Viral Replication Process Research Articles

Sphingolipids: Effectors and Achilles Heals in Viral Infections?

As viruses are obligatory intracellular parasites, any step during their life cycle strictly depends on successful interaction with their particular host cells. In particular, their interaction with cellular membranes is of crucial importance for most steps in the viral replication cycle. Such interactions are initiated by uptake of viral particles and subsequent trafficking to intracellular compartments to access their replication compartments which provide a spatially confined environment concentrating viral and cellular components, and subsequently, employ cellular membranes for assembly and exit of viral progeny. The ability of viruses to actively modulate lipid composition such as sphingolipids (SLs) is essential for successful completion of the viral life cycle. In addition to their structural and biophysical properties of cellular membranes, some sphingolipid (SL) species are bioactive and as such, take part in cellular signaling processes involved in regulating viral replication. It is especially due to the progress made in tools to study accumulation and dynamics of SLs, which visualize their compartmentalization and identify interaction partners at a cellular level, as well as the availability of genetic knockout systems, that the role of particular SL species in the viral replication process can be analyzed and, most importantly, be explored as targets for therapeutic intervention.

Identification of Potential Inhibitors against Epstein–Barr Virus Nuclear Antigen 1 (EBNA1): An Insight from Docking and Molecular Dynamic Simulations

Epstein-Barr virus (EBV), a known tumorigenic virus, is associated with various neuropathies, including multiple sclerosis (MS). However, there is no anti-EBV FDA-approved drug available in the market. Our study targeted EBV protein EBV nuclear antigen 1 (EBNA1), crucial in virus replication and expressed in all the stages of viral latencies. This dimeric protein binds to an 18 bp palindromic DNA sequence and initiates the process of viral replication. We chose phytochemicals and FDA-approved MS drugs based on literature survey followed by their evaluation efficacies as anti-EBNA1 molecules. Molecular docking revealed FDA drugs ozanimod, siponimod, teriflunomide, and phytochemicals; emodin; protoapigenone; and EGCG bound to EBNA1 with high affinities. ADMET and Lipinski's rule analysis of the phytochemicals predicted favorable druggability. We supported our assessments of pocket druggability with molecular dynamics simulations and binding affinity predictions by the molecular mechanics generalized Born surface area (MM/GBSA) method. Our results establish a stable binding for siponimod and ozanimod with EBNA1 mainly via van der Waals interactions. We identified hot spot residues like I481', K477', L582', and K586' in the binding of ligands. In particular, K477' at the amino terminal of EBNA1 is known to establish interaction with two bases at the major groove of the DNA. Siponimod bound to EBNA1 engaging K477', thus plausibly making it unavailable for DNA interaction. Computational alanine scanning further supported the significant roles of K477', I481', and K586' in the binding of ligands with EBNA1. Conclusively, the compounds showed promising results to be used against EBNA1.

Correlative multi-scale cryo-imaging unveils SARS-CoV-2 assembly and egress

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events – e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.

Functional implications of residues of the B' motif of geminivirus replication initiator protein in its helicase activity.

Geminivirus replication initiator protein (Rep) is a multifunctional viral protein required for replication. During the process of viral replication, Rep acts as a site- and strand-specific endonuclease, ligase, ATPase, and helicase. B' motif and β-hairpin loop of the geminivirus Rep are conserved and important for Rep-mediated helicase activity required for viral replication. To dissect the roles of various amino acid residues of the B' motif and β-hairpin loop of the geminivirus Rep helicase in its process of unwinding DNA, we investigated eight conserved residues near the ATP active site or the ssDNA contact channel. Our strategy was to mutate these residues to alanines and investigate the effects of these mutations on various biochemical activities associated with DNA unwinding. We looked into the ATP binding, ATP hydrolysis, DNA binding, and DNA unwinding activities of the wild-type and mutant Rep proteins. These investigations showed four residues (Arg279, Asp280, Tyr287, and Pro290) affecting the DNA unwinding activity. A structural model analysis confirmed the B' loop and ssDNA binding loop to be connected through a β-hairpin structure, suggesting that changes on one loop might affect the other and that these residues function by acting in concert. Viral genomes containing Rep proteins having these mutations in the B' motif did not replicate in planta. Taken together, these results indicated all four residues to be implicated in helicase activity mediated by Rep and demonstrated the significance, for viral replication, of the B' motif and β-hairpin loop of the C-terminal region of the Rep protein.

