Myosin is a molecular motor protein that is critical for using stored energy to yield contraction in both skeletal muscle and in the heart. Mutations in myosin can result in inherited human hypertrophic cardiomyopathy, wherein the heart walls become thickened. This results in inability to adequately pump enough oxygenated blood to meet bodily demands, and can lead to abnormal heart beating patterns and the possibility of sudden death. Typically, over-active myosin can be causative for this disease. Here, we used the common fruit fly to study one of these cardiomyopathy-causing mutations by expressing the mutant protein in the indirect flight muscle of the fly. Surprisingly, we found that the mutation and associated mutations decrease myosin and muscle function instead of enhancing it. We show that at least some of these defects result from improper interactions within the myosin protein. This suggests that appropriate communications within myosin are critical for its function and further indicates that certain aspects of indirect flight muscle myosin are functionally distinct from those of myosin in the human heart. By understanding the intricate interactions within the myosin motor protein that are important for its normal role, therapeutics for improving mutant myosin function can be developed.Drosophila melanogaster indirect flight muscle myosin yields mechanical and locomotory defects. Here, we use transgenic Drosophila mutants to demonstrate that residue R249 serves as a critical communication link within myosin that controls both ATPase activity and myofibril integrity. R249 is located on a β-strand of the central transducer of myosin, and our molecular modeling shows that it interacts via a salt bridge with D262 on the adjacent β-strand. We find that disrupting this interaction via R249Q, R249D or D262R mutations reduces basal and actin-activated ATPase activity, actin in vitro motility and flight muscle function. Further, the R249D mutation dramatically affects myofibril assembly, yielding abnormalities in sarcomere lengths, increased Z-line thickness and split myofibrils. These defects are exacerbated during aging. Re-establishing the β-strand interaction via a R249D/D262R double mutation restores both basal ATPase activity and myofibril assembly, indicating that these properties are dependent upon transducer inter-strand communication. Thus, the transducer plays an important role in myosin function and myofibril architecture. The R249Q mutation in human β-cardiac myosin results in hypertrophic cardiomyopathy. We previously showed that inserting this mutation into  Myosin II is an ATP-dependent molecular motor of muscle. Myosin-containing thick filaments are interdigitated with actin-containing thin filaments in repeating sarcomeric units of muscle myofibrils. The myosin heads act as cross-bridges during the actomyosin mechanochemical cycle. Rotation of the myosin lever arm in attached cross-bridges produces the force necessary for muscle contraction (reviewed by ). DuringDrosophila melanogaster disables flight due to decreased muscle kinetics and power production arising from a reduced number of cross-bridges bound to actin [Mutation R249Q in strand 6 of the transducer’s seven-stranded β-sheet in β-cardiac myosin II leads to hypertrophic cardiomyopathy ,6,7. Thito actin .Here, we examine the in vitro properties of full-length R249Q IFM myosin and report reductions in actin-activated ATPase activity and in vitro actin filament motility, suggesting that the mutation yields an under-active myosin. This contrasts with the enhanced activity of human β-cardiac R249Q myosin, which is proposed to arise from disruption of the interacting heads motif of the myosin dimer, wherein the molecule generally does not hydrolyze ATP rapidly or interact with actin ,11. Furthttp://swissmodel.expasy.org/; accessed on 15 May 2013) [The near-rigor human β-cardiac myosin crystal structure (PDB: 4P7H) was used as a template to analyze the predicted protein structure of the R249 and D262 mutants. The wild-type indirect flight muscle isoform (IFI) myosin S1 amino acid sequence as well as versions containing R249D, D262R or R249D-D262R were modeled onto the human β-cardiac myosin backbone using the SWISS-MODEL homology modeling server (ay 2013) ). StructDrosophila P element-containing Mhc transgene. Construction of R249Q was previously described [EagI to produce two subclones, pwMhc-5′ and pMhc-3′. The pwMhc-5′ subclone contains an 11.3-kb EagI Mhc fragment that was cloned into the pCasper vector [EagI Mhc fragment that was cloned into the pBluescriptKS EagI site . The pwMhc-5′ subclone was further digested with XhoI and AvrII. A 6.8 kb XhoI-AvrII-digested fragment from pwMhc-5′ was gel isolated and ligated into an XhoI-AvrII site in the pLitmus 28I vector , to produce the pMhc-5′-XA subclone. This was digested with PstI and AvrII. A 4.3 kb PstI-AvrII fragment was gel isolated and ligated into a PstI-AvrII site in vector pLitmus 28I, to produce the pMhc-5′-PA subclone. This was further digested with AgeI and BamHI, and the resulting 370 bp fragment was gel isolated and ligated into an AgeI-BamH I site in the pLitmus 28I vector to produce the pMhc-5′-AB370 subclone. This subclone was subjected to site-directed mutagenesis using the QuickChange II kit . For R249D, the primer 5′- CAAGGGTAAATTCATCGACATCCACTTCGGACCC -3′ containing the R249D nucleotide mutation (underlined) was used to yield pR249D-5′AB370. For D262R, the primer 5′- TAAACTGGCTGGTGCTCGTATTGAGACCTGTAAGT -3′ containing the D262R nucleotide mutation (underlined) was used to yield pD262R-5′AB370. For the R249D-D262R construct, the pR249D-5′AB370 clone was used as the template, and the primer 5′- TAAACTGGCTGGTGCTCGTATTGAGACCTGTAAGT -3′ containing the D262R nucleotide mutation (underlined) was employed to yield pR249D-D262R-5′AB370. Upon sequence confirmation of the R249D, D262R and R249D-D262R site-directed mutagenesis products, the pR249D-5′AB370, pD262R-5′AB370 and pR249D-D262R-5′AB370 subclones were separately and sequentially cloned back into the intermediate subclones from which they originated. The resulting subclones, pwMhcR249D-5′, pwMhcD262R-5′ and pwMhcR249D-D262R-5′ were digested with EagI. The 12.5-kb pMhc-3′ fragment was digested with EagI, gel isolated and ligated into the EagI site of each of these subclones to yield pwMhcR249D, pwMhcD262R and pwMhcR249D-D262R. The entire coding region and all ligation sites of the final plasmids were confirmed by DNA sequencing  before P element transformation.Site-directed mutagenesis was used to introduce a series of mutations  into a escribed . For proescribed  was diger vector , and theDrosophila lines for pwMhcR249D, pwMhcD262R and pwMhcR249D-D262R were generated using P element-mediated germline transformation [pwMhcR249D-6 and pwMhcR249D-12; pwMhcD262R-1, pwMhcD262R-6 and pwMhcD262R-7; pwMhcR249D-D262R-2, pwMhcR249D-D262R-4 and pwMhcR249D-D262R-5. Each of these transgenes were crossed into the Mhc10 background that eliminates endogenous myosin expression in the IFM and jump muscles [Transgenic ormation  by BestG muscles . Mhc transcripts from transgenic lines were spliced correctly and contained the appropriate site-directed nucleotide changes. RNA was prepared from upper thoraces of 2-day-old female flies using the LiCl2 extraction method [RT-PCR was used to confirm that the n method . The Prohttps://imagej.nih.gov/ij/ accessed on 1 June 2013). Mean value ± SEM of myosin/actin ratio for each transgenic line relative to pwMhc2 wild-type control are reported. To determine myosin expression levels relative to actin accumulation for each transgenic line, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used as previously described . BrieflyMore than 100 flies of each genotype were assessed for flight ability in small groups  at either two or seven days post-eclosion. Flies were released into a Plexiglas box with a light at the top  and clas2HPO4, 1.06 mM KH2PO4, 154.0 mM NaCl) the next day . Specimens were laid supine on a glass slide and snap-frozen in liquid nitrogen for 10 s. Frozen thoraces were immediately bisected down the mid-sagittal plane using a razor blade. Hemithoraces were stained with mouse anti-α-actinin antibody (1:50 in 1× PBS-T: PBS with 0.1% Triton-X 100) overnight at 4 °C. Samples were rinsed in 1× PBS the next day , before being incubated with a mixture of secondary antibody  and Alexa-568 phalloidin  for 2 h at room temperature. Samples were rinsed in 1× PBS  before being mounted on a glass slide with Vectashield (Vector Laboratories) and viewed using a 10× air lens to assess gross muscle morphology, and a 100× oil immersion lens (1.25 NA) to assess myofibrillar morphology on a Leica TCS SPE RGBV confocal microscope .Two-day-old flies were anesthetized using FlyNap (Carolina), and heads and abdomens were removed. Thoraces were fixed overnight in 4% formaldehyde at 4 °C and rinsed in 1× Phosphate Buffered Saline  was used to graph phosphate levels  vs. actin concentration to yield a Michaelis–Menten plot. maxV and mK values were calculated using the Michaelis–Menten equation and are expressed as mean ± SD. Values among genotypes were compared using Welch’s one-way ANOVA followed by Dunnett’s T3 multiple comparisons test. Statistical significance was assumed for p < 0.05. Steady-state Mg-ATPase assays were carried out using 2 µg of myosin, [γ-escribed ,23,24. Rp < 0.05.In vitro motility analyses were carried out in the presence of ATP using fluorescently labeled F-actin interacting with myosin bound to nitrocellulose-coated coverslips, as previously described ,23. VideDrosophila muscle dysfunction [Drosophila IFM myosin heavy chain sequence onto the human β-cardiac myosin motor domain (PDB: 4P7H), as shown in Drosophila myosins [Drosophila melanogaster transgenic flies, which allow biochemical, structural and functional dissection of myosin mutation phenotypes [The R249 residue is critical to myosin function based on the observations that the R249Q mutation causes human cardiac disease ,6,7 and function . Our molfunction . To stud myosins . The wilenotypes . R249D, D262R and R249D-D262R mutations into the single Drosophila muscle Mhc gene. DNA sequence analysis confirmed the correct nucleotide base changes for each DNA construct. We then employed P element-mediated transformation to stably insert the transgenes into the Drosophila germline and mapped the chromosomal location of each insertion. At least two independent transgenic lines for each construct that mapped to the third, fourth or X chromosome were examined in detail. Transgenic lines that mapped to the second chromosome were not studied since the endogenous myosin gene is located on that chromosome. Each transgene was crossed into the Mhc10 background, which eliminates endogenous myosin