Aedes aegypti. As RNA viruses, DENV rely on RNA-binding proteins (RBPs) to complete their life cycle. Alternatively, RBPs can act as restriction factors that prevent DENV multiplication. While the importance of RBPs is well-supported in humans, there is a dearth of information about their influence on DENV transmission by mosquitoes. Such knowledge could be harnessed to design novel, effective interventions against DENV. Here, we successfully adapted RNA-affinity chromatography coupled with mass spectrometry–a technique initially developed in mammalian cells–to identify RBPs in Ae. aegypti cells. We identified fourteen RBPs interacting with DENV serotype 2 3’UTR, which is involved in the viral multiplication and produces subgenomic flaviviral RNA (sfRNA). We validated the RNA affinity results for two RBPs by confirming that AePur binds the 3’UTR, whereas AeStaufen interacts with both 3’UTR and sfRNA. Using in vivo functional evaluation, we determined that RBPs like AeRan, AeExoRNase, and AeRNase have pro-viral functions, whereas AeGTPase, AeAtu, and AePur have anti-viral functions in mosquitoes. Furthermore, we showed that human and mosquito Pur homologs have a shared affinity to DENV2 RNA, although the anti-viral effect is specific to the mosquito protein. Importantly, we revealed that AeStaufen mediates a reduction of gRNA and sfRNA copies in several mosquito tissues, including the salivary glands and that AeStaufen-mediated sfRNA reduction diminishes the concentration of transmission-enhancing sfRNA in saliva, thereby revealing AeStaufen’s role in DENV transmission. By characterizing the first RBPs that associate with DENV2 3’UTR in mosquitoes, our study unravels new pro- and anti-viral targets for the design of novel therapeutic interventions as well as provides foundation for studying the role of RBPs in virus-vector interactions.Dengue viruses (DENV) are expanding global pathogens that are transmitted through the bite of mosquitoes, mostly  Dengue viruses are important human pathogens transmitted by mosquitoes. Currently, there are no effective control measures for dengue. The RNA-binding proteins (RBPs) in mosquitoes, which bind to the dengue virus genome to regulate viral multiplication, could serve as new targets for developing therapeutic interventions. In this study, we pioneered the use of RNA-affinity chromatography–a technique that identifies proteins binding to specific RNA sequences–in mosquito cells. This led to the detection of fourteen RBPs that associate with the 3’UTR of dengue virus serotype 2. We validated our results using immunoprecipitation method. Furthermore, we demonstrated that 6 of the 14 RBPs influence viral multiplication in mosquitoes. Among these six RBPs, we showed that the AePur mosquito and human homologs share an affinity to dengue virus RNA, whereas the anti-viral function is specific to the mosquito homolog. Importantly, we revealed that AeStaufen mediates a reduction of genomic and subgenomic flaviviral RNAs in multiple mosquito tissues. We also showed that the reduction of subgenomic flaviviral RNA in salivary glands diminishes the secretion of salivary subgenomic RNA, which facilitates infection at the bite site, thereby unveiling the function of AeStaufen in the virus transmission. By characterizing the first mosquito RBPs that interact with dengue virus genome, our study paves the way for leveraging these proteins as potential targets to block virus transmission. Aedes aegypti species  using a bead mill homogenizer . Homogenates were kept on ice for 30 min and centrifuged at 13,000 rpm for 15 min at 4°C. Cleared lysates were sonicated in an ultrasound bath cleaner  for 15 sec and placed on ice for 15 sec. The sonication procedure was repeated three times.For AePur RIP, mosquitoes were orally infected by feeding them blood containing 10AeStaufen cDNA synthesized by Genscript was cloned into pIZT/V5-His (Invitrogen). The same plasmid containing Chloramphenicol acetyltransferase (CAT) that was provided in the kit was used as expression control (5 C6/36 cells using TransIT-2020 transfection reagent (Mirus) for 4 h. After washing, cells were infected with DENV2 at multiplicity of infection (MOI) = 5. Two days post-infection, cells were collected in 150 μl of lysis buffer .To perform AeStaufen RIP,  control . Four μg2 (Sigma), and 0.01% Igepal CA-630] and incubated with 5 μg of rabbit anti-V5 antibody (Sigma), anti-Pur (Bethyl Laboratories), or normal rabbit IgG (Merck) at 4°C overnight. Bound complexes were captured into 50 μl of Dynabeads-protein G (Thermo Fisher Scientific) by rotation for 2 h at 4°C and washed four times in NT2 buffer. Immunoprecipitates were analyzed by western blot and gRNA and sfRNA were quantified by RT-qPCR.For RIP, 500 μg of protein lysate were diluted in 500 μl of NT2 buffer  with silica beads (BioSpec) using mini-beadbeater (BioSpec). Saliva-containing mixture and cells were lysed in 350 μl of TRK lysis buffer without bead homogenization. Total RNA was extracted using the E.Z.N.A. Total RNA kit I protocol and eluted with 30 μl DEPC-treated water (Ambion).gRNA was quantified by RT-qPCR using the iTaq Universal probe one-step kit (Bio-Rad) with primers and probe targeting the DENV2 envelope . The 12.To absolutely quantify gRNA and sfRNA/3’UTR, we amplified templates encompassing either the gRNA or the sfRNA/3’UTR targets using forward T7-tagged primers; for gRNA we used 5’-CAGGATAAGAGGTTCGTCTG-3’ and 5’-TTGACTCTTGTTTATCCGCT-3’, resulting in a 453 bp fragment; for sfRNA we used 5’-AGAAGAGGAAGAGGCAGGA-3’ and 5’-CATTGTTGCTGCGATTTGT-3’, resulting in a 319 bp fragment. Templates were transcribed using the MegaScript T7 kit and purified using the E.Z.N.A total RNA extraction kit. As we did not treat the template samples with DNase, there may be some DNA left after RNA purification. Both DNA and RNA were simultaneously quantified at 260 nm absorbance using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific). Dilutions of specific quantities of RNA fragments were used to generate absolute standard curves.sfRNA copy number was calculated by subtracting the number of gRNA fragments (estimated using the envelope primers) from the combined number of sfRNA and 3’UTR copies (estimated using the 3’UTR/sfRNA1 primers). For infected samples that contained detectable amount of sfRNA, sfRNA: gRNA ratio was calculated by dividing the number of sfRNAs over the number of gRNAs.E. coli LacZ was used as the template for generating dsRNA control (dsCtl). The PCR fragments were transcribed using the MegaScript T7 transcription kit and the resulting dsRNA was purified using the E.Z.N.A. Total RNA kit I (Omega) and folded by heating to 95°C for 2 min and slowly cooling down at 0.1°C per second using a thermocycler (Bio-Rad). DsRNA concentrations were adjusted to 3 or 14 μg / μl using SpeedVac Concentrator (Thermo Scientific) and their integrity was checked on agarose gels. The dsRNA was then injected into the thorax of two- to four-day-old cold-anesthetized female mosquitoes. 69 nl of 3 μg / μl dsRNA was used for functional evaluation of the 14 3’UTR-interacting proteins in whole mosquitoes and 138 nl of 14 μg/μl of AeStaufen dsRNA was used for functional evaluation of AeStaufen in different tissues, including the salivary glands. We injected a higher quantity of dsRNA against AeStaufen to have an effective silencing in salivary glands. The same amounts of dsLacZ were injected as control in both experiments.The DNA templates used to generate dsRNA against the 14 3’UTR-interacting proteins were synthesized using T7-tagged primer pairs as detailed in Actin, a house-keeping gene, was quantified to normalize mRNA amount. Quantification was conducted on a CFX96 Touch Real-Time PCR Detection System with the following thermal profile: 95°C for 1 min and 40 cycles of 95°C for 10 sec and 60°C for 15 sec, followed by melting curve analysis. Three repeats were conducted.Total RNA was extracted from 10 mosquitoes or 10 organs using the E.Z.N.A. Total RNA kit I, DNase-treated using the TURBO DNA-free kit (Thermo Fisher Scientific) and reverse-transcribed using the iScript cDNA Synthesis Kit (Bio-Rad). Gene expression was quantified using the SensiFAST SYBR No-ROX Kit (Bioline) with the primers listed in 5 C6/36 cells for 1 h, with gentle rocking at every 15 min. The inoculum was then removed and 1 ml of 1% carboxymethyl cellulose (CMC)  and 2% FBS diluted in RPMI medium was added. After 3 days, the CMC medium was removed and the cells were fixed with 4% paraformaldehyde (Merck), permeabilized with 0.5% Triton X-100 (Sigma), blocked with 2% FBS, and stained using 1:400 mouse monoclonal anti-envelope antibody (4G2) and 1:20,000 secondary anti-mouse Dylight 680 (Rockland). Focus forming units (ffu) were counted using the Odyssey Clx imaging system (LI-COR) in three replicates per dilution and the average ffu per sample was calculated. Infection rate corresponded to the number of samples with at least one ffu over the total number of tested samples.Single whole mosquitoes were homogenized in 500 μl of RPMI medium with silica beads (BioSpec) using mini-beadbeater (BioSpec). Homogenized tissues and saliva were sterilized by passing them through 0.22 μm filter (Sartorius). Samples were subjected to a 10-fold serial dilution in RPMI medium and 150 μl of each dilution was incubated with 1.5 x 105 Huh7 cells. Forty-eight hours post-transfection, cells were infected with DENV NGC at a MOI of 0.1. At 24 hours post-infection (hpi) plaque assay was performed on the supernatant as previously detailed  and siRNAs targeting HsPURB  were obtained from Qiagen. The siRNA negative control (siNC) ON-TARGETplus siRNA#2 was obtained from Dharmacon (Horizon Discovery). A final concentration of 5 nM siRNA was complexed with 2.5 μl of Lipofectamine RNAiMax (Invitrogen) and incubated in 12-well plates for 15 minutes prior to the plating of 1.3 x 10HsPURA and HsPURB, respectively, were designed with an online CRISPR tool (crispr.mit.edu). The sgRNAs were cloned into pSpCas9-BB-2A-GFP (Adgene) as previously detailed , where p is the proportion and n the number of observations. Differences in percentages were evaluated with the comparative error test. To justify pooling results from different experiments and ruling out any uncontrolled variable, we validated that controls were not significantly different. Z-test, unpaired T-test, and Dunnett’s test were conducted with Prism 8.0.2 (GraphPad). Copies of gRNA and titers (FFU) were log-transformed before statistical analysis to meet normal distribution.S1 FigMosquitoes were injected with either dsRNA against AePur or a dsRNA control against LacZ. Eleven days post-injection, eight mosquitoes were homogenized in RIPA and protein quantity was quantified with microBCA. The same quantity of protein was used for WB and revealed with anti-human PurB. Analysis was done in triplicate and each column represents a replicate.