Viral RNA Accumulation Research Articles

Antiviral modes of action of the novel compound GLY-15 containing pyrimidine heterocycle and moroxydine skeleton against tobacco mosaic virus.

Plant virus diseases are difficult to prevent and control, causing serious economic losses to the agricultural production world. To develop new pesticides with antiviral activity, a serial of compounds containing the structure of pyrimidine and moroxydine were synthesized, among which GLY-15 exhibited good antiviral activity against tobacco mosaic virus (TMV), while the mechanism of antiviral activity remains to be clarified. GLY-15 treatment significantly inhibited the formation of necrotic spots caused by TMV in Nicotiana glutinosa, and effectively suppressed the systemic transportation of TMV expressing a reporter gene (p35S-30B:GFP) in N. benthamiana and markedly reduced the accumulation of a movement deficient TMV in plants as well as viral RNA accumulation in tobacco protoplasts. The results of RNA sequencing showed that GLY-15 induced significant differential expression of genes or pathways involved in the stress response, defense response and signal transduction, phytohormone response and metabolism. Among them, real-time quantitative PCR validated that the expression of 12 critical genes such as heat shock protein, receptor kinase, cell-wall-related protein, disease-related protein and glucan endo-1,3-β-glucosidase were significantly up-regulated. In addition, GLY-15 triggered reactive oxygen species (ROS) production and induced the activity of several crucial defense related enzymes in plants. The results of molecular docking showed potential binding ability of GLY-15 with TMV helicase and the coat protein. This study provide valuable insights into antiviral mechanism of action for GLY-15, which is expected to be applied as a pesticide for the management of plant viruses. © 2022 Society of Chemical Industry.

Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency

BackgroundThe generation of over 69 spliced HIV-1 mRNAs from one primary transcript by alternative RNA splicing emphasizes the central role that RNA processing plays in HIV-1 replication. Control is mediated in part through the action of host SR proteins whose activity is regulated by multiple SR kinases (CLK1-4, SRPKs).MethodsBoth shRNA depletion and small molecule inhibitors of host SR kinases were used in T cell lines and primary cells to evaluate the role of these factors in the regulation of HIV-1 gene expression. Effects on virus expression were assessed using western blotting, RT-qPCR, and immunofluorescence.ResultsThe studies demonstrate that SR kinases play distinct roles; depletion of CLK1 enhanced HIV-1 gene expression, reduction of CLK2 or SRPK1 suppressed it, whereas CLK3 depletion had a modest impact. The opposing effects of CLK1 vs. CLK2 depletion were due to action at distinct steps; reduction of CLK1 increased HIV-1 promoter activity while depletion of CLK2 affected steps after transcript initiation. Reduced CLK1 expression also enhanced the response to several latency reversing agents, in part, by increasing the frequency of responding cells, consistent with a role in regulating provirus latency. To determine whether small molecule modulation of SR kinase function could be used to control HIV-1 replication, we screened a GSK library of protein kinase inhibitors (PKIS) and identified several pyrazolo[1,5-b] pyridazine derivatives that suppress HIV-1 gene expression/replication with an EC50 ~ 50 nM. The compounds suppressed HIV-1 protein and viral RNA accumulation with minimal impact on cell viability, inhibiting CLK1 and CLK2 but not CLK3 function, thereby selectively altering the abundance of individual CLK and SR proteins in cells.ConclusionsThese findings demonstrate the unique roles played by individual SR kinases in regulating HIV-1 gene expression, validating the targeting of these functions to either enhance latency reversal, essential for “Kick-and-Kill” strategies, or to silence HIV protein expression for “Block-and-Lock” strategies.

Functional Analysis of BcSNX3 in Regulating Resistance to Turnip Mosaic Virus (TuMV) by Autophagy in Pak-choi (Brassica campestris ssp. chinensis)