expression only in the indirect flight and jump muscles [We used in vitro mutagenesis to introduce  muscles . RT-PCR To determine myosin expression levels for the transgenic lines, we extracted proteins from upper thoraces of two-day-old adult female flies. SDS-PAGE and densitometric analysis allowed for determination of the ratio of myosin heavy chain to actin levels. The ratio of expression of myosin:actin for each transgenic line was essentially equal to wild-type levels .Mhc10 background , the mutant lines yielded mean values of 0 to 1.7, with flies displaying mostly downward flight or no flight at all . A summary of the results obtained for each myosin and a listing of statistically significant differences are shown in To examine the effects of the p < 0.001). Interestingly, combining these two mutations in R249D-D262R myosin yielded activity that was significantly higher  than either individual mutation and not significantly different from the wild-type control. This indicates that the R249D and D262R mutations suppress each other’s defective basal Mg-ATPase activity. Basal Mg-ATPase activity  was not maxV values does not arise from rebuilding the salt bridge between the two stands of the β-sheet of the myosin transducer. Likewise, the mK values that signify the amount of actin that yields half-maximal myosin ATPase activity were significantly reduced by more than 50% for all mutant myosins, including R249D-D262R, indicating that the double mutation does not improve this property. Actin-stimulated ATPase activities were significantly reduced by more than 65% for all three single-mutant myosins relative to the wild-type control A. This dp < 0.0001; As with actin-stimulated ATPase, the velocity of actin filaments moving over myosin molecules was reduced by ~65% or more for each of the mutant myosins (1.66 ± 0.11–2.54 ± 0.25 µm/s) relative to wild-type control . In thir output ,34, apper output ). The IHr output , whereinr output . The molr output ,34 and tr output . In thesDrosophila IFM myosin to readily form the IHM motif might explain the lack of enhanced function for its R249Q mutation. Electron microscopy demonstrated that Drosophila IFM myosin dimers are less capable of forming the IHM compared to Drosophila embryonic myosin [Drosophila thick filaments showed myosin heads in random orientations [Lethocerus [Drosophila IFM thick filaments failed to yield a structure for the S1 domains, suggesting that they are randomly oriented rather than engaged in an ordered IHM on the thick filament backbone [Lethocerus IFM [Drosophila thick filament backbone, a version could still occur without backbone interaction [Drosophila IFM myosin activity may not be influenced by the standard IHM conformation, suggesting that enhanced myosin function might not be expected from the R249Q mutation in the Drosophila model. Future analysis in the Drosophila system could focus on whether embryonic myosin, which is more capable of forming the IHM [Drosophila, with its strong escape response and fast wing beat frequency may take this to the extreme for IFM myosin by eliminating the IHM substantially or entirely. It should also be noted that while the contractile properties of human cardiac muscle and Drosophila IFM have similarities [Drosophila flightin) as well as differences in cellular architecture that may play a role in the differential responses of these two muscle types to the R249Q mutation. The failure of c myosin . Furtherntations , rather thocerus  or in tathocerus . Finallybackbone . Notablyerus IFM . Note theraction . Overall the IHM , yields  the IHM . Drosophlarities , there aDrosophila melanogaster. One of these residues is causative of HCM when mutated in human β-cardiac myosin. Mutating either amino acid residue of the salt bridge had severe effects upon basal ATPase activity, actin-activated ATPase activity, actin in vitro motility, myofibril assembly and stability, and flight muscle function. Restoring the salt bridge with compensatory mutations rescued basal ATPase activity and improved myofibril assembly and stability. Thus, while the salt bridge connecting the strands of the transducer is critical for these latter parameters, additional roles played by the wild-type residues are necessary for actin-based ATPase/motility, complete myofibril stability and flight muscle function. We examined the importance of maintaining the integrity of a salt bridge connecting two strands of the β-sheet within the transducer domain of muscle myosin heavy chain in


Searching for novel compounds with antibiotic activity and understanding theirmechanism of action is extremely important. The ribosome is one of the maintargets for antibiotics in bacterial cells. Even if the molecule does not suitthe clinical application for whatever reasons, an investigation of itsmechanism of action can deepen our understanding of the ribosome function. Suchdata can inform us on how the already used translational inhibitors can bemodified. In this study, we demonstrate that 1-(2-oxo-2-((4-phenoxyphenyl) The coronavirus pandemic has highlighted the problem of human vulnerability topathogens. The outlook for the expansion of antibiotic resistance in bacteriais also unfavorable. According to the Organization for Economic Cooperation andDevelopment (OECD), approximately 17% of infectious diseases in OECD membercountries were rooted in antibiotic resistance of bacteria in 2015. In theRussian Federation, the percentage of such diseases exceeds 40% [1]. As of2016, ~700,000 people die each year from infections caused byantibiotic-resistant bacteria. According to a prediction published in 2016, thenumber of deaths caused by resistant bacteria may be as high as 10 millionpeople by 2050 [2]. Hence, modern science currently faces the challenge ofcoming up with novel antibiotics.The protein synthesis occurring on ribosomes is the vital process through whichthe genetic information in the mRNA is translated into the amino acid sequenceof a protein. The bacterial ribosome consists of three ribosomal RNAs 16S,23S, and 5S) and more than 50 proteins, forming two subunits; the small, 30S,subunit and the large, 50S, one; they combine to form the 70S ribosome. Each ofthese subunits and the exit tunnel through which the newly synthesized peptideis released are targets for a large number of antibiotics S, and 5S, streptoApplication of a dual reporter system for analyzing the mechanism ofaction of antibioticsEscherichia coli JW5503 cellsATPpolynucleotide kinase  according to the manufacturer’sprotocol. Next, The resulting samples were purified using a QIAquick PCR purification kit.The sequence mixtures were prepared using a USB® Thermo Sequenase CycleSequencing Kit  according to the manufacturer’s protocol.Electrophoresis was carried out in 6% polyacrylamide gel (60 × 40 ×0.03 cm) containing 19% acrylamide, 1% N,N’-methylenebisacrylamide, and 7M urea in TBE buffer for 2–3 h. The specimens and products of thesequencing reactions  were applied onto thegel.The gel was transferred onto 3-mm paper, dried, and exposed to a sensory screenfor 18 h. The screen was scanned using a Typhoon FLA 9500 Biomolecular Imager.The RST1 template for this experiment was obtained by PCR amplification using a Taq-DNApolymerasekit (ThermoFisher), according to the standard protocol. The template sequence was as follows:ACTAATACGACTCACTATAGGGCTTAAGTATAAGGAGGAAAACATATGTATTGGGTAACCTCACGTCAGCCGAATATGCTGAAAATCCATGGCTTCGAAGACTGCGCCTAATAATAATAAAAAAAGTGATAGAATTCTATCGTTAATAAGCAAAATTCATTATAAC. The forward primer GTAAAACGACGGCCAGT, reverse primerCAGGAAACAGCTATGAC, and primer for reverse transcription GGTTATAATGAATTTTGCTTATTAAC were used.High-throughput screening for compounds exhibiting antimicrobialactivityE. coli JW5503 strain with tolC deletionimidazol-1-iumchloride, is shownin Fig. 3A (STOCK4S-33513).During screening, two analogs of this molecule were tested:1-amino)-2-oxoethyl)-3-(p-tolyl)-6,7-dihydro-5H-pyrroloimidazol-1-ium chloride  and1-(2-((2-methoxyphenyl)amino)-2-oxoethyl)-3-(p-tolyl)-6,7-dihydro-5H-pyrroloimidazol-1-ium chloride. These molecules didnot yield growth inhibition zones for the E. coli JW5503strain transformed with plasmid pDualrep2 in the solid agar medium test;therefore, further experiments were conducted using exclusively1-(2-oxo-2-((4-phenoxyphenyl)amino)ethyl)-3-(p-tolyl)-6,7-dihydro-5H-pyrroloimidazol-1-ium chloride.During the high-throughput screening libraries of the chemical compoundsprovided by InterBioScreen Ltd., among the compounds with antimicrobialactivity, we found a molecule that both inhibited growth of the  E. coli BW25113strain transformed with the reporter plasmid.This compound was inactive in the test for theMeasuring the minimum inhibitory concentrationE. coli JW5503 strain with tolCdeletion imidazol-1-ium chloride is 3.1 µg/mL. It is comparable with that oferythromycin, a natural protein synthesis inhibitor whose minimum inhibitoryconcentration for this strain was 3.1 µg/mL.The minimum inhibitory concentration was measured using the serial dilutionmethod for the deletion . The minMeasuring cytotoxicity in eukaryotic cellsTable.The toxicity of this compound was tested in several human cell cultures usingthe MTT assay. Unfortunately, it was found to be more toxic to human cellscompared to bacterial ones; so, this compound cannot be used as a drug, atleast in the tested form. The data are summarizedin Translation in the cell-free systemE. coli S30 Extract System for Linear Templates kit (Promega).Synthesis of firefly luciferase in this experiment was determined using thereaction of luciferin oxidation to oxyluciferin. If the reaction mixturecontains a translation inhibitor, luciferase is not synthesized and luciferinis not degraded. The results of each experiment were normalized with respect tothe added solvent (dimethyl sulfoxide), whose volume was identical to that ofthe test compound. The data for1-(2-oxo-2-((4-phenoxyphenyl)amino)ethyl)-3-(p-tolyl)-6,7-dihydro-5H-pyrroloimidazol-1-ium chloride (STOCK4S-33513) arepresented in Fig. 4.The translation reaction in the cell-free system was carried out using ap-tolyl)-6,7-dihydro-5H-pyrroloimidazol-1-ium chloride (STOCK4S-33513) is a translation inhibitor.According to the results, it appears fair to say that the compound1-(2-oxo-2-((4-phenoxyphenyl)amino)ethyl)-3-. Thesecodons were 1-AUG (M), 2-UAU (Y), and 8-CAG (Q). However, in the controlexperiment without the DMSO antibiotic added, one can see the same short pauses(but less pronounced) at the same spots. Therefore, a hypothesis can be putforward that this translation inhibitor can affect the kinetics of proteinsynthesis at mRNA regions that are difficult for the ribosome to traverse.Not only does the toeprinting assay allow one to verify whether a compoundinhibits protein synthesis, or not, but it also makes it