Characterization of SARS-CoV-2 replication in human H1299/ACE2 cells: A versatile and practical infection model for antiviral research and beyond.

A range of cell culture infection models have been used to study SARS-CoV-2 and perform antiviral drug research. Commonly used African green monkey Vero, human lung-derived Calu-3 and ACE2+TMPRSS2-expressing A549 cells, each have their limitations. Here, we describe human ACE2-expressing H1299 lung cells as a more efficient and robust model for SARS-CoV-2 research. These cells are as easy to handle as Vero cells, support SARS-CoV-2 replication to high titers, display a functional innate immune response and are suitable for plaque assays, microscopy, the production of (genetically stable) virus stocks and antiviral assays. H1299/ACE2-based (CPE reduction) assays can be performed without adding a P-gP drug efflux pump inhibitor, which is often required in Vero-based assays. Moreover, H1299/ACE2 cells allowed us to perform CPE reduction assays with omicron variants that did not work in Vero-based assays. In summary, H1299/ACE2 cells are a versatile infection model to study SARS-CoV-2 replication in the context of antiviral drug development and virus-host interaction studies.

SARS-CoV-2-infected human airway epithelial cell cultures uniquely lack interferon and immediate early gene responses caused by other coronaviruses.

Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a member of a class of highly pathogenic coronaviruses. The large family of coronaviruses, however, also includes members that cause only mild symptoms, like human coronavirus-229E (HCoV-229E) or OC43 (HCoV-OC43). Unravelling how molecular (and cellular) pathophysiology differs between highly and low pathogenic coronaviruses is important for the development of therapeutic strategies. Methods: Here, we analysed the transcriptome of primary human bronchial epithelial cells (PBEC), differentiated at the air-liquid interface (ALI) after infection with SARS-CoV-2, SARS-CoV, Middle East Respiratory Syndrome (MERS)-CoV and HCoV-229E using bulk RNA sequencing. Results: ALI-PBEC were efficiently infected by all viruses, and SARS-CoV, MERS-CoV and HCoV-229E infection resulted in a largely similar transcriptional response. The response to SARS-CoV-2 infection differed markedly as it uniquely lacked the increase in expression of immediate early genes, including FOS, FOSB and NR4A1 that was observed with all other coronaviruses. This finding was further confirmed in publicly available experimental and clinical datasets. Interfering with NR4A1 signalling in Calu-3 lung epithelial cells resulted in a 100-fold reduction in extracellular RNA copies of SARS-CoV-2 and MERS-CoV, suggesting an involvement in virus replication. Furthermore, a lack in induction of interferon-related gene expression characterised the main difference between the highly pathogenic coronaviruses and low pathogenic viruses HCoV-229E and HCoV-OC43. Conclusion: Our results demonstrate a previously unknown suppression of a host response gene set by SARS-CoV-2 and confirm a difference in interferon-related gene expression between highly pathogenic and low pathogenic coronaviruses.

Voclosporin and the Antiviral Effect Against SARS-CoV-2 in Immunocompromised Kidney Patients.

Introduction: Immunocompromised kidney patients are at increased risk of prolonged SARS-CoV-2 infection and related complications. Preclinical evidence demonstrates a more potent inhibitory effect of voclosporin on SARS-CoV-2 replication than tacrolimus in vitro. We investigated the potential antiviral effects of voclosporin on SARS-CoV-2 in immunocompromised patients. Methods: First, we conducted a prospective, randomized, open-label, proof-of-concept study in 20 kidney transplant recipients (KTRs) on tacrolimus-based immunosuppression who contracted mild to moderate SARS-CoV-2 infection. Patients were randomized to continue tacrolimus or switch to voclosporin. Second, we performed a post hoc analysis on SARS-CoV-2 infections in 216 patients with lupus nephritis (LN) on standard immunosuppression who were randomly exposed to voclosporin or placebo as part of a clinical trial that was conducted during the worldwide COVID-19 pandemic. Results: The primary end point was clearance of SARS-CoV-2 viral load and that did not differ between voclosporin-treated KTRs (median 12 days, interquartile range [IQR] 8-28) and tacrolimus-treated KTRs (median 12 days, IQR 4-16) nor was there a difference in clinical recovery. Pharmacokinetic analyses demonstrated that, when voclosporin trough levels were on-target, SARS-CoV-2 viral load dropped significantly more (ΔCt 7.7 [3.4-10.7]) compared to tacrolimus-treated KTRs (ΔCt 2.7 [2.0-4.3]; P = 0.035). In voclosporin-exposed patients with LN, SARS-CoV-2 infection was detected in 6% (7/116) compared to 12% (12/100) in placebo-exposed patients (relative risk [RR] 1.4 [0.97-2.06]). Notably, no voclosporin-exposed patients with LN died from severe SARS-CoV-2 infection compared to 3% (3/100) in placebo-exposed patients (RR 2.2 [1.90-2.54]). Conclusion: This proof-of-concept study shows a potential positive risk-benefit profile for voclosporin in immunocompromised patients with SARS-CoV-2 infection. These results warrant further investigations on voclosporin to establish an equipoise between infection and maintenance immunosuppression.