Sorting nexin protein is a class of highly conserved eukaryotic proteins containing the PX domain. Recent studies related to SNX in plants have focused on the regulation of abiotic stress processes, and there are few studies on the involvement of SNX in biological stress processes in plants. In this paper, a YTH assay and BiFC experiments were conducted twice to show that BcSNX3 (Brassica campestris Sorting nexin 3) interacted with CP and VPg of TuMV, and the interaction between BcATG8h (Brassica campestris autophagy-related gene 8h) and BcSNX3 was also found by YTH and BiFC. The colocalization of BcSNX3 and BcATG8b (Brassica campestris autophagy-related gene 8b) revealed BcSNX3 and autophagosome at the same place in the cell. QRT-PCR analysis showed that TuMV infection promotes the expression of BcSNX3, and the overexpression of this gene hinders the expression of autophagy-related genes and facilitates TuMV infection. VIGS was used to repress the expression level of the BcSNX3 gene in pak-choi to further study the function of BcSNX3 in the infection process of TuMV. After inoculation with TuMV, it was found that the accumulation of viral RNA in BcSNX3-gene-silenced plants was significantly less than in control plants. The accumulation of TuMV virus in the Arabidopsis snx3 knockout mutant was also less than in the wild type after TuMV inoculation. These results suggest that TuMV infection facilitates the expression of BcSNX3, and this gene may promote virus infection by inhibiting autophagy degradation of the virus and interacting with the CP and VPg of the virus. These results lay the foundation for the TuMV resistance breeding of pak-choi.

Pseudomonas aeruginosa isolate PM1 effectively controls virus infection and promotes growth in plants.

A bacterial isolate PM1 obtained from the rhizosphere of healthy plants was identified as Pseudomonas aeruginosa by biochemical characteristics and 16S rRNA gene sequence (GenBank ID OL321133.1). It induced resistance in Nicotiana tabacum cv. Xanthi-nc and Cyamopsis tetragonoloba, against Tobacco mosaic virus (TMV) and Sunn-hemp rosette virus (SRV), respectively. Foliar treatment with isolate PM1 curbed TMV accumulation in susceptible N. tabacum cv. White Burley. PM1 was more effective as a foliar than a root/soil drench treatment, evident through a comparative decrease in ELISA values, and reduced viral RNA accumulation. Foliar and soil drench treatment with PM1 resulted in a disease index of 48 and 86 per cent, and a control rate of 48.9 and 8.5 per cent, respectively. PM1 exhibited phosphate solubilization, produced siderophores, auxins, HCN, and ammonia, all important plant growth-promoting traits. Foliar treatment with PM1 enhanced growth in tobacco, while its volatiles significantly promoted seedling growth in C. tetragonoloba. Of the several metabolites produced by the isolate, many are known contributors to induction of systemic resistance, antibiosis, and growth promotion in plants. Soluble metabolites of PM1 were less effective in inducing antiviral resistance in N. tabacum cv. Xanthi-nc in comparison with its broth culture. PM1 and its metabolites were antagonistic to Gram-positive Bacillus spizizenii and Staphylococcus aureus, and fungi Fusarium oxysporum, Aspergillus niger, and Rhizopus stolonifer. Its volatiles were inhibitory to F. oxysporum and R. stolonifer. Thus, PM1 exhibited considerable potential for further evaluation in plant virus control and production of diverse metabolites of use in agriculture and medicine.

TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction.

Ebola virus (EBOV) causes highly pathogenic disease in primates. Through screening a library of human interferon-stimulated genes (ISGs), we identified TRIM25 as a potent inhibitor of EBOV transcription-and-replication-competent virus-like particle (trVLP) propagation. TRIM25 overexpression inhibited the accumulation of viral genomic and messenger RNAs independently of the RNA sensor RIG-I or secondary proinflammatory gene expression. Deletion of TRIM25 strongly attenuated the sensitivity of trVLPs to inhibition by type-I interferon. The antiviral activity of TRIM25 required ZAP and the effect of type-I interferon was modulated by the CpG dinucleotide content of the viral genome. We find that TRIM25 interacts with the EBOV vRNP, resulting in its autoubiquitination and ubiquitination of the viral nucleoprotein (NP). TRIM25 is recruited to incoming vRNPs shortly after cell entry and leads to dissociation of NP from the vRNA. We propose that TRIM25 targets the EBOV vRNP, exposing CpG-rich viral RNA species to restriction by ZAP.

