Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca+2 binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A2 enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity.In the present study a major protein has been purified from the venom of Indian  Haemostasis, one of the most important physiological processes of vertebrates, involves four crucial steps for sustaining equilibrium, namely, (i) vasoconstriction, to reduce blood flow from the site of injury (ii) platelet activation, aggregation and adherence leading to the platelet plug formation at the injured site (iii) initiation of coagulation cascade involving the extrinsic, intrinsic and the common pathway, forming fibrin mesh on the platelet plug and (iv) fibrinolysis leading to the dissolution of the clot formed, in order to restore normal blood flow . Any malDaboia russelii russelii (Daboia r. russelii), a venomous snake of the Indian subcontinent, contains an intricate blend of various biologically active protein families. Consumptive coagulopathy is one of the most manifested systemic pathological condition observed in viper envenomated victims  was purchased from Bioworld . The Edman sequencing reagents were purchased from Applied Biosystems chemicals . The rest of the reagents and chemicals were of analytical grade and obtained from Merck Millipore  or Sigma .Crude venom of 2-D-CHG-Gly-Arg-pNA.AcOH) was purchased from Sekisui Diagnostics , rest of the substrates like S-2366 (pyroGlu-Pro-Arg-pNA•HCl), S-2302 (H-D-Pro-Phe-Arg-pNA•2HCl), S-2222 (Bz-IIe-Glu(γ-OR)-Gly-Arg-pNA•HCl), S-2765 (Z-D-Arg-Gly-Arg-pNA•2HCl)S-2238 (H-D-Phe-Pip-Arg-pNA•2HCl) and S-2288 (H-D-Ile-Pro-Arg-pNA•2HCl) were procured from Chromogenix .Serine proteases factor XIa (FXIa), factor IXa (FIXa), factor VIIa (FVIIa), factor X (FX), factor Xa (FXa) and Russell’s viper venom-X activator (RVV-X) were obtained from Haematologic Technologies Inc. , factor XIIa (FXIIa) was procured from Merck Calbiochem , factor VIII (FVIII) from Creative Biomart , phospholipid blend from Avanti Polar Lipids Inc.  and tissue factor Innovin from Siemens . The chromogenic substrates namely spectrozyme (MeSOHuman embryonic kidney (HEK)-293 and Michigan Cancer Foundation (MCF) -7 cell lines were purchased from National Centre for Cell Science . Dulbecco modified Eagle’s media (DMEM) was obtained from Himedia , 3--2,5-diphenyltetrazolium bromide (MTT) and trypan blue from Sigma Aldrich , fetal bovine serum (FBS), streptomycin-penicillin (Strep-Pen), trypsin/EDTA solution and dimethyl sulfoxide (DMSO) were procured from Thermo Fischer .TM 16/600 Superdex 75 prep grade and Hiprep CM FF 16/10 columns were purchased from GE Healthcare life Sciences . Jupiter C18 column was procured from Phenomenex  and Hypersil Gold C18 column was obtained from Thermo Scientific .HiloadDaboia r. russelii venom was fractionated on HiloadTM 16/600 Superdex 75 prep grade column (1x120 ml) pre-equilibrated with 50 mM of Tris-Cl pH 7.4 using Äkta Purifier HPLC system, GE Healthcare . Fractionation was carried out at a flow rate of 1 ml/min under isocratic conditions with the same buffer. Each eluted fraction was assayed for PLA2 and anticoagulant activity. P6 with highest anticoagulant and PLA2 activity was further subjected to cation exchange chromatography on Hiprep CM FF 16/10 (1.6x10 cm) using Äkta Purifier HPLC system. Elution was carried out at a flow rate of 2.25 ml/min with a linear gradient of 50 mM Tris-Cl, pH 7.4 containing 0.8 M NaCl. Ion exchange fraction, CM-II with highest PLA2 and anticoagulant activity was subjected to Rp-HPLC using Jupiter C18 column . The column was pre-equilibrated with milli q containing 0.1% trifluoro acetic acid (TFA) and fractionated using 80% acetonitrile (MeCN) containing 0.1%TFA on Äkta Purifier HPLC system. The homogeneity of the purified protein (5 μg of the protein treated with 0.45 μl of β-mercaptoethanol) was validated on SDS-PAGE and stained using PierceTM silver staining kit, Life technologies, Thermo Fischer Scientific  ..Daboia r2 enzymes which are basic, like vurtoxin which is a strong anticoagulant enzyme reported from Vipera renardi and FXa binding enzymes like DPLA2 from Daboia russelii pulchella, AtxA and AtxC from V. a. ammodytes with a bootstrap value of 99 [Daboxin P showed close phylogenetic relation with the reported anticoagulant PLAue of 99 –61  and neutral pH (7.4) showed stable structural conformation. However, the structure was distorted completely at alkaline pH (12.0) with residual α-helix of 5.51% & β-sheet of 23.75% . On the Daboxin P exhibited catalytic activity on diheptanoylthio-phosphatidylcholine (PC) in a dose dependent manner . The MicDaboia r. russelii has shown prominent cytotoxic effect on HEK-293 and MCF-7 cells in a dose dependent manner  in the time-to-occlusion in comparison to the saline treated mice (8.29 ± 2.61 min) (in-vitro anticoagulant assays on human plasma and highlighted the anticoagulant property of daboxin P under in-vivo condition.Daboxin P exhibited anticoagulant effect in the carotid artery of mouse treated with FeCl.61 min) . This reScreening for inhibitory effect of daboxin P on the amidolytic activity of various serine proteases involved in the extrinsic (FVIIa), intrinsic  and common (FXa) pathway revealed that it did not inhibit any of these serine proteases when assayed using respective synthetic chromogenic substrates . However+2 ions did not show any effect on the quenching spectra for both FX and FXa when treated with daboxin P (data not shown).The fluorescence emission spectrum of FX and