(TIFF)Click here for additional data file.S2 Fig(A) Illustration of the AeStaufen-pIZT/V5 plasmid construct. (B) Western Blot of AeStaufen-V5 (AeStau-V5) at 24, 48 and 72h post-transfection in C6/36 cells. Chloramphenicol Acetyltransferase (CAT)-V5 provided in the kit used to control for expression. Actin was used as loading control.(TIFF)Click here for additional data file.S3 FigAe. aegypti mosquitoes were intrathoracically injected with dsRNA targeting the indicated genes or a dsRNA control targeting LacZ gene. Gene expression was quantified by RT-qPCR four days later in pools of five whole mosquitoes. Data show mean ± s.e.m. from three independent repeats. Reduction in percentage is indicated on the column for the targeted genes. *, p-value < 0.05; ***, p-value < 0.001, as determined by unpaired T-test.Adult female (TIFF)Click here for additional data file.S4 FigA. aegypti mosquitoes were intrathoracically injected with dsRNA targeting the indicated genes or a dsRNA control targeting the LacZ gene. Four days post-dsRNA injection, the mosquitoes were exposed to a blood meal containing 106 pfu/ml of DENV. (A) Blood feeding rate. (B) Survival rate determined at 7 days post-feeding. Bars indicate percentage ± s.e. N, number of mosquitoes used for oral feeding in several biological repeats.Adult female (TIFF)Click here for additional data file.S5 FigThe three repeats of PUR domain are indicated. Conserved amino acids are highlighted. Color coding as follows: yellow, aromatic ; red, acidic ; blue, basic , orange, non-polar ; and green, polar .(TIF)Click here for additional data file.S6 FigActin expression levels were used for normalization. Bars show mean ± s.e.m. from three repeats.AeStaufen gene expression was quantified at 10 days post-oral infection in pools of ten whole mosquitoes, carcasses, midguts or salivary glands. (TIF)Click here for additional data file.S7 Fig(A) Positions of the RNA-binding domains (RBD) in Staufen homologs. TBD, Tubulin-binding domain. SSM, Staufen-swapping motif. (B-F) Alignment of RBD1-5 in different species.(TIFF)Click here for additional data file.S1 TableUniprot IDs, fold changes and p-values are systematically provided.(XLS)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file. 19 Apr 2022Dear Dr Pompon,Thank you very much for submitting your manuscript "Characterization of dengue virus 3’UTR RNA binding proteins in mosquitoes reveals that AeStaufen reduces subgenomic flaviviral RNA in saliva" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.While I have considered this a 'minor' revision - one of the reviewers listed a number of major concerns that could be rectified with additional data/analysis. You will need to address at least some of these concerns.Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.When you are ready to resubmit, please upload the following:[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Elizabeth A. McGraw, PhDAssociate EditorPLOS PathogensSonja BestSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************While I have considered this a 'minor' revision - one of the reviewers listed a number of major concerns that could be rectified with additional data/analysis. You will need to address at least some of these concerns.Reviewer Comments :Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: The host factors that positively or negatively modulate dengue virus infection in mosquitoes are still poorly characterized. The authors used RNA-affinity chromatography and mass spectrometry to identify RNA-binding proteins interacting with the dengue virus 3’UTR in Aedes aegypti cells, followed by their functional validation in mosquitoes in vivo. This is a well conducted study, and the manuscript is clearly organized and written. The topic is relevant and the approach is innovative. My (mostly minor) suggestions to improve the paper are below.Reviewer #2: The genome and the replicative forms of Dengue viruses, as is the case with other RNA viruses, is able to interact with RNA binding proteins (RBPs). While the importance of RBPs has been documented in humans, there is poor information about the relevance of these factors in DENV transmission by mosquitoes. In the present manuscript authors identified RBPs which interact with DENV2 3’UTR in Ae. aegypti cells. Authors confirmed the interaction of AePur to the 3’UTR, whereas AeStaufen interacts with both 3’UTR and sfRNA. The proviral function of AeRan, AeExoRNase, and AeRNase was evaluated by in vivo functional assay, whereas AeGTPase, AeAtu, and AePur have anti-viral functions in mosquitoes.Authors also found that AeStaufen mediates a reduction of gRNA andsfRNA copies in several mosquito tissues, revealing AeStaufen’s role in DENV transmission.The manuscript is interesting and relevant, however there are some aspects that should considered to fully support authors conclusions.Reviewer #3: “Characterization of dengue virus 3’UTR RNA binding proteins in mosquitoes reveals that AeStaufen reduces subgenomic flaviviral RNA in saliva” by Shih-Chia Yeh et al.,The main findings: DENV shuttle between vertebrate hosts and mosquitoes. Although many human RNA-binding proteins (RBPs) have been identified with important roles during DENV life cycle in human cells, the role of mosquito RBPs in regulating DENV infection cycle is less investigated. Using RNA-affinity chromatography followed by mass spectrometry, the authors report in this paper on the identification of fourteen Aedes aegypti proteins which associate with DENV2 3’UTR. They then choose two candidates to verify these associations. They further conduct in vivo functional analysis of these candidates in mosquito and identify 3 pro-viral candidates  and 3 anti-viral candidates . Interestingly, though human homologs of AePur also associate with DENV2, these human proteins do not have a role in DENV2 infection in the cultured cells. Whereas the initial investigation by RNAi in mosquito does not identify a role of AeStaufen in DENV2 infection, detailed analyses of AeStaufen function in mosquito tissues show that it suppresses both gRNA and sfRNA levels in several tissues including the salivary gland. Of interest, AeStaufen reduces sfRNA:gRNA ratio in the saliva without affecting viral particles.Novelty/significance: As a single-strained RNA virus, the life cycle of DENV in mosquito is tightly regulated by mosquito factors. Among these factors, RNA binding proteins play important roles in controlling DENV gRNA translation, degradation, replication, and virion assembly. Although similar approach has already being developed to identify Aedes proteins which bind to 3’UTR of Zika virus, this study is the first attempt to identify Aedes proteins associate with DENV2 3’UTR and provides some useful information for follow up study. Overall, the study is well designed and this is an interesting piece of work with novel aspects. It offers some insights on how DENV2 infection and replication in mosquito is regulated via its 3’UTR associated proteins, which warrant further investigation.Weakness: Some methodology is not well explained. Some texts are a bit confusing and need to be rephrased. Additional data could be considered to support the authors’ claim on the “transmission inhibition” role of AeStaufen. Addressing/clarifying these would strengthen the conclusion present in this manuscript.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: (No Response)Reviewer #2: Major concerns1. Although the RNA-affinity chromatography coupled with mass spectrometry (MS) using mosquito cell lysates is an appropriate strategy to analyze RBPs, authors only used uninfected mosquito cell lysates. It is possible that the RBPs identified from uninfected cell lysates may differ from the ones identified in infected cell lysates. The validation of the RBPs should be performed in the presence of infected cell lysates.2. How do the authors know that the RBPs identified are bound directly to the RNA? Is it possible that the proteins are not binding directly to the RNA and are part of a complex which is bound to the RNA?3. Have the authors confirmed the interaction of the identified proteins with gRNA of sfRNA by mobility shift assays?4. Have the authors analyzed the location of the identified RBPs in infected cells? Have these proteins the same location than viral RNA?5. Have the authors confirmed some of the interactions using the 3’UTR of another DENV serotype? Have the authors analyzed the role of AeStaufe in the infection with others DENV serotypes in mosquito?Reviewer #3: In several places , the authors state that “AeStaufen impacts on viral transmission”. However, data present in this manuscript are in line with their claim that “AeStaufen reduces subgenomic flaviviral RNA in saliva” (in the title) but do not lend support on its role on viral transmission. Interestingly, saliva from AeStaufen knockdown mosquito show higher sfRNA:gRNA ratio without affecting viral titers. This provides a unique situation to test whether AeStaufen has an impact on viral transmission (via controlling the sfRNA:gRNA ratio). The authors could use the infection assay established in their ref 28 to infect human cells with saliva collected from control or AeStaufen knockdown mosquitoes. If saliva derived from AeStaufen knockdown is more infectious than saliva from control knockdown, it will not only demonstrate the role of AeStaufen during DENV2 infection cycle but also give strong support to the role of sfRNA during virus infection in general.