that PB2, M, and NP are the key virulence genes, together with the surface hemagglutinin (HA) and neuraminidase (NA) proteins, contributing to the high infectivity of the H7N9 virus in humans. IMPORTANCE: To date, the novel H7N9 influenza A virus has caused 437 human infections, with approximately 30% mortality. Previous work has primarily focused on the two viral surface proteins, HA and NA, but the contribution of the six internal genes to the high pathogenicity of H7N9 has not been systematically studied. Here, the H9N2 virus was used as a genetic backbone to evaluate the virulence genes of H7N9 virus in vitro and in vivo. Our data indicate that the PB2, M, and NP genes play important roles in viral infection in mice and, together with HA and NA, contribute to the high infectivity of the H7N9 virus in humans.

StralSV: assessment of sequence variability within similar 3D structures and application to polio RNA-dependent RNA polymerase.

BACKGROUND: Most of the currently used methods for protein function prediction rely on sequence-based comparisons between a query protein and those for which a functional annotation is provided. A serious limitation of sequence similarity-based approaches for identifying residue conservation among proteins is the low confidence in assigning residue-residue correspondences among proteins when the level of sequence identity between the compared proteins is poor. Multiple sequence alignment methods are more satisfactory--still, they cannot provide reliable results at low levels of sequence identity. Our goal in the current work was to develop an algorithm that could help overcome these difficulties by facilitating the identification of structurally (and possibly functionally) relevant residue-residue correspondences between compared protein structures. RESULTS: Here we present StralSV (structure-alignment sequence variability), a new algorithm for detecting closely related structure fragments and quantifying residue frequency from tight local structure alignments. We apply StralSV in a study of the RNA-dependent RNA polymerase of poliovirus, and we demonstrate that the algorithm can be used to determine regions of the protein that are relatively unique, or that share structural similarity with proteins that would be considered distantly related. By quantifying residue frequencies among many residue-residue pairs extracted from local structural alignments, one can infer potential structural or functional importance of specific residues that are determined to be highly conserved or that deviate from a consensus. We further demonstrate that considerable detailed structural and phylogenetic information can be derived from StralSV analyses. CONCLUSIONS: StralSV is a new structure-based algorithm for identifying and aligning structure fragments that have similarity to a reference protein. StralSV analysis can be used to quantify residue-residue correspondences and identify residues that may be of particular structural or functional importance, as well as unusual or unexpected residues at a given sequence position. StralSV is provided as a web service at http://proteinmodel.org/AS2TS/STRALSV/.

A novel and inexpensive application of RNAi technology to protect shrimp from viral disease.

Large-scale production of long dsRNA is needed if antiviral applications of RNAi are to succeed in shrimp farm operations. A novel hairpin-RNA expression vector was developed based on the RNA-dependent RNA polymerase (RdRp) gene of yellow head virus (YHV), the cause of a lethal shrimp disease. Using transformed RNase-deficient Escherichia coli, large amounts (approximately 5 mg dsRNA from 130 ml bacterial culture) of long dsRNA (>300 nt) were produced. Large-scale in vivo dsRNA production was approximately one-fourth the cost of production of a commercial in vitro transcription kit. The hairpin-RNA consisted of the target RdRp sequence ("forward") and a 100-base shortened version of its inverted repeat ("reverse") to introduce a loop and bypass the difficulty of including a small "loop" connector into the "carrier" vector. A test group of whiteleg shrimp Penaeus (Litopenaeus) vannamei (approximately 10-15 g) was injected with 25 microg of this dsRNA 1-day prior to YHV challenge while control groups were injected with NaCl solution or similarly prepared dsGFP-RNA. The group injected with YHV-specific dsRNA did not develop yellow head disease during 14-day of observation after YHV challenge, whereas the control groups injected with NaCl and dsGFP-RNA developed gross signs of yellow head disease and died within 7-10 days after challenge. Quantitative RT-PCR and immunohistochemistry revealed that both viral mRNA and viral proteins were suppressed in the protected shrimp.

Nucleotide synthetase ribozymes may have emerged first in the RNA world.

Though the "RNA world" hypothesis has gained a central role in ideas concerning the origin of life, the scenario concerning its emergence remains uncertain. It has been speculated that the first scene may have been the emergence of a template-dependent RNA synthetase ribozyme, which catalyzed its own replication: thus, "RNA replicase." However, the speculation remains uncertain, primarily because of the large sequence length requirement of such a replicase and the lack of a convincing mechanism to ensure its self-favoring features. Instead, we propose a nucleotide synthetase ribozyme as an alternative candidate, especially considering recent experimental evidence suggesting the possibility of effective nonenzymatic template-directed synthesis of RNA. A computer simulation was conducted to support our proposal. The conditions for the emergence of the nucleotide synthetase ribozyme are discussed, based on dynamic analysis on a computer. We suggest the template-dependent RNA synthetase ribozyme emerged later, perhaps after the emergence of protocells.

Pause sites and regulatory role of secondary structure in RNA replication.

We analyze the correlation between pause sites and changes in enzyme boundaries within the elongation complex in template-instructed MDV-1 RNA replication. A Monte-Carlo simulation is carried out to follow the refolding events in the replica and in the portion of template strand upstream of the site where nucleotide incorporation takes place. We introduce and verify the hypothesis that the refolding events upstream of the replication fork are involved in the regulation of replication by leading to a partial relaxation of interaction between the enzyme and the growing chain. This relaxation is carried out by replacing enzyme-product binding by intra-chain pairing of the bases involved.