Highly Potent Antisense Oligonucleotides Locked Nucleic Acid Gapmers Targeting the SARS-CoV-2 RNA Genome.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the current worldwide pandemic and the associated coronavirus disease 2019 with potentially lethal outcome. Although effective vaccines strongly contributed to reduce disease severity, establishing a toolbox to control current and newly emerging coronaviruses of epidemic concern requires the development of novel therapeutic compounds, to treat severely infected individuals and to prevent virus transmission. Here we present a therapeutic strategy targeting the SARS-CoV-2 RNA genome using antisense oligonucleotides (ASOs). We demonstrate that selected locked nucleic acid gapmers have the potency to reduce the in vitro intracellular viral load by up to 96%. Our promising results strongly support the case for further development of our preselected ASOs as therapeutic or prophylactic antiviral agents.

Novel approaches for the rapid development of rationally designed arbovirus vaccines.

Vector-borne diseases, including those transmitted by mosquitoes, account for more than 17% of infectious diseases worldwide. This number is expected to rise with an increased spread of vector mosquitoes and viruses due to climate change and man-made alterations to ecosystems. Among the most common, medically relevant mosquito-borne infections are those caused by arthropod-borne viruses (arboviruses), especially members of the genera Flavivirus and Alphavirus. Arbovirus infections can cause severe disease in humans, livestock and wildlife. Severe consequences from infections include congenital malformations as well as arthritogenic, haemorrhagic or neuroinvasive disease. Inactivated or live-attenuated vaccines (LAVs) are available for a small number of arboviruses; however there are no licensed vaccines for the majority of these infections. Here we discuss recent developments in pan-arbovirus LAV approaches, from site-directed attenuation strategies targeting conserved determinants of virulence to universal strategies that utilize genome-wide re-coding of viral genomes. In addition to these approaches, we discuss novel strategies targeting mosquito saliva proteins that play an important role in virus transmission and pathogenesis in vertebrate hosts. For rapid pre-clinical evaluations of novel arbovirus vaccine candidates, representative in vitro and in vivo experimental systems are required to assess the desired specific immune responses. Here we discuss promising models to study attenuation of neuroinvasion, neurovirulence and virus transmission, as well as antibody induction and potential for cross-reactivity. Investigating broadly applicable vaccination strategies to target the direct interface of the vertebrate host, the mosquito vector and the viral pathogen is a prime example of a One Health strategy to tackle human and animal diseases.