Structure of the poxvirus decapping enzyme D9 reveals its mechanism of cap recognition and catalysis

Poxviruses encode decapping enzymes that remove the protective 5' cap from both host and viral mRNAs to commit transcripts for decay by the cellular exonuclease Xrn1. Decapping by these enzymes is critical for poxvirus pathogenicity by means of simultaneously suppressing host protein synthesis and limiting the accumulation of viral double-stranded RNA (dsRNA), a trigger for antiviral responses. Here we present a high-resolution structural view of the vaccinia virus decapping enzyme D9. This Nudix enzyme contains a domain organization different from other decapping enzymes in which a three-helix bundle is inserted into the catalytic Nudix domain. The 5' mRNA cap is positioned in a bipartite active site at the interface of the two domains. Specificity for the methylated guanosine cap is achieved by stacking between conserved aromatic residues in a manner similar to that observed in canonical cap-binding proteins VP39, eIF4E, and CBP20, and distinct from eukaryotic decapping enzyme Dcp2.

Effects of Elevated Temperature on the Susceptibility of Capsicum Plants to Capsicum Chlorosis Virus Infection.

Capsicum, an important vegetable crop in Queensland, Australia, is vulnerable to both elevated temperatures and capsicum chlorosis virus (CaCV). Thus, it is imperative to understand the genetic responses of capsicum plants (Capsicum annuum) to CaCV under elevated temperature conditions. Here, we challenged susceptible plants (cv. Yolo Wonder) with CaCV and investigated the effects of elevated temperature on symptom expression, the accumulation of virus-derived short interfering RNA (vsiRNA) and viral RNA, and the expression of plant defense-associated genes. CaCV-inoculated plants initially showed more severe symptoms and higher viral concentrations at a higher temperature (HT, 35 °C) than at ambient temperature (AT, 25 °C). However, symptom recovery and reduced viral RNA accumulation were seen in the CaCV-infected plants grown at HT at later stages of infection. We also observed that HT enhanced the accumulation of vsiRNAs and that, concurrently, RNA interference (RNAi)-related genes, including Dicer-like2 (DCL2), DCL4, RNA-dependent RNA polymerase 1 (RdRp1), RdRp6, and Argonaute2 (AGO2), were upregulated early during infection. Moreover, continuous high levels of vsiRNAs were observed during later stages of CaCV infection at HT. Overall, our investigation suggests that HT facilitates CaCV replication during early infection stages. However, this appears to lead to an early onset of antiviral RNA silencing, resulting in a subsequent recovery from CaCV in systemic leaves.

Poly(rC)-Binding Protein 1 Limits Hepatitis C Virus Virion Assembly and Secretion.

The hepatitis C virus (HCV) co-opts numerous cellular elements, including proteins, lipids, and microRNAs, to complete its viral life cycle. The cellular RNA-binding protein, poly(rC)-binding protein 1 (PCBP1), was previously reported to bind to the 5′ untranslated region (UTR) of the HCV genome; however, its importance in the viral life cycle has remained unclear. Herein, we sought to clarify the role of PCBP1 in the HCV life cycle. Using the HCV cell culture (HCVcc) system, we found that knockdown of endogenous PCBP1 resulted in an overall decrease in viral RNA accumulation, yet resulted in an increase in extracellular viral titers. To dissect PCBP1’s specific role in the HCV life cycle, we carried out assays for viral entry, translation, genome stability, RNA replication, as well as virion assembly and secretion. We found that PCBP1 knockdown did not directly affect viral entry, translation, RNA stability, or RNA replication, but resulted in an overall increase in infectious particle secretion. This increase in virion secretion was evident even when viral RNA synthesis was inhibited, and blocking virus secretion could partially restore the viral RNA accumulation decreased by PCBP1 knockdown. We therefore propose a model where endogenous PCBP1 normally limits virion assembly and secretion, which increases viral RNA accumulation in infected cells by preventing the departure of viral genomes packaged into virions. Overall, our findings improve our understanding of how cellular RNA-binding proteins influence viral genomic RNA utilization during the HCV life cycle.

Differential Synergistic Interactions Among Four Different Wheat-Infecting Viruses.