**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: Main comments:In the RNA immunoprecipitation assays , the sfRNA/gRNA ratio in immunoprecipitates could simply reflect their ratio in the protein lysate, not the differential binding affinity of candidate RBPs. This analysis should account for the original ratio, which is presumably different between live mosquitoes and C6/36 cells.The authors claim that they “validated gene silencing” (line 195) but the data presented in Figure S2 indicate that silencing efficiency was highly variable among target genes. It would be useful to report the % reduction in gene expression level and its statistical significance, as well as discuss the implications of incomplete silencing for some of the candidate genes.There are some statistical issues with the analysis of proportions  such as those shown in Figure 3A and Figure S3. In Figure 3A, the use of Z test does not seem appropriate to compare infection rates because Z test is only valid for a continuous variable with a large sample size and a normally distributed residual variance. By nature, percentages cannot be normally distributed because they have lower and upper limits (0 and 100). Moreover, calculating the average of percentages is incorrect unless the three experimental repeats had the exact same sample size, or if the average was weighted. In general percentages are more safely analyzed as a binary variable with a 95% confidence interval, which directly accounts for the sample size.Pooling experimental repeats  could be misleading if the repeats were inconsistent. In general, pooling is only justified when a potential experiment effect (or experiment x treatment interaction effect) has been ruled out.The authors could have discussed to which extent the choice of DENV type and strain (DENV2 NGC) could have influenced the results. All four DENV types share conserved functional RNA structures in their 3’UTR (except for DENV4), however the 3’UTR differs in sequence length and nucleotide composition between DENV types.Minor comments:Line 106: “interphase”, do you mean “interface”?Line 210: Expressing a proportion as percentage of reduction is ambiguous because it could represent the absolute difference in percentage or the percentage of reduction, relative to the control. Please clarify.Lines 249-250: Since the authors did not test HsPurG this statement could be softened.Line 396: “non-mediated”, do you mean “nonsense-mediated”?Line 506: Is the blood meal titer stable for 1.5 hours?Reviewer #2: NoneReviewer #3: 1. Among the fourteen proteins identified, some of them (such as AeGTPase) do not contain defined/known RNA binding domain. These proteins may associate with DENV2 3’UTR indirectly via other proteins and do not necessarily have a direct interaction with DENV2 3’UTR. Thus, it is more appropriate to use “association” than “binding” to describe the relation between these identified proteins and DENV2 3’UTR.2. Following sentences are a bit confusing and misleading. The authors might like to rephrase them.• Line 141-142 “AeMaleless, an ATP-dependent helicase involved in dosage compensation [30]”. This sentence gives the impression that AeMaleless plays a role in dosage compensation. In fact, the role of AeMaleless has not been established. The role of Maleless in dosage compensation is reported in Drosophila melanogaster.• Similarly, in Line 142-142 “AeSex-lethal, an RNA-binding protein also involved in dosage compensation” need to be rephrased.3. For each of fourteen candidate genes, ~0.2 ug of dsRNA was injected into mosquito to evaluate their potential function, while ~1.9 ug of dsRNA was delivered to address AeStaufen function in different tissues. The authors should explain the reason of using different amount of dsRNA in these experiments.4. Line 201-202, “we controlled that survival was similar across the various silencing conditions”. How did the authors control the survival rate without affecting the knockdown efficiency? Similarly, in Line 270, “we controlled that blood feeding and survival rates were not affected by AeStaufen depletion…”. How was this experimented conducted without affecting knockdown efficiency? Line 298 “we controlled that the salivation rate was not affected by AeStaufen silencing…”. How was salivation rate experimentally controlled without affecting knockdown efficiency? The authors need to add the methodology used for these experiments.5. For the RIP experiment, anti-human PurB antibody is used to pull down AePur, it’d be great if the authors could conduct dsRNA knockdown experiment in cells to verify the specificity of this antibody toward AePur.6. There is a discrepancy of DENV2 titer used for AePur RIP experiment. It is mentioned as 10^6 PFU/mL for AePer RIP (line 502-505), but it is stated as 10^7 PFU/mL. The authors need to correct one mistake here.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. 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Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. 9 Aug 2022AttachmentAsnwers to Reviewer comments.docxSubmitted filename: Click here for additional data file. 9 Sep 2022Dear Dr Pompon,We are pleased to inform you that your manuscript 'Characterization of dengue virus 3’UTR RNA binding proteins in mosquitoes reveals that AeStaufen reduces subgenomic flaviviral RNA in saliva' has been provisionally accepted for publication in PLOS Pathogens.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Elizabeth A. McGraw, PhDAssociate EditorPLOS PathogensSonja BestSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens <span style="font-family: "Noto Sans", sans-serif; font-size: 12px;"> orcid.org/0000-0002-7699-2064 </span> ***********************************************************While most of the suggestions do not rise to the level of a major revision, there are many things raised by the reviewers. Please attempt to address them all.Reviewer Comments : 14 Sep 2022Dear Dr Pompon,We are delighted to inform you that your manuscript, "Characterization of dengue virus 3’UTR RNA binding proteins in mosquitoes reveals that AeStaufen reduces subgenomic flaviviral RNA in saliva," has been formally accepted for publication in PLOS Pathogens.We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. 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The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards,Kasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064


Flavivirus is a large group of positive, single-stranded RNA viruses that includes several important human pathogens, such as West Nile, Dengue and Zika virus. The genome of flaviviruses bears a type 1 cap structure at its 5′ end, needed for the main translation initiation mechanism. Several members of the genus also use a cap-independent translation mechanism. The present work provides evidence that the WNV 5′ end also promotes a cap-independent translation initiation mechanism in mammalian and insect cells, reinforcing the hypothesis that this might be a general strategy of flaviviruses. In agreement with previous reports, we show that this mechanism depends on the presence of the viral genomic 3′ UTR. The results also show that the 3′ UTR of the WNV genome enhances translation of the cap-dependent mechanism. Interestingly, WNV 3′ UTR can be replaced by the 3′ UTR of other flaviviruses and the translation enhancing effect is maintained, suggesting a molecular mechanism that does not involve direct RNA-RNA interactions to be at work. In addition, the deletion of specific structural elements of the WNV 3′ UTR leads to increased cap-dependent and cap-independent translation. These findings suggest the 3′ UTR to be involved in a fine-tuned translation regulation mechanism.Viruses rely on the cellular machinery of host cells to synthesize their proteins, and have developed different mechanisms enabling them to compete with cellular mRNAs for access to it. The genus  Flavivirus use the cap-dependent translation initiation mechanism [7GpppN) is required for recruiting the eukaryotic translation initiation factor complex eIF4F, a pre-requisite for binding to the 40S ribosomal subunit and subsequent scanning for an appropriate translation initiation codon [The regulation of translation is probably the main process controlling gene expression in positive, single-stranded RNA viruses. Like any other virus, RNA viruses possess no translation machinery of their own. Viral translation thus relies on the usurpation of the host cell’s machinery, requiring infecting virus particles use molecular mechanisms that allow them to compete efficiently with cellular mRNAs. Like many other viruses, and most cellular mRNAs, the members of the genus echanism ,2. The con codon . In addion codon ,6,7.Flavivirus has many members, including numerous human pathogens. Their genome consists of a single-stranded RNA molecule about 11,000 nt long that bears a single open reading frame (ORF) flanked by untranslated regions (UTRs). These UTRs vary in length from one type of flavivirus to another, but all are composed of universally conserved structural elements with essential functional roles in the viral cycle . Tview see . This deview see ,14,15. Eview see . More review see . There iview see ,17,18. Fview see ,20.The present work provides evidence that West Nile Virus (WNV), a well-studied mosquito-borne flavivirus, also uses a 5′ cap-independent translation initiation mechanism which is dependent on the 3′ UTR of the viral genome. This further supports the idea that this might be a mechanism used by all flaviviruses. Interestingly, the WNV 3′ UTR can be substituted by the genomic 3′ UTR of other flaviviruses and cap-independent translation initiated, suggesting that the molecular mechanism behind the enhancement function of the genomic 3′ UTR involves no direct interaction between the two ends of the viral genome. Evidence that the 3′ UTR of flaviviruses functions as a general enhancer of translation is also provided.7G-WNV, consisted of a 5′ capped RNA containing the coding sequence of firefly luciferase (FLuc) flanked by the 5′ end (nt positions 1–160) and the complete 3′ UTR of the NY99-flamingo382-99 WNV strain . The m7G-WNVΔ3′UTR RNA lacked the WNV 3′ UTR , was also synthesized. The translation efficiency of each of these constructs was measured in Vero cells. Cells were cotransfected with each individual RNA construct and cap-dependent RLuc mRNA (m7G-RLuc) to normalize transfection efficiency.To explore the idea that cap-independent translation might be a general strategy followed by flaviviruses, the ability of the 5′ end of WNV to initiate translation was tested in the absence of the 5′ cap. For this, a series of subgenomic viral RNAs was constructed. The series-head RNA construct, named mV 3′ UTR a. Both R7G-WNV did so five times as efficiently as the control m7G-FLuc-3′WNV  IRES c in Vero7G-DIC, which carries a first ORF encoding the reporter RLuc protein (the synthesis of which is cap-dependent) followed by the cassette including the 5′ UTR of WNV directing the synthesis of the FLuc protein, ending with the WNV 3′ UTR; (ii) the RNA m7G-DIC_del3′UTR, which lacks the WNV 3′ UTR; (iii) the RNA m7G-DIC_del5′, which only bears nt 1–95 of the 5′ UTR, and lacks the AUG start codon; and (iv) the RNA m7G-DIC_del5′AUG, which lacks the 3′ end of the 5′ UTR but retains the AUG codon at position 96. The AUG of the fluc gene was deleted in all these constructs in order to examine the role of the 5′ UTR in cap-independent translation.To further examine the putative existence of an IRES at the 5′ end of the WNV RNA genome, a series of dicistronic constructs were assayed : (i) RNA7G-DIC_del5′AUG also revealed