FXa was quenched by daboxin P with increasing incubation time . The preThe electrophoretic profile of the flow through for both FX and FXa did not show any protein bands suggesting the binding of these proteins to the daboxin P immobilized CNBr matrix. Nevertheless, the eluent at 1.0 M NaCl showed prominent protein bands for both the serine proteases suggesting the disturbance of the protein-protein interaction at high salt concentration .The predicted 3D model of daboxin P has shown structural similarity to the crystal structure of AtxA (PDB: 3G8G) with a RMSD value of 0.9 Å which validates the predicted model  of 472.62 kcal/mol.The best protein-protein docked model of daboxin P and FXa chosen in terms of minimum free energy and maximum surface contact area, was obtained from PatchDock web server . Daboxin2. The docked complex was stabilized by molecular interactions like hydrogen bonding and non- bonded contacts. The interface amino acid residues of FXa (chain B) and daboxin P (chain C) involved in the interaction are shown in The interface and possible interacting residues across the interface of the complex were predicted by PDBsum. The total number of interface residues in protein-protein complex was found to be 31 and the interface area for each chain involved in the interaction was observed to be more than ~500 Å+2 binding loop; Asp48, Tyr51, Gly52 of helix C; Asn58 of the anticoagulant region and Phe113 of C-terminal region interact with residues Thr132, Arg165, Lys169, Asn166, Leu170, Tyr225 and Arg125 of heavy chain of FXa (The docked complex shows interaction of daboxin P with the heavy chain (Chain B) of FXa at eight critical regions some of which are reported to be crucial for FVa binding . The dabn of FXa .2 with sixteen residues of the heavy chain of FXa while only four residues of daboxin P are found to share a contact area greater than the threshold limit (8 Å2) with three residues of the light chain of FXa. This suggests the interaction of daboxin P with the light and heavy chain of FXa ; helix C , anticoagulant region (Asn58) and C-terminal region (Phe113) of daboxin P with the heavy chain of FXa  but not with its light chain. The residues of helix A , helix B  and the anticoagulant region (Arg68) of AtxA are reported to be involved in the interaction with the light chain of FXa [+2 binding loop , helix C , anticoagulant region , and the C-terminal region  of daboxin P  of the peptide (protonated molecular ions), Z represents the number of charges a peptide carries after ionization, Score implies the sum of all peptide cross correlation (Xcorr) values. Figure A. The Phylogenetic relationship of daboxin P with the reported anticoagulant PLA2enzymes from snake venom. The phylogenetic tree was constructed by neighbour joining (NJ) tree using Mega 5 software with a bootstrap value of 1000. Anticoagulant PLA2 enzymes with complete sequence were obtained from Pubmed Protein Database (http://www.ncbi.nlm.nih.gov/pubmed) and aligned using ClustalW. The sequences used in the study were CBc , CB crotoxin , CBb , PLA2 F17 , CBa2 , Cdc-9 , Cdc-10 , bID-PLA2 (Bothrops leucurus), Bothropstoxin (Bothrops jararacussu), DPLA2 (Daboia russellii russellii), vurtoxin (Vipera renardi), AtxA (Vipera ammodytes ammodytes), AtxC (Vipera ammodytes ammodytes), bAhp , BP III (Protobothrops flavoviridis), II-BP (Protobothrops flavoviridis), Ts-K49b (Trimeresurus stejnegeri), MTxII (Bothrops asper), PLA2 homology (Bothrops atrox), BthA-I-PLA2 (Bothrops jararacussu), Vur PL2B (Vipera renardi), EC-I-PLA2 (Echis carinatus), PLa2Vb (Vipera berus berus), CbII (Pseudocerastes fieldi), HDP-1P (Vipera nikolskii), HD-2P (Vipera nikolskii), Caudoxin , APLA2-2 (Ophiophagus hannah); CM-III/CM-IV (Naja nigricollis); CM-III (Naja mossambica); CM-II (Naja mossambica) and CM-I (Naja mossambica).Figure B. Effect of daboxin P on thrombin time and fibrinogen. (i): Different amount of daboxin P  were pre-incubated with 50 μl platelet poor plasma for 3 min at 37°C. 50 μl of thrombin (10 u/ml) was added to initiate clot formation and monitored on Tulip Coastat-1 coagulo analyser. Clotting time in presence of Tris buffer  was taken as normal clotting time (NCT). The results are mean ± SD of three independent experiments. (ii): Assessment of fibrinogenolytic activity on SDS-PAGE, different amount of daboxin P  were pre-incubated with 300 μl of 2 mg/ml fibrinogen for 24 h at 37°C. Fibrinogen with thrombin (3 μl of 10 units/ml) was considered as the positive control and fibrinogen with buffer  was taken as the negative control. Fibrinogen degradation products were analyzed on 12.5% glycine SDS-PAGE, stained with CBB R-250. . Figure C. Three dimensional (3D) molecular modelling (i): The 3D ribbon structure of daboxin P  was superimposed with X-ray crystallographic structure of AtxA (PDB 3G8G) (in yellow), (ii): 3D ribbon model of daboxin P as predicted by I-TASSER server. The Ca+2 binding loop (YGCYCGCGG in torquise blue), anticoagulant region (53NLPDCNNKSKRYRYKK68) (in yellow) and the active site His residue (in stick) are highlighted. Table B. Summary of interface plot statistics of the docked model of daboxin P and FXa. Table C. List of displayed residue to residue contact area of the light (A) and heavy chain (B) of FXa with daboxin P (C). Figure D. Sequence alignment of daboxin P with ammodytoxin A. The amino acid residues varying in daboxin P with respect to ammodytoxin A are underlined. The predicted anticoagulant region in both the PLA2 enzymes is highlighted in red.(DOCX)Click here for additional data file.