Honokiol Inhibits SARS-CoV-2 Replication in Cell Culture at a Post-Entry Step.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019, and the resulting pandemic has already caused the death of over 6 million people. There are currently few antivirals approved for treatment of the 2019 coronavirus disease (COVID-19), and more options would be beneficial, not only now but also to increase our preparedness for future coronavirus outbreaks. Honokiol is a small molecule from magnolia trees for which several biological effects have been reported, including anticancer and anti-inflammatory activities. Honokiol has also been shown to inhibit several viruses in cell culture. In this study, we determined that honokiol protected Vero E6 cells from SARS-CoV-2-mediated cytopathic effect, with a 50% effective concentration of 7.8 µM. In viral load reduction assays, honokiol decreased viral RNA copies as well as viral infectious progeny titers. The compound also inhibited SARS-CoV-2 replication in the more relevant human A549 cells expressing angiotensin converting enzyme 2 and transmembrane protease serine 2. Time-of-addition and other assays showed that honokiol inhibited virus replication at a post-entry step of the replication cycle. Honokiol was also effective against more recent variants of SARS-CoV-2, including Omicron, and it inhibited other human coronaviruses as well. Our study suggests that honokiol is an interesting molecule to be evaluated further in animal studies and, when successful, maybe even in clinical trials to investigate its effect on virus replication and pathogenic (inflammatory) host responses. IMPORTANCE Honokiol is a compound that shows both anti-inflammatory and antiviral effects, and therefore its effect on SARS-CoV-2 infection was assessed. This small molecule inhibited SARS-CoV-2 replication in various cell-based infection systems, with up to an ~1,000-fold reduction in virus titer. In contrast to earlier reports, our study clearly showed that honokiol acts on a postentry step of the replication cycle. Honokiol also inhibited different recent SARS-CoV-2 variants and other human coronaviruses (Middle East respiratory syndrome CoV and SARS-CoV), demonstrating its broad spectrum of antiviral activity. The anticoronavirus effect, combined with its anti-inflammatory properties, make honokiol an interesting compound to be further explored in animal coronavirus infection models.

Mathematical modeling of plus-strand RNA virus replication to identify broad-spectrum antiviral treatment strategies.

Plus-strand RNA viruses are the largest group of viruses. Many are human pathogens that inflict a socio-economic burden. Interestingly, plus-strand RNA viruses share remarkable similarities in their replication. A hallmark of plus-strand RNA viruses is the remodeling of intracellular membranes to establish replication organelles (so-called "replication factories"), which provide a protected environment for the replicase complex, consisting of the viral genome and proteins necessary for viral RNA synthesis. In the current study, we investigate pan-viral similarities and virus-specific differences in the life cycle of this highly relevant group of viruses. We first measured the kinetics of viral RNA, viral protein, and infectious virus particle production of hepatitis C virus (HCV), dengue virus (DENV), and coxsackievirus B3 (CVB3) in the immuno-compromised Huh7 cell line and thus without perturbations by an intrinsic immune response. Based on these measurements, we developed a detailed mathematical model of the replication of HCV, DENV, and CVB3 and showed that only small virus-specific changes in the model were necessary to describe the in vitro dynamics of the different viruses. Our model correctly predicted virus-specific mechanisms such as host cell translation shut off and different kinetics of replication organelles. Further, our model suggests that the ability to suppress or shut down host cell mRNA translation may be a key factor for in vitro replication efficiency, which may determine acute self-limited or chronic infection. We further analyzed potential broad-spectrum antiviral treatment options in silico and found that targeting viral RNA translation, such as polyprotein cleavage and viral RNA synthesis, may be the most promising drug targets for all plus-strand RNA viruses. Moreover, we found that targeting only the formation of replicase complexes did not stop the in vitro viral replication early in infection, while inhibiting intracellular trafficking processes may even lead to amplified viral growth.

R-Propranolol Has Broad-Spectrum Anti-Coronavirus Activity and Suppresses Factors Involved in Pathogenic Angiogenesis.

The SARS-CoV-2 pandemic highlighted the need for broad-spectrum antivirals to increase our preparedness. Patients often require treatment by the time that blocking virus replication is less effective. Therefore, therapy should not only aim to inhibit the virus, but also to suppress pathogenic host responses, e.g., leading to microvascular changes and pulmonary damage. Clinical studies have previously linked SARS-CoV-2 infection to pathogenic intussusceptive angiogenesis in the lungs, involving the upregulation of angiogenic factors such as ANGPTL4. The ß-blocker propranolol is used to suppress aberrant ANGPTL4 expression in the treatment of hemangiomas. Therefore, we investigated the effect of propranolol on SARS-CoV-2 infection and the expression of ANGPTL4. SARS-CoV-2 upregulated ANGPTL4 in endothelial and other cells, which could be suppressed with R-propranolol. The compound also inhibited the replication of SARS-CoV-2 in Vero-E6 cells and reduced the viral load by up to ~2 logs in various cell lines and primary human airway epithelial cultures. R-propranolol was as effective as S-propranolol but lacks the latter's undesired ß-blocker activity. R-propranolol also inhibited SARS-CoV and MERS-CoV. It inhibited a post-entry step of the replication cycle, likely via host factors. The broad-spectrum antiviral effect and suppression of factors involved in pathogenic angiogenesis make R-propranolol an interesting molecule to further explore for the treatment of coronavirus infections.