that the domains located downstream of the WNV AUG start codon are critical for preserving cap-independent protein synthesis, which is in good agreement with previous results indicating the importance of the cHP domain for ribosomal positioning [These RNAs were used to transfect both Vero and C6/36 cells. At 4 h post-transfection, both cell lines showed slight but significant FLuc activity resembling that observed in assays with monocistronic constructs lacking the cap, thus demonstrating that cap-independent internal translation initiation occurs at the 5′ UTR of WNV . Importantly, this translational activity was extremely dependent on the presence of the 3′ UTR b, as demitioning .Taken together, these results highlight the critical role of the 3′ UTR as a translation enhancer in WNV, and reveal the existence of a minor, but detectable, internal translation initiation that supports cap-independent translational activity at the WNV 5′ end, which is dependent on the 3′ UTR.7G-WNV, m7G-WNVΔ3′UTR or m7G-RLuc. In all cell lines, the presence of the genomic WNV 3′ UTR resulted in a significant enhancement of 5′ end translation initiation   . These r7G-WNV-3′DENV and m7G-WNV-3′YFV, respectively; In an attempt to determine the molecular mechanism involving the 3′ UTR of WNV in enhancing translation at the genomic 5′ end, a series of chimeric RNA constructs were obtained with the genomic 3′ UTRs of DENV or YFV instead of the 3′ end of WNV , the 3′ UTR of YFV was the strongest enhancer of the corresponding uncapped RNA . A slight increase (compared to the complete 3′ UTR) was also seen for the construct lacking the PK1 element. In the absence of the 5′ cap, the translation enhancing effect was greater still for all the mutants; all were associated with greater translation efficiency than the WNV construct—by a factor of 4 for the dSLIII mutant  with respect to the control mI mutant b. The ef7G-WNV, with that for dPK1 returning values similar to those recorded for dSLIII, while the dPK3 construct was associated with less translation activity than m7G-WNV—which is in good agreement with the data reported for 3′ UTR deletion mutants of DTMUV [The series of mutant capped constructs was also tested in mosquito C6/36 cells, and similar results were returned c. The deThe observation that translation of the DENV genome is resistant to cap-dependent translation arrest conditions led to the hypothesis of the existence of a general cap-independent translation mechanism in flaviviruses . Indeed,The present results show that the WNV cap-independent translation mechanism works both in mammalian and insect cells , althougFlaviviridae [7G-WNV, both in Vero and mosquito cells  is reminiscent of that associated with the 5′BSL3.2 structural element at the 3′ end of the HCV genome ,44. In eThe enhancement in translation initiation induced by the wild type 3′ UTR was similar in both the capped and uncapped context. However, greater translation efficiency was observed for all deletion variants when compared to the corresponding wild type construct in the cap-independent context . The reaIn conclusion, the present results clearly show that the genomic 3′ UTR of flaviviruses is a translation enhancer. Its effect may be the result of balancing the participation in translation initiation of structural elements with different and even opposing functions. The 3′ UTR enhances both the cap-dependent and cap-independent initiation translation mechanisms. The 5′ end of WNV retains a cap-independent initiation of translation mechanism, as observed in other flaviviruses, the function of which is dependent on the presence of a flavivirus genomic 3′ UTR.KpnI restriction enzyme site followed by the promoter sequence of the T7 RNA polymerase at the 5′ end of the amplification product, and a HindIII restriction enzyme site at its 3′ end. The primers used to amplify the coding sequence of the genomic 3′ UTR  introduced an XbaI and a BamHI site at the 5′ and 3′ ends of the amplification product, respectively. The plasmid pGLWNV for the WNV RNA construct was generated in three steps: first the 5′ PCR amplification product was cloned in the KpnI-HindIII sites of the pGL3 Basic vector . The 3′ amplification product was then introduced into the XbaI-BamHI sites of the resulting plasmid. Finally, the luciferase AUG codon was removed by site-directed mutagenesis using the Phusion Site-Directed Mutagenesis Kit . In the resulting pGLWNV plasmid, the FLuc coding sequences is in phase with the WNV AUG, and is flanked by the 5′ end and the 3′ UTR of the NY99-flamingo382–99 WNV strain. The WNV RNA construct was obtained by in vitro transcription from the BamHI linearized pGLWNV plasmid. Plasmid linearization with XbaI yielded the template for the WNVΔ3′UTR construct.The WNV genomic fragments used in this study were obtained by PCR amplification of the NY99-flamingo382–99 strain with the modifications described by Martín-Acebes and Saiz (GeneBank access AF196835) ,46. The XbaI-BamHI fragment of the pGL-ICU plasmid [XbaI-BamHI fragment encompassing the WNV 3′ UTR from plasmid pGLWNV. DNA templates encoding the chimeric WNV_3′DENV and WNV_3′YFV RNA constructs were obtained by replacing the 3′ UTR of plasmid pGLWNV by the 3′ UTR of DENV and YFV, respectively. DNA fragments encoding the 3′ UTR of DENV and YFV were purchased from Integrated DNA Technologies . Plasmid DNA encoding the chimeric subgenomic RNA construct was obtained as follows. First, the WNV 3′ UTR coding sequence was deleted from the plasmid pGLWNV using the Phusion Site-Directed Mutagenesis Kit (Thermo Scientific) following the manufacturer’s instructions, and using an appropriate pair of primers that added BamHI and SalI restriction enzymes sites at the 3′ end of the FLuc coding sequence. The fragments encoding the 3′ UTRs of DENV and YFV were introduced by recombination between the BamHI and SalI sites using the In-Fusion® HD Cloning Kit  to yield plasmids pGLWNV-3′DENV and pGLWNV-3′YFV. SalI digestion of the plasmids yielded DNA templates for the corresponding chimeric RNA constructs.DNA templates encoding the FLuc-WNV were obtained by PCR from the plasmid pGLWNV using primer T7pFLuc  and the  plasmid  by the X® HD Cloning Plus kit from Takara, following the manufacturer’s instructions. This construct bears the RLuc protein coding sequence followed by the 5′ UTR of the WNV fused to the FLuc protein coding sequence, in which the FLuc AUG start codon has been deleted. The 3′ UTR of the WNV genome is attached to this cassette at the latter’s 3′ end. To generate the DNA, the pRL-SV40 receptor plasmid (Promega) was digested with the endonucleases XbaI and BamHI. The cassette bearing the 5′UTR-FLuc-3′UTR was then obtained by PCR from the pGLWNV DNA template. Primers were designed to include flanking sequences that would facilitate homologous recombination with the receptor plasmid, avoiding the recognition of the XbaI site. Thus, only one XbaI site at the 3′ end of the FLuc gene remains in the resulting DNA construct.The DNA template for the dicistronic construct pRL-WNV was obtained by recombination using the In-FusionAUG were obtained from the parental DNA pRL-WNV by site-directed mutagenesis using appropriate primers and the Phusion Site-Directed Mutagenesis Kit (Thermo Scientific) following the manufacturer’s instructions. The pRL-WNV_del5′ plasmid codes for a deleted version of the 5′ UTR (comprising only nt 1–95) that lacks the starting AUG, whereas pRL-WNV_del5′AUG bears nt1–98, thus incorporating this start codon sequence.DNA templates pRL-WNV_del5′ and pRL-WNV_del5′AUG plasmids were digested with the restriction enzyme BamHI and in vitro transcribed from the T7 promoter sequence attached at the 5′ end of the rluc gene using T7 RNA polymerase, to yield the m7G-DIC, m7G-DIC_del5′ and the m7G-DIC_del5′AUG dicistronic RNAs, respectively. Alternatively, the pRL-WNV DNA was digested with the endonuclease XbaI to generate a truncated version lacking the WNV 3′ UTR, named m7G-DIC_del3′UTR. These transcription reactions were performed in the presence of a cap analogue nucleotide (see below).The pRL-WNV, pRL-WNV_del5′ and pRL-WNV_del5′BamHI restriction enzyme to yield the DNA templates for the corresponding RNA constructs. The DNA template for the RNA-100 was obtained as previously described [The DNA templates encoding the mutant RNA constructs dPK1, dPK2, dPK3 and dSLIII with deletions of different structural elements were generated by site-directed mutagenesis (using the Phusion Site-Directed Mutagenesis Kit) from the pGLWNV template plasmid using appropriate pairs of primers. The dPK1 construct lacked the structural elements involved in the formation of the PK1 structure ; the dPK2 construct lacked the structural elements involved in the formation of the PK2 structure , and the dPK3 construct lacked the structural elements involved in the formation of the PK3 structure . The dSLIII construct lacked the entire SL-III element . The resulting DNA plasmids were digested with the escribed .The primer sequences used in this work can be provided upon request.All RNAs were synthesized using the HighYield T7 RNA Synthesis kit  following the manufacturer’s instructions. Synthesis of the RNA RLuc and the addition of the 5′ cap was as previously described . All tra® by LifeTechnologies™, Invitrogen, Waltham, MA, USA), 2 mM L-Glutamine  and 1 mM sodium pyruvate (Sigma). Baby hamster kidney (BHK-21) cells were maintained in Glasgow Minimum Essential Medium (G-MEM) supplemented with 10% heat-inactivated foetal bovine serum (FBS), 0.1 mM tryptose phosphate and 10 mM HEPES buffer (Sigma). Human embryonic kidney cells (HEK-293T) and human cervical cancer cells (HeLa) were maintained in Dulbecco’s Modified Eagle Medium (DMEM-LG) supplemented with 10% heat-inactivated foetal bovine serum, 2 mM L-Glutamine and 0.1 mM non-essential amino acids (Gibco® by LifeTechnologies™). Cells were incubated at 37 °C in a 5% CO2 atmosphere.African green monkey kidney cells (Vero) were maintained in Minimum Essential Medium (MEM) supplemented with 5% heat-inactivated foetal bovine serum  and 2 µL of transfection reagent  for well-to-well normalisation. This lipid–RNA complex was added to the cell culture.Cell transfections were essentially performed as previously described . BrieflyTranslational efficiency was determined by measuring firefly and Renilla luciferase activities using the Dual-Luciferase Reporter Assay Kit  following the manufacturer’s instructions. All measurements were performed at 4 h post-transfection, and relative translation efficiency was calculated from the FLuc/RLuc ratio and then referred to that obtained by the reference RNA construct indicated in each assay.