ADIPOQ) polymorphisms and type 2 diabetes mellitus (T2DM) in a Chinese population. Methods: We designed a case-control study involving 340 normal glucose tolerant (NGT) subjects and 340 type 2 diabetes patients. Three SNPs  were genotyped by TaqMan methods. Results: We found that rs7627128, rs1501299 and rs182052 were significantly associated with T2DM. Haplotypes analysis indicated that the frequency of the haplotypes A-A-T was frequent in T2DM patients , but G-A-T was more frequent in the control group than in the T2DM group . Conclusion: The ADIPOQ genetic polymorphisms were associated with type 2 diabetes in a Chinese population.Objective: The manuscript investigates the relation between adiponectin gene ( C5L2 gene  in our study. Finally, the relatively small sample size may overestimate the OR value during the analysis of the risk for T2DM.ADIPOQ gene polymorphisms. The A-A-T haplotype appear to be a useful genetic marker, and the G-A-T haplotype might be a protective factor from T2DM in Chinese people.In conclusion, the present results indicate that T2DM is associated with the


Influenza A viruses (IAVs) inhibit host gene expression by a process known as host shutoff. Host shutoff limits host innate immune responses and may also redirect the translation apparatus to the production of viral proteins. Multiple IAV proteins regulate host shutoff, including PA-X, a ribonuclease that remains incompletely characterized. We report that PA-X selectively targets host RNA polymerase II (Pol II) transcribed mRNAs, while sparing products of Pol I and Pol III. Interestingly, we show that PA-X can also target Pol II-transcribed RNAs in the nucleus, including non-coding RNAs that are not destined to be translated, and reporter transcripts with RNA hairpin structures that block ribosome loading. Transcript degradation likely occurs in the nucleus, as PA-X is enriched in the nucleus and its nuclear localization correlates with reduction in target RNA levels. Complete degradation of host mRNAs following PA-X-mediated endonucleolytic cleavage is dependent on the host 5’->3’-exonuclease Xrn1. IAV mRNAs are structurally similar to host mRNAs, but are synthesized and modified at the 3’ end by the action of the viral RNA-dependent RNA polymerase complex. Infection of cells with wild-type IAV or a recombinant PA-X-deficient virus revealed that IAV mRNAs resist PA-X-mediated degradation during infection. At the same time, loss of PA-X resulted in changes in the synthesis of select viral mRNAs and a decrease in viral protein accumulation. Collectively, these results significantly advance our understanding of IAV host shutoff, and suggest that the PA-X causes selective degradation of host mRNAs by discriminating some aspect of Pol II-dependent RNA biogenesis in the nucleus. All viruses depend on host components to convert viral mRNAs into proteins. Several viruses, including influenza A virus, encode factors that trigger RNA destruction. The influenza A virus factor that serves in this capacity is known as PA-X. PA-X limits accumulation of host mRNAs and proteins in infected cells and suppresses host responses to infection, but to date its precise mechanism of action remains obscure. Here we report that PA-X selectively targets cellular mRNAs, while sparing viral mRNAs, thereby compromising host gene expression and ensuring priority access of viral mRNAs to the protein synthesis machinery. We demonstrate that complete degradation of mRNAs cut by PA-X is dependent on the host factor Xrn1 and that PA-X likely works in the cell’s nuclei. Interestingly, PA-X targeting appears to be selective for products of host RNA polymerase II, and canonical mRNA processing is required for cleavage. Even though viral mRNAs are spared from PA-X-mediated degradation, PA-X-deficient viruses displayed defects in the synthesis of certain viral mRNAs and decreased viral protein accumulation. Thus, PA-X-mediated host shutoff influences the efficiency of viral gene expression. These studies significantly advance our understanding of this important viral host shutoff protein and may provide future opportunities to limit the pathogenesis of influenza A virus infection. Influenza A virus (IAV) has long been known to carry out host shutoff, and multiple shutoff mechanisms have been reported for this virus, including translation blockade , inhibitnscripts –15. PA-Xnscripts –19, but IAV is a negative strand RNA virus with a genome consisting of eight segments. PA-X protein is encoded on IAV genome segment 3, which also produces polymerase acidic protein (PA), one of the three subunits of the viral RNA-dependent RNA polymerase (RdRp). It is generated by ribosomal pausing on a rare CGU codon that results in a +1 frameshift and read-through of an alternative open reading frame (ORF) ,20. PA-X7G) cap and a 3’ poly-adenylate (poly(A)) tail. 5’ m7G caps are acquired by “cap-snatching”, whereby the PA subunit cleaves nascent host Pol II-transcribed RNAs at a position 10–14 nucleotides downstream from the 5’ cap [7G caps and poly(A) tails allows efficient loading of ribosomes and translation of IAV mRNAs; however, the similarity between host and viral mRNAs raises the question of their susceptibility to PA-X-mediated degradation.RdRp-transcribed IAV mRNAs share essential features with host mRNAs like a 5’ 7-methyl guanosine , whereas Pol I transcribes the rRNA precursor 47S and Pol III transcribes a variety of short ncRNAs including the 5S rRNA. To determine whether PA-X could trigger the degradation of distinct host RNA species, we transfected HEK 293T cells with plasmid reporters encoding an RFP gene driven by a Pol II promoter and a GFP gene under the control of a Pol I-, Pol II-, or Pol III-driven promoter. We observed that ectopic expression of PA-X derived from the A/PuertoRico/8/34 H1N1 (PR8) virus together with these reporters consistently inhibited the accumulation of Pol II-driven RFP and GFP transcripts, whereas Pol I and Pol III-driven GFP transcripts accumulated to high levels . These dTo determine whether PA-X could similarly selectively target endogenous Pol II-driven transcripts, we constructed HEK 293T- and A549-based cell lines that stably express PA-X in a doxycycline-inducible manner (iPA-X cells). In comparison to an inducible RFP control, PA-X expression