Field-grown wheat (Triticum aestivum L.) plants can be co-infected by multiple viruses, including wheat streak mosaic virus (WSMV), Triticum mosaic virus (TriMV), brome mosaic virus (BMV), and barley stripe mosaic virus (BSMV). These viruses belong to four different genera in three different families and are, hence, genetically divergent. However, the impact of potential co-infections with two, three, or all four of them on the viruses themselves, as well as the wheat host, has yet to be examined. This study examined bi-, tri-, and quadripartite interactions among these viruses in wheat for disease development and accumulation of viral genomic RNAs, in comparison with single virus infections. Co-infection of wheat by BMV and BSMV resulted in BMV-like symptoms with a drastic reduction in BSMV genomic RNA copies and coat protein accumulation, suggesting an antagonism-like effect exerted by BMV toward BSMV. However, co-infection of either BMV or BSMV with WSMV or TriMV led to more severe disease than singly infected wheat, but with a decrease or no significant change in titers of interacting viruses in the presence of BMV or BSMV, respectively. These results were in stark contrast with exacerbated disease phenotype accompanied with enhanced virus titers caused by WSMV and TriMV co-infection. Co-infection of wheat by WSMV, TriMV, and BMV or BSMV resulted in enhanced synergistic disease accompanied by increased accumulation of TriMV and BMV but not WSMV or BSMV. Quadripartite interactions in co-infected wheat by all four viruses resulted in very severe disease synergism, leading to the death of the most infected plants, but paradoxically, a drastic reduction in BSMV titer. Our results indicate that interactions among different viruses infecting the same plant host are more complex than previously thought, do not always entail increases in virus titers, and likely involve multiple mechanisms. These findings lay the foundation for additional mechanistic dissections of synergistic interactions among unrelated plant viruses.

Nuclear Exportin 1 (XPO1) Binds to the Nuclear Localization/Export Signal of the Turnip Mosaic Virus NIb to Promote Viral Infection.

The nuclear localization signal (NLS) and nuclear export signal (NES) are key signatures of proteins for controlling nuclear import and export. The NIb protein of turnip mosaic virus (TuMV) is an RNA-dependent RNA polymerase (RdRP) that is absolutely required for viral genome replication. Previous studies have shown that NIb is a nucleocytoplasmic shuttling protein and contains four putative NES and four putative NLS motifs. Here, we analyzed the function of these NESs and NLSs, and identified two functional NESs and one functional NLS. Mutation of the identified functional NESs or NLS inhibited viral RNA accumulation and systemic infection. Exportin 1 (XPO1) is a nuclear export receptor that binds directly to cargo proteins harboring a leucine-rich NES and translocates them to the cytoplasm. We found that XPO1 contains two NIb-binding domains, which recognize the NLS and NES of NIb, respectively, to mediate the nucleocytoplasmic transport of NIb and promote viral infection. Taken together, these data suggest that the nucleocytoplasmic transport of NIb is modulated by XPO1 through its interactions with the functional NLS and NES of NIb to promote viral infection.

Effect of mutations in the 2b protein of tomato aspermy virus on RNA silencing suppressor activity, virulence, and virus-induced gene silencing.

Tomato aspermy virus (TAV) and cucumber mosaic virus (CMV) belong to the genus Cucumovirus in the family Bromoviridae. The cucumovirus 2b protein is one of the first identified viral suppressors of RNA silencing (VSR). The cucumovirus 2b protein contains a conserved amino acid motif consisting of several highly conserved amino acid residues. Here, we demonstrate that the TAV 2b protein N-terminal region, Arg46, Ser40, and Ser42 as well as the CMV 2b protein are essential for VSR activity, virulence, and viral RNA accumulation. Furthermore, we developed the first TAV-induced gene silencing (VIGS) vector. In contrast to other cucumovirus vectors, such as CMV and peanut stunt virus, the TAV vector did not induce a silencing phenotype in Nicotiana benthamiana when 2b protein VSR activity was retained. These findings suggest that the cucumovirus 2b proteins share amino acid residues for VSR activity but may have different roles in VIGS induction.

Identification of broad anti-coronavirus chemical agents for repurposing against SARS-CoV-2 and variants of concern