Impact of Changes in Human Airway Epithelial Cellular Composition and Differentiation on SARS-CoV-2 Infection Biology.

The consequences of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can range from asymptomatic to fatal disease. Variations in epithelial susceptibility to SARS-CoV-2 infection depend on the anatomical location from the proximal to distal respiratory tract. However, the cellular biology underlying these variations is not completely understood. Thus, air-liquid interface cultures of well-differentiated primary human tracheal and bronchial epithelial cells were employed to study the impact of epithelial cellular composition and differentiation on SARS-CoV-2 infection by transcriptional (RNA sequencing) and immunofluorescent analyses. Changes of cellular composition were investigated by varying time of differentiation or by using specific compounds. We found that SARS-CoV-2 primarily infected not only ciliated cells but also goblet cells and transient secretory cells. Viral replication was impacted by differences in cellular composition, which depended on culturing time and anatomical origin. A higher percentage of ciliated cells correlated with a higher viral load. However, DAPT treatment, which increased the number of ciliated cells and reduced goblet cells, decreased viral load, indicating the contribution of goblet cells to infection. Cell entry factors, especially cathepsin L and transmembrane protease serine 2, were also affected by differentiation time. In conclusion, our study demonstrates that viral replication is affected by changes in cellular composition, especially in cells related to the mucociliary system. This could explain in part the variable susceptibility to SARS-CoV-2 infection between individuals and between anatomical locations in the respiratory tract.

The alphavirus nonstructural protein 2 NTPase induces a host translational shut-off through phosphorylation of eEF2 via cAMP-PKA-eEF2K signaling.

Chikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host cell factors at many levels and is expected to have a profound effect on cellular physiology. To obtain more insight into host responses to infection, stable isotope labeling with amino acids in cell culture and liquid chromatography-tandem mass spectrometry were used to assess temporal changes in the cellular phosphoproteome during CHIKV infection. Among the ~3,000 unique phosphorylation sites analyzed, the largest change in phosphorylation status was measured on residue T56 of eukaryotic elongation factor 2 (eEF2), which showed a >50-fold increase at 8 and 12 h p.i. Infection with other alphaviruses (Semliki Forest, Sindbis and Venezuelan equine encephalitis virus (VEEV)) triggered a similarly strong eEF2 phosphorylation. Expression of a truncated form of CHIKV or VEEV nsP2, containing only the N-terminal and NTPase/helicase domains (nsP2-NTD-Hel), sufficed to induce eEF2 phosphorylation, which could be prevented by mutating key residues in the Walker A and B motifs of the NTPase domain. Alphavirus infection or expression of nsP2-NTD-Hel resulted in decreased cellular ATP levels and increased cAMP levels. This did not occur when catalytically inactive NTPase mutants were expressed. The wild-type nsP2-NTD-Hel inhibited cellular translation independent of the C-terminal nsP2 domain, which was previously implicated in directing the virus-induced host shut-off for Old World alphaviruses. We hypothesize that the alphavirus NTPase activates a cellular adenylyl cyclase resulting in increased cAMP levels, thus activating PKA and subsequently eukaryotic elongation factor 2 kinase. This in turn triggers eEF2 phosphorylation and translational inhibition. We conclude that the nsP2-driven increase of cAMP levels contributes to the alphavirus-induced shut-off of cellular protein synthesis that is shared between Old and New World alphaviruses. MS Data are available via ProteomeXchange with identifier PXD009381.

SARS-CoV-2-specific CD4+ and CD8+ T cell responses can originate from cross-reactive CMV-specific T cells.