in the HEK 293T cell lines dramatically reduced endogenous mRNA levels for actin, EEF1A, GAPDH, GUSB, HIST1H3C, tubulin and RPS6, but had a much smaller effect on RPS18 and POLR2A mRNAs . By contin vitro data [We previously found that herpesvirus and coronavirus host shutoff endonucleases employ analogous mechanisms to trigger widespread RNA degradation, whereby endonuclease cleavage of target RNA is followed by processive 5’->3’ degradation by the host exonuclease Xrn1 . To detetro data .Many Pol II-transcribed RNAs are translated. Association with components of the translation apparatus has been shown or proposed to be a determinant of selective mRNA targeting by other host-shutoff RNases, such as HSV-1 vhs ,31 and KIn addition to mRNAs, Pol II also transcribes several ncRNAs, including the long ncRNAs MALAT1 and TP53TG1 and the precursor of the small nuclear RNA U2. Based on our reporter data , we hypoTo determine whether PA-X selectively targets Pol II-transcribed RNA in the context of IAV infection, A549 cells were infected with PR8 or a mutant PR8-PA(fs) virus that should not produce PA-X due to codon optimization that prevents ribosome pausing and frameshifting . As we pWhen determining the titers of recombinant PR8-PA(fs) virus stocks generated in our laboratory, we consistently observed reduction in plaque size compared to the parental wild-type PR8 strain . This super se are not sufficient or necessary for PA-X targeting of mRNAs. Instead they hint at a more universal feature of the 3’ end processing of the Pol II transcribed RNAs that earmark target RNAs for PA-X mediated cleavage. Since mRNA processing and maturation are linked to nuclear export, we compared the PA-X mediated changes in reporter transcript levels between cytoplasmic and nuclear RNA fractions. Importantly, PA-X was able to target both the cytoplasmic and the nuclear pools of the susceptible reporter RNAs (poly(A)-tailed GFP and GFP-hisL), while we failed to detect down-regulation of the HR bearing constructs in either fraction  tail at the 3’ end of the RNA, which is absent from Pol I and Pol III transcripts, and the canonical polyadenylation signal serves to direct termination, cleavage of the RNA and polyadenylation. An alternative stem loop termination signal is used for histone mRNAs , whereasfraction . This deet al. [Although all other host shutoff RNases are proposed to work in the cytoplasm, our results link PA-X targeting to 3’ end processing  and showet al.  highlighet al. . FinallyOur study provides new mechanistic insights into the specificity of PA-X, the most recently identified viral host shutoff nuclease. We demonstrate here for the first time that PA-X is recruited to the nucleus, selectively targets a subset of host transcripts, and is not active against viral mRNAs. Moreover, we uncover a novel route of target discrimination by a viral RNase, which takes advantage of the divergent mRNA biogenesis mechanisms that generate viral and host transcripts. We demonstrate that PA-X shares some specificity features with other host shutoff nucleases, as it selectively targets products of cellular RNA Pol II, while sparing Pol I and Pol III transcripts, and requires host RNases to complete RNA degradation. Interestingly, the mechanism for PA-X targeting of Pol II transcripts is not related to the translatability of the mRNAs. Instead, PA-X targeting is directly linked to synthesis by RNA Pol II complex or early processing events unique to Pol II transcripts. Moreover, we find that PA-X likely degrades RNAs in the nucleus, because it accumulates in this compartment and affects both the nuclear and cytoplasmic fraction of its target RNAs. Both the host shutoff activity of PA-X and the nuclear localization function of the C-terminal X-ORF are dependent on the presence of a set of basic residues in the X-ORF, indicating a correlation between nuclear localization and RNA targeting. Thus, the mechanism of PA-X targeting may be different from other shutoff RNases because it is tightly linked to the mechanism of biogenesis of host and viral mRNAs in the nucleus.Like other host shutoff endonucleases SOX, vhs and nsp1 ,29,37, Pper se is not required. Our data point instead to the processing of Pol II transcripts in the nucleus as the link between PA-X and its target. Importantly, our examination of endogenous and reporter Pol II transcripts that differ in their 3’ end processing suggests that this step in biogenesis may be recognised by PA-X to find its targets. In our experiments, GFP reporters bearing the canonical polyadenylation signal (PAS) or a histone stem loop at the 3’ end were efficiently inhibited by PA-X. By contrast, the same reporter with a templated poly(A) stretch followed by a self-cleaving hammerhead ribozyme sequence was not degraded by PA-X despite being translated -myc-SV40-3’UTR. Clonal populations were selected and several lines were used for all experiments. PA-X expression was induced by addition of doxycycline  for approximately 18 h prior to harvesting, with the exception of half-life experiments, in which doxycycline was added for 4.5 h before actinomycin D addition. HEK 293T shNS  and shXrn1 cells [Mouse embryonic fibroblasts , HeLa Tet-Off (Clontech), HEK 293A (ATCC), HEK 293T (ATCC), A549 (ATCC), and their derivative 293T and A549 “iPA-X” cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  at 37°C in 5% COn1 cells  were genThe A/PuertoRico/8/34/(H1N1) (PR8) and the recombinant mutant PR8-PA(fs) viruses are described in . Virus sCells grown on glass coverslips were fixed and immunostained according to the protocol in  using moFor steady-state measurements of RNA levels in cells ectopically expressing PA-X protein, RNA was harvested 1 day after transfection or 18 h after PA-X induction in iPA-X cells. For half-life measurements, PA-X (wt or D108A) was induced in A549 iPA-X cells for 4.5 h, followed by addition of actinomycin D at 10 μg/ml final concentration. RNA was collected 0, 2, 4, 6 h after actinomycin D addition, as well as prior to doxycycline treatment.RNA for Northern blotting was harvested and extracted using Trizol reagent (Life Technologies) following manufacturer’s protocol. To isolate cytoplasmic vs. nuclear fractions, cells were lysed in 0.1% NP-40 in Dulbecco’s phosphate buffered saline (DPBS) and the