Multiple RNA Viruses Research Articles

Novel quinolone derivatives targeting human dihydroorotate dehydrogenase suppress Ebola virus infection in vitro

Ebola virus (EBOV) has emerged as a significant public health concern since the 2013–2016 outbreak in West Africa. Currently, no effective antiviral treatments have been approved for clinical use. Compound 1 RYL-634 is a quinolone-derived compound that can inhibit dihydroorotate dehydrogenase, a rate-limiting enzyme in the de novo pyrimidine synthesis pathway and it exhibited antiviral activity against multiple RNA virus infection. In this study, we evaluated the efficacy of a panel of newly developed compounds based on RYL-634 against EBOV infection. Our data showed that RYL-634 as well as its derivatives are effective against EBOV transcription- and replication-competent virus-like particle (trVLP) infection and authentic EBOV infection in vitro at low nanomolar IC50 values and relatively high CC50. Of note, the new derivative RYL-687 had the lowest IC50 at approximately 7 nM and was almost 6 times more potent than remdesivir (GS-5734). Exogenous addition of different metabolites in the pyrimidine de novo synthesis pathway confirmed DHODH as the target of RYL-687. These data provide evidence that such quinolone-derived compounds are promising therapeutic candidates against EBOV infection.

An isoform of Dicer protects mammalian stem cells against multiple RNA viruses.

In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses-including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells.

PERK Is Critical for Alphavirus Nonstructural Protein Translation

Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes encephalitis. Previous work indicated that VEEV infection induced early growth response 1 (EGR1) expression, leading to cell death via the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) arm of the unfolded protein response (UPR) pathway. Loss of PERK prevented EGR1 induction and decreased VEEV-induced death. The results presented within show that loss of PERK in human primary astrocytes dramatically reduced VEEV and eastern equine encephalitis virus (EEEV) infectious titers by 4–5 log10. Loss of PERK also suppressed VEEV replication in primary human pericytes and human umbilical vein endothelial cells, but it had no impact on VEEV replication in transformed U87MG and 293T cells. A significant reduction in VEEV RNA levels was observed as early as 3 h post-infection, but viral entry assays indicated that the loss of PERK minimally impacted VEEV entry. In contrast, the loss of PERK resulted in a dramatic reduction in viral nonstructural protein translation and negative-strand viral RNA production. The loss of PERK also reduced the production of Rift Valley fever virus and Zika virus infectious titers. These data indicate that PERK is an essential factor for the translation of alphavirus nonstructural proteins and impacts multiple RNA viruses, making it an exciting target for antiviral development.

Serum IgG Profiling of Toddlers Reveals a Subgroup with Elevated Seropositive Antibodies to Viruses Correlating with Increased Vaccine and Autoantigen Responses.

PurposeThe human antibody repertoire forms in response to infections, the microbiome, vaccinations, and environmental exposures. The specificity of such antibody responses was compared among a cohort of toddlers to identify differences between seropositive versus seronegative responses.MethodsAn assessment of the serum IgM and IgG antibody reactivities in 197 toddlers of 1- and 2-years of age was performed with a microfluidic array containing 110 distinct antigens. Longitudinal profiling was done from years 1 to 2. Seropositivity to RNA and DNA viruses; bacteria; live attenuated, inactive, and subunit vaccines; and autoantigens was compared. A stratification was developed based on quantitative variations in the IgG responses. Clinical presentations and previously known genetic risk alleles for various immune system conditions were investigated in relation to IgG responses.ResultsIgG reactivities stratified toddlers into low, moderate, and high responder groups. The high group (17%) had elevated IgG responses to multiple RNA and DNA viruses (e.g., respiratory syncytial virus, Epstein-Barr virus, adenovirus, Coxsackievirus) and this correlated with increased responses to live attenuated viral vaccines and certain autoantigens. This high group was more likely to be associated with gestational diabetes and an older age. Genetic analyses identified polymorphisms in the IL2RB, TNFSF4, and INS genes in two high responder individuals that were associated with their elevated cytokine levels and clinical history of eczema and asthma.ConclusionSerum IgG profiling of toddlers reveals correlations between the magnitude of the antibody responses towards viruses, live attenuated vaccines, and certain autoantigens. A low responder group had much weaker responses overall, including against vaccines. The serum antibody screen also identifies individuals with IgG responses to less common infections (West Nile virus, parvovirus, tuberculosis). The characterization of the antibody responses in combination with the identification of genetic risk alleles provides an opportunity to identify children with increased risk of clinical disease.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10875-021-00993-w.