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific CD4+ and CD8+ T cells in SARS-CoV-2-unexposed donors has been explained by the presence of T cells primed by other coronaviruses. However, based on the relatively high frequency and prevalence of cross-reactive T cells, we hypothesized cytomegalovirus (CMV) may induce these cross-reactive T cells. Stimulation of pre-pandemic cryo-preserved peripheral blood mononuclear cells (PBMCs) with SARS-CoV-2 peptides revealed that frequencies of SARS-CoV-2-specific T cells were higher in CMV-seropositive donors. Characterization of these T cells demonstrated that membrane-specific CD4+ and spike-specific CD8+ T cells originate from cross-reactive CMV-specific T cells. Spike-specific CD8+ T cells recognize SARS-CoV-2 spike peptide FVSNGTHWF (FVS) and dissimilar CMV pp65 peptide IPSINVHHY (IPS) presented by HLA-B*35:01. These dual IPS/FVS-reactive CD8+ T cells were found in multiple donors as well as severe COVID-19 patients and shared a common T cell receptor (TCR), illustrating that IPS/FVS-cross-reactivity is caused by a public TCR. In conclusion, CMV-specific T cells cross-react with SARS-CoV-2, despite low sequence homology between the two viruses, and may contribute to the pre-existing immunity against SARS-CoV-2.

Mathematical modeling of plus-strand RNA virus replication to identify broad-spectrum antiviral treatment strategies.

Plus-strand RNA viruses are the largest group of viruses. Many are human pathogens that inflict a socio-economic burden. Interestingly, plus-strand RNA viruses share remarkable similarities in their replication. A hallmark of plus-strand RNA viruses is the remodeling of intracellular membranes to establish replication organelles (so-called "replication factories"), which provide a protected environment for the replicase complex, consisting of the viral genome and proteins necessary for viral RNA synthesis. In the current study, we investigate pan-viral similarities and virus-specific differences in the life cycle of this highly relevant group of viruses. We first measured the kinetics of viral RNA, viral protein, and infectious virus particle production of hepatitis C virus (HCV), dengue virus (DENV), and coxsackievirus B3 (CVB3) in the immuno-compromised Huh7 cell line and thus without perturbations by an intrinsic immune response. Based on these measurements, we developed a detailed mathematical model of the replication of HCV, DENV, and CVB3 and show that only small virus-specific changes in the model were necessary to describe the in vitro dynamics of the different viruses. Our model correctly predicted virus-specific mechanisms such as host cell translation shut off and different kinetics of replication organelles. Further, our model suggests that the ability to suppress or shut down host cell mRNA translation may be a key factor for in vitro replication efficiency which may determine acute self-limited or chronic infection. We further analyzed potential broad-spectrum antiviral treatment options in silico and found that targeting viral RNA translation, especially polyprotein cleavage, and viral RNA synthesis may be the most promising drug targets for all plus-strand RNA viruses. Moreover, we found that targeting only the formation of replicase complexes did not stop the viral replication in vitro early in infection, while inhibiting intracellular trafficking processes may even lead to amplified viral growth. Author summary: Plus-strand RNA viruses comprise a large group of related and medically relevant viruses. The current global pandemic of COVID-19 caused by the SARS-coronavirus-2 as well as the constant spread of diseases such as dengue and chikungunya fever show the necessity of a comprehensive and precise analysis of plus-strand RNA virus infections. Plus-strand RNA viruses share similarities in their life cycle. To understand their within-host replication strategies, we developed a mathematical model that studies pan-viral similarities and virus-specific differences of three plus-strand RNA viruses, namely hepatitis C, dengue, and coxsackievirus. By fitting our model to in vitro data, we found that only small virus-specific variations in the model were required to describe the dynamics of all three viruses. Furthermore, our model predicted that ribosomes involved in viral RNA translation seem to be a key player in plus-strand RNA replication efficiency, which may determine acute or chronic infection outcome. Furthermore, our in-silico drug treatment analysis suggests that targeting viral proteases involved in polyprotein cleavage, in combination with viral RNA replication, may represent promising drug targets with broad-spectrum antiviral activity.

The Cyclophilin-Dependent Calcineurin Inhibitor Voclosporin Inhibits SARS-CoV-2 Replication in Cell Culture.