nuclear pellet was spun down. The supernatant was collected as the cytoplasmic fraction. The pellet was washed in 0.1% NP-40 in DPBS, then it was collected as the nuclear fraction. The RNA was extracted from both fractions using Trizol (Life Technologies) following manufacturer’s protocol.The RNA for northern blotting was run on a 1.8% agarose/formaldehyde gel and transferred by capillary action onto nitrocellulose membrane (Bio-Rad) using 10x SSC buffer. Northern blots were probed with probes against the SV40 3’ UTR present in pd2-eGFP-N1 , the firTotal cellular RNA for RT-qPCR in iPA-X and transfected cells was harvested and isolated using Zymo mini-prep kit (Zymo Research) following manufacturer’s protocol. cDNA was generated using iScript (Bio-Rad) from DNase-treated RNA.For analyses of mRNA and vRNA levels in A549 cells infected with IAV, total RNA was isolated at 6, 9, 12, and 15 h post-infection using Qiagen RNeasy Plus kit. For cDNA synthesis, Thermo Maxima H Minus kit was used with gene-specific primer for 18S rRNA combined with either oligo(dT) (for mRNA targets) or influenza vRNA-specific Uni12 primer . To minit-test was used to analyze values for significant differences.Quantitative PCR analysis was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) and Ct values were analyzed using the BioRad CFX Connect Real-Time System qPCR and Bio-Rad CFX Manager 3.1 program analysis. Metabolic labeling and isolation of nascent RNA in A549 cells infected with wild-type PR8 or mutant PR8-PA(fs) viruses was performed using Molecular Probes Click-iT Nascent RNA Capture Kit. At 8 hpi, 0.4 mM Click-iT nucleotide analogue 5-ethynyl uridine (EU) was added to the infection media for 1 hr at 37°C. At 9 hpi monolayers were washed twice with PBS and total RNA was isolated as described using Qiagen RNeasy Plus kit. Biotinylation and subsequent purification of EU-labelled RNA was performed according to the kit manufacturer protocol. cDNA synthesis and qPCR was performed on streptavidin beads as directed by the kit manufacturer protocol and described in methods section above.Total cellular protein was collected in protein lysis buffer ) unless specified otherwise in the text. Proteins were separated on SDS-PAGE gels and transferred onto PVDF membranes (Millipore). Western blots were performed in PBST with 5% milk or TBST with 4% bovine serum albumin. The following antibodies were used: Xrn1 , IAV NS1  < 0.05, 0.01, 0.001 vs. 293T iRFP  or A549 iPA-X D108A #8, shown in (A-C) HEK 293T (A) or A549 (B-C) cells expressing doxycycline-inducible wild-type PA-X-myc (“iPA-X wt”), a catalytically inactive mutant (“iPA-X D108A”) or RFP (“iRFP”) were treated with doxycycline for 18 h to induce expression of PA-X. Total cellular RNA was extracted and RT-qPCR was performed to measure levels of the indicated endogenous RNAs or the PA-X mRNA in panel C. Gene expression normalized by 18S rRNA levels is plotted as fold change in induced (PA-X- or RFP-expressing) vs. uninduced cells. The numbers indicate the clones of HEK293T and A549 iPA-X cells used for this panel. *,**,*** = (EPS)Click here for additional data file.S2 Figp value (Student’s t-test) < 0.05, 0.01, 0.001 vs. 293T iRFP  or A549 iPA-X D108A #8, shown in (A) 293T cells were transfected with Pol II-driven GFP reporters and either PA-X or an empty vector. The hp-GFP construct contains a hairpin close to the 5’ end of the GFP mRNA that blocks its translation. The levels of expression of GFP protein were examined by western blotting. Tubulin is included as a loading control. A representative blot is shown. (B) 293T iPA-X cells (line #7) were transfected with a T7 polymerase expressing construct and an EMCV IRES-containing luciferase reporter driven by the T7 promoter, which results in T7-mediated RNA transcription. PA-X expression was induced 5 h after transfection by doxycycline addition for 18 h. Luciferase activity was measured using a luminescence assay and is plotted as fold change relative to background luminescence levels in cells transfected with the EMCV-luciferase construct alone. Error bars = std. dev. (C-D) HEK 293T (C) or A549 (D) cells expressing doxycycline-inducible wild-type PA-X-myc (“iPA-X wt”), a catalytically inactive mutant (“iPA-X D108A”) or RFP (“iRFP”) were treated with doxycycline for 18 h to induce expression of PA-X. Total cellular RNA was extracted and RT-qPCR was performed to measure levels of the indicated endogenous RNAs. Gene expression normalized by 18S rRNA levels is plotted as fold change in induced (PA-X- or RFP-expressing) vs. uninduced cells. The numbers indicate the clones of HEK293T and A549 iPA-X cells used for this panel. Error bars = s.e.m. *,**,*** = (EPS)Click here for additional data file.S3 Fig(A) PA-X targets both the cytoplasmic and the nuclear pools of susceptible reporter RNAs, and does not degrade the HR bearing constructs in either fraction. HEK 293Ts were transfected with indicated Pol-II driven GFP reporters and either PA-X or empty vector. Total cellular RNA was extracted from nuclear and cytoplasmic fraction and GFP mRNA, 7SL and U2 ncRNA levels were measured by northern blotting. A representative blot is shown. (B) Relative levels of pre-mRNAs and MALAT1 ncRNA in the nuclear vs. cytoplasmic fraction confirm that they are enriched in the nucleus (control for (EPS)Click here for additional data file.S4 Fig(A and B) Spontaneously immortalized mouse embryonic fibroblast (MEF) cells were co-transfected with the NF-kB promoter-driven Firefly luciferase reporter , thymidine kinase promoter-driven Renilla luciferase reporter , and the indicated GFP-fusion protein expression constructs. At 24 h post-transfection, (A) the cells were fixed with 4% paraformaldehyde, stained with Hoechst dye to visualize cell nuclei, and analyzed by fluorescence microscopy. Asterisk indicates 2-times longer exposure for cells transfected with the PA-X-GFP fusion construct due to low level of expression. Scale bar = 20 um. Alternatively, (B) the cells were used to measure Firefly (FF-luc) and Renilla luciferase expression using Dual-Luciferase Reporter Assay kit (Promega). Values are normalized to vector-transfected cells. (C) HeLa Tet-Off cells (Clontech) were co-transfected with tetracycline response element (TRE2) promoter-driven Firefly (FF-luc) and Renilla (Renilla) luciferase reporters and the indicated GFP-fusion protein expression constructs. Luciferase activity was measured at 24 h post-transfection as described in (B). Values are normalized to those in cells transfected with the GFP-X41 fusion construct. In (B) and (C) the error bars represent standard deviation between measurements from 2 independently transfected wells.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