The rocaglate CR-31-B (−) inhibits SARS-CoV-2 replication at non-cytotoxic, low nanomolar concentrations in vitro and ex vivo

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, a severe respiratory disease with varying clinical presentations and outcomes, and responsible for a major pandemic that started in early 2020. With no vaccines or effective antiviral treatments available, the quest for novel therapeutic solutions remains an urgent priority. Rocaglates, a class of plant-derived cyclopenta[b]benzofurans, exhibit broad-spectrum antiviral activity against multiple RNA viruses including coronaviruses. Specifically, rocaglates inhibit eukaryotic initiation factor 4A (eIF4A)-dependent mRNA translation initiation, resulting in strongly reduced viral RNA translation. Here, we assessed the antiviral activity of the synthetic rocaglate CR-31-B (−) against SARS-CoV-2 using both in vitro and ex vivo cell culture models. In Vero E6 cells, CR-31-B (−) inhibited SARS-CoV-2 replication with an EC50 of ~1.8 nM. In primary human airway epithelial cells, CR-31-B (−) reduced viral titers to undetectable levels at a concentration of 100 nM. Reduced virus reproduction was accompanied by substantially reduced viral protein accumulation and replication/transcription complex formation. The data reveal a potent anti-SARS-CoV-2 activity by CR-31-B (−), corroborating previous results obtained for other coronaviruses and supporting the idea that rocaglates may be used in first-line antiviral intervention strategies against novel and emerging RNA virus outbreaks.

Emergence, Transmission, and Potential Therapeutic Targets for the COVID-19 Pandemic Associated with the SARS-CoV-2.

The pandemic of the severe acute respiratory syndrome coronavirus (SARS-CoV)-2 at the end of 2019 marked the third outbreak of a highly pathogenic coronavirus affecting the human population in the past twenty years. Cross-species zoonotic transmission of SARS-CoV-2 has caused severe pathogenicity and led to more than 655,000 fatalities worldwide until July 28, 2020. Outbursts of this virus underlined the importance of controlling infectious pathogens across international frontiers. Unfortunately, there is currently no clinically approved antiviral drug or vaccine against SARS-CoV-2, although several broad-spectrum antiviral drugs targeting multiple RNA viruses have shown a positive response and improved recovery in patients. In this review, we compile our current knowledge of the emergence, transmission, and pathogenesis of SARS-CoV-2 and explore several features of SARS-CoV-2. We emphasize the current therapeutic approaches used to treat infected patients. We also highlight the results of in vitro and in vivo data from several studies, which have broadened our knowledge of potential drug candidates for the successful treatment of patients infected with and discuss possible virus and host-based treatment options against SARS-CoV-2.

Identification of a Broad-Spectrum Viral Inhibitor Targeting a Novel Allosteric Site in the RNA-Dependent RNA Polymerases of Dengue Virus and Norovirus