Translationally controlled tumour protein: A protein necessary for potyvirus intracellular multiplication that supports plant infection by unrelated viruses

AbstractThe multifunctional protein translationally controlled tumour protein (TCTP) was previously identified as necessary for infection by the potyvirus pepper yellow mosaic virus. Using turnip mosaic virus (TuMV) as a model to study potyvirus biology, we confirmed that TCTP has a positive effect on virus infection. Living cell confocal microscopy demonstrated that TCTP colocalises with 6K2‐tagged replication vesicles and with a perinuclear globular structure typically observed during potyvirus infection. Also, TCTP silenced protoplasts showed reduced virus accumulation, quantified by qRT‐PCR, which suggests an effect on virus replication, translation or other intracellular process. Finally, TCTP silencing in plants reduced the accumulation of two species belonging to Orthotospovirus and a Begomovirus genus, which are not closely related to potyviruses. The results suggest that TCTP is a general susceptibility factor to several unrelated viruses.

SARS-CoV-2: Origin, Evolution, and Targeting Inhibition.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused an outbreak in Wuhan city, China and quickly spread worldwide. Currently, there are no specific drugs or antibodies that claim to cure severe acute respiratory diseases. For SARS-CoV-2, the spike (S) protein recognizes and binds to the angiotensin converting enzyme 2 (ACE2) receptor, allowing viral RNA to enter the host cell. The main protease (Mpro) is involved in the proteolytic process for mature non-structural proteins, and RNA-dependent RNA polymerase (RdRp) is responsible for the viral genome replication and transcription processes. Owing to the pivotal physiological roles in viral invasion and replication, S protein, Mpro, RdRp are regarded as the main therapeutic targets for coronavirus disease 2019 (COVID-19). In this review, we carried out an evolutionary analysis of SARS-CoV-2 in comparison with other mammal-infecting coronaviruses that have sprung up in the past few decades and described the pathogenic mechanism of SARS-CoV-2. We displayed the structural details of S protein, Mpro, and RdRp, as well as their complex structures with different chemical inhibitors or antibodies. Structural comparisons showed that some neutralizing antibodies and small molecule inhibitors could inhibit S protein, Mpro, or RdRp. Moreover, we analyzed the structural differences between SARS-CoV-2 ancestral S protein and D614G mutant, which led to a second wave of infection during the recent pandemic. In this context, we outline the methods that might potentially help cure COVID-19 and provide a summary of effective chemical molecules and neutralizing antibodies.

LncRNA GAS5‑mediated miR‑23a‑3p promotes inflammation and cell apoptosis by targeting TLR4 in a cell model of sepsis.

Sepsis is a syndrome characterized by organ dysfunction and an abnormal immune response to infection. A growing body of research has shown the importance of long non-coding RNAs (lncRNAs) in tumorigenesis, virus replication, inflammatory injury and other pathological processes. The aim of the present study was to explore the role and potential mechanism of the lncRNA growth arrest-specific 5 (GAS5) in the lipopolysaccharide (LPS)-induced inflammation and apoptosis of THP-1 cells. An in vitro sepsis model was established by treating THP-1 cells with LPS. Apoptosis was detected by flow cytometry. The expression levels of IL-6, IL-1β and TNF-α were detected using reverse transcription-quantitative PCR (RT-qPCR) and ELISA, and those of GAS5, microRNA (miR)-23a-3p and Toll-like receptor 4 (TLR4) were detected by RT-qPCR. The changes in the biological activity of THP-1 cells induced by the silencing of GAS5 and overexpression of miR-23a-3p and TLR4 were investigated. The relationships among GAS5, miR-23a-3p and TLR4 were analyzed using luciferase reporter assays. The results revealed that LPS increased the expression of GAS5 in THP-1 cells, and GAS5 knockdown effectively inhibited inflammation and cell apoptosis in the LPS-induced sepsis model. In addition, the results of the luciferase reporter assays indicated that both GAS5 and TLR4 directly target miR-23a-3p. The expression of miR-23a-3p was downregulated whereas that of TLR4 was upregulated in the septic cells. Further experiments showed that the overexpression of TLR4 attenuated the suppressive effects of miR-23a-3p overexpression and GAS5 knockdown on LPS-induced inflammation and apoptosis. In conclusion, the present study indicates that GAS5 strengthens LPS-induced inflammation and apoptosis via the miR-23a-3p/TLR4 pathway.