Kidney transplant recipients (KTRs) are at increased risk for a more severe course of COVID-19, due to their pre-existing comorbidity and immunosuppression. Consensus protocols recommend lowering immunosuppression in KTRs with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but the optimal combination remains unclear. Calcineurin inhibitors (CNIs) are cornerstone immunosuppressants used in KTRs and some have been reported to possess antiviral activity against RNA viruses, including coronaviruses. Here, we evaluated the effect of the CNIs tacrolimus, cyclosporin A, and voclosporin (VCS), as well as other immunosuppressants, on SARS-CoV-2 replication in cell-based assays. Unexpected, loss of compound due to plastic binding and interference of excipients in pharmaceutical formulations (false-positive results) complicated the determination of EC50 values of cyclophilin-dependent CNI's in our antiviral assays. Some issues could be circumvented by using exclusively glass lab ware with pure compounds. In these experiments, VCS reduced viral progeny yields in human Calu-3 cells at low micromolar concentrations and did so more effectively than cyclosporin A, tacrolimus or other immunosuppressants. Although, we cannot recommend a particular immunosuppressive regimen in KTRs with COVID-19, our data suggest a potential benefit of cyclophilin-dependent CNIs, in particular VCS in reducing viral progeny, which warrants further clinical evaluation in SARS-CoV-2-infected KTRs.

SHAPE-guided RNA structure homology search and motif discovery.

The rapidly growing popularity of RNA structure probing methods is leading to increasingly large amounts of available RNA structure information. This demands the development of efficient tools for the identification of RNAs sharing regions of structural similarity by direct comparison of their reactivity profiles, hence enabling the discovery of conserved structural features. We here introduce SHAPEwarp, a largely sequence-agnostic SHAPE-guided algorithm for the identification of structurally-similar regions in RNA molecules. Analysis of Dengue, Zika and coronavirus genomes recapitulates known regulatory RNA structures and identifies novel highly-conserved structural elements. This work represents a preliminary step towards the model-free search and identification of shared and conserved RNA structural features within transcriptomes.

Ultralarge Virtual Screening Identifies SARS-CoV-2 Main Protease Inhibitors with Broad-Spectrum Activity against Coronaviruses.

Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.

Synthesis, Structure-Activity Relationships, and Antiviral Profiling of 1-Heteroaryl-2-Alkoxyphenyl Analogs as Inhibitors of SARS-CoV-2 Replication.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has led to a pandemic, that continues to be a huge public health burden. Despite the availability of vaccines, there is still a need for small-molecule antiviral drugs. In an effort to identify novel and drug-like hit matter that can be used for subsequent hit-to-lead optimization campaigns, we conducted a high-throughput screening of a 160 K compound library against SARS-CoV-2, yielding a 1-heteroaryl-2-alkoxyphenyl analog as a promising hit. Antiviral profiling revealed this compound was active against various beta-coronaviruses and preliminary mode-of-action experiments demonstrated that it interfered with viral entry. A systematic structure-activity relationship (SAR) study demonstrated that a 3- or 4-pyridyl moiety on the oxadiazole moiety is optimal, whereas the oxadiazole can be replaced by various other heteroaromatic cycles. In addition, the alkoxy group tolerates some structural diversity.

Tomatidine reduces Chikungunya virus progeny release by controlling viral protein expression.

Tomatidine, a natural steroidal alkaloid from unripe green tomatoes has been shown to exhibit many health benefits. We recently provided in vitro evidence that tomatidine reduces the infectivity of Dengue virus (DENV) and Chikungunya virus (CHIKV), two medically important arthropod-borne human infections for which no treatment options are available. We observed a potent antiviral effect with EC50 values of 0.82 µM for DENV-2 and 1.3 µM for CHIKV-LR. In this study, we investigated how tomatidine controls CHIKV infectivity. Using mass spectrometry, we identified that tomatidine induces the expression of p62, CD98, metallothionein and thioredoxin-related transmembrane protein 2 in Huh7 cells. The hits p62 and CD98 were validated, yet subsequent analysis revealed that they are not responsible for the observed antiviral effect. In parallel, we sought to identify at which step of the virus replication cycle tomatidine controls virus infectivity. A strong antiviral effect was seen when in vitro transcribed CHIKV RNA was transfected into Huh7 cells treated with tomatidine, thereby excluding a role for tomatidine during CHIKV cell entry. Subsequent determination of the number of intracellular viral RNA copies and viral protein expression levels during natural infection revealed that tomatidine reduces the RNA copy number and viral protein expression levels in infected cells. Once cells are infected, tomatidine is not able to interfere with active RNA replication yet it can reduce viral protein expression. Collectively, the results delineate that tomatidine controls viral protein expression to exert its antiviral activity. Lastly, sequential passaging of CHIKV in presence of tomatidine did not lead to viral resistance. Collectively, these results further emphasize the potential of tomatidine as an antiviral treatment towards CHIKV infection.