In recent years, humanity has had to face a critical pandemic due to SARS-CoV-2. In the rapid search for effective drugs against this RNA-positive virus, the repurposing of already existing nucleotide/nucleoside analogs able to stop RNA replication by inhibiting the RNA-dependent RNA polymerase enzyme has been evaluated. In this process, a valid contribution has been the use of in silico experiments, which allow for a rapid evaluation of the possible effectiveness of the proposed drugs. Here we propose a molecular dynamic study to provide insight into the inhibition mechanism of Penciclovir, a nucleotide analog on the RNA-dependent RNA polymerase enzyme. Besides the presented results, in this article, for the first time, molecular dynamic simulations have been performed considering not only the RNA-dependent RNA polymerase protein, but also its cofactors  and double-strand RNA. During recent years, severe infective diseases have been caused by RNA-positive viruses, including acute hepatitis A, chronic hepatitis C, hemorrhagic fevers, meningitis, encephalitis, paralytic poliomyelitis, Ebola virus (EBV), severe acute respiratory syndrome coronavirus (SARS-CoV), and the more recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Like Ebola virus, coronavirus has already been characterized and well-studied since 2003. Like every RNA virus, both SARS-CoV and SARS-CoV-2 use, to replicate their genetic material the RNA-dependent RNA polymerase (RdRp), which therefore plays a key role in the replication and transcription processes during the life of the RNA virus.The RNA-dependent RNA polymerase (RdRp) derives from a common gene encoded by the RNA virus , and theAlthough viral RdRps are different in size and structural organization, all of them share a well-conserved sequence motif (about 500 residues) of the core structure , dividedIn the case of SARS-CoV-2, its replication is performed by the RdRp C terminal domain (residues 398-815) of the non-structural protein nsp12.The whole replicase function is exerted by not only the RNA polymerase , but also a helicase (nsp13) and rare or even unique domains involved in mRNA capping  and fidelity control (nsp14). Several smaller subunits nsp7-nsp10) act as crucial cofactors of these enzymes [-nsp10 ac2+) inside the active site [A multi-step mechanism for the cyclic addition of nucleotides in the RNA replication of the RdRp has been proposed  based onive site ,9. One oive site .The knowledge of the RNA replication mechanisms in RdRp is of primary importance in looking for new, highly efficient drugs such as nucleotide/nucleoside analogs (NAs), an important class of small molecule compounds targeting various types of polymerases, and allows a rapid investigation, experimentally or in silico, about the capability of the repurposing of antiviral, antibacterial, or antifungal drugs. In more detail, in cases in which no therapeutic agents are rapidly available, as occurred in 2019 during the COVID-19 pandemic, already existing drugs have been evaluated against the new therapeutic target, which, in the case of SARS-CoV-2, is the RdRp nsp12 protein. This is the case, for example, of Remdesivir, Sofosbuvir, Tenofovir, and Ribavirin ,12,13.The reuse of existing small molecule antiviral drugs is, indeed, an important and promising strategy for addressing the COVID-19 epidemic. If the existing drugs can be repurposed for the treatment of SARS-CoV-2 infection, preclinical research  and early clinical research can be bypassed, and the drugs can directly enter phase II or III clinical trials .In this context, for example, molecular docking allows for the evaluation of binding energies and the mode of physicochemical interaction of the active site of the protein with the drugs ,16 in a In this work we present molecular dynamics (MD) simulations of a potential drug against SARS-CoV-2, that is, Penciclovir triphosphate PENT, , during This article is to obtain a twofold purpose. The first is the design of molecular dynamics simulations that, for the first time, to the best of our knowledge, include a stable ds-RNA in which template and product strands are present .Moreover, the other novelty with respect to the literature is that in the MD simulations, not only are nsp7 and nsp8 cofactors included, but also RNA double strands ,21,22,27Furthermore, importantly, this study opens the way for MD approaches to determine, in silico, possible mechanisms of action for other NA drugs.In this paper, structural, dynamical, and mechanism insights were obtained regarding the action of Penciclovir towards RdRp of SARS-CoV-2.As described in the In order to study how the drug may influence the various steps of the RNA elongation process, with the aim of identifying the key features of the RNA duplication inhibition mechanism by PENT, the major differences between the replication complex with the drug PENT and the native binding partner GTP during this process were analyzed.In this context, indeed, in the literature, in the unbound state or bonded in the +1 or -1 position, various residues of protein complex in the active site can interact with the drug/base  by means of hydrogen bonds and electrostatic interactions determining the overall stability of the complex ,29. MoreIn this work, the starting point was the APO structure, that is, the protein/dsRNA complex without any drug inside, obtained after MD simulation. Several configurations of proteins/dsRNA/drug complex were simulated  or PENT (or PENC) in the complex with amino acids present in the active site, with the adjacent base in the product strand and with the complementary base in the template strand are analyzed and presented in the following. Moreover, changes are also monitored in the distance of protein groups not directly involved in the interaction of the drug (or base) and the protein/dsRNA complex, which causes key variation in the overall asset of the protein complex .Taking these in mind, simulations show that GTP (unbound state) is located in the active site, firmly stabilized by a complex network of hydrogen bonds, in accordance with the literature ,3,8,31. In the case of PENT, instead the drug is inserted in the active site but is not firmly stabilized by hydrogen bond networks (HBs) because it lacks of the ribose moiety, thus lacking the ability to form these bonds with D623 and N691, as shown in Nevertheless, interestingly, in this case, the arrangement of the ions and residues involved in the binding event is similar to that obtained in the case of nbGTP B. By comAs reported in As reported above, in the unbound state, GTP is stabilized by a huge number of interactions; this is not the case for G24, as shown in Summarizing, whilst GUA in the bounded form in the +1 position shows a lack of interaction with amino acids of the active site and at the same time is strongly linked to the template stand by three HBs, this is not the case for PENC. In the first case, therefore, translocation may occur easily, while, for PEN24, a weak interaction with the complementary base of the product strand and strong interactions with amino acids in the active site represent disfavorable factors for translocation. It is plausible, therefore, that inhibition could occur at this stage.Notwithstanding results reported in the ζ of R555 is 10.3 ± 2.4 Å), far apart from G23, and thus able to coordinate an incoming NTP. All of these data indicate their availability to accommodate an incoming NTP.First of all, in the G23 complex A, D618 dFinally, as in the G24 state, GUA is still strongly involved in HBs with the complementary base in the template strand , and thus is not more able to exercise its role, which is the interaction with the triphosphate group of a new incoming NTP into the active site. This means that Penciclovir bound after translocation can act as a blocker for the two metal ions and R555, which are important features for the addition of the nucleotides in the product strand [α in motif A residues (motif A contains D618 and D623 amino acids), reported in α in motif A residues in PENs24 and G23 are also reported , respectively, resulting in an NPT ensemble. Long range Lennard-Jones interactions were treated with a cut-off of length 1.4 nm, and electrostatic interactions were calculated with the Particle Mesh Ewald algorithm with the same cut-off [All MD simulations were performed using the software Gromacs19 with the version of Force-Field Charmm36 implemented in February 2021 (see below). In detail, the molecular system was inserted into a periodic cubic box and solvated with a suitable number of water molecules  the number of times in which the distance between the pair of atoms considered was between 2.5 and 3.0 Å . The percentage was obtained from the ratio between this persistence time and the total simulation time. Images were obtained by VMD .In this article, we indicate at which stage of the replication process Penciclovir exerts its inhibition action against RdRp of SARS-CoV-2 by MD simulation. The importance of this article resides not only in this result, but also in the fact that, to obtain this, for the first time to the best of our knowledge, MD simulations have been performed including a stable ds-RNA and, as regards the RdRp complex, not only nsp12, but also the nsp7 and nsp8 cofactors. Furthermore, not only we have characterized the non-covalently bound drug within the active site, but also in the bound pre- and post-translocation state. MD simulations indeed indicate that the drug, similar to the natural base, can enter into the active site and bind to the product strand. This indicates that Penciclovir does not inhibit these stages. On the contrary, after bonding to the product strand, a strong stabilization of the drug in the active site can disfavor the translocation, differently to Guanine. This feature suggests that this is the main cause of inhibition. Moreover, MD simulations performed when PENC or GUA is in the -1 position indicate that, in the presence of the drug, unlike GUA, the active site remains in an arrangement unfavorable to the entrance of an incoming NTP, thus strengthening the PENC inhibition. Finally, at the same time, these results open the way for up-to-date MD simulations to determine possible mechanisms of action for other NA drugs.