All RNA viruses encode the RNA-dependent RNA polymerase (RdRp) which replicates and transcribes viral RNA. This essential viral enzyme does not exist in mammalian cells, thus presents a main target for the development of antiviral drugs with potential pan-antiviral activity. In this study, we take advantage of the structurally equivalent site in the dengue virus (DENV) RdRp, the N-pocket, and in the human norovirus (hNV) RdRp, the B-site, and performed a parallel structure-based virtual screening to discover compounds that can inhibit the RdRps of both hNV and DENV. We successfully identified a small molecule called Entrectinib (RAI-13) as a potent inhibitor of both hNV and DENV infection. Specifically, RAI-13 binds directly to hNV and DENV RdRps, effectively inhibits the polymerase activity in the in vitro biochemical assays, and exhibits does-responsive inhibition of murine norovirus (MNV) and DENV2 infection with IC50 values of 2.01 and 2.43 μM, respectively. Most promisingly, RAI-13 inhibits hepatitis C virus (HCV) infection by 95% at the 2 μM concentration. We have therefore discovered a small molecule compound that targets an allosteric site that is shared by different viral RdRps and strongly inhibits multiple pathogenic RNA viruses, thus holding the potential of being developed into a broad-spectrum antiviral drug.

Endogenous viral elements integrated into the genome of the soybean aphid, Aphis glycines

Sequence analysis of the genomic DNA isolated from four biotypes of the soybean aphid, Aphis glycines (AG), revealed that in addition to the commonly observed retrovirus-related retrotransposons, viral sequences derived from multiple RNA and DNA viruses have integrated into the genome. Notably, sequences of more than 60 nudiviral genes were identified from de novo assembled DNA contigs, and mapped to assembled genomic scaffolds of AG, indicating that an ancient nudivirus, named Aphis glycines endogenous nudivirus (AgENV), had integrated into the AG genome. Furthermore, sequences derived from a similar endogenous nudivirus, Melanaphis sacchari endogenous nudivirus (MsENV), were identified from the genomic scaffolds of the sugarcane aphid, Melanaphis sacchari. Analysis of transcriptome and small RNA sequence data derived from AG did not provide evidence for transcription of the integrated AgENV genes. Hence, the genes of AgENV may be present as pseudogenes. Phylogenetic analysis based on nudivirus core genes indicated that these aphid ENVs belong to the genus Alphanudivirus.

A Microfluidic Diagnostic Device Capable of Autonomous Sample Mixing and Dispensing for the Simultaneous Genetic Detection of Multiple Plant Viruses.

As an efficient approach to risk management in agriculture, the elimination of losses due to plant diseases and insect pests is one of the most important and urgent technological challenges for improving the crop yield. Therefore, we have developed a polydimethylsiloxane (PDMS)-based microfluidic device for the multiplex genetic diagnosis of plant diseases and pests. It offers unique features, such as rapid detection, portability, simplicity, and the low-cost genetic diagnosis of a wide variety of plant viruses. In this study, to realize such a diagnostic device, we developed a method for the autonomous dispensing of fluid into a microchamber array, which was integrated with a set of three passive stop valves with different burst pressures (referred to as phaseguides) to facilitate precise fluid handling. Additionally, we estimated the mixing efficiencies of several types of passive mixers (referred to as chaotic mixers), which were integrated into a microchannel, through experimental and computational analyses. We first demonstrated the ability of the fabricated diagnostic devices to detect DNA-based plant viruses from an infected tomato crop based on the loop-mediated isothermal amplification (LAMP) method. Moreover, we demonstrated the simultaneous detection of RNA-based plant viruses, which can infect cucurbits, by using the reverse transcription LAMP (RT-LAMP) method. The multiplex RT-LAMP assays revealed that multiple RNA viruses extracted from diseased cucumber leaves were successfully detected within 60 min, without any cross-contamination between reaction microchambers, on our diagnostic device.