Modeling poliovirus replication dynamics from live time-lapse single-cell imaging data

Viruses experience selective pressure on the timing and order of events during infection to maximize the number of viable offspring they produce. Additionally, they may experience variability in cellular environments encountered, as individual eukaryotic cells can display variation in gene expression among cells. This leads to a dynamic phenotypic landscape that viruses must face to replicate. To examine replication dynamics displayed by viruses faced with this variable landscape, we have developed a method for fitting a stochastic mechanistic model of viral infection to time-lapse imaging data from high-throughput single-cell poliovirus infection experiments. The model’s mechanistic parameters provide estimates of several aspects associated with the virus’s intracellular dynamics. We examine distributions of parameter estimates and assess their variability to gain insight into the root causes of variability in viral growth dynamics. We also fit our model to experiments performed under various drug treatments and examine which parameters differ under these conditions. We find that parameters associated with translation and early stage viral replication processes are essential for the model to capture experimentally observed dynamics. In aggregate, our results suggest that differences in viral growth data generated under different treatments can largely be captured by steps that occur early in the replication process.

An autoantigen profile of human A549 lung cells reveals viral and host etiologic molecular attributes of autoimmunity in COVID-19

We aim to establish a comprehensive COVID-19 autoantigen atlas in order to understand autoimmune diseases caused by SARS-CoV-2 infection. Based on the unique affinity between dermatan sulfate and autoantigens, we identified 348 proteins from human lung A549 cells, of which 198 are known targets of autoantibodies. Comparison with current COVID data identified 291 proteins that are altered at protein or transcript level in SARS-CoV-2 infection, with 191 being known autoantigens. These known and putative autoantigens are significantly associated with viral replication and trafficking processes, including gene expression, ribonucleoprotein biogenesis, mRNA metabolism, translation, vesicle and vesicle-mediated transport, and apoptosis. They are also associated with cytoskeleton, platelet degranulation, IL-12 signaling, and smooth muscle contraction. Host proteins that interact with and that are perturbed by viral proteins are a major source of autoantigens. Orf3 induces the largest number of protein alterations, Orf9 affects the mitochondrial ribosome, and they and E, M, N, and Nsp proteins affect protein localization to membrane, immune responses, and apoptosis. Phosphorylation and ubiquitination alterations by viral infection define major molecular changes in autoantigen origination. This study provides a large list of autoantigens as well as new targets for future investigation, e.g., UBA1, UCHL1, USP7, CDK11A, PRKDC, PLD3, PSAT1, RAB1A, SLC2A1, platelet activating factor acetylhydrolase, and mitochondrial ribosomal proteins. This study illustrates how viral infection can modify host cellular proteins extensively, yield diverse autoantigens, and trigger a myriad of autoimmune sequelae. Our work provides a rich resource for studies into “long COVID” and related autoimmune sequelae.

Modelling studies reveal the importance of the C-terminal inter motif loop of NSP1 as a promising target site for drug discovery and screening of potential phytochemicals to combat SARS-CoV-2

COVID-19 pandemic causative SARS-CoV-2 coronavirus is still rapid in progression and transmission even after a year. Understanding the viral transmission and impeding the replication process within human cells are considered as the vital point to control and overcome COVID-19 infection. Non-structural Protein 1, one among the proteins initially produced upon viral entry into human cells, instantly binds with the human ribosome and inhibit the host translation process by preventing the mRNA attachment. However, the formation of NSP1 bound Ribosome complex does not affect the viral replication process. NSP1 plays an indispensable role in modulating the host gene expression and completely steals the host cellular machinery. The full-length structure of NSP1 is essential for the activity in the host cell and importantly the loop connecting N and C-terminal domains are reported to play a role in ribosome binding. Due to the unavailability of the experimentally determined full-length structure of NSP1, we have modelled the complete structure using comparative modelling and the stability and conformational behaviour of the modelled structure was evaluated through molecular dynamics simulation. Interestingly, the present study reveals the significance of the inter motif loop to serves as a potential binding site for drug discovery experiments. Further, we have screened the phytochemicals from medicinal plant sources since they were used for several hundred years that minimizes the traditional drug development time. Among the 5638 phytochemicals screened against the functionally associated binding site of NSP1, the best five phytochemicals shown high docking score of −9.63 to −8.75 kcal/mol were further evaluated through molecular dynamics simulations to understand the binding affinity and stability of the complex. Prime MM-GBSA analysis gave the relative binding free energies for the top five compounds (dihydromyricetin, 10-demethylcephaeline, dihydroquercetin, pseudolycorine and tricetin) in the range of −45.17 kcal/mol to −37.23 kcal/mol, indicating its binding efficacy in the predicted binding site of NSP1. The density functional theory calculations were performed for the selected five phytochemicals to determine the complex stability and chemical reactivity. Thus, the identified phytochemicals could further be used as effective anti-viral agents to overcome COVID-19 and as well as several other viral infections.