N1-methyladenosine (m1A) is a prevalent and reversible post-transcriptional RNA modification that decorates tRNA, rRNA and mRNA. Recent studies based on technical advances in analytical chemistry and high-throughput sequencing methods have revealed the crucial roles of m1A RNA modification in gene regulation and biological processes. In this review, we focus on progress in the study of m1A methyltransferases, m1A demethylases and m1A-dependent RNA-binding proteins and highlight the biological mechanisms and functions of m1A RNA modification, as well as its association with human disease. We also summarize the current understanding of detection approaches for m1A RNA modification. N1-methyladenosine(m1A) is a reversible methylation involving the addition of a methyl group at the N1 position of adenosine in cellular transcripts [1A-modified RNAs are also altered under physiological conditions [1A can be installed by methyltransferases, removed by demethylases and recognized by m1A-dependent RNA-binding proteins [1A RNA modification affects RNA metabolism, including RNA structure, stability and mRNA translation, thereby regulating gene expression and several fundamental cellular processes [Cellular RNAs contain more than 170 different types of chemical modifications across species [nscripts . The metnscripts . The secnditions . As a dyproteins ,5. m1A Rrocesses .1A RNA modification has been found with high abundance in transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) but at low levels in messenger RNAs (mRNAs) [1A9, m1A14, m1A16, m1A22, m1A57, and m1A58, respectively) [1A RNA modification occurs at five different positions  [1A14 has only been identified in cyt(tRNA)Phe from mammals, m1A22 has only been identified in bacteria tRNAs, and m1A57 has been identified in archaea existing only transiently as an intermediate of 1-methylinosine (m1I) [1A9 is quite abundant and found in 14 species of mt-tRNA, while m1A58 is a minor modification with a 17% frequency found in four species of mt-tRNAs [1A16 is unique to human mt-tRNAArg, and its frequency is approximately 20% [1A645 in 25S rRNA and m1A1322 in 28S rRNA located in the peptidyl transfer center of the ribosome are conserved in budding yeast and humans, respectively [1A is conserved at position 947 of 16S rRNA in the mitochondrial ribosome of vertebrates [1A in mRNA accounts for approximately 0.015–0.054% of all adenosines in mammalian cell lines and 0.05–0.16% in mammalian tissues [1A sites are usually located near the translation start site and the first splice site of mRNA, and they are associated with the translation of coding transcripts [m (mRNAs) ,10,11,12ctively) . In cyto and 58) . Among tne (m1I) . In mitoectively ,19, and  tissues . m1A sit1A RNA-modifying proteins that specifically install, remove, and bind to the m1A base. We also discuss the recent progress in understanding the biological mechanisms involving m1A in post-transcriptional gene expression regulation and the biological functions of m1A. Further, we review the current approaches for transcriptome-wide and single-base resolution m1A detection.In this review, we describe mammalian m1A methylomes in nuclear- and mitochondrial-encoded transcripts are achieved via the dynamic regulation of m1A RNA-modifying proteins . The characterization of m1A-modifying proteins is crucial for understanding the mechanisms underlying m1A-mediated gene regulation and the biological roles of m1A RNA modification. To date, several m1A RNA-modifying proteins responsible for nuclear- and mitochondrial-encoded transcripts have been identified in humans -N(1))-methyltransferase non-catalytic subunit TRM6(Trm6) and Trm61, respectively [1A58 modification in cytoplasmic tRNAs [mectively . Trm61 iectively . The conic tRNAs . Demethyic tRNAs ,26.1A modification has been founded in the large subunit of nuclear-encoded rRNAs in yeast and humans [1A645 and m1A2142 modifications, respectively, of 25S rRNA [1A modification of 28S rRNA in human and mouse cells [1A-dependent RNA-binding protein in HEK293T and RAW264.7 cells, because it showed the highest ability to bind to a human 28S rRNA-derived m1A probe. A subsequent RNA immunoprecipitation analysis confirmed that m1A-modified RNA was bound by YTHDF3, but not YTHDF1/2, in trophoblast HTR8/SVneo cells [rRNAs act as scaffolds for ribosomal proteins and as ribozymes for peptide bond formation in ribosomes; these functions are partly regulated by post-transcriptional modifications . The m1Ad humans . Yeast 225S rRNA ,29. The se cells ,31. NML se cells . YTH domeo cells .1A-modifying proteins are also responsible for the m1A modification of mRNAs. For example, the TRMT6/TRMT61A complex mediates the m1A addition to mRNA. TRMT6/61A also requires a consensus GUUCRA motif and a tRNA T-loop-like structure for substrate recognition in mRNA, which is similar to tRNA recognition [1A in mRNA can be demethylated by ALKBH3 [1A peaks were reported to be specifically detected in ALKBH3-knockout HEK293T cells, and these peaks were enriched in the 5′UTR [1A-dependent RNA-binding proteins in mRNA; these proteins can directly bind to an m1A-modified RNA probe (sequence from the human transcription factor SOX-18 mRNA) in HEK293T cells. It was also found that Trp432 in YTHDF2 is a key binding residue [1A RNA modification was reported to favor a GA-rich motif, and transcripts containing this motif were increased after ALKBH3 depletion [1A RNA modification was placed in a GAGGm1AG sequence. It could be recognized by YTHDF1 and YTHDF2 in HeLa cells, but YTHDC1 was not detected in this system [Studies have shown that tRNA mognition . In conty ALKBH3 ,10,33. I residue . m1A RNAepletion . Therefos system .1A9 of mt tRNALys and m1A1374 of mitochondrial NADH–ubiquinone oxidoreductase chain 5 (ND5) mRNA [1G9 modification in mt tRNAs [1A58 of mt tRNALeu (UUR) and m1A947 of mt 16S rRNA [1A sites from mt RNAs. The level of m1A16 of mt-tRNAArg and m1A58 of mt-tRNALys was found to be increased upon ALKBH1 knockout, indicating the role for ALKBH1 in the m1A of mt RNAs [1A in Ile and Leu1 pre-tRNA in the mitochondria [tRNA methyltransferase 10 homolog C (TRMT10C) acts as a catalytic core in the TRMT10C/SDR5C1  methylase complex, which is responsible for the mD5) mRNA ,36. In a16S rRNA ,37. Two  mt RNAs . The humchondria .1A RNA modification as a chemical modification of RNAs, efforts have been taken to understand the functional characterization of this dynamic methylation in RNA metabolism and gene expression regulation.Since the discovery of m1A RNA modification is a pivotal regulator of RNA metabolism, including RNA structure alteration, decay and translation  modification [insulin like growth factor 1 receptor (IGF1R) mRNA [The regulation of mfication . In addi1R) mRNA .1A modification varies among different RNA types. The m1A demethylases ALKBH1 and FTO have been reported to control specific tRNA m1A demethylation and decrease translation initiation [1A-methylated tRNAs are enriched in polysomes, indicating the role of m1A RNA modification in translation activation [1A58 of tRNA3Lys acted as a stop site that contributed to genome integration [1A undergo translation repression because of interfered Watson–Crick base pairing [Translational regulation by mitiation ,46. Eukativation . During egration . Further pairing ,48.1A RNA modification in this field. In a high-temperature-sensitive Thermococcus kodakarensis strain, decreased m1A58 and melting temperature of tRNA were observed, suggesting the relevance of m1A58 and the growth ability of this strain at high temperatures [1A RNA modification was found to exhibit its protective ability of RNAs under stress conditions. During heat shock, m1A-harbouring transcripts were found to preferentially accumulate in stress granules, subsequently resulting in a shorter time to restore the translation state during recovery [1A modification in RNAs and orchestrated translational suppression by recruiting the ASCC damage repair complex  [Aplysia central nervous system showed increased m1A RNA modification levels in animals after behavioral training [1A RNA modification during defensive reflex-associated behavioral sensitization. Petunia TRMT61A catalyzed m1A RNA modification in mRNAs, and the knockdown of TRMT61A decreased the chlorophyll content and changed chlorotic and wrinkled leaf phenotype [1A demethylase ALKBH3 functioned as a negative regulator of ciliogenesis by removing the m1A sites on Aurora A mRNA (a key regulator of cilia disassembly) in mammalian cells, which was further involved in cilia-associated developmental processes in zebrafish [Post-transcriptional modifications are involved in various biological processes, and recent evidence showed the importance of meratures . m1A RNArecovery . Alkylatcomplex) . The tRNtraining ; this wahenotype . A recenebrafish .1A RNA modification as a pathological feature has mainly focused on tumor progression  along with suppressed nascent protein in pancreatic cancer cells [1A demethylation of macrophage colony-stimulating factor 1 (CSF1) mRNA enhanced its mRNA stability and thus promoted the invasion of breast and ovarian cancer cells [1A RNA modification of tRNAGlyGCC to promote tRNA cleavage by angiogenin. The generation of excessive tRNA-derived small RNAs may affect ribosome assembly and apoptosis in HeLa cells [1A modification were upregulated in hepatocellular carcinoma (HCC) tissues. The TRMT6/TRMT61A complex mediated increased m1A58 levels in tRNA, which then triggered peroxisome proliferator-activated receptor delta (PPARδ) mRNA translation in HCC stem cells. PPARδ promoted cholesterol biogenesis to activate the Hedgehog pathway, thereby initiating the self-renewal of HCC stem cells [The limited exploration of mgression . It was er cells . The ALKer cells . In addiLa cells . FurtherLa cells . A recenem cells .1A modifications in RNAs. m1A can be detected by bi-dimensional thin-layer chromatography (2D-TLC) or high-pressure liquid chromatography (HPLC), both of which utilize the differential retention properties for the separation of various nucleosides [1A level in RNAs [1A is generally lost.Several methods have been developed for the detection of mleosides . Further in RNAs . The dra1A distribution map and dynamics in the transcriptome, and developed tools provide easy ways for m1A site-specific detection. Here, we summarize the high-throughput sequencing approaches for m1A RNA-modification detection , the removal of m1A, m3C (N3-methylcytidine), or m1G (N1-methylguanosine) modifications by AlkB treatment facilitates the production of full-length cDNAs from previously modified templates, producing a ratio of reads in treated versus untreated samples that can be used to identify methylated RNAs [1A-specific base mutations, thus providing a direct and rapid method for mapping m1A modification [Several enzyme-based strategies have been developed for detecting mted RNAs . RT-1306fication . One pos1A antibody-dependent RNA immunoprecipitation has several advantages in m1A-modification detection. m1A antibody-dependent methylated RNA immunoprecipitation sequencing (m1A-MeRIP-seq) combined with AlkB demethylation (m1A-ID-seq) has been used to describe m1A dynamics in the human transcriptome [1A-seq’ that combined MeRIP-seq and Dimroth rearrangement to obtain the mRNA m1A methylome in human cells [1A sites—with m1A antibody was shown to provide a feasible way to map m1A at a single-base resolution (m1A-seq-TGIRT and m1A-seq-SS) [1A (m1A-MAP) has improved the previous m1A-ID-seq by utilizing TGIRT and AlkB treatment. m1A-MAP has been used to identify a total of 740 m1A sites in the transcriptome and showed a GUUCRA consensus motif located in a subset of tRNA substrates of the TRMT6/61A complex [1A antibodies, which may cause false positives.mcriptome . Dominisan cells . Moreove-seq-SS) . The mis complex . A major1A-induced Watson–Crick base-pairing disruption, some approaches have been developed for site-specific m1A evaluation. m1A induces the truncation or mutation of cDNA during reverse transcription, so primer extension can be applied to compare m1A modification levels based on the intensity of the truncated and full-length cDNA bands [1A-modified RNAs [1A base. It can easily detect the m1A state at a specific site by analyzing the PCR amplification products from nick ligation [1A sites rather than the identification of novel modification sites.Based on mNA bands . The cluied RNAs . Anotherligation . These m1A modification have been reported, some key questions remain to be addressed. For instance, studies on m1A-dependent RNA-binding proteins are limited. With the advances in the sensitivity and throughput of proteomics techniques, it is critical to further characterize m1A-dependent RNA-binding proteins involved in m1A recognition and RNA metabolism, as well as to determine how they recognize the m1A sites in their target transcripts. In addition, the reported methyltransferases and demethylases may target different transcripts in different cell types or biological processes; therefore, the context-dependent roles and regulatory mechanisms of m1A-modifying proteins are also interesting and important to explore. Further, optimized technologies are still needed for transcriptome-wide and single-base resolution m1A detection. These will greatly improve our understanding of the biological roles of m1A in RNA. Finally, more work is needed to understand the roles of the m1A RNA modification in human diseases.Although findings related to the biological functions of the m