possible tohypothesize regarding the stage at which translation was stalled. The workingprinciple of the method is as follows: in a cell-free system that is based onindividually isolated translation components, a short peptide is synthesized inthe presence of the test compound. A radiolabeled primer (complementary to the3’-terminus of mRNA), RNA-dependent DNA polymerase, and2’-deoxynucleoside-5’-triphosphates are added to the reactionmixture. Next, template RNA-directed primer extension takes place untilRNA-dependent DNA polymerase either meets the ribosome or reaches the templateend. If a protein synthesis inhibitor is added to the mixture, the ribosomewill stall on the template and will not allow RNA-dependent DNA polymerase toreach the end of the template; so, the cDNA fragment will be short. The exactlength of the cDNA fragment and the ribosome stall site on mRNA can becalculated according to the RNA sequence and position of the reversetranscription product in the gel with respect to the Sanger sequencing productsbeing separated in the respective gel lanes. In a typical experimental run, wealso compared the sites of ribosome stalling induced by the novel and alreadyknown translation inhibitors. The distance between the first nucleotide of theP site of the ribosome blocked on mRNA and the last synthesized cDNA nucleotideis 16 nucleotides long. It is convenient to use the thiostrepton antibiotic forcomparison as it is known to induce ribosome stalling at the first translationstep, right when the start codon AUG resides at the ribosomal P site. Based onthese data, we have performed computations for the codons residing at the Psite at the instant of ribosomal stalling(p-tolyl)-6,7-dihydro-5H-pyrroloimidazol-1-ium chloride, retrieved from the chemical library. This compound was shown toinduce the reporter system and act as an in vivo translationinhibitor. It was revealed that it can inhibit in vitrotranslation and potentiate ribosomal stalling during the synthesis of smallpeptides. Although this compound is highly toxic to human cells and, thus,cannot be used as a drug, a more thorough examination of this molecule mayprovide a deeper insight into the functioning of such an important molecularmachine as ribosome.We have investigated a novel inhibitor of bacterial translation,1-(2-oxo-2-((4-phenoxyphenyl)amino)ethyl)-3-(


The COVID-19 pandemic revealed a need for new understanding of the mechanisms regulating host–pathogen interactions during viral infection. Transfer RNA-derived RNAs (tDRs), previously called transfer RNA fragments (tRFs), have recently emerged as potential regulators of viral pathogenesis. Many predictive studies using bioinformatic approaches have been conducted providing a repertoire of potential small RNA candidates for further analyses; however, few targets have been validated to directly bind to SARS-CoV-2 sequences. In this study, we used available data sets to identify host tDR expression altered in response to SARS-CoV-2 infection. RNA-interaction-prediction tools were used to identify sequences in the SARS-CoV-2 genome where tDRs could potentially bind. We then developed luciferase assays to confirm direct regulation through a predicted region of SARS-CoV-2 by tDRs. We found that two tDRs were downregulated in both clinical and in vitro cell culture studies of SARS-CoV-2 infection. Binding sites for these two tDRs were present in the 3′ untranslated region (3′UTR) of the SARS-CoV-2 reference virus and both sites were altered in Variants of Concern (VOCs) that emerged later in the pandemic. These studies directly confirm the binding of human tDRs to a specific region of the 3′UTR of SARS-CoV-2 providing evidence for a novel mechanism for host–pathogen regulation. In late 2019, a novel zoonotic virus, a beta coronavirus now known as SARS-CoV-2, was identified in Wuhan, China . The dishttp://trna.ucsc.edu/tDRnamer/docs (accessed on 12 November 2022). While small noncoding RNAs with antiviral activity are a well-defined phenomenon in plants and invertebrates, only recently have studies implicated such noncoding RNAs in the antiviral response in mammals [Small noncoding RNAs (sncRNAs) are short RNA molecules <100 nt) that have been implicated in regulating a variety of context-dependent cellular processes . Two wel0 nt that mammals ,14. Smal mammals . Viruses mammals . Viral e mammals ,18.The expression of a large number of microRNAs, produced by both host and virus, have been identified as altered in SARS-CoV-2 infected cells, and yet limited knowledge of their function exists ,20. In aIn this study we accessed available RNA sequencing data to identify tDRs that are differentially expressed in the plasma of patients with severe COVID-19 compared to healthy controls . Many sthttp://genome.bioch.virginia.edu/trfdb/ (accessed on 17 January 2022) (We utilized an available data set to identify tDRs in plasma samples from patients with a confirmed infection of SARS-CoV-2 compared to those without infection . These dry 2022) . p < 0.05) in patients diagnosed with COVID-19 compared to uninfected individuals .To refine the search for human tDRs of interest, we analyzed another available data set. The second data set was originally established to identify small noncoding RNA using small RNA-seq in Calu-3 cells infected with SARS-CoV-2 compared to mock infected cells. These data were accessed at NCBI (GEO: GSE148729). In the original analysis, these data were examined for altered expression of the host miRNAs . Using tWe found that the expression of several of the same human tDRs from the plasma samples was also significantly altered in response to direct infection by SARS-CoV-2 . Two tDRhttp://trna.ucsc.edu/tDRnamer/docs/ accessed 12 November 2022).The choice of specific tDRs for further experimentation was based on those tDRs identified in both the patient plasma and Calu-3 cell line NGS data as significantly decreased in expression in response to infection ,26. ThusUsing the sequences for tDR-Gly (5′-GCGTTGGTGGTATAGTGGTGAGCATAGCTG) and for tDR-Val (5′-GTTTCCGTAGTGTAGTGGTTATCACGTTCGC), we examined the predicted RNA binding of these two tDRs to SARS-CoV-2 and the variants of concern (VOCs) Alpha, Delta, Omicron subvariants BA.1, and BA.2, by using RNAhybrid  followedtDR-Gly and tDR-Val are predicted to hybridize in multiple regions across the panel of SARS-CoV-2 genomes tested . To narrG to U change is outside of the predicted binding site, while the binding of tDR-Val is expected to be reduced  response by the host in SARS-CoV-2-infected cells. The concept that RNAi is part of the immune response in mammals has been controversial, even though it is well documented as part of the anti-viral response in plants and insects . RNAi reSmall noncoding RNAs have previously been studied in SARS-CoV-2-infected patient samples and cell lines . HoweverTo study the direct regulation of this region of SARS-CoV-2 by the two tDRs, we developed sponge constructs to soak up the specific tDRs . Spongeshttp://genome.bioch.virginia.edu/trfdb/ accessed 17 January 2022). The unique sequences were then assembled into a single annotated reference sequence in Geneious Prime 2022.0.2 . The full SRA dataset was downloaded from NCBI (Bioproject PRJNA702830) as fastq files. The adapter sequences were right end trimmed using the BBDuk Geneious Prime plugin (https://sourceforge.net/projects/bbmap/ accessed 21 March 2022) . Reads were evaluated for quality before and after trimming using FastQC v. 0.11.9 [p-value of p < 0.05 was used to determine the differentially expressed tDRs.All 3′ and 5′ human tDR sequences were accessed from tRFdb (. 0.11.9  (FastQC:. 0.11.9 . Read co. 0.11.9 . An adjuhttps://sourceforge.net/projects/bbmap/ accessed 21 March 2022). For the first pass, the Illumina adapters were trimmed using the parameters k = 12, trimq = 13, minlength = 12 and all other default parameters. In the second pass, the poly-A tails were trimmed. All further analysis was completed as described above.The full SRA dataset was downloaded from NCBI (Bioproject PRJNA702830) as fastq files. The sequences were right end trimmed in two passes using the BBDuk Geneious Prime plugin , were used to search the full sequence of SARS-CoV-2 (NC_045512.2) for hybridization using RNAhybrid version 2.2 . Once thrameters . tDR 500A region of the 3′-UTR of SARS-CoV-2 from the reference sequence, including predicted binding sites for tDR-Gly and tDR-Val, was cloned into the psiCheck 2 dual luciferase vector using XhoI and NotI restriction enzymes and the sequence was confirmed by submission for sequencing to ACGT Inc. . The sequence used from SARS-CoV-2 for psi-reference is 5′AGGGAGGACTTGAAAGAGCCACCACATTTTCACCGAGGCCACGCGGAGTACGATCGAGTGTACAGTGAACAATGCTA. In addition, the same region with either the one base-pair change observed in the Delta variant (psi-Delta) 5′ AGGGAGGACTTGAAAGAGCCACCACATTTTCACCGAGGCCACTCGGAGTACGATCGAGTGTACAGTGAACAATGCTA or the region missing 26 base pairs (psi-BA.2) 5′AGGGAGGACTTGAAAGAGCCACCACATTTTCACCTACAGTGAACAATGCTA was cloned into psiCheck 2 at the Xho1 and NotI restriction sites using the methods previously described . These pthermofisher.com accessed on 29 August 2022). The quantification of cells was conducted on triplicate wells using ImageJ software [The EGFP sponge plasmids were constructed by placing two antisense sequences with a spacer in between shown in  and clonsoftware . The HEKt-test was used to compare the differences between the triplicate wells. A p-value less than 0.05 is considered to be significant in this study.Statistical analysis for transfection studies: All the data are expressed as a percent of the control with standard deviations. The Student’s