Endemic human coronaviruses (hCoVs) 229E and OC43 cause respiratory disease with recurrent infections, while severe acute respiratory syndrome (SARS)-CoV-2 spreads across the world with impact on health and societies. Here, we report an image-based multicycle infection procedure with α-coronavirus hCoV-229E-eGFP in an arrayed chemical library screen of 5440 clinical and preclinical compounds. Toxicity counter selection and challenge with the β-coronaviruses OC43 and SARS-CoV-2 in tissue culture and human airway epithelial explant cultures (HAEEC) identified four FDA-approved compounds with oral availability. Methylene blue (MB, used for the treatment of methemoglobinemia), Mycophenolic acid (MPA, used in organ transplantation) and the anti-fungal agent Posaconazole (POS) had the broadest anti-CoV spectrum. They inhibited the shedding of SARS-CoV-2 and variants-of-concern (alpha, beta, gamma, delta) from HAEEC in either pre- or post exposure regimens at clinically relevant concentrations. Co-treatment of cultured cells with MB and the FDA-approved SARS-CoV-2 RNA-polymerase inhibitor Remdesivir reduced the effective anti-viral concentrations of MB by 2-fold, and Remdesivir by 4 to 10-fold, indicated by BLISS independence synergy modelling. Neither MB, nor MPA, nor POS affected the cell delivery of SARS-CoV-2 or OC43 (+)sense RNA, but blocked subsequent viral RNA accumulation in cells. Unlike Remdesivir, MB, MPA or POS did not reduce the release of viral RNA in post exposure regimen, thus indicating infection inhibition at a post-replicating step as well. In summary, the data emphasize the power of unbiased, full cycle compound screens to identify and repurpose broadly acting drugs against coronaviruses.

MicroRNA 122 Affects both the Initiation and the Maintenance of Hepatitis C Virus Infections.

A liver-specific microRNA, miR-122, anneals to the hepatitis C virus (HCV) genomic 5' terminus and is essential for virus replication in cell culture. However, bicistronic HCV replicons and full-length RNAs with specific mutations in the 5' untranslated region (UTR) can replicate, albeit to low levels, without miR-122. In this study, we have identified that HCV RNAs lacking the structural gene region or having encephalomyocarditis virus internal ribosomal entry site (EMCV IRES)-regulated translation had reduced requirements for miR-122. In addition, we found that a smaller proportion of cells supported miR-122-independent replication compared a population of cells supporting miR-122-dependent replication, while viral protein levels per positive cell were similar. Further, the proportion of cells supporting miR-122-independent replication increased with the amount of viral RNA delivered, suggesting that establishment of miR-122-independent replication in a cell is affected by the amount of viral RNA delivered. HCV RNAs replicating independently of miR-122 were not affected by supplementation with miR-122, suggesting that miR-122 is not essential for maintenance of an miR-122-independent HCV infection. However, miR-122 supplementation had a small positive impact on miR-122-dependent replication, suggesting a minor role in enhancing ongoing virus RNA accumulation. We suggest that miR-122 functions primarily to initiate an HCV infection but has a minor influence on its maintenance, and we present a model in which miR-122 is required for replication complex formation at the beginning of an infection and also supports new replication complex formation during ongoing infection and after infected cell division. IMPORTANCE The mechanism by which miR-122 promotes the HCV life cycle is not well understood, and a role in directly promoting genome amplification is still debated. In this study, we have shown that miR-122 increases the rate of viral RNA accumulation and promotes the establishment of an HCV infection in a greater number of cells than in the absence of miR-122. However, we also confirm a minor role in promoting ongoing virus replication and propose a role in the initiation of new replication complexes throughout a virus infection. This study has implications for the use of anti-miR-122 as a potential HCV therapy.

Administration of Exogenous Surfactant and Cytosolic Phospholipase A2α Inhibitors may Help COVID-19 Infected Patients with Chronic Diseases