Next generation sequencing of RNA molecules (RNA-seq) has become a common tool to characterize the expression profiles of RNAs and their regulations in normal physiological processes and diseases. Although increasingly accumulating RNA-seq data are widely available through publicly accessible sites, most of the data for short non-coding RNAs (sncRNAs) have been obtained for microRNA (miRNA) analyses by standard RNA-seq, which only capture the sncRNAs with 5′-phosphate (5′-P) and 3′-hydroxyl (3′-OH) ends. The sncRNAs with other terminal formations such as those with a 5′-hydroxyl end (5′-OH), a 3′-phosphate (3′-P) end, or a 2′,3′-cyclic phosphate end  cannot be efficiently amplified and sequenced by standard RNA-seq. Due to the invisibility in standard RNA-seq data, these non-miRNA-sncRNAs have been a hidden component in the transcriptome. However, as the functional significances of these sncRNAs have become increasingly apparent, specific RNA-seq methods compatible with various terminal formations of sncRNAs have been developed and started shedding light on the previously unrecognized sncRNAs that lack 5′-P/3′-OH ends. In this review, we summarize the expanding world of sncRNAs with various terminal formations and the strategic approaches of specific RNA-seq methods to distinctively characterize their expression profiles. These datasets can be redirected from various sites such as the cBioPortal for Cancer Genomics [Next generation sequencing of RNA molecules (RNA-seq) has revolutionized transcriptome analyses and has become a common tool to identify RNA expression profiles. Increasingly, accumulating RNA-seq data have become widely available through publicly accessible sites, such as the National Center for Biotechnology Information (NCBI)’s Sequence Read Archive (SRA), not only for basic molecular, cellular, and computational biology research, but also for broader application with clinical contexts. In addition to studies from individual research groups, consortiums of large-scale transcriptome projects have been collecting numerous RNA-seq datasets. For example, The Cancer Genome Atlas (TCGA), aiming to compile cancer-associated genetic/epigenetic information, encompasses a total of 43 “microRNA (miRNA)-seq” projects, which have generated 461,071 RNA-seq datasets from 17,866 cases  whose lengths are shorter than mature transfer RNA (tRNA) molecules (less than ~60 nucleotides (nt)). Most previous studies targeting the sncRNAs, such as those in TCGA project, have focused on miRNAs by using standard small RNA-seq method. However, it has become increasingly apparent that not only miRNAs but also other types of sncRNAs are abundantly expressed as functional molecules. Many of these sncRNAs cannot be efficiently captured by standard small RNA-seq mainly due to the method’s incompatibility with their terminal structures, thus underrepresenting the non-miRNA-sncRNAs in many of the current transcriptome analyses.O-methyl group (2′-O-Me) at the 3′-terminal ribose, further differentiate terminal forms of RNA molecules , a monophosphate (P), or a 2′,3′-cyclic phosphate  at their termini A. The 2′olecules A. The cuC. elegans [The first distinct class of functional sncRNAs was discovered in the studies of  elegans ,5, follo elegans . While tO-Me end [miRNAs are bound by Argonaute (AGO) proteins to form RNA-Induced Silencing Complex (RISC) and act as guide molecules to target complementary regions of messenger RNAs (mRNAs) and other ncRNAs, silencing their expression through translational repression and/or promotion of RNA decay ,7. miRNAO-Me end ,14 , a similar yet distinct type of sncRNA. esiRNAs are also bound by AGO proteins and regulate transcription from transposons or post-transcriptionally silence gene expression ,16. esiRO-Me end ,16,17,18O-Me ends [Argonaute family proteins are divided into AGO and PIWI subclades. While AGO subclades bind to miRNAs and esiRNAs, PIWI subclades bind to a distinct class of longer 23–34-nt) sncRNAs, termed PIWI-interacting RNAs (piRNAs) ,20. Unli–34-nt sn-Me ends ,30 , a member of the RNase A superfamily, is the enzyme responsible for the anticodon cleavage ,36. ANG-O-Me ends, respectively. tRFs further have a variety of functions, such as regulating gene expression via modulation of mRNA stability or translation, preventing cell apoptosis, and promoting virus infection, and their dysregulations are involved in various diseases [tRFs are generally shorter than tRNA halves and comprise the rest of tRNA-derived sncRNAs. tRFs can mainly be subclassified into 5′-tRFs, 3′-tRFs, and i-tRFs ,32,33,34diseases ,33,34,50As in the case of tRFs, sdRNAs, the sncRNAs derived from small nucleolar RNAs (snoRNAs), have been reported to function as miRNAs by binding to AGO proteins and silencing the expression of target mRNAs ,52,53. WRibosomal RNAs (rRNAs) and mRNAs are also utilized as substrates for sncRNAs ,56,57,58The representative RNA-seq workflow includes: (1) extraction/purification of RNAs from cells/tissues, (2) construction of cDNA library, (3) next generation sequencing, and (4) bioinformatics analysis. Various strategies have been developed, mainly in the second step, to target specific sncRNAs with different terminal structures as follows.The standard method of cDNA library construction for sncRNAs utilizes adaptor (AD) ligations to the both 5′- and 3′-ends of sncRNAs. The ligated 5′- and 3′-ADs provide uniform hybridization sites for the primers in subsequent reverse transcription (RT) and PCR amplification. Commercially available kits for this standard method have been previously summarized . GeneralCircularizing AD-ligated RNA molecules is an alternative approach to AD ligation-based protocol . The metWhile the above two methods are based on AD ligation, there is a ligation-free sncRNA sequencing method that uses polyadenylation and template switching . In thisO-Me end, because the 3′-AD ligation or poly (A) addition to the 2′-O-Me end of RNAs are significantly inefficient [O-Me end) will be significantly underrepresented in standard RNA-seq data. To overcome this issue and focus on the 2′-O-Me-containing sncRNAs, periodate oxidation has been used to enrich the 2′-O-Me-containing sncRNAs as substrates of 3′-AD ligation. Treatment of RNA fraction with sodium periodate (NaIO4) disrupts the 3′-OH ends of RNAs by transforming 2′,3′-cis diol moiety into 2′,3′-dialdehyde, which is no longer available for 3′-AD ligation. Therefore, the periodate treatment prior to AD ligations makes 2′-O-Me-containing RNAs sole RNA substrates for 3′-AD ligation. Even if the 3′-AD ligation to the 2′-O-Me end is inefficient, the enrichment step enables amplifying and sequencing 2′-O-Me-containing RNAs as major species  has been used [Saccharomyces cerevisiae Tpt1 for efficient RT [As an alternative method to selectively capture 2′,3′-cP-containing sncRNAs, een used ,73. Unlieen used . Therefocient RT . The seqcient RT ,73.Escherichia coli RtcB RNA ligase, which can specifically ligate 3′-P-containing 5′-AD to the 5′-OH ends of RNAs, leading to specific amplification and sequencing of 5′-OH-containing RNAs , N3-methylcytidine (m3C), and N1-methylguanosine (m1G) residues, improving the efficiency of RT-PCR for the modified RNAs by erasing these RT-impairing modifications. Panoramic RNA display by overcoming RNA modification aborted sequencing (PANDORA-seq) combined the AlkB and T4-PNK treatments to fully capture sncRNAs [7G cap structure and 5′-triphosphates [N2,N2-dimethylguanosine (m22G), and because the biases generated by additional enzymatic treatments remain to be assessed in detail, the recent attempts to fully capture sncRNAs have shed light on previously unrecognized and uncharacterized sncRNAs.Not only terminal structures, but also internal post-transcriptional modifications of RNA molecules, can greatly affect sequencing efficiency. Some modifications block Watson–Crick base pairings and thus can interfere with RT, making the modified RNA molecules underrepresented in RNA-seq data. To overcome this issue, sequencing procedures for tRNAs (the most heavily-modified RNAs) and their fragments, such as DM-tRNA-seq  and AlkBred AlkB ,78 can r sncRNAs . Cap-Cliosphates . AlthougAlthough the RNA-seq of T4 PNK-treated samples has an ability to capture broader sncRNA species compared to standard RNA-seq without any pre-treatment, it does not necessarily mean that the T4 PNK pretreatment of RNA is always required in all experiments. The information of terminal phosphates and modifications on RNA molecules are essential to understand the biogenesis mechanism and molecular function of the RNAs. T4 PNK treatment erases the terminal phosphate information of RNA molecules in sequencing data. It is also a possibility that the increased amounts and variations of the sncRNAs in the T4 PNK-treated RNA sequence data could mask the expression profiles of RNAs with relatively low abundance. For example, when the main research focus is on miRNAs, standard RNA-seq could be a better method than RNA-seq with T4 PNK treatment, because T4 PNK treatment vastly increases the abundance of non-miRNA reads , which could lessen the depths of the miRNA reads. These considerations speak to the importance of understanding the biogenesis pathways and resultant properties of each sncRNA. Although increasing accumulation of sequencing data from standard and modified RNA-seq has identified greater numbers of sncRNA species, the biogenesis pathways and responsible RNases remain unknown for large parts of the sncRNAs. Without knowing the biogenesis enzymes and their generating terminal phosphate states of the targeted sncRNAs, it is difficult to determine an appropriate sequencing method for the RNAs and accurately interpret RNA-seq data. More importantly, to advance our understanding of the expanding realm of sncRNA, it is imperative to distinguish functional sncRNAs from random degradation products. Further research to accumulate the data of previously “hidden” sncRNA from the above-described modified versions of RNA-seq to capture specific sncRNAs or whole sncRNAome will not only reveal expressional regulation of wider repertoires of sncRNAs, but also help their functional characterization, potentially clarifying substantially greater biological significance of sncRNAs.