Here, we present evidence that certain CMV strains have evolved polymorphisms in gO to evade trimer-specific neutralizing antibodies. Using luciferase-tagged variants of strain TB40/E in which the native gO is replaced by gOs from other strains, we tested the effects of gO polymorphisms on neutralization by monoclonal antibodies (mAbs) targeting four independent epitopes in gH/gL that are common to both trimer and pentamer. Neutralization of fibroblast entry by three mAbs displayed a range of potencies that depended on the gO type, a fourth mAb failed to neutralize fibroblast entry regardless of the gO type, while neutralization of epithelial cell entry by all four mAbs was potent and independent of the gO type. Thus, specific polymorphisms in gO protect the virus from mAb neutralization in the context of fibroblast but not epithelial cell entry. No influence of gO type was observed for protection against CMV hyperimmune globulin or CMV-seropositive human sera, suggesting that antibodies targeting protected gH/gL epitopes represent a minority of the polyclonal neutralizing repertoire induced by natural infection.Human cytomegalovirus (CMV) utilizes different glycoproteins to enter into fibroblast and epithelial cells. A trimer of glycoproteins H, L, and O (gH/gL/gO) is required for entry into all cells, whereas a pentamer of gH/gL/UL128/UL130/UL131A is selectively required for infection of epithelial, endothelial, and some myeloid-lineage cells, but not of fibroblasts. Both complexes are of considerable interest for vaccine and immunotherapeutic development but present a conundrum: gH/gL-specific antibodies have moderate potency yet neutralize CMV entry into all cell types, whereas pentamer-specific antibodies are more potent but do not block fibroblast infection. Which cell types and neutralizing activities are important for protective efficacy  Human cytomegalovirus (CMV) is transmitted primarily through saliva and urine and infects a relatively high prevalence of human populations worldwide. CMV infections can cause pneumonitis, retinitis, gastrointestinal disfunction, encephalitis, myocarditis, and mortality in immunocompromised patients, often leading to graft rejection in solid organ transplant recipients . CMV is Treatment options for congenital CMV infections are limited. Currently approved antivirals are associated with dose-limiting hematologic or renal toxicities, and consequently their use is proscribed during pregnancy . Howeverin vivo, and while initial virion attachment is believed to occur via interactions between cell surface glycosaminoglycans and a heterodimer of glycoproteins M and N (gM/gN), subsequent entry mechanisms are cell type-dependent and mediated by different glycoprotein complexes on the viral envelope [CMV infects a variety of cell types envelope ,12,13. Tenvelope ,18,19,20envelope ,24,25,26UL74 open reading frame, is heavily glycosylated and highly polymorphic; consequently, CMV strains cluster into eight distinct gO genotypes with the greatest sequence variability residing in the first 100 residues [During fibroblast entry, the gH/gL/gO trimer interacts with its receptor, platelet-derived growth factor receptor-alpha (PDGFRα) . The gO residues ,29,30,31In previous studies , we obseEvidence in support of this hypothesis, at least with regard to the linear gH epitope, has been recently reported . In the ®) was a gift from CSL Behring, King of Prussia, PA, USA. Sera were acquired from two normal healthy adults with CMV seropositivity confirmed by gB-ELISA [gB-ELISA . ResearcHuman MRC-5 lung fibroblasts (ATCC CCL-171) and ARPE-19 retinal pigment epithelium epithelial cells (ATCC CRL-2302) were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s Minimum Essential Medium  with 10% fetal bovine serum (Gemini) and 1% Penicillin Streptomycin Glutamine .UL74 sequences encoding gO in the parental strain GT1c were fully replaced by UL74 sequences from strains Towne, AD169, BE/29/2011, or Han16, respectively, and chimera GT1c/3, which contains UL74 sequences encoding amino acids 1–107 from strain TB40/E with the remaining 111 C-terminal amino acids from strain Han16. Genome sequences were obtained using GenBank accession numbers for CMV strains AD169 (X17403.1), Towne (GQ121041.1), BE/29/2011(KP745672.1), Han16 (JX512204.1), Merlin (NC_006273.2), and for the TB40-BAC4 variant of strain TB40/E (EF999921).E. coli strain SW102 cells using a Gene Pulser X cell electroporation system . Colonies containing BAC GT1c were isolated by selection on plates containing 12.5 µg/mL chloramphenicol. Using plasmid pGalK [E. coli SW102 cells containing BAC GT1c were prepared as described [galK insertion was confirmed by PCR and targeted sequencing (eurofins).Chimera GT1c/4 was constructed using GalK recombineering  and encoid pGalK  as a temescribed  followinUL74 sequences from strain Towne flanked by 50-bp homologies to sequences flanking the galk insertion was amplified using primers UL74-Towne-FW (TCTAAATTATTCTTTATTATATCACTGACTGTCCTGTTATTTTCTATAATAAACTGTAAGATCGCGGTAGCGCGTTTTCGAGTAAAGAGTCAGAAAGCAAAAGAGGAAGAGAGGCAACTA) and UL74-Towne-RV (GGTCATATTCATAGTAAGATTTTTAACGTGTTGCCTAGTCATATTGAAGTATTTTGTATAATCACCTGTTTTTGACGCTAGTTCTTGCAGTATACGTAATTTTAGTTGCCTCTCTTCCTC). The product was purified using the MinElute PCR purification kit  and transformed as above into induced/competent E. coli SW102 cells containing the galK insertion mutant of BAC GT1c. Clones in which galK was replaced by Towne UL74 sequences were isolated by counter-selection on plates containing 0.2% 2-Deoxy-D-galactose  and 12.5 µg/mL chloramphenicol. Correct insertion of Towne sequences in the resulting BAC clone, designated GT1c/4, was confirmed by PCR and targeted sequencing (eurofins). GT1c/4 BAC DNA was prepared as described above and transfected into MRC-5 cells using effectene transfection reagent , as previously described [Using Towne DNA as a template, a PCR product containing escribed ,43. ViruApproximately 300 PFU of each chimeric virus were incubated with mAbs at a final concentration of 100 μg/mL for one hour (h) at 37 °C, then transferred to black-walled clear-bottom 96-well plates (Corning) containing confluent MRC-5 or ARPE-19 cell monolayers. Immunofluorescence staining was performed as previously described . After iTM HTX Multi-Mode Microplate Reader . The amounts of each virus stock needed to generate a signal of 10,000–20,000 RLU at 48 h post-infection (hpi) were determined from the resulting dose–response curves. This amount of each virus was then mixed with serial dilutions of mAbs, HIG, or human sera, incubated for 1 h at 37 °C, and transferred in triplicate to 96-well cultures containing confluent MRC-5 or ARPE-19 monolayers. After 48 h RLU were measured as above.Serial dilutions of each virus stock were used to infect black-walled clear-bottom 96-well plates containing confluent MRC-5 or ARPE-19 cell monolayers. After incubation for 48 h at 37 °C, 100 μL of Steady-Glo Luciferase substrate  was added to each well and after incubation at RT for an additional 10 min, relative luminescence units (RLU) were measured using a Synergy50) values were determined using Prism 5  as the inflection points of four-parameter curves fitted by non-linear regression to plots of mean relative fluorescent units (from triplicate wells) vs. Log (antibody concentration) as described previously [50 values were reported as the antibody concentration at which the fitted curve intersected the 50% inhibition line. Where neutralization did not reach 50%, IC50 values were reported as >50 μg/mL, the highest antibody concentration used. Luciferase-based assays were repeated at least twice with similar results.The 50% inhibitory concentration (ICeviously . Where nUL74 sequences (encoding gO) from various CMV strains were genetically swapped for the native UL74 sequences in parental virus GT1c, a BAC-cloned virus derived from CMV strain TB40/E [In a previous study  a panel UL74 sequence from TB40/E, or against GT4, a variant derived from GT1c in which the native UL74 was replaced with UL74 from strain Towne , Merlin (GT5), Han 16 (GT3), or BE/29/2011 (GT2b) (As illustrated in 1 (GT2b) B. Lucife223.4, 124.4, and 3–16, while GT4 (encoding Towne gO) was consistently among the most resistant to all three antibodies A. IntereWhile the overall amino acid identity between TB40/E and Towne gO is 76%, the majority of polymorphisms lie within the first 100 residues, where the amino acid identity is only 43% , red boxCMV is among the most common pathogens that cause opportunistic diseases in immunocompromised individuals such as transplant and AIDS patients . Diseasetrans to protect neutralizing epitopes in the gH/gL dimer. These results are consistent with earlier observations that resistance to inhibition of spread by gH-specific mAbs C2 and 14–4b correlates with strain differences in gO but not gH [UL74 gene enhances sensitivity to inhibition of spread by gH-specific mAb 14–4b and gB-specific mAb C23 [trans to four distinct gH/gL epitopes and gOs representing six different subtypes. Moreover, the results from gO chimeras determined that the region of gO that confers epitope protection in trans corresponds to the highly polymorphic N-terminal 98 amino acids, with residues 31–64 from gO of strain Towne being sufficient to confer resistance.In the current study, gO sequences from various CMV strains were found to act in t not gH , and tha mAb C23 . In a mo mAb C23 . As AP86 mAb C23 , the res mAb C23  it is noBased on negative staining electron microscopy of mAb-gH/gL complexes , site 5 Negative staining electron microscopy and mAb binding competition experiments indicate that sites 5 and 6 are adjacent or partially overlap . Site 8 While it is possible that the gO type could alter the levels of trimer in virions, thus impacting the neutralizing activities of trimer-specific neutralizing antibodies, previous studies have shown that gO-chimeric viruses retain the ability to infect epithelial cells and fibroblasts  and thatThe N-terminus of gO is important for trimer binding to its cellular receptor PDGFRα on fibroblasts ,66,67. Atrans. The mechanism of protection has been linked to glycosylation, as mutations in gN that reduce its glycosylation enhanced sensitivity to antibodies targeting epitopes in gB and gH, in addition to gN [trans involves glycan shielding, as proposed for gN [Like gO, gN is heavily glycosylated, exhibits extensive amino acid sequence polymorphism, and mediates neutralizing epitope protection in on to gN . Recent on to gN , suggeston to gN ,71. Howeon to gN ,72,73, id for gN , or extetrans by gO may allow escape from neutralizing antibodies targeting gH/gL epitopes induced by a previous infection, and therefore provide advantages during reinfection. As the epitopes are presumably protected regardless of their polymorphisms, this mechanism of antibody evasion would be equally effective in the context of prior immunity induced by strains with the same or similar epitopes or by the same strain, as occurs when CMV reactivates from latency. Why certain strains such as Towne have acquired this mechanism of neutralizing antibody evasion while others, such as TB40/E have not, remains a mystery. However, it is possible that the TB40/E gO provides other advantages; for example, it may protect other epitopes in gH/gL that have not as yet been identified.Protection in in vivo. Consequently, their impact may only manifest in the context of population dynamics over multiple host infections. Nevertheless, that CMV has evolved elaborate mechanisms to avoid neutralization by mAbs targeting gH/gL, and specifically to protect fibroblast and not epithelial cell entry, suggests that fibroblast infection may have special importance for the natural history of CMV infections.While the importance or clinical relevance of protecting epitopes in gH/gL is not known, that the linear epitope at site 8 is polymorphic  suggests