Structure-based identification of SARS-CoV-2 main protease inhibitors from anti-viral specific chemical libraries: an exhaustive computational screening approach

Worldwide coronavirus disease 2019 (COVID-19) outbreak is still threatening global health since its outbreak first reported in the late 2019. The causative novel virus has been designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although COVID-19 emergent with significant mortality, there is no availability of definite treatment measures. It is now extremely desirable to identify potential chemical entities against SARS-CoV-2 for the treatment of COVID-19. In the present study, a state-of-art virtual screening protocol was implemented on three anti-viral specific chemical libraries against SARS-CoV-2 main protease (Mpro). Particularly, viewing the large-scale biological role of Mpro in the viral replication process it has been considered as a prospective anti-viral drug target. Herein, on collected 79,892 compounds, hierarchical multistep docking followed by relative binding free energy estimation has been performed. Thereafter, implying a user-defined XP-dock and MM-GBSA cut-off scores as −8.00 and −45.00 kcal/mol, chemical space has been further reduced. Exhaustive molecular binding interactions analyses and various pharmacokinetics profiles assessment suggested four compounds (ChemDiv_D658-0159, ChemDiv_F431-0433, Enamine_Z3019991843 and Asinex_LAS_51389260) as potent inhibitors/modulators of SARS-CoV-2 Mpro. In-depth protein–ligand interactions stability in the dynamic state has been evaluated by 100 ns molecular dynamics (MD) simulation studies along with MM-GBSA-based binding free energy estimations of entire simulation trajectories that have revealed strong binding affinity of all identified compounds towards Mpro. Hence, all four identified compounds might be considered as promising candidates for future drug development specifically targeting the SARS-CoV-2 Mpro; however, they also need experimental assessment for a better understanding of molecular interaction mechanisms.Graphic abstract

Progress in developing MNK inhibitors

The MNKs (mitogen-activated protein kinase-interacting protein kinases) phosphorylate eIF4E (eukaryotic initiation factor 4E) at serine 209; eIF4E plays an important role in the translation of cytoplasmic mRNAs, all of which possess a 5' 'cap' structure to which eIF4E binds. Elevated levels of eIF4E, p-eIF4E and/or the MNK protein kinases have been found in many types of cancer, including solid tumors and leukemia. MNKs also play a role in metabolic disease. Regulation of the activities of MNKs (MNK1 and MNK2), control the phosphorylation of eIF4E, which in turn has a close relationship with the processes of tumor development, cell migration and invasion, and energy metabolism. MNK knock-out mice display no adverse effects on normal cells or phenotypes suggesting that MNK may be a potentially safe targets for the treatment of various cancers. Several MNK inhibitors or 'degraders' have been identified. Initially, some of the inhibitors were developed from natural products or based on other protein kinase inhibitors which inhibit multiple kinases. Subsequently, more potent and selective inhibitors for MNK1/2 have been designed and synthesized. Currently, three inhibitors (BAY1143269, eFT508 and ETC-206) are in various stages of clinical trials for the treatment of solid cancers or leukemia, either alone or combined with inhibitors of other protein kinase. In this review, we summarize the diverse MNK inhibitors that have been reported in patents and other literature, including those with activities invitro and/or invivo.

Molecular dynamics and in silico mutagenesis on the reversible inhibitor-bound SARS-CoV-2 main protease complexes reveal the role of lateral pocket in enhancing the ligand affinity