RNAi‐Mediated Silencing of Endogenous Wheat Genes EIF(Iso)4E‐2 and EIF4G Induce Resistance to Multiple RNA Viruses in Transgenic Wheat

ABSTRACTWheat (Triticum aestivum L.) lacks sources of effective resistance to the potyviruses Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV). Potyviruses use host eukaryotic initiation factors (eIFs) to facilitate initial translation of their genomes, and many plant virus resistance genes are due to mutations in eIFs. Therefore, we hypothesized that silencing TaeIF(iso)4E and TaeIF4G would provide effective and broad‐spectrum virus resistance. Three transgenic wheat lines with an RNA interference (RNAi) hairpin construct targeting TaeIF(iso)4E and four lines with a hairpin construct targeting TaeIF4G were recovered through wheat transformation. Early generation lines were found to be resistant to WSMV and TriMV, and co‐inoculation of both. The lines were selfed to the T5 generation to insure transgene stability, as well as crossed onto the hard red winter wheat cultivar ‘Karl 92’ to test the RNAi constructs in a more adapted genetic background. Phenotypically, transgenic lines were found to be resistant to WSMV, TriMV, and mixed infections of both, were resistant to Soil‐borne wheat mosaic virus, and demonstrated a significant reduction in Barley yellow dwarf virus infection. The RNAi effectiveness on viral RNA was evaluated using reverse transcription quantitative polymerase chain reaction (PCR). An 18‐fold reduction in WSMV and TriMV viral RNA was found in the T5 transgenic lines when compared with control plants. Viral RNA reduction was also found in the F1, and BC1F1 crosses to Karl 92. These results demonstrate that a single, endogenously derived transgene in wheat can provide resistance to multiple viruses and provides a proof of concept for future gene editing.

Selected nucleos(t)ide-based prescribed drugs and their multi-target activity

Nucleos(t)ide analogues play pivotal roles as antiviral, cytotoxic or immunosuppressive agents. Here, we review recent reports of nucleoside analogues that exhibit broad-spectrum activity towards multiple life-threatening RNA and DNA viruses. We also present a discussion about nucleoside antimetabolites—approved antineoplastic agents—that have recently been shown to have antiviral and/or antibacterial activity. The approved drugs and drug combinations, as well as recently identified candidates for investigation and/or experimentation, are discussed. Several examples of repurposed drugs that have already been approved for use are presented. This strategy can be crucial for the first-line treatment of acute infections or coinfections and for the management of drug-resistant strains.

NAC1 Potentiates Cellular Antiviral Signaling by Bridging MAVS and TBK1.

Intracellular viral RNAs are recognized by the RIG-I-like receptors (RLRs), which signal through the mitochondrial antiviral signaling protein MAVS. MAVS recruits and activates TBK1 kinase, which further phosphorylates and activates the transcription factor IRF3, leading to the induction of type I IFN and downstream antiviral genes. We identified human nucleus accumbens-associated 1 (NAC1), a member of the BTB/POZ family, as a bridge for MAVS and TBK1 that positively regulates the RLR-mediated induction of type I IFN. Overexpression or knockdown of NAC1 could, respectively, enhance or impair Sendai virus-triggered activation of TBK1 and IRF3, as well as induction of IFN-β. NAC1 also significantly boosted host antiviral responses against multiple RNA viruses. NAC1 was able to interact with MAVS and TBK1 upon viral infection. The BTB/POZ domain (aa 1-133) of NAC1 interacted with MAVS, and the remainder of NAC1 bound to TBK1. Furthermore, NAC1 could promote the recruitment of TBK1 to MAVS. In contrast, knockdown of NAC1 attenuated the interaction between TBK1 and MAVS. Collectively, our study characterizes NAC1 as an important component of RLR-mediated innate immune responses and uncovers a previously unrecognized function of the BTB/POZ family proteins.

A Human Long Non-coding RNA LncATV Promotes Virus Replication Through Restricting RIG-I-Mediated Innate Immunity.