Mycobacterium tuberculosis (Mtb) infection remains a major health problem worldwide. Although the Bacillus Calmette-Guérin (BCG) vaccine is the most widely used vaccination for preventing tuberculosis (TB), its efficacy is limited. We previously developed a new recombinant BCG (rBCG)-based vaccine encoding the Ag85B protein of M. kansasii (Mkan85B), termed rBCG-Mkan85B, and its administration is followed by boosting with plasmid DNA expressing the Ag85B gene (DNA-Mkan85B). Previously, we identified MHC-I (H2-Kd)-restricted epitopes that highly cross-react with those of Mtb in BALB/c (H2d) and CB6F1 (H2b/d) mice. We also reported that the rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination protocol protected CB6F1 mice against M. kansasii infection. In this study, to investigate the protective effect of our novel rBCG against Mtb infection, CB6F1 mice were either left unimmunized or immunized with the BCG, rBCG-Mkan85B, or rBCG-Mkan85B/DNA-Mkan85B vaccine for 10 weeks prior to inhalation exposure to the virulent Mtb Erdman strain for another 6 weeks. Compared with the BCG and rBCG-Mkan85B vaccinations, the rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination protocol significantly reduced the numbers of pulmonary colony-forming units (CFUs). Moreover, the rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination induced antigen-specific polyfunctional CD4+ and CD8+ T cells. These results suggest that CD8+ T-cell immunity to immunodominant epitopes of Mtb is enhanced by rBCG vector-based immunization. Thus, rBCG vector-based vaccinations may overcome the limited ability of the current BCG vaccine to elicit TB immunity. Mycobacterium tuberculosis (Mtb) continues to cause major health concerns worldwide, and the development of a more effective vaccine is needed. The WHO estimates that one-quarter of the world’s population is infected with Mtb and that approximately 9.9 million people have developed tuberculosis (TB). Several respiratory infections, such as influenza, decreased in frequency during the COVID-19 pandemic, and although the number of new TB infections appeared to decrease in 2020, this figure increased again in 2021. Recently, TB has become an even more serious public health problem worldwide with the emergence of multidrug-resistant (MDR), extensively resistant (XDR), and totally resistant (TDR) Mtb strains.M. kansasii (rBCG-Mkan85B) antigen 85B (Ag85B) protein. We used plasmid DNA expressing the same M. kansasii Ag85B gene (DNA-Mkan85B) as a booster vaccination [M. tuberculosis and M. leprae [+ T cells and CD8+ T cells [M. avium, M. kansasii, and M. leprae, in both BALB/c (H2d) mice and CB6F1 (H2b/d) mice [M. kansasii [M. tuberculosis and evaluated the protective effect and cellular immunity.We previously developed a new recombinant BCG (rBCG) vaccine encoding the cination . The Ag8cination . The Ag8. leprae ,4. We re. leprae . We also T cells  and iden4 pulmonary CFUs per animal (p < 0.01) (p < 0.05–0.01)  . Naïve Cr animal . All thr < 0.01) . The mos05–0.01) .+ T cells in both the lung and spleen  method. Immune cells isolated from the lungs and splenocytes were obtained from each mouse infected with Mtb either unvaccinated or vaccinated with BCG-Tokyo, BCG-Mkan85B, or rBCG-Mkan85B/DNA-Mkan85B. The cells were stimulated with 9-mer Ag85B CD8 epitope peptides (Pep8) ,5 and thd spleen A,B. In ccination A,B. M. bovis, is the only available vaccine for Mtb. Although the BCG vaccine has been used for approximately 100 years, researchers have reached a consensus that the effectiveness of the BCG vaccine in preventing TB infection is limited. Therefore, the development of a novel TB vaccine is urgently needed. The development of various new TB vaccines has been widely pursued, and several different rBCG vaccines and immunization strategies have been explored. Various methods have been attempted to construct new rBCG vaccines, including inserting mycobacteria-derived antigens into the conventional BCG vaccine [The Bacillus Calmette-Guérin (BCG) vaccine, which is a live attenuated strain of  vaccine ,8,9,10 o vaccine ,12 and m vaccine . Some of vaccine . In addi vaccine . In addi vaccine .+ T cells, the induction of antigen-specific CD8+ T cells is poor. Therefore, some novel rBCG vaccines have been designed to induce TB antigen-specific CD8+ T cells in addition to TB antigen-specific CD4+ T cells [+ T cells, than the BCG vaccine alone [+ T cells in both the lungs and spleen, this vaccination strategy could not induce polyfunctional CD8+ T cells . All animal studies were conducted in accordance with institutional guidelines approved by the Nihon University Animal Care and Use Committee , the Institutional Committee for Gene Recombination Experiments (2018MED21), and the Biorisk Management and Control Committee (30-10-4). The institutional animal experimental guidelines comply with the Institute of Laboratory Animal Resources (ILAR) Guide. The mice were allowed free access to sterile water and standard mouse food, and their physiological conditions were assessed every few days. We used the previously prepared rBCG-Mkan85B strain and DNA-Mkan85B plasmid ,5 in thi6 CFUs, or 0.1 mg of bacilli, intradermally (i.d.), and 100 µg of plasmid DNA or control DNA in saline intramuscularly (i.m.) three times (CB6F1 mice were immunized with either the BCG or rBCG-Mkan85B vaccine at a concentration of 4 × 10ee times . We usedThe virulent Mtb Erdman strain was grown in Middlebrook 7H9 broth (BD Difco) supplemented with albumin dextrose complex (ADC) enriched (BD Difco) with 0.05% Tween 80 at 37 °C and harvested at the stage of exponential growth stage. The bacilli were passed through a 5.0 µm filter to obtain a single-cell suspension and frozen at −80 °C until use. Thawed aliquots were diluted in PBS containing 0.05% Tween 20 to the desired inoculum concentrations. Two weeks after the final immunization, the mice were exposed to Mtb in an Inhalation Exposure System . Based on a preliminary study estimating appropriate counts of primary tuberculoma in the lung at autopsy, we adjusted the dose to deliver 62.5 ± 33.0 CFUs of bacilli to the lungs of each animal after Mtb challenge infection. The thoracic and abdominal cavities of the animals were then opened aseptically, and the lungs and spleens were removed. Both the lungs and the spleens were homogenized with a gentleMACS dissociator . Tenfold serial dilutions of homogenized lung tissues were inoculated onto duplicate 7H10-OADC agar plates (Difco) to determine the bacterial loads. The colonies were counted after incubation for 3 weeks at 37 °C. The Mtb challenge experiments were conducted in the ABSL3 facility of the Research Institute of Tuberculosis, Tokyo, Japan with the approval from its Animal Research Committee.+ and CD8+ T cells is shown in Intracellular cytokine staining was performed as previously reported ,5. In brAll comparisons between the control group and the vaccinated groups and among the vaccinated groups were conducted by one-way ANOVA with the Tukey-Kramer post hoc test using JMP software . The data are expressed as the means ± SDs.


High viremia in HIV+ individuals was related to increased HLA-E expression, and changes in NK subpopulations, especially a reduction of the CD56bright as well as an increase in adaptive NK subpopulation. Uncontrolled HIV infection was also characterized by a reversion of the NKG2A/NKG2C expression ratio and a loss of positive and negative regulation of NK mediated by HLA-E. This was reflected in a lower cytotoxic, degranulation and cytokine production capacity, especially in CD56bright and adaptive NK. In line with these results, HLA-E expression showed a positive correlation with viral growth inhibition in an in vitro model of acute infection at day 7, which was lost after 14 days of culture. Using HLA-E expressing K562 cells, we determined that only one out of 11 described HIV-derived HLA-E epitopes increased HLA-E surface stability. In spite of that, eight of the 11 epitopes were capable of increasing degranulation and three drove differences in NK-cell mediated cell lysis or cytokine secretion. In conclusion, our results indicate that HLA-E molecules presenting HIV-derived epitopes may sensitize target cells for NK lysis in early HIV infection. However, prolonged exposure to elevated HLA-E expression levels in vivo may lead to NK cell dysfunction and reduced viral control In chronic infection.The contribution of the HLA-E/NKG2X axis in NK-mediated control of HIV infection remains unclear. We have studied the relationship between HLA-E expression and phenotypical as well as functional characteristics of NK cells, in the context of chronic HIV infection and in an  In brief, NK cells were first classified into two subpopulations based on the expression of CD56 and CD16, named CD56 subsets . In addi subsets –5. To da subsets .In general, NK-cell stimulation is the result of a balance between activating and inhibitory signals to which they are exposed, signals that can vary depending on the nature of the binding cell . NK cellHLA-E is a non-classical class Ib histocompatibility molecule with very limited polymorphism. Only two alleles (HLA-E*01:01 and HLA-E*01:03) have been demonstrated to be properly expressed on the cell surface and together cover >99% of the global allelic diversity of HLA-E . Given tvia the missing self-axis -expressing NK cells  or ii) high HIV plasma viral loads  of >50,000 HIV RNA copies/mL  and CD4 counts that ranged from 11 to 726 cells/mm3 (median: 284 cells/ mm3) (HIV-infected subjects (n=51) recruited at the Hospital Germans Trias i Pujol  and the IMPACTA clinics in Lima (Peru) were included in this study. These included individuals that were not receiving antiretroviral treatment and had i) low HIV plasma viral loads  of <10,000 HIV RNA copies/mL  and CD4 counts that ranged from 434 to 1,343 cells/mmA total of 14 HLA-E binding epitopes Table 1 A molecular model was constructed for HLA-E*01:01 and HLA-E*01:03 bound to PM9 and RL9 peptides based on the 6GL1 X-ray crystal structure. The initial molecular model of HLA-E-peptides interacting with NKG2A/2C-CD94 and TCR was constructed based on available X-ray crystal structure in the Protein Data Bank (3CDG and 5W1V). The initial models were Energy Minimized using Gromacs 5.0 .2 for three hours, prior to being pulsed with 20 µg/mL of canonical and non-canonical HIV-derived peptides, for 16 hours at 26°C 5% CO2. Cells were then washed twice with I10 medium and rested for 2 hours at 37°C 5% CO2. Cells were then stained with 3D12 PE anti-human HLA-E (BioLegend) and fixed . At least 100,000 events were collected on a LSRII BD cytometer and analyzed using FlowJo v10 software. HLA-E surface stabilization was expressed as (% HLA-E+ cells in peptide-pulsed cells/% HLA-E+ cells in non-pulsed cells) and (HLA-E