The 2019 novel coronavirus pandemic caused by SARS-CoV-2 remains a serious health threat to humans and there is an urgent need to develop therapeutics against this deadly virus. Recent scientific evidences have suggested that the main protease (Mpro) enzyme in SARS-CoV-2 can be an ideal drug target due to its crucial role in the viral replication and transcription processes. Therefore, there are ongoing research efforts to identify drug candidates against SARS-CoV-2 Mpro that resulted in hundreds of X-ray crystal structures of ligand-bound Mpro complexes in the Protein Data Bank (PDB) describing the interactions of different fragment chemotypes within different sites of the Mpro. In this work, we performed rigorous molecular dynamics (MD) simulation of 62 reversible ligand–Mpro complexes in the PDB to gain mechanistic insights about their interactions at the atomic level. Using a total of over 3 µs long MD trajectories, we characterized different pockets in the apo Mpro structure, and analyzed the dynamic interactions and binding affinity of ligands within those pockets. Our results identified the key residues that stabilize the ligands in the catalytic sites and other pockets of Mpro. Our analyses unraveled the role of a lateral pocket in the catalytic site in Mpro that is critical for enhancing the ligand binding to the enzyme. We also highlighted the important contribution from HIS163 in the lateral pocket towards ligand binding and affinity against Mpro through computational mutation analyses. Further, we revealed the effects of explicit water molecules and Mpro dimerization in the ligand association with the target. Thus, comprehensive molecular-level insights gained from this work can be useful to identify or design potent small molecule inhibitors against SARS-CoV-2 Mpro.

Factor and 6th Polynomial Regression Analyses of COVID19 Data from 35 Countries Reveal 4 Patterns of Epidemic Evolution that Includes up to 4 Sequential Waves.

COVID Pandemics is now one year old, and virus not only spread across the world but also drastically changes by means of a high mutation rate, many of them appearing simultaneously at different geographic regions. Because of this mutation capacity the virus cannot be considered single entity as proposed current epidemiologic models, but has to be assumed as a fuzzy being that expresses itself as a family of variants, requiring its dynamics to be understood by means of new theoretical approaches and new epidemic data analyses. The purpose of the present paper is to discuss actual knowledge about COVID19 to better model its dynamics and analyze worldwide epidemic data under this point of view. To accomplish this, actual knowledge about the virus genome structure and replication process, as well as about its known mutations are reviewed, resulting in the proposal that: SARS Cov2 evolved from animals and/or other Human CoVs following the rules of natural selection due to its high rate of mutations that transformed its ancestors to better infect humans using ACE2 receptors, simultaneously in many distinct and widely distributed geographic regions, and it continues, nowadays, this kind of evolution across the world, largely independent from human mobility. Therefore, COVID19 concurrently appeared at many different world regions and have not one but multiple concurrent ancestors. Because of this, the theory of Formal Languages is proposed as an adequate tool to model the structure of COVID19 genome; its mutations and actual virus mutational evolution. This model justifies the use of Factor Analysis as a statistical tool to investigate patterns of covariation in a large number of variables and to determine if information may be condensed into small sets of these variables called principal components . Results shows that 4 patterns explain 75% of covariance of the Daily New Cases reported by 35 countries, indicating the existence of 4 worldwide epidemic patterns. 6th Polynomial regression was used to further investigation the national particularities of the epidemics of the different countries associated to each of these patterns. Results suggest that COVID19 high mutational rate creates simultaneously a large number of virus lineages across the world that submitted to natural selection, promotes distinct epidemic evolution in different countries, mostly dictated by the local virus mutational development rather than influenced by foreigner lineage importation. Based on this, actual Pandemic Dogmatic Management based on lockdown; stay home orders and indiscriminate mobility restriction is discussed and alternative solutions are suggested.

Co-opting of nonATP-generating glycolytic enzymes for TBSV replication

Positive-strand RNA viruses build viral replication organelles (VROs) with the help of co-opted host factors. The energy requirement of intensive viral replication processes is less understood. Previous studies on tomato bushy stunt virus (TBSV) showed that tombusviruses hijack two ATP-producing glycolytic enzymes to produce ATP locally within VROs. In this work, we performed a cDNA library screen with Arabidopsis thaliana proteins and the TBSV p33 replication protein. The p33 - plant interactome contained highly conserved glycolytic proteins. We find that the glycolytic Hxk2 hexokinase, Eno2 phosphopyruvate hydratase and Fba1 fructose 1,6-bisphosphate aldolase are critical for TBSV replication in yeast or in a cell-free replicase reconstitution assay. The recruitment of Fba1 is important for the local production of ATP within VROs. Altogether, our data support the model that TBSV recruits and compartmentalizes possibly most members of the glycolytic pathway. This might allow TBSV to avoid competition with the host for ATP.

Potential Antiviral Target for SARS-CoV-2: A Key Early Responsive Kinase during Viral Entry

Currently, there is no effective antiviral medication for coronavirus disease 2019 (COVID-19) and the knowledge on the potential therapeutic target is in great need. Guided by a time-course transmi...