Background : Coronavirus-19 (COVID-19) pandemic is a worldwide public health problem causing 347,070 deaths from December 25, 2019, till May 25, 2020. Phospholipids are structural components of mammalian cytoskeleton and cell membranes. Phosphatidylglycerol is an anionic lipid found in mammalian membranes in low amounts (1–2%) of the total phospholipids. Also, phosphatidylglycerol suppresses viral attachment to the plasma membrane and subsequent replication in lung cells. Phosphatidylglycerol depletion caused by over expression of cytosolic phospholipase A2α induces lipid accumulation in lung alveoli and promotes acute respiratory distress syndrome (ARDS). An exogenous-surfactant replacement has been successfully achieved in ARDS and improved oxygenation and lung mechanics. Inhibition of cytosolic phospholipase A2alpha impairs an early step of COVID-19 replication. Aim: The present study was carried out to explain the correlation between the administration of exogenous artificial surfactant as well as cytosolic phospholipase A2α inhibitors to improve oxygenation and lung mechanics and inhibit COVID-19 replication. Methods: Database research was carried out on Medline, Embase, Cochrane Library, country-specific journals, and following-up WHO reports published between December 25, 2019 – May 25, 2020. Results: Till 25 May 2020, coronavirus cases were 5,307,298, with 347,070 deathsand 2,314,849 recovered cases. According to the WHO reports, most COVID-19 deaths seen are in people who suffered from other chronic diseases characterized by phospholipidosis and phosphatidylglycerol deficiency, including hypertension, liver, heart, and lung diseases and diabetes. Phospholipases A2 (PLA2) catalyze the cleavage of fatty acids esterified at the sn-2 position of glycerophospholipids leading to enhanced inflammation and lung damage. Also, cytosolic phospholipase A2α inhibitors may reduce the accumulation of viral proteins and RNA. In addition, administration of exogenous phospholipid surfactant may help COVID-19 infected patients with ARDS to remove inflammatory mediators. Conclusion: The present study showed a relation between phosphatidylglycerol deficiency in COVID-19 infected patients with ARDS and/or chronic diseases and their mortality. These findings also showed an important approach for the prevention and treatment of COVID-19 infections by using cytosolic phospholipase A2α inhibitors and exogenous administration of a specific phospholipid surfactant.

Nitrate-inducible MdBT2 acts as a restriction factor to limit apple necrotic mosaic virus genome replication in Malus domestica.

Apple necrotic mosaic virus (ApNMV) is highly associated with the occurrence of apple mosaic disease in China. However, ApNMV–host interactions and defence mechanisms of host plants against this virus are poorly studied. Here, we report that nitrate treatment restrains ApNMV genomic RNA accumulation by destabilizing viral replication protein 1a through the MdBT2‐mediated ubiquitin‐proteasome pathway. MdBT2, a nitrate‐responsive BTB/TAZ domain‐containing protein, was identified in a yeast two‐hybrid screen of an apple cDNA library using viral protein 1a as bait, and 1a was further confirmed to interact with MdBT2 both in vivo and in vitro. It was further verified that MdBT2 promoted the ubiquitination and degradation of viral protein 1a through the ubiquitin‐proteasome pathway in an MdCUL3A‐independent manner. Viral genomic RNA accumulation was reduced in MdBT2‐overexpressing transgenic apple leaves but enhanced in MdBT2‐antisense leaves compared to the wild type. Moreover, MdBT2 was found to interfere with the interaction between viral replication proteins 1a and 2apol by competitively interacting with 1a. Taken together, our results demonstrate that nitrate‐inducible MdBT2 functions as a limiting factor in ApNMV viral RNA accumulation by promoting the ubiquitination and degradation of viral protein 1a and interfering with interactions between viral replication proteins.

Heterogeneity in the Response of Different Subtypes of Drosophila melanogaster Midgut Cells to Viral Infections.

Single-cell RNA sequencing (scRNA-seq) offers the possibility to monitor both host and pathogens transcriptomes at the cellular level. Here, public scRNA-seq datasets from Drosophila melanogaster midgut cells were used to compare the differences in replication strategy and cellular responses between two fly picorna-like viruses, Thika virus (TV) and D. melanogaster Nora virus (DMelNV). TV exhibited lower levels of viral RNA accumulation but infected a higher number of cells compared to DMelNV. In both cases, viral RNA accumulation varied according to cell subtype. The cellular heat shock response to TV and DMelNV infection was cell-subtype- and virus-specific. Disruption of bottleneck genes at later stages of infection in the systemic response, as well as of translation-related genes in the cellular response to DMelNV in two cell subtypes, may affect the virus replication.