MFI in peptide-pulsed cells/HLA-E MFI in non-pulsed cells).HLA-E*(01:03) transfected K562 cell lines (HLA-E/VL9-K562), co-expressing the HLA-B*0702 signal sequence derived VL9 peptide (VMAPRTVLL) , were ki2 with calcein-AM at 10 nM and then cultured in R10 medium , together with the different ratios of NK cells isolated from the HIV seronegative individuals, for 4 hours at 37°C and 5% CO2. Cultures were then run on a Canto II BD Cytometer and the percentage of killing of labelled target cells calculated as 100-% calcein-AM+ K562 cells. In each condition, an exponential regression curve was determined by the percentage of killing resulting from the different effector to target ratios in the co-culture. Results were expressed in lytic units  were isolated from cryopreserved leukocyte fractions using Lymphoprep™ (Stemcell) density gradient. NK cells were isolated by magnetic bead separation (MACS Miltenyi Biotec) from PBMC, following the manufacturer’s instructions. NK cytotoxic capacity was measured at 10:1, 5:1, 2.5:1, and 1:1 NK effector to target cell ratio (HLA-E*0103/VL9-K562 targets). Target cells were pulsed with canonical and non-canonical HIV-derived peptides and labeled using calcein-AM to perform a retention assay as previously described . Brieflyic units  calculatNK degranulation activity was measured by flow cytometry at a fix 5:1 NK effector to target cell ratio. Targets were HLA-E*01:03/VL9-K562 cells pulsed with canonical and non-canonical (HIV-derived) peptides. NK cells sorted from cryopreserved PBMC and peptide-pulsed target cells were co-cultured for 4 hours in the presence of Golgi Stop, Golgi Plug and APC anti-human CD107 (Biolegend) and stained for surface markers: SK7 Amcyan anti-CD3 (BD), 5.1H11 BV785 anti-CD56 and B73.1 BV-605 anti-CD16 (Biolegend). Subsequently, cells were fixed, permeabilized  and stained with 6401.1111 FITC anti-human TNF-α and B27 PE-Cy7 anti-human IFN-γ (BD). Approximately 20,000 events per samples were acquired on a Canto II BD Cytometer and analyzed using FlowJo v10 software.-CD16- (CD3-CD56-CD16-CD57-), (ii) CD56dim (CD3-CD56dimCD16+CD57-), (iii) adaptive NK cells (CD3-CD56dimCD16+CD57+) and (iv) CD56bright (CD3-CD56brightCD16-CD57-). NKG2A and NKG2C surface expression were evaluated in all NK subpopulations, by the gating strategy summarized in To discriminate different NK cell subpopulations, isolated NK cells from cryopreserved PBMC from HIV seronegative (n=12), HIV-high (n=8) and HIV-low (n=8) individuals were stained with SK7 Amcyan anti-CD3 (BD), 5.1H11 BV-785 anti-CD56, B73.1 BV-605 anti-CD16, HNK-1 PerCP-Cy5 anti-CD57 (Biolegend), 131411 PE anti-NKG2A and 134591 APC anti-NKG2C (R&D Systems). Samples were run on a LSRII BD Cytometer and analyzed using FlowJo v10 software. NK cell subpopulations were classified as (i) CD562 after magnetic sorting in R10 medium, supplemented with 50 U/mL of IL-2 and 1 ng/mL of IL-15. After one week (day 7 of co-culture), half of the medium was collected and replaced with 50 µL of R10 supplemented with 100 U/mL of IL-2 and 2 ng/mL of IL-15. At day 14, the culture supernatant was harvested and NK cells from the long-term culture characterized by flow cytometry (as described before). HIV-1 p24 Gag concentration in the supernatant was measured at day 7 and day 14 by commercially available HIV-1 p24 ELISA kit (Innogenetics) in accordance with manufacturer’s instructions. Samples were read on a Perkin Elmer Ensight Plate reader. NK-mediated inhibition of viral replication was calculated as %inhibition = [-(HIV-1 p24 Gag concentration in CD4+ T-cells cultured with NK cells)]/x100.NK cell mediated viral inhibition activity was measured in 12 HIV-negative individuals. Briefly, the CD4+ T cell enriched fraction was obtained from freshly isolated PBMC using NK and CD8+ T-cell specific magnetic beads depletion  and stimulated with PHA (5 µg/mL) in R10. HLA-E surface expression was measured on the CD3+ CD4+ population by flow cytometry (as mentioned before) after three days of stimulation. Gating strategy is shown in Total HIV-1 DNA was quantified in PBMC lysates by droplet digital PCR (ddPCR) in duplicate as described previously . Briefly-ΔCt (where Ct is the median threshold cycle from 3 replicates).DNA and RNA were simultaneously extracted using the AllPrep DNA/RNA Mini kit (QIAGEN) from cell pellets from the 51 HIV infected individuals and the 12 HIV seronegative individuals. Retrotranscription (SuperScript II first-strand synthesis Supermix) was performed on those samples and the resulting cDNA was used for reverse transcription-PCR using TaqMan gene expression assay for detection of HLA-E (Hs03045171_m1) and TBP (Hs99999910_m1) all from Applied Biosystems in a 7500 Real-Time PCR Systems (Applied Biosystems™). Relative expression was calculated as 2An in-house developed strategy based on Next Generation Sequencing (NGS) was used for HLA-E genotyping of human PBMC and HLA-E expressing K562 cell lines at the Laboratori d’Histocompatibilitat i Immunogenètica, Banc de Sang i Teixits (BST). This strategy consists of a long-range PCR to amplify the HLA-E gene from 5’ UTR to 3’ UTR. Library preparation  was performed using the NGSgo-LibrX and NGSgo-IndX kits (GenDx) according to the manufacturer’s instructions. The final denatured library was sequenced using a MiSeq system with a 300-cycle MiSeq Reagent Kit (Illumina). HLA-E genotype determination was performed using NGSengine software  and the IPD-IMGT/HLA database as a reference (version 3.38.0). Raw sequencing data are available at the European Nucleotide Archive under the accession number ERA18504000.2 analysis was used to detect differences in the frequency of individuals with plasma viral loads below the detection level. Multiple comparisons were performed by one-way ANOVA, two-way ANOVA for unpaired data and Friedman’s test (F) for paired data with post-hoc false discovery rate correction (FDR). HLA-E mRNA expression levels measured by RT-PCR were corrected for CD4+ T cell counts when possible. Correlation between two measures was analyzed using the non-parametric Spearman rank test, the corresponding correlation p-value was also calculated. Statistical significance criteria were set at p-value <0.05 and q-value <0.15.GraphPad Prism version 7 for Mac OS X  was used for statistical analyses. For comparison between groups, non-parametric Mann-Whitney (MW) test was performed for unpaired data and Wilcoxon test (W) for paired data. χ+ T cell counts. Significantly different HLA-E mRNA levels were observed between HIV-high and HIV-low groups, even when correcting for CD4+ T cell counts . However, when we compared the impact of the allele polymorphisms between HLA-E*01:01 and *01:03 on plasma viral load only in individuals with lower HIV viral load , a significantly higher proportion of individuals with undetectable plasma viral load (<50 HIV RNA copies/mL) were HLA-E heterozygous , HIV-low (HL) and HIV-high (HH) individuals.-CD16-CD57-) was significantly more abundant in HH compared to SN  and HL . By contrast, cluster 2 (CD56bright NKG2A++) was significantly reduced in HH compared to SN  and HL . Cluster 5 (CD56-CD16-CD57+NKG2A+NKG2C-) was increased in HL compared to HH  and cluster 8 (CD56-CD16+CD57+) was increased in HL compared to SN . Cluster 10, corresponding to CD16+ adaptive NK cells (CD56+CD16++CD57+++NKG2C+) were less abundant in SN and HL compared to HH , linking the expansion of these cells to chronic uncontrolled HIV infection , CD56dim (CD56dimCD16+) and adaptive NK (CD56dimCD16+CD57+) populations, together with their expression of the HLA-E receptors NKG2A and NKG2C in SN, HL and HH were determined. The median % of these subpopulations in HL, HH and SN is represented as a radar chart in bright NK cells compared to HL  and SN . In addition, in total NK cells, the NKG2A/NKG2C ratio was significantly increased in SN compared to HH , highlighting the reduced expression of NKG2A in chronic uncontrolled HIV infection. The subpopulation analysis indicated this difference to stem mainly from CD56bright NK cells  and adaptive NK cells . The shift towards NKG2C expression was less pronounced in HL compared to SN. Still, a significant reduction was observed in NKG2A/NKG2C ratios in CD56bright compared to SN individuals  . Plasma viral load correlated negatively with NKG2A expression in total NK cells  as well as with the preservation of CD56bright subpopulation . Of note, HLA-E mRNA correlated positively with NKG2C expression in total NK cell fraction  and CD56bright  NK cells while it correlated negatively with NKG2A expression in adaptive NK cells . As a consequence, HLA-E expression was correlated with an imbalance in the NKG2A/C ratio in adaptive NK cells , indicating a link between the increased expression of NKG2C, its ligand HLA-E and uncontrolled HIV infection. Furthermore, the results indicate that NK cell subsets and the expression of NKG2X receptors are severely impacted by chronic HIV infection since HIV infected individuals, and individuals with high viral loads  in particular, showed a loss of CD56bright and an increase of the terminally-differentiated adaptive NK cells (characterized here as CD56dimCD16+CD57+). The HLA-E/NKG2X axis also showed severe alterations depending on CD4 count and plasma viral load, suggesting that the impaired antiviral activity of these NK-subsets could be driven by high levels of HLA-E expression during uncontrolled HIV infection.We next tested for potential relationships between NK cell subsets size, the relative levels of the NKG2A and NKG2C expression in each of these subset as well as the NKG2A/NKG2C ratio and the CD4 count, plasma viral load and HLA-E mRNA levels (+ NK cells) and cytokine production (IFN-γ+ and TNF-α+ NK cells) after coculture with HLA-null K562 cells. When testing total NK cells derived from SN, HL and HH, we observed a significant reduction in NK cytotoxic capacity in HIV infected individuals compared with SN, true for both HL  and, especially, for HH , degranulation  and IFN-γ production , degranulation , secrete IFN-γ  or produce TNF-α . No significant differences in CD56bright functional responses were observed between HL and HH, although this subset was significantly reduced in HH. Similarly, the adaptive NK subset showed a significantly higher IFN-γ  and TNF-α production  in uninfected individuals compared to chronically HIV infected individuals. Interestingly, degranulation activity was not reduced in this adaptive NK subset which was also not reduced in size in HIV infected individuals. In line with the data obtained in the total NK population, HLA-E mRNA expression in the source PBMC was negatively corelated , IFN-γ and TNF-α production in CD56bright  and adaptive NK cells production of IFN-γ and TNF-α . Overall, these results indicate that higher levels of