Structural Insights into the Mechanisms of Action of Functionally Distinct Classes of Chikungunya Virus Nonstructural Protein 1 Inhibitors.

Chikungunya virus (CHIKV) nonstructural protein 1 (nsP1) harbors the methyltransferase (MTase) and guanylyltransferase (GTase) activities needed for viral RNA capping and represents a promising antiviral drug target. We compared the antiviral efficacies of nsP1 inhibitors belonging to the MADTP, CHVB, and FHNA series (6'-fluoro-homoneplanocin A [FHNA], its 3'-keto form, and 6'-ß-fluoro-homoaristeromycin). Cell-based phenotypic cross-resistance assays revealed that the CHVB and MADTP series had similar modes of action that differed from that of the FHNA series. In biochemical assays with purified Semliki Forest virus and CHIKV nsP1, CHVB compounds strongly inhibited MTase and GTase activities, while MADTP-372 had a moderate inhibitory effect. FHNA did not directly inhibit the enzymatic activity of CHIKV nsP1. The first-of-their-kind molecular-docking studies with the cryo-electron microscopy (cryo-EM) structure of CHIKV nsP1, which is assembled into a dodecameric ring, revealed that the MADTP and CHVB series bind at the S-adenosylmethionine (SAM)-binding site in the capping domain, where they would function as competitive or noncompetitive inhibitors. The FHNA series was predicted to bind at the secondary binding pocket in the ring-aperture membrane-binding and oligomerization (RAMBO) domain, potentially interfering with the membrane binding and oligomerization of nsP1. Our cell-based and enzymatic assays, in combination with molecular docking and mapping of compound resistance mutations to the nsP1 structure, allowed us to group nsP1 inhibitors into functionally distinct classes. This study identified druggable pockets in the nsP1 dodecameric structure and provides a basis for the rational design, optimization, and combination of inhibitors of this unique and promising antiviral drug target.

Genome-scale deconvolution of RNA structure ensembles.

RNA structure heterogeneity is a major challenge when querying RNA structures with chemical probing. We introduce DRACO, an algorithm for the deconvolution of coexisting RNA conformations from mutational profiling experiments. Analysis of the SARS-CoV-2 genome using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and DRACO, identifies multiple regions that fold into two mutually exclusive conformations, including a conserved structural switch in the 3' untranslated region. This work may open the way to dissecting the heterogeneity of the RNA structurome.

Genome-wide mapping of SARS-CoV-2 RNA structures identifies therapeutically-relevant elements.

SARS-CoV-2 is a betacoronavirus with a linear single-stranded, positive-sense RNA genome, whose outbreak caused the ongoing COVID-19 pandemic. The ability of coronaviruses to rapidly evolve, adapt, and cross species barriers makes the development of effective and durable therapeutic strategies a challenging and urgent need. As for other RNA viruses, genomic RNA structures are expected to play crucial roles in several steps of the coronavirus replication cycle. Despite this, only a handful of functionally-conserved coronavirus structural RNA elements have been identified to date. Here, we performed RNA structure probing to obtain single-base resolution secondary structure maps of the full SARS-CoV-2 coronavirus genome both in vitro and in living infected cells. Probing data recapitulate the previously described coronavirus RNA elements (5' UTR and s2m), and reveal new structures. Of these, ∼10.2% show significant covariation among SARS-CoV-2 and other coronaviruses, hinting at their functionally-conserved role. Secondary structure-restrained 3D modeling of these segments further allowed for the identification of putative druggable pockets. In addition, we identify a set of single-stranded segments in vivo, showing high sequence conservation, suitable for the development of antisense oligonucleotide therapeutics. Collectively, our work lays the foundation for the development of innovative RNA-targeted therapeutic strategies to fight SARS-related infections.