Pattern recognition receptors sense pathogen components and initiate the host antiviral innate immune response, such as inducing interferons (IFNs). Long non-coding RNAs (lncRNAs) are emerging regulators of multiple biological processes. However, their role in antiviral response, especially through regulating the human innate immune, is largely unexplored. Here we characterized that lncATV, a human specific lncRNA, was up-regulated upon type I/III IFN stimulations and virus infection. LncATV was cytoplasmic localized and relatively high expressed in human monocytes, erythroleukemia cells and hepatoma cells. Notably, lncATV knockdown significantly inhibited the replication of multiple RNA viruses, such as hepatitis C virus, Zika virus, Newcastle disease virus, and Sendai virus. Mechanistically, RIG-I antiviral signaling and IFN effective pathway were enhanced when lncATV expression was knocked down but inhibited by overexpressed lncATV. RNA immunoprecipitation results demonstrated an association between LncATV and RIG-I. Collectively, our findings reveal the functional role of a novel human specific lncATV as a regulatory lncRNA restricting virus associated innate immune response.

Synthesis and Structure-Activity Relationships of 3,5-Disubstituted-pyrrolo[2,3- b]pyridines as Inhibitors of Adaptor-Associated Kinase 1 with Antiviral Activity.

There are currently no approved drugs for the treatment of emerging viral infections, such as dengue and Ebola. Adaptor-associated kinase 1 (AAK1) is a cellular serine-threonine protein kinase that functions as a key regulator of the clathrin-associated host adaptor proteins and regulates the intracellular trafficking of multiple unrelated RNA viruses. Moreover, AAK1 is overexpressed specifically in dengue virus-infected but not bystander cells. Because AAK1 is a promising antiviral drug target, we have embarked on an optimization campaign of a previously identified 7-azaindole analogue, yielding novel pyrrolo[2,3- b]pyridines with high AAK1 affinity. The optimized compounds demonstrate improved activity against dengue virus both in vitro and in human primary dendritic cells and the unrelated Ebola virus. These findings demonstrate that targeting cellular AAK1 may represent a promising broad-spectrum antiviral strategy.

Basal expression of interferon regulatory factor 1 drives intrinsic hepatocyte resistance to multiple RNA viruses.

Current paradigms of cell intrinsic immunity to RNA viruses center on virus-triggered inducible antiviral responses initiated by RIG-I-like receptors (RLRs) or Toll-like receptors (TLRs) that sense pathogen-associated molecular patterns, and signal downstream through interferon regulatory factors (IRFs), transcription factors that induce synthesis of type I and type III interferons (IFNs)1. RNA viruses have evolved sophisticated strategies to disrupt these signaling pathways and evade elimination by cells, attesting to their importance2. Less attention has been paid how IRFs maintain basal levels of protection against viruses. Here, we depleted antiviral factors linked to RLR and TLR signaling in order to map critical host pathways restricting positive-strand RNA virus replication in immortalized hepatocytes and identified an unexpected role for IRF1. We show constitutively expressed IRF1 acts independently of MAVS, IRF3, and STAT1-dependent signaling to provide intrinsic antiviral protection in actinomycin D-treated cells. IRF1 localizes to the nucleus, where it maintains basal transcription of a suite of antiviral genes that protect against multiple pathogenic RNA viruses, including hepatitis A and C viruses (HAV and HCV), dengue virus (DENV) and Zika virus (ZIKV). Our findings reveal an unappreciated layer of hepatocyte intrinsic immunity to these positive-strand RNA viruses, and identify previously unrecognized antiviral effector genes.

Host Lipids in Positive-Strand RNA Virus Genome Replication.

Membrane association is a hallmark of the genome replication of positive-strand RNA viruses [(+)RNA viruses]. All well-studied (+)RNA viruses remodel host membranes and lipid metabolism through orchestrated virus-host interactions to create a suitable microenvironment to survive and thrive in host cells. Recent research has shown that host lipids, as major components of cellular membranes, play key roles in the replication of multiple (+)RNA viruses. This review focuses on how (+)RNA viruses manipulate host lipid synthesis and metabolism to facilitate their genomic RNA replication, and how interference with the cellular lipid metabolism affects viral replication.

Rapid Sequencing of Multiple RNA Viruses in Their Native Form.