HLA-E expression in chronic HIV infection are not only associated with shifts in the NKG2A to NKG2C expression ratios, but also with reductions in cytotoxic, degranulation and cytokine production effector functions in the most affected NK subsets.To gain further insights whether HLA-E expression was associated with dysregulated NK function in specific NK subsets, we repeated the effector function analysis focusing on the two NK subsets showing the most significant alterations in chronic HIV infection, namely CD56in vitro infection model. Using this model, we assessed antiviral activity of NK cells in an in vitro viral replication inhibition assay (NK-VIA) by coculturing sorted NK cells from HIV-negative individuals with in vitro HIV infected autologous CD4+ T-cells. For the two HIV strains tested, a strong inhibitory capacity of NK cells was observed at day 7  and HIV-1BaL. . In parallel, the 14-day culture showed marked changes in NK subpopulations, with a loss of CD56dim and a gain of CD56bright and CD56neg subsets  and HIV-1BaL . The level of inhibition also correlated with the basal HLA-E mRNA expression in the PBMC used to isolate autologous NK and CD4+ T cells . This was in line with the direct correlation between HLA-E mRNA level in the sample and surface HLA-E expression  co-cultures with infected CD4 T cells, drives NKG2C expression which, in turn, may cause reduced NK functions due to its diminished affinity for HLA-E presented peptides compared to NKG2A , using HLA-E*01:03 transfected K562 cells, co-expressing the canonical peptide VL9-B7 and pulsed with canonical and non-canonical HLA-E binding peptides. We measured HLA-E surface stability as well as HLA-E restricted NK cytotoxicity, degranulation and cytokine production (IFN-γ and TNF-α). The effect of each peptide compared to the canonical, endogenously expressed, VL9-B7 epitope was determined and compared between peptides Figure 6The presence of the described canonical HLA-E binding epitopes increased the surface expression when compared with VL9-B7 endogenous expression . The same was seen for the non-canonical EBV-derived SL9 peptide . Interestingly, only one of the 11 HIV-derived candidate epitopes (H RL9) increased the surface expression  presented by HLA-E*01:03 stimulated NK cells and increased three of the four cellular functions measured . These data indicate that HLA-E*01:03 might be sensitive to changes in the HLA-E repertoire and undergo changes that can be recognized by NKG2X receptors on NK cells.In order to further understand the interplay between HLA-E alleles (*01:01 or *01:03), NKG2X receptors and HIV-derived peptides that triggered NK responses, a structural model was constructed. The structural model of HLA-E-CD94-NKG2X Figure 7Thus, our structural model identifies interactions between non-canonical HIV-1-derived HLA-E presented peptides and NKG2X receptors in an allele-dependent manner, providing the potential explanation for differential NK-cell recognition of infected cells through the HLA-E/NKG2X axis. In this model, Position 5 in the presented epitope may further determine the affinity to the activator (NKG2C) or inhibitory (NKG2A) receptor.NK cells play a crucial role in antiviral immunity, a role evidenced by the increased susceptibility to viral infections when NK cells are impaired or depleted , 40, 50.+ T-cells had an increased surface HLA-E expression compared to their uninfected counterparts  sigterparts  and iii)terparts . Such anImportantly, in our study we also controlled for HLA-E allelic differences in terms of HIV disease progression and NK cells activation. This aspect has been largely neglected in the past, probably due to the fact that the two worldwide distributed alleles, HLA-E*01:01 and *01:03, only differ in one amino acid. However, it is well known that such small differences can cause functional differences and, for instance in the case of HLA-B*58, have been associated with opposite risk for HIV disease progression . Indeed,A past study by Natterman et al. also demonstrated that HIV infection led to reduced NK cell activity . To undein vitro stimulation of NK cells through the HLA-E/NKG2X axis, using plate-bound agonistic antibodies, promoted the expansion of adaptive NK cells and induced the expression NKG2C and CD57. These cells also present an epigenetic reprogramming that causes an exhausted phenotype and profound NK dysfunction . We confirmed that HIV-high showed a profound loss of CD56bright NK cells and identified a significant reversion of the NKG2A/NKG2C balance in total NK cells from HIV infected individuals’ cells, which was mainly driven by the CD56bright and adaptive subsets. While some preceding studies reported NKG2C+NKG2A- NK cells as a marker of lower viral set point  through the ASILP/SIISP motif on NGK2X that interacts with position P5 of the bound epitope and which could directly determine the affinity to the activator (NKG2C) or inhibitory (NKG2A) receptor to the HLA-E/epitope complex; (2) through E/K 197 residues that map close to presented epitopes, such as PM9 and RL9, and (3) through Asp202 that interacts with Arg 108, located in close proximity to Arg107 and representing the only change between HLA-E*01:01 and *01:03. Some of these structural particularities may support the benefits of HLA-E heterozygosity in HIV control as heterozygous might have a broader and more differentiated ability to present more/different epitopes and thus trigger differential NK responses. The expanded structural insights in the present study may also explain the reported 6-fold lower affinity for the HLA-E-peptide complex to NKG2C and contribute to the observed switch from NKG2A to NKG2C expression, with its downstream functional consequences on NK-mediated antiviral activity.bright NK cells towards a functionally less active profile. These CD56bright NK cells are traditionally considered as regulatory and their role in HIV control could go beyond the direct killing of infected cells  repository, accession number ERA18504000.The studies involving human participants were reviewed and approved by Comitè Ètic d’Investigació Clínica of Hospital Germans Trias i Pujol (CEIC EO-12-042). The patients/participants provided their written informed consent to participate in this study.Conceptualization: L-RM, MR-R, CB, AO. Methodology: LR-M, CD-C, MO, MR-U. Investigation: LR-M, CD-C, MR-U. Visualization: LR-M, CB, AO. Supervision: CB, AO. Resources: BM, CB, DH-O'C. Writing – original draft: LR-M. Writing – review & editing: CD-C, MO, MR-U, MR-R, JS, BM, DH-O'C, AO, CB. All authors contributed to the article and approved the submitted version.The present study was supported by the USA National Institutes of Health (NIH) - National Institute of Allergy and Infectious Diseases - P01 Research Program Project Grant 1P01AI131568-01 (CB) and funding from the European Union’s Horizon 2020 research and innovation program under grant European AIDS Vaccine Initiative 2020 (EAVI2020) #GA681137 (CB). The work was also partly supported by grant PI17/01465 (AO) from the Instituto de Salud Carlos III, co-financed by the Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa”. CB is a senior ICREA research professor.We would like to thank the team of Dr. J. Miguel López-Botet  for their advice starting this project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Recent work suggests that Ras small GTPases interact with the anionic lipid phosphatidylserine (PS) in an isoform-specific manner, with direct implications for their biological functions. Studies on PS-Ras associations in cells, however, have relied on immuno-EM imaging of membrane sheets. To study their spatial relationships in intact cells, we have combined the use of Lact-C2-GFP, a biosensor for PS, with multicolor super resolution imaging based on DNA-PAINT. At ~20 nm spatial resolution, the resulting super resolution images clearly show the nonuniform molecular distribution of PS on the cell membrane and its co-enrichment with caveolae, as well as with unidentified membrane structures. Two-color imaging followed by spatial analysis shows that KRas-G12D and HRas-G12V both co-enrich with PS in model U2OS cells, confirming previous observations, yet exhibit clear differences in their association patterns. Whereas HRas-G12V is almost always co-enriched with PS, KRas-G12D is strongly co-enriched with PS in about half of the cells, with the other half exhibiting a more moderate association. In addition, perturbations to the actin cytoskeleton differentially impact PS association with the two Ras isoforms. These results suggest that PS-Ras association is context-dependent and demonstrate the utility of multiplexed super resolution imaging in defining the complex interplay between Ras and the membrane. The inner membrane Ras small GTPases function as molecular switches in canonical signaling pathways by cycling between an active GTP-bound and an inactive GDP-bound state, and in so doing regulate cell growth, proliferation, and metabolism ,2. As keIt has been well established that in addition to GTP loading, Ras molecules have to be positioned on the membrane in order to be biologically active ,5. MembrIn healthy mammalian cells, PS localizes to the inner leaflet of the plasma membrane, making up 20% of all membrane lipids ,27. Its Previous efforts to map the intracellular distribution of PS culminated in the discovery of Lact-C2-GFP as a specific PS sensor . Using tThe recent advent of super resolution microscopy (SRM) has allowed spatial mapping of biological molecules within intact cells with 10–20 nm lateral resolution ,33,34,35Here, we report dual-color SRM imaging of both PS and Ras (HRas-G12V and KRas-G12D) at nanoscale resolution in intact cells and the analysis of their spatial associations. Using the sensor Lact-C2-GFP in combination with DNA-PAINT, we obtained images showing the molecular distribution of PS. SRM images of PS showed that it is universally expressed across the entirety of the cell membrane and is nonuniformly distributed at the ~100 nm scale or below. Two-color DNA-PAINT-ERS imaging of both PS and Ras followed by spatial analysis showed that PS co-organizes with both KRas and HRas. HRas seems to have a stronger co-organization with PS than KRas. Perturbation of the actin cytoskeleton suggests actin involvement in the regulation of PS–Ras interactions. Our results demonstrate the utility of multiplexed super resolution imaging in defining the complex interplay between Ras and PS, and complement past studies by showing that PS–Ras association is context-dependent (including isoform).All cell culture reagents and supplies were purchased from Thermo Fisher Scientific  unless otherwise specified. Primers and DNA oligonucleotides were synthesized by Integrated DNA Technologies .Lact-C2-GFP (#22852) was