The Effect of Point Mutations on Structure and Dynamics of SARS-CoV-2 Main Protease Mutants

The SARS-CoV-2 main protease (Mpro) is a serine-type protease with catalytic residues His-41 and Cys-145. These residues cleave highly specific protein sequences, making Mpro a vital part of the viral replication process and an attractive target for inhibitor design. As the virus spreads across the globe, sequence mutations occur, creating an additional challenge for robust inhibitor design. In previous work we have reported results from sequence analysis, structure prediction, and molecular modeling of the first seventy-nine Mpro variants, which revealed patterns in residue substitutions and a distribution of cohesion and constraint in the active site due to mutations.

Cover Image: 3D vacuole structure during viral attack

Viruses need to establish replication factories upon entry into the cells. In this issue, Wang et al. (pp. 319–330) report the 3D model of Cucumber mosaic virus (CMV) replication factories in Nicotiana benthamiana cells. The authors showed that CMV remodels the membrane of vacuole (dark blue) and multivesicular bodies (brilliant blue) to form spherules (yellow) where CMV replication occurs, and crystalline arrays of icosahedral viral particles (red), which appeared to be surrounded by putative ER membrane (purple). These findings enhance our understanding of the viral replication processes and would help to develop antiviral strategies in plants.

Stenoparib, an Inhibitor of Cellular Poly(ADP-Ribose) Polymerase, Blocks Replication of the SARS-CoV-2 and HCoV-NL63 Human Coronaviruses In Vitro.

By late 2020, the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had caused tens of millions of infections and over 1 million deaths worldwide. A protective vaccine and more effective therapeutics are urgently needed. We evaluated a new poly(ADP-ribose) polymerase (PARP) inhibitor, stenoparib, that recently advanced to phase II clinical trials for treatment of ovarian cancer, for activity against human respiratory coronaviruses, including SARS-CoV-2, in vitro Stenoparib exhibits dose-dependent suppression of SARS-CoV-2 multiplication and spread in Vero E6 monkey kidney and Calu-3 human lung adenocarcinoma cells. Stenoparib was also strongly inhibitory to the human seasonal respiratory coronavirus HCoV-NL63. Compared to remdesivir, which inhibits viral replication downstream of cell entry, stenoparib impedes entry and postentry processes, as determined by time-of-addition (TOA) experiments. Moreover, a 10 μM dosage of stenoparib-below the approximated 25.5 μM half-maximally effective concentration (EC50)-combined with 0.5 μM remdesivir suppressed coronavirus growth by more than 90%, indicating a potentially synergistic effect for this drug combination. Stenoparib as a stand-alone or as part of combinatorial therapy with remdesivir should be a valuable addition to the arsenal against COVID-19.IMPORTANCE New therapeutics are urgently needed in the fight against COVID-19. Repurposing drugs that are either already approved for human use or are in advanced stages of the approval process can facilitate more rapid advances toward this goal. The PARP inhibitor stenoparib may be such a drug, as it is currently in phase II clinical trials for the treatment of ovarian cancer and its safety and dosage in humans have already been established. Our results indicate that stenoparib possesses strong antiviral activity against SARS-CoV-2 and other coronaviruses in vitro. This activity appears to be based on multiple modes of action, where both pre-entry and postentry viral replication processes are impeded. This may provide a therapeutic advantage over many current options that have a narrower target range. Moreover, our results suggest that stenoparib and remdesivir in combination may be especially potent against coronavirus infection.

Mutations designed to modify the NS gene mRNA secondary structure affect influenza A pathogenicity in vivo

The influenza A virus genome consists of eight segments of negative-sense RNA that encode up to 18 proteins. During the process of viral replication, positive-sense (+)RNA (cRNA) or messenger RNA (mRNA) is synthesized. Today, there is only a partial understanding of the function of several secondary structures within vRNA and cRNA promoters, and splice sites in the M and NS genes. The most precise secondary structure of (+)RNA has been determined for the NS segment of influenza A virus. The influenza A virus NS gene features two regions with a conserved mRNA secondary structure located near splice sites. Here, we compared 4 variants of the A/Puerto Rico/8/1934 strain featuring different combinations of secondary structures at the NS segment (+)RNA regions 82-148 and 497-564. We found that RNA structures did not affect viral replication in cell culture. However, one of the viruses demonstrated lower NS1 and NEP expression levels during early stage cell infection as well as reduced pathogenicity in mice compared to other variants. In particular, this virus is characterized by an RNA hairpin in the 82-148 region and a stable hairpin in the 497-564 region.