Long-read nanopore sequencing by a MinION device offers the unique possibility to directly sequence native RNA. We combined an enzymatic poly-A tailing reaction with the native RNA sequencing to (i) sequence complex population of single-stranded (ss)RNA viruses in parallel, (ii) detect genome, subgenomic mRNA/mRNA simultaneously, (iii) detect a complex transcriptomic architecture without the need for assembly, (iv) enable real-time detection. Using this protocol, positive-ssRNA, negative-ssRNA, with/without a poly(A)-tail, segmented/non-segmented genomes were mixed and sequenced in parallel. Mapping of the generated sequences on the reference genomes showed 100% length recovery with up to 97% identity. This work provides a proof of principle and the validity of this strategy, opening up a wide range of applications to study RNA viruses.

Understanding the selectivity of inhibitors toward PI4KIIIα and PI4KIIIβ based molecular modeling.

Type III phosphatidylinositol 4 kinases (PI4KIIIs) are essential enzymes that are related to the replication of multiple RNA viruses. Understanding the interaction mechanisms of molecular compounds with the alpha and beta isoforms of PI4KIII (PI4KIIIα and PI4KIIIβ) is of significance in the development of inhibitors that can bind to these two enzymes selectively. In this work, molecular dynamics (MD) simulations and binding free energy calculations were combined to investigate the binding modes of seven selected compounds to PI4KIIIα and PI4KIIIβ. Analyses based on MD trajectories provide detailed interaction mechanisms of these compounds with PI4KIIIα and PI4KIIIβ at the atomic level, and indicate that the selectivity of these compounds is mainly due to the structural difference of the binding pockets. It is expected that the detailed binding information found in this study can provide useful help for the structure-based design of selective inhibitors toward PI4KIIIα and PI4KIIIβ.

Silvestrol Inhibits Chikungunya Virus Replication

Silvestrol, a natural compound that is isolated from plants of the genus Aglaia, is a specific inhibitor of the RNA helicase eIF4A, which unwinds RNA secondary structures in 5′-untranslated regions (UTRs) of mRNAs and allows translation. Silvestrol has a broad antiviral activity against multiple RNA virus families. Here, we show that silvestrol inhibits the replication of chikungunya virus (CHIKV), a positive single-stranded RNA virus. Silvestrol delayed the protein synthesis of non-structural (nsPs) and structural proteins, resulting in a delayed innate response to CHIKV infection. Interferon-α induced STAT1 phosphorylation was not inhibited nor did eIF2α become phosphorylated 16 h post infection in the presence of silvestrol. In addition, the host protein shut-off induced by CHIKV infection was decreased in silvestrol-treated cells. Silvestrol acts by limiting the amount of nsPs, and thereby reducing CHIKV RNA replication. From our results, we propose that inhibition of the host helicase eIF4A might have potential as a therapeutic strategy to treat CHIKV infections.

LY6E mediates an evolutionarily conserved enhancement of virus infection by targeting a late entry step

Interferons (IFNs) contribute to cell-intrinsic antiviral immunity by inducing hundreds of interferon-stimulated genes (ISGs). In a screen to identify antiviral ISGs, we unexpectedly found that LY6E, a member of the LY6/uPAR family, enhanced viral infection. Here, we show that viral enhancement by ectopically expressed LY6E extends to several cellular backgrounds and affects multiple RNA viruses. LY6E does not impair IFN antiviral activity or signaling, but rather promotes viral entry. Using influenza A virus as a model, we narrow the enhancing effect of LY6E to uncoating after endosomal escape. Diverse mammalian orthologs of LY6E also enhance viral infectivity, indicating evolutionary conservation of function. By structure-function analyses, we identify a single amino acid in a predicted loop region that is essential for viral enhancement. Our study suggests that LY6E belongs to a class of IFN-inducible host factors that enhance viral infectivity without suppressing IFN antiviral activity.