a gift from Dr. Sergio Grinstein   and pGEXEscherichia coli BL21 (#C600003) and protein expression was induced by addition of 1 mM IPTG (#15529019) to exponential-phase bacteria (OD600 = 0.5). After 5 h at 30 °C with constant shaking, the bacteria were harvested by centrifugation . During the following procedures, cells and protein solutions were kept at 4 °C at all times to avoid protein degradation. Bacteria were lysed by sonication (4 × 30 s bursts at 300 W with 30 s cooling periods) in lysis buffer  on ice. After centrifugation , a clear lysate was obtained by collecting the supernatant. The protein lysate was incubated with Ni-NTA Agarose  with mechanical rotation for 2 h. The lysate–Ni-NTA mixture was transferred into a column and washed with wash buffer . Protein was eluted by centrifugation with elution buffer . To remove excess imidazole, desalting was performed using size exclusion chromatography on an ÄKTA Pure system  with a 5 mL Cytiva HiTrap HP desalting prepacked column .The His-tagged pET21b-GFP-nanobody plasmid was transformed into Anti-GFP-nanobodies were conjugated to docking strand (DS) oligos via dibenzocyclooctyne (DBCO)-azide click chemistry. DS sequences contained a 5′ amino modifier C6 and a 3′ Cy3 fluorophore. To first conjugate DS with DBCO-PEG4-NHS ester, the latter was added to DNA in 20 × molar excess. A 50 μL reaction volume of DBCO-ester, DS, and ultra-pure water with pH adjusted to 8.5 using sodium bicarbonate was incubated on a shaker for 3 h at room temperature. Ethanol precipitation was carried out twice with 0.3 M sodium acetate at −80 °C to purify the DS-DBCO product. The DS-DBCO product was then reacted in 10 × molar excess with the azide groups of the anti-GFP–nanobody via copper-free click chemistry overnight on a shaker at room temperature. The final anti-GFP-nanobody-DS product was purified using a HiTrap Q HP anion exchange chromatography column  with an ÄKTA Pure system.® Ultra Columns  to remove excess unconjugated nanobodies. The final anti-GFP-nanobody-DS was resuspended in PBS and stored at −20 °C until sample labeling was carried out. Intermediate products were run on Bris-Tris gradient gels  and stained with InstantBlue® Coomassie Protein Stain .The resulting product was applied to 10 kDa Amicon2, passaged every 3–4 days, and used below passage 20. Prior to experiments, cells were plated in eight-well Lab-Tek II chambers (#155360). To induce expression of HRas-G12V and KRas-G12D, cells were exposed to 50 ng/mL doxycycline  for 48 h. Cells were transfected with 200 ng Lact-C2-GFP plasmid and Lipofectamine 3000 transfection reagent (#L3000015) according to the manufacturer’s instructions in OptiMEM reduced serum medium (#31985062) for 5–6 h. Afterwards, the transfection reagent was replaced with fresh DMEM supplemented with 10% FBS and 50 ng/mL doxycycline. At 24 h after transfection, Lact-C2-GFP was expressed at desired levels and cells were treated with either 2 μM Latrunculin A  for 10 min or 15 μM Jasplakinolide  for 30 min at 37 °C. Subsequently, cells were fixed with 3.7% paraformaldehyde in 1 × PHEM buffer .Halo-tagged HRas-G12V and SNAP-tagged KRas-G12D coding sequences were placed under a CMV promoter regulated by the TetOn operon. Both constructs were transduced into an isogenic U2OS-tetR cell line that constitutively expressed the tet repressor (tetR) derived from the parent U2OS  using lentivirus. Single-cell clones expressing HRas-G12V and KRas-G12D fusion proteins under doxycycline regulation were isolated and used for experiments. Cells were maintained in Gibco DMEM (#11995073) supplemented with 10% FBS (#26140079) at 37 °C and 5% COAfter fixation, cells were quenched with 300 mM glycine (Sigma #W328712) and permeabilized with 0.2% saponin  in PBS for 40 min at room temperature. Cells were incubated with Image-iT™ FX Signal Enhancer (#I36933) for 40 min in the dark before blocking with 2% bovine serum albumin  supplemented with 5% salmon sperm DNA (#15632011) in PBS for 40 min. Subsequently, cells were incubated with anti-GFP-nanobody-DS to stain PS and either SNAP- or Halo-tag-substrate-DS to stain KRas and HRas, respectively, in 2% BSA supplemented with 5% salmon sperm DNA in PBS for 2 h on a rocker in the dark. Cells were post-fixed with 3.7% PFA supplemented with 0.2% glutaraldehyde.−2.All SRM data were obtained with a custom-built single molecule imaging system. In brief, lasers emitting at wavelengths of 488 nm, 561 nm, and 647 nm were combined and introduced into the back of a Nikon Ti-U microscope. An f = 400 mm lens was used to focus the collimated laser light to the back aperture of a 60X TIRF objective to achieve total internal reflection illumination of the sample. Images were acquired with strict TIRF illumination to probe membrane proteins appropriately. During data acquisition a custom-built focus stabilization system based on the detection of the reflected excitation laser was used. A multi-edge polychroic mirror and emission filters FF01-525/45, FF01-605/64, and FF01-708/75  were used to reflect laser light into the objective and clean up fluorescent signals. Images were collected using an iXon Ultra electron-multiplied charge coupled device  with an EM gain of 200 and 30–40 ms exposure time. The power density of the 647 nm laser for DNA-PAINT(ERS) imaging was typically ~500 W cmhttps://micro-manager.org/ accessed on 10 February 2020) and saved as TIF files. For HRas and KRas, targets of 60,000–80,000 frames were acquired. For PS, we generally acquired 120,000–160,000 raw image frames, corresponding to an average of 5 × 106 localizations. In-house Matlab scripts were used for single-molecule localization, typically at a spatial resolution of ~20 nm, as well as for localization filtering, sorting, and rendering for visualization , a, a41], aFor DNA-PAINT imaging, the GFP-Nb is conjugated to a single-stranded DNA oligo designated as the docking strand  at the artificial amino acid 4-azido-phenylalanine (AzPhe) via a copper-free click reaction . The AzPUsing this strategy, we studied the distribution of PS in the plasma membrane of U2OS cells. The imaging was confined to the membrane and the immediate cytoplasmic space by illuminating the sample under strict total internal reflection (TIR). Compared with the apparent diffusive distribution of PS across the whole bottom membrane, zoom-in views of the super-resolved images show a more heterogenous molecular distribution of PS. Between the sub-µm to nanoscales . The SNAP tag is similar in size to GFP, thus offering a similar advantage to using the GFP/GFP-Nb strategy, as in the case of PS labeling A. U2OS cWe note that this lack of pixel-to-pixel overlap is typical in SRM and EM images of supposedly ‘colocalizing’ targets ,44. In cColoc-Tesseler analyzes ‘colocalizations’ in SMLM images as spatial associations between molecular targets C. This iColoc-Tesseler computes the normalized density of the immediate (first rank) neighbor polygons based on the PS (green) and the KRas-G12D (purple) Voronoï diagram and compares this to the average density of a reference distribution of spatial randomness for each channel independently F. AutomaAs such, PS and KRas-G12D localizations are organized into four different orthogonal classes F,G that Previous immuno-EM studies have suggested that PS is a component of both HRas and KRas nanoclusters. Ras isoforms interact with the membrane in an isoform-specific manner, which applies to Ras-PS interactions as well, and it remains to be seen how the two Ras isoforms interact and/or spatially associate with PS in cells. We therefore studied the co-organization of HRas-G12V and PS using a similar imaging strategy. We genetically fused HRas-G12V to the Halo-tag and stably expressed the fusion protein in U2OS cells followed by transient transfection with Lact-C2-GFP, as previously described. The Halo-tag was reacted with a chloroalkane linker substrate conjugated to DS3 to perform dual-color exchange-PAINT imaging, with Halo-HRas-G12V imaged using the corresponding imager strand IS3.2 area on average) ROIs in a number of Lact-C2-GFP positive cells. Somewhat surprisingly, the Manders A coefficients (HRas-G12V overlapping with PS) were almost always close to 1, suggesting extensive co-enrichment of HRas-G12V with PS  and latrunculin A (LA), to modulate actin dynamics and assess their impacts on Ras–PS associations. Short dynamic actin filaments are key to PS immobilization and clustering on the membrane ,47. Jas U2OS cells expressing Lact-C2-GFP and either SNAP-tagged KRas-G12D or Halo-tagged HRas-G12V were treated with either 15 μM Jas for 30 min or 2µM LA for 10 min to promote or inhibit actin polymerization, respectively. Depolymerization of the actin cytoskeleton upon treatment with LA was confirmed using stochastic optical reconstruction microscopy (STORM) imaging after labeling with Alexa Fluor™ 647 Phalloidin ,51 Figu. STORM iActin stabilization with Jas had a much stronger effect on the spatial association be-tween KRas-G12D with PS than HRas-G12V. Jas treatment shifted the Manders A coefficient for KRas-G12D and PS from 73% towards 96% (nearly complete association) for all cells analyzed B. The MaIn comparison, LA treatment had a much smaller effect on Ras–PS associations compared with that observed for Jas. LA treatment led to a less pronounced increase of Manders A from 73% to 82% in KRas-G12D and PS co-organization, whereas the Manders B coefficient for KRas-G12D was unaffected by LA treatment. Disassembling actin with LA treatment had no effect on the colocalization of HRas-G12V with PS and an intermediate effect on the association of PS with HRas-G12V (49%). Manders B is higher for cells expressing HRas-G12V compared to KRas-G12D, which is an indication that PS prefers co-organization with HRas-G12V compared to KRas-G12D. While the membrane plays a critical role in regulating Ras activity, how Ras interacts with various membrane compartments remains unclear. In this work, we used dual-color DNA-PAINT(ERS) super resolution imaging to map PS and Ras (HRas-G12V or KRas-G12D) at the nanoscale on the native plasma membrane . By combUsing exchange-PAINT, we confirmed that KRas-G12D co-enriches with PS on the cell membrane . InteresPS was identified as a component of the nanoclusters of both KRas and HRas in previous work , althougThe actin cytoskeleton facilitates diverse cellular processes and is highly involved in the spatiotemporal organization of the membrane by providing a barrier that restricts diffusion of proteins and lipids . PS direAside from PS, other anionic lipids such as PIP2 have been found to interact with Ras, although current studies have not reached a consensus as to whether and to what extent PIP2 (and other PI lipids) is important in the spatial organization and function of Ras ,14,15,58