Salicylic acid and the viral virulence factor 2b regulate the divergent roles of autophagy during cucumber mosaic virus infection

ABSTRACT Macroautophagy/autophagy is a conserved intracellular degradation pathway that has recently emerged as an integral part of plant responses to virus infection. The known mechanisms of autophagy range from the selective degradation of viral components to a more general attenuation of disease symptoms. In addition, several viruses are able to manipulate the autophagy machinery and counteract autophagy-dependent resistance. Despite these findings, the complex interplay of autophagy activities, viral pathogenicity factors, and host defense pathways in disease development remains poorly understood. In the current study, we analyzed the interaction between autophagy and cucumber mosaic virus (CMV) in Arabidopsis thaliana. We show that autophagy is induced during CMV infection and promotes the turnover of the major virulence protein and RNA silencing suppressor 2b. Intriguingly, autophagy induction is mediated by salicylic acid (SA) and dampened by the CMV virulence factor 2b. In accordance with 2b degradation, we found that autophagy provides resistance against CMV by reducing viral RNA accumulation in an RNA silencing-dependent manner. Moreover, autophagy and RNA silencing attenuate while SA promotes CMV disease symptoms, and epistasis analysis suggests that autophagy-dependent disease and resistance are uncoupled. We propose that autophagy counteracts CMV virulence via both 2b degradation and reduced SA-responses, thereby increasing plant fitness with the viral trade-off arising from increased RNA silencing-mediated resistance.

MiR-122–based therapies select for three distinct resistance mechanisms based on alterations in RNA structure

Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with a liver-specific microRNA called miR-122. miR-122 binds to two sites in the 5' untranslated region of the viral genome and promotes HCV RNA accumulation. This interaction is important for viral RNA accumulation in cell culture, and miR-122 inhibitors have been shown to be effective at reducing viral titers in chronic HCV-infected patients. Herein, we analyzed resistance-associated variants that were isolated in cell culture or from patients who underwent miR-122 inhibitor-based therapy and discovered three distinct resistance mechanisms all based on changes to the structure of the viral RNA. Specifically, resistance-associated variants promoted riboswitch activity, genome stability, or positive-strand viral RNA synthesis, all in the absence of miR-122. Taken together, these findings provide insight into the mechanism(s) of miR-122-mediated viral RNA accumulation and provide mechanisms of antiviral resistance mediated by changes in RNA structure.

Macrolides May Prevent Severe Acute Respiratory Syndrome Coronavirus 2 Entry into Cells: A Quantitative Structure Activity Relationship Study and Experimental Validation.

The global pandemic caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is threatening the health and economic systems worldwide. Despite the enormous efforts of scientists and clinicians around the world, there is still no drug or vaccine available worldwide for the treatment and prevention of the infection. A rapid strategy for the identification of new treatments is based on repurposing existing clinically approved drugs that show antiviral activity against SARS-CoV-2 infection. In this study, after developing a quantitative structure activity relationship analysis based on molecular topology, several macrolide antibiotics are identified as promising SARS-CoV-2 spike protein inhibitors. To confirm the in silico results, the best candidates were tested against two human coronaviruses (i.e., 229E-GFP and SARS-CoV-2) in cell culture. Time-of-addition experiments and a surrogate model of viral cell entry were used to identify the steps in the virus life cycle inhibited by the compounds. Infection experiments demonstrated that azithromycin, clarithromycin, and lexithromycin reduce the intracellular accumulation of viral RNA and virus spread as well as prevent virus-induced cell death, by inhibiting the SARS-CoV-2 entry into cells. Even though the three macrolide antibiotics display a narrow antiviral activity window against SARS-CoV-2, it may be of interest to further investigate their effect on the viral spike protein and their potential in combination therapies for the coronavirus disease 19 early stage of infection.

The aryl hydrocarbon receptor facilitates the human cytomegalovirus-mediated G1/S block to cell cycle progression

The tryptophan metabolite, kynurenine, is known to be produced at elevated levels within human cytomegalovirus (HCMV)-infected fibroblasts. Kynurenine is an endogenous aryl hydrocarbon receptor (AhR) ligand. Here we show that the AhR is activated following HCMV infection, and pharmacological inhibition of AhR or knockdown of AhR RNA reduced the accumulation of viral RNAs and infectious progeny. RNA-seq analysis of infected cells following AhR knockdown showed that the receptor alters the levels of numerous RNAs, including RNAs related to cell cycle progression. AhR knockdown alleviated the G1/S cell cycle block that is normally instituted in HCMV-infected fibroblasts, consistent with its known ability to regulate cell cycle progression and cell proliferation. In sum, AhR is activated by kynurenine and perhaps other ligands produced during HCMV infection, it profoundly alters the infected-cell transcriptome, and one outcome of its activity is a block to cell cycle progression, providing mechanistic insight to a long-known element of the virus-host cell interaction.