A41L, B15R, C6L, or C12L open reading frames (ORFs) enhanced cellular immune responses in recombinant MVA (rMVA) by up to 2-fold. However, previously, we showed that deletion of the C12L, A44L, A46R, B7R, or B15R ORFs from rMVA, using MVA-BAC recombineering technology, did not enhance rMVA immunogenicity at either peak or memory cellular immune responses. Here, we extend our previous study to examine the effect of deleting clusters of genes on rMVA cellular immunogenicity. Two clusters of fifteen genes were deleted in one rMVA mutant that encodes either the 85A antigen of Mycobacterium tuberculosis or an immunodominant H2-Kd-restricted murine malaria epitope (pb9). The deletion mutants were tested in prime only or prime and boost vaccination regimens. The responses showed no improved peak or memory CD8+ T cell frequencies. Our results suggest that the reported small increases in MVA deletion mutants could not be replicated with different antigens, or epitopes. Therefore, the gene deletion strategy may not be taken as a generic approach for improving the immunogenicity of MVA-based vaccines, and should be carefully assessed for every individual recombinant antigen.Modified vaccinia virus Ankara (MVA) is a highly attenuated strain of vaccinia virus, which has been used as a recombinant vaccine vector in many vaccine development programmes. The loss of many immunosuppressive and host-range genes resulted in a safe and immunogenic vaccine vector. However it still retains some immunomodulatory genes that may reduce MVA immunogenicity. Earlier reports demonstrated that the deletion of the  B15R, encoding the IL-1β binding protein [A41L, encoding a CC-chemokine binding protein [Modified Vaccinia virus Ankara (MVA) is a highly attenuated strain of vaccinia virus, the smallpox vaccine. This attenuation was achieved by more than five hundred passages in chick embryo fibroblast (CEF) cells . The res protein , or A41L protein .A41L , B15R, or both [+ T cell responses upon stimulation with VACV-infected cells or MVA-specific peptides. The immune responses to the recombinant antigens were also increased to a similar extent. In the double deletion ΔA41L\B15R [+ T cell response to one of the pools (GPN-pool) in the rMVA mutant vaccinated group. This mutant also induced more CD8+ T cells than CD4+ T cells in the Env-pool. In another study, the deletion of C6L gene from MVA-B (ΔC6-MVA-B) increased memory CD4+ and CD8+ T cell responses, and although this mutant did not shift the immune responses towards particular peptide pools, the increased CD4+ or CD8+ T cells were only observed in one peptide pool (the GPN-pool), but not in the Env- or Gag-pool [C6L and K7R in the same MVA-B vaccine (ΔC6/K7-MVA-B) showed that the ΔC6-MVA-B, included as a control, had no impact on the peak immune responses [+ T cells, but not CD4+ T cells; and upon comparison with the ΔC6-MVA-B, the increase seemed to be mainly an effect of C6 absence and not due to the K7R deletion [+ T cells specific to the MVA vector or to encoded antigens were also reported upon the single deletion of F1L , N2L , or C12L (MVA008L) [It has been reported that deletion of some of these genes improved innate, adaptive memory cellular or humoral immune responses to the vector (MVA) or to the encoded recombinant antigens, and enhanced the protective capacity of MVA following a challenge with VACV WR. The deletion of the ORF  or both , showed 41L\B15R , the MVAGag-pool . AlthougMVA008L)  in rMVA A41L, B15R, or the double deletion of both had no impact on the antibody induction at the memory phase, the deletion of C6L or N2L increased the memory antibody-mediated responses by around 2-fold in the total anti-recombinant antigen antibody titres. Of note, the C6L deletion enhanced the anti-gp120 antibody titres [C6L deletion mutant, in a later study, along with the double deletion C6L/K7R mutant showed no significant difference in the anti-gp120 antibody titres by those two mutants compared to the control (MVA-B vaccine) [1L and C12L studies. Finally, the deletion of A35R showed small non-significant increase in the cellular immunogenicity but increased the anti-VACV antibody titres by 2-fold with isotype switching to more IgG and its subclasses IgG1 and IgG2a [ex vivo re-stimulation (e.g. VACV-infected cells vs. E3 or F2(G) peptides), and the time for harvesting the spleens to determine memory responses .In terms of improving humoral immunogenicity in these studies, while the deletion of y titres , but thend IgG2a . OverallC12L (MVA008L), B15R (MVA184R), A41L (MVA153L), and A46R (MVA159R) from MVA encoding HIV-1 clade C consensus Gag and Env immunogen, given twice with a 9 week interval, resulted in increased CD4+ and CD8+ T cells either after the prime (4-fold) or after the boost dose (5-fold); and around 25-fold increase in the anti-gp120 antibody titres [udg) gene, in addition to those four genes, resulted in a five-gene deletion rMVA mutant that did not further enhance the improved immunogenicity of the four-gene deletion mutant. The deletion of the fifth gene, udg, was expected to improve the immune responses, as a previous report by the same group showed that the deletion of udg gene alone in rMVA inhibited the late MVA gene expression, reduced the antigen complexity of MVA, and improved the elicited CD4+ and CD8+ T cells (2-fold) in a similar regimen, but with Gag gene from HIV clade B, which is a different recombinant antigen [Nevertheless, a study in macaques reported that deleting four genes;  antigen .C12L, A44L, A46R, B7R, or B15R, from MVA did not enhance the cellular immunogenicity of the TIP model antigen  using seven immunodominant MVA epitopes in two strains of mice at the peak cellular responses. Of the memory responses (day 56 post-MVA injection), only the ΔB15R-MVA showed a small (1.5-fold) but significant increase in the CD8+ T cell frequencies [M. tuberculosis [d-restricted murine malaria epitope (pb9) from the Plasmodium berghei circumsporozoite protein [Previously, we reported that the single deletion of quencies . Here, wrculosis  or the Trculosis  that enc protein . The MVA protein .