RNA-dependent RNA polymerase (RdRP) is essential for the replication and expression of RNA viral genomes. This class of viruses comprise a large number of highly pathogenic agents that infect essentially all species of plants and animals including humans. Infections often lead to epidemics and pandemics that have remained largely out of control due to the lack of specific and reliable preventive and therapeutic regimens. This unmet medical need has led to the exploration of new antiviral targets, of which RdRP is a major one, due to the fact of its obligatory need in virus growth. Recent studies have demonstrated the ability of several synthetic nucleoside analogs to serve as mimics of the corresponding natural nucleosides. These mimics cause stalling/termination of RdRP, or misincorporation, preventing virus replication or promoting large-scale lethal mutations. Several such analogs have received clinical approval and are being routinely used in therapy. In parallel, the molecular structural basis of their inhibitory interactions with RdRP is being elucidated, revealing both traditional and novel mechanisms including a delayed chain termination effect. This review offers a molecular commentary on these mechanisms along with their clinical implications based on analyses of recent results, which should facilitate the rational design of structure-based antiviral drugs. Mononegavirales is an order of RNA viruses with negative-strand nonsegmented genomes and consists of eleven families [Flaviviridae family, belonging to the Order Amarillovirales, contain positive-strand nonsegmented RNA genomes. Since the metazoan cells by and large lack RdRP activity, the RNA viruses encode their own RdRP, as it is essential for viral transcription and replication. In corollary, the inhibition of viral RdRP offers a lucrative target for designing specific antiviral drugs with minimal host toxicity.Important examples of highly pathogenic RNA viruses, responsible for severe diseases in humans, are shortlisted here . In thisfamilies , some ofOver the years, various ribonucleoside and deoxyribonucleoside analogs have been used in vitro and in vivo to inhibit transcription and replication, respectively. For example, the triphosphate derivative of cordycepin , i.e., 3’dATP, mimics ATP and is incorporated instead of A. However, the lack of an -OH group at the 3’ position of ribose prevents the formation of the 3’-5’ phosphodiester bond with the 5’ end of the next nucleotide, which terminates RNA synthesis . RecentlIn using the analogs ex vivo or in vivo , their triphosphate forms are not useful, since the triphosphates are labile and the cell membranes in general lack transport mechanisms for organic phosphates. Instead, the nucleosides without phosphate groups, and often conjugated with other moieties for desirable properties, are employed, using what is often called “ProTide” technology, detailed later . The freNucleoside analogs have received particular attention as antivirals due to the fact of their ability to serve as substrates for viral RNA-dependent RNA polymerase (RdRP) by acting as nucleoside mimics ,6,7. ColAs mentioned before, the active form of a nucleoside analog (henceforth abbreviated as NA) is the triphosphate, but it is administered as a prodrug that is inactive and activated metabolically. The steps for the activation are very similar, as illustrated later, for remdesivir, molnupiravir, and sofosbuvir. In brief, the prodrug is synthesized with substitutions that increase the lipophilicity and improve the bioavailability. When administered in the body, the substitutions are removed in multiple steps of catalytic and spontaneous reactions to produce the monophosphate form. The latter is phosphorylated further by cellular kinases to generate the final triphosphate form that is active and recognized by RdRP as a nucleotide analog. In an alternative pathway, the monophosphate is dephosphorylated to generate the nucleoside, which can be fully phosphorylated to the triphosphate. It is fair to say that the renewed awareness of the antiviral effect of the NAs owes its origin to remdesivir, an adenosine analog (See later), when it was repurposed to inhibit SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2), the causative agent of the deadly pandemic COVID-19 (coronavirus disease 2019). Remdesivir, sold under the brand name Veklury, was previously developed for the treatment of hepatitis C virus . Highly +2 ion and interaction with ribonucleotides and polynucleotide chains. The viral RdRPs also display conserved 3D structures that have been architecturally likened to a human right hand with palm, thumb, and finger domains, where the seven motifs, located in specific domains, play their respective roles in RNA replication. A snapshot of these motifs in selected flaviviral RdRPs are shown here, but there are several outstanding reviews that present them in much more detail, which the readers can consult [At this point, it is relevant to describe the conserved sequence features of viral RdRP, especially the sequence motifs of the active site ,31. Spec consult ,33,34.The various derivatives of remdesivir and their bioconversion are depicted in The efficacy of remdesivir against the Ebola virus (EBOV) in West Africa  led to aMuch of our knowledge in this area is owed to the establishment of a partially reconstituted coronavirus (CoV) transcription in vitro. The RdRP holoenzyme of CoV is a large multisubunit complex consisting of 16 nonstructural proteins (Nsps), produced by the proteolytic processing of two large polyproteins early in the virus infection, resulting from the translation of the positive-sense genome RNA by the host translational machinery ,38,39,40Several studies using this system have suggested that the predominant mechanism of remdesivir action is “delayed” chain termination following incorporation of the remdesivir monophosphate RMP;  in the pThis is clearly different from the action of 3’-deoxy analogs, such as cordycepin, where the termination is immediate and the next nucleotide cannot be added, as mentioned earlier. In the case of remdesivir, elongation appears to gradually slow down following incorporation; kinetic studies in vitro, using a synthetic RNA template and purified RdRP of SARS-CoV-2, demonstrated stalling of the RdRP translocation after 3–4 additional nucleotides are incorporated following the first RMP moiety ,10,48. DWhen increasing concentrations of regular nucleotides were added in the reaction, remdesivir-induced termination was gradually abolished, and the RdRP eventually transcribed the full-length product ,11,51. TInterestingly, these studies unraveled a second mechanism in which remdesivir was capable of causing misincorporation . In thesThe majority of the SARS-CoV transcription reactions that showed the inhibitory activity of NAs in vitro employed the minimal heterotrimeric complex, composed of Nsp12, and the accessory factors Nsp7 and Nsp8, without the Nsp14-Nsp10 3’-exonuclease. Unfortunately, when the exonucleases were present, they proofread and removed incorporated NAs, including the delayed terminator, remdesivir, and the immediate terminator, sofosbuvir, although the latter was somewhat more resistant to exonuclease, probably due to the fact of its structural distinction from remdesivir . The relAs stated earlier, the mechanism of the transcription inhibition by remdesivir was also investigated in several other RNA viruses, whereby most of them appeared to follow the SARS-CoV-2 paradigm of delayed termination. In a recent study, remdesivir and 4’-fluorouridine  both inhibited in vitro transcriptions, reconstituted with the minimal RdRP of RSV and SARS-CoV-2 . While tLike remdesivir, sofosbuvir (GS-331077), a uridine analog, terminates transcription when incorporated into the viral RNA. Initially licensed for use in HCV infection , sofosbuUnlike the delayed stalling effect of remdesivir, sofosbuvir stops the elongating RdRP immediately without allowing the next nucleotide to be added. In the absence of a 3D structure of the sofosbuvir–RdRP complex, the molecular mechanism of how exactly sofosbuvir promotes termination remains unclear. Nonetheless, a similar nucleotide analog, 2’-deoxy-2’-α-fluoro-β-C-methylcytidine, or 2’F-2’C-Me-CTP for short , acts as a transcription terminator and inhibits flaviviruses likely because its 2’ methyl group (on ribose) causes a steric clash with an incoming NTP , which l2OH). In the architecture of the active site, S282 is positioned directly below the terminal nucleotide of the primer strand, such that a Thr in this position blocks the incoming 2’-C-Me-CTP more than the normal CTP [Several other amino acid residues in the RdRP active site are also relevant in the NA mechanism. Of these, Ser282 of HCV is the best studied and deserves mention here, especially in regard to the antiviral activity of the C-analog, 2’-C-Me-CTP, which showed proof-of-concept clinical efficacy in HCV-infected patients ,65. Nonermal CTP . 2’-O-mermal CTP . In thisrmal CTP .The prime location of Ser282 has drawn the attention of antiviral researchers. Moreover, it is located in Motif B, which is highly conserved in all RdRPs, particularly in flaviruses, as shown in the multiple sequence alignment . Based opubchem.ncbi.nlm.nih.gov (accessed on 24 September 2022); Compound CID 45375808). In general, S351 was far from sofosbuvir, making it unlikely that it would clash with sofosbuvir. For example, the distances between S351 and sofosbuvir were DENV2, 19.8 and WNV, 17.2  appear in DENV and other flaviviruses in the future, it will be worthwhile to map its location in the primary structure and, at the same time, determine the 3D structure of cocrystals, obtained by cryo-EM or crystallography to complement the studies.Remdesivir and sofosbuvir hold the distinction of having received fast-track “emergency authorization” for clinical use in COVID-19- and HCV-infected patients, respectively. A query of their off-target effects led to the search for host genes that are differentially expressed in treated versus untreated individuals. The premise that the drugs may inhibit mitochondrial function received special attention in view of the distant similarity of the active sites of viral RdRP and mitochondrial RNA polymerase (mtRP). However, an assay of the diverse mitochondria function revealed little inhibition by remdesivir at clinically relevant concentrations ,75. In tAlthough the evolutionary origin of RdRP genes remains unknown, it occurs primarily in nonretroviral RNA viruses, as presented here. In eukaryotic cells, the RdRP activity is responsible for the amplification step of RNA interference (RNAi), found in plants and fungi ,78. RdRPMolnupiravir (EIDD-2801) ,79 is reUnlike remdesivir, the NHC-monophosphate, when incorporated in the product RNA, does not terminate transcription ,63,80 buBased on what we know so far, in order for a nucleoside analog (NA) to target viral RdRP and act as an antiviral, it needs to meet two fundamental criteria.(i) Acceptance by RdRP: The NA should be able to enter the NTP-binding domain of the RdRP where the normal nucleotide binds for transcription. Obviously, NAs with bulky substitutions or very different chemical properties will be unacceptable. This is why the prodrug does not work. First, it has a large substituent, introduced for improved pharmacology ; second,(ii) Incorporation in product RNA: The NA needs to be incorporated in the growing RNA chain, following which one of several things may happen: (a) The incorporated NA prevents joining with the next NTP due to the lack of 3’-OH . For the same reason, 3’-OH cannot be derivatized, since it is necessary for the 3’-5’ phosphodiester bond. (b) Alternatively, the NA with a normal, extendable 3’-OH can block transcription for several other reasons, such as a spatial clash with an amino acid side chain in the catalytic tunnel of the RdRP, as in remdesivir and sofosbuvir. (c) Lastly, it allows for transcript elongation, but its base-pairing property is different from that of the original nucleotide that it replaced. Subsequently, this leads to mutant viral RNA, with a lethal effect on the virus.(iii) Generalized ProTide technology: The prodrug forms of NA inhibitors, as described here, and other similar derivatives are often referred to as ProTide (Prodrug-Nuclotide) technology ,83,84,85Lastly, regardless of all our efforts, drug-resistant viral mutations have appeared throughout history including many against NAs; some of them have been described before, such as the S282T mutant of HCV and the sofosbuvir-resistant ZIKV mutant. Currently, this is circumvented by multidrug combination therapy and screening with novel analogs. It is fair to assume that this evolutionary battle between virus and man will continue. It is hoped that this critical review, which includes structural discussions, will inspire the design and evaluation of additional nucleoside analogs as antiviral candidates for use in the clinic as well as in the research laboratory.Mammalian cells are traditionally cultured in two-dimensional monolayers for initial studies of virus infection and drug sensitivity screening. However, it has been increasingly acknowledged that a three-dimensional cell culture may better approximate metazoan tissues and organs and may be suitable for diverse studies such as “epithelial–mesenchymal transition”, drug uptake, and virus growth ,90,91,92