+ T cell frequencies. This unchanged immunogenicity was observed using either the synthetic model TIP antigen or the naturally occurring 85A antigen. The responses at 28, 56, or 84 (long-term) days post-MVA injection showed no improved memory CD8+ T cell frequencies, except to one peptide (E3) at day 28. However, at day 84, or at day 56 in the prime-boost vaccination, this increase in E3-specific responses was very marginal and not statistically significant. This support the earlier studies showing that the increase could be observed with only few peptides or peptide pools.None of the derived rMVA mutants showed improved immunogenicity either to the MVA or to encoded TIP antigens. The large MVA deletion mutant (lacking fifteen genes) was tested in prime only or in prime-boost regimens and showed no improved CD8Our results suggest that the reported increases in MVA deletion mutants, which were small increases in many cases (around 2-fold), could not be replicated with different antigens and that the approach of gene deletion to improve MVA-vectored vaccines should be carefully assessed for every recombinant antigen rather than taken as a generic approach for improving MVA immunogenicity.+ T cells, specific to MVA-peptides (E3 and F2(G)) or to the TIP-specific peptide (pb9) were then measured. There were no significant differences in response to the MVA-E3, MVA-F2(G), or pb9 peptide between any of the mutant groups and the control  or with different recombinant MVA mutants . The presence of either 85A or TIP antigens were screened with PCR; and the absence of the MVA wild type was also confirmed by PCR.As there was no improved immunogenicity with various deletion mutants, we constructed a large MVA mutant, lacking fifteen genes (Δ15-MVA) described in 6 pfu via i.m route, and the immune responses followed for three months. First, all mice were bled at day 7 post-immunization and the PBMCs were screened in blood-ELISpot using the pb9 peptide, or the vector-specific E3 and F2(G) peptides. The CD8+ T cell immune responses were similar between the MVA-TIP and the mutant (Δ15-MVA-TIP). At day 28 post-immunization, five mice from each group were sacrificed, spleen harvested, and ICS was performed using the same three peptides. Only E3-specific CD8+ T cell responses were enhanced (in two independent experiments) in the mutant group while there were no differences in the recombinant epitope (pb9) or in the other vector-specific F2(G) peptide. The remaining of the mice were sacrificed at day 84 post-immunization and showed no improved CD8+ T cell frequencies, even the increased responses with the E3 peptide at day 28 was not observed at day 84, suggesting it was transient enhancement  10ancement .7 pfu, via i.p. route, with either the Δ15-MVA-TIP mutant or the control (MVA-TIP). Five mice were sacrificed at day 7 post-boost to determine the peak immune responses with the remaining mice sacrificed at day 56 post-boost to determine the memory responses. There were no improved CD8+ T cell responses either at the peak or at the memory responses to pb9, E3, or F2(G) peptide stimulations  and has been shown to induce CD4+ and CD8+ T cell responses and when used to boost BCG-vaccinated adult or infant humans [wt), non-mutant MVA-BAC-85A, and the MVA85A  was performed to determine the cellular immune responses using two 85A peptide pools, specific for CD4+ or CD8+ T cells, or E3 and F2(G) MVA-peptides specific for CD8+ T cells. The ICS did not show any difference in the frequencies of IFN-γ secreting CD4+ or CD8+ T cells between the mutant and any of the control viruses. The comparison of MVAwt and the MVA-BAC-85A showed similar vector-specific immune responses using CD8+ T cell-specific MVA peptides (E3 and F2(G) peptides). Moreover, the conventional MVA85A induced similar anti-vector and anti-rAg85A responses as the MVA-BAC-85A. Taking together, this supports our earlier observation [+ or CD8+ T cells by ELISpot and observed a similar result to the ICS  epitopes. The use of TIP model antigen, which harbours the immunodominant pb9 epitope, enabled us to follow the immune responses for three months, but did not show any improvement at the long-term memory CD8+ T cell responses (for three months). Many previous studies showed that the single deletion of C6L [N2L [C12 [A41L/B15R and C6L/K7R from MVA vectors enhanced the cellular immunogenicity in DNA-prime MVA-boost vaccination regimens. This regimen is also relevant to MVA clinical testing applications; MVA is used as a boosting agent in many vaccine clinical trials [A41L and B15R are deleted in the Δ15-MVA-TIP mutant, and therefore this mutant would be expected to elicit stronger cellular immune responses, especially in this regimen as reported before. However, our result did not show any improved immunogenicity with the Δ15-MVA-TIP in prime-only or prime-boost vaccinations. Garber et al (2012) reported that the deletion of four immunomodulatory genes enhanced MVA cellular immunogenicity in macaques, but the deletion of a fifth gene did not extend this improved immunogenicity [A41L or B15R, was also noted when the A41L or B15R were singly deleted, as we previously published [A41L or B15R, or both of them was reported to enhance MVA cellular immune responses, but this enhancement was 2-fold at best [A41L or B15R, or both, the inability to reproduce this improved immunogenicity could suggest that improving MVA immunogenic profiles could be very difficult to achieve through gene deletions, especially as MVA does not replicate in vivo, and its gene expression declines within 12 hours, and vanishes less than 48 hours post-inoculation in vivo [Here we investigated the effect of deleting some immunomodulatory or unknown non-essential genes from MVA, in different combinations , on the n of C6L , N2L [17N2L [C12 ; or the l trials . Thus, wer et al 12 reportgenicity . It coulublished . Moreove at best –13. Alth in vivo , 28.+ T and CD8+ T cell immunogenicity. Thus, we replaced TIP with 85A antigen in the large MVA deletion mutant (deriving Δ15-MVA-85A), confirmed that no other changes had occurred in the genome by sequencing, then tested it in mice, measuring both CD4+ T and CD8+ T cell responses with peptide pools by ICS or two strong individual peptides (specific for CD4+ or CD8+ T cells) by ELISpot. Here, three control viruses were included; the MVA85A vaccine that is used for clinical testing against TB, the MVA-BAC-85A , and the MVA wild type (MVAwt), which is non-recombinant and non-mutant control. The results showed a continued lack of improved immunogenicity even with this natural immunogenic antigen. Although we were able to test CD4+ T cell responses, there was no significant difference in the magnitudes of 85A-specific CD4+ T cell between the mutant group and any of the control groups.The concern over the use of non-natural model antigen like TIP should not apply to a “real” immunogenic antigen. The TB 85A is one of the most advanced candidates in clinical trials and showed strong CD4E3L, which is difficult to grow in CEFs[+ and CD8+ T cell responses to the rAg-specific peptide pools by ICS or to the individual strong peptides measured by ELISpot as well as similar CD8+ T cell responses to the vector-specific E3 and F2(G) peptides. Moreover, this was also supported by the result of MVAwt compared to the MVA-BAC-85A that showed similar responses to the vector specific E3 and F2(G) peptides. This MVA-BAC system was also shown to have no altering affect on the immunogenicity of MVA [+ T cells was the main readout as it would be more relevant for the improvement required for clinical testing of MVA-based vaccines.It could be speculated that the MVA-BAC system or deriving MVA in BHk-21 cell lines had some effect on these mutants, which could prevent them from enhancing the immune responses. The BHK-21 cell line was reported to allow for the replication of some MVA mutant lacking w in CEFs. This waw in CEFs. The BHKw in CEFs. The PKRy of MVA . The MVAC12L and C6L, which were reported to enhance cellular immunogenicity when deleted from MVA [C12L [C6L (data not shown) failed to enhance the immune responses to TIP.The ΔC11-K3/B15-MVA lacks from MVA , 18, didVA [C12L  or C6L (+ T cell responses. The memory responses at 28 or 84 (long-term) days post-MVA immunization showed no improved memory CD8+ T cell frequencies, except to one peptide (E3) at day 28. At day 56, in prime-boost regimen, this increase was observed but it was very marginal and insignificant. Even replacing TIP with TB 85A “real” antigen (Δ15-MVA-85A) and testing CD8+ as well as CD4+ T cells magnitudes did not detect any differences compared to various control viruses. This study suggests that the previously reported immunogenicity increases in MVA deletion mutant, which were small increases in many cases (2-fold), may not be replicated with different antigens. The approach of gene deletion to improve MVA-vectored vaccines should be carefully assessed for every recombinant antigen, and epitope (peptide), and may not be taken as a generic approach for improving MVA immunogenicity.In conclusion, none of the derived rMVA mutants showed improved immunogenicity either to the MVA antigens or to the encoded TIP antigen. Focusing on the large deletion Δ15-MVA-TIP mutant, neither prime only nor prime-boost regimens showed any improved CD8All animal procedures were performed in accordance with the terms of the UK Animals (Scientific Procedures) Act (ASPA) for the project licenses 30/2414 or 30/2889 and were approved by the University of Oxford Animal Care and Ethical Review Committee. All mice were housed at least 7 days for settlement prior to any procedure in the University animal facility, Oxford, UK under Specific Pathogen Free (SPF) conditions.d restricted murine CD8+ T cell epitope SYIPSAEKI (pb9) from the Plasmodium berghei circumsporozoite protein [Mycobacterium tuberculosis 85A antigen (TB 85A), deriving the Δ15-MVA-85A. The 85A was also inserted into two other viruses to serve as controls; first, into a non-mutated MVA-BAC using MVA-BAC recombineering (virus named MVA-BAC-85A); second, into MVA wild type using the conventional recombinant vaccinia virus construction .GalK recombineering [GalK resistance gene under a prokaryotic promoter. This was amplified with Phusion (Finnzymes) as a targeting DNA for recombineering by using long oligonucleotide primers (Eurofins MWG Operon) to add 50bp homology arms (matching two regions within the TK ORF) to the 5’ and 3’ ends. Primers were designed to insert the TIP-GalK cassette into the TK locus. These targeting constructs were used for MVA-BAC recombineering as previously described [GalK from MVA-BAC that contains TIP-GalK, leaving only the TIP into the TK locus. Therefore, GalK was then amplified with long oligonucleotide primers with 50 bp homology arms, matching the flanks of the targeted ORFs, deleting and replacing them with GalK. The colonies on GalK-positive plates were re-streaked three times before the recombineered MVA-BAC constructs are then confirmed by identity and purity PCR. The presence of the TIP was also confirmed by PCR and sequencing.Construction and generation of MVA-BAC and generation of rMVA deletion mutants using ineering  has beenineering . To geneescribed . Next, MGalK bacterial marker gene was only removed from mutants that required a multiple deletions such as the Δ15-MVA-TIP, for the mutants with only one gene or one region of deleted genes, the GalK was not removed. BACs and derived viruses were checked for identity and purity by PCR and the sequences of the homology arms and transgenes were confirmed at both stages. BAC-derived rMVAs were plaque-picked three times to ensure purity, as a precautionary measure, since the bacterial colonies were re-streaked three times prior to rescue. The viruses were amplified in 1500cm2 of BHK cell monolayers, purified over sucrose cushions and titred in BHK-21 cells according to standard practice, and purity and identity were again verified by PCR. Since MVA-BAC has a GFP marker gene under control of the Fowlpox virus p4B promoter [The recombineered MVA-BACs were rescued to recombinant MVA in BHK cells using a fowlpox virus helper as previously described . The Galpromoter , all theDNA samples of the Δ15-MVA-85A from bacterial LB culture (for MVA-BAC DNA) or from BHK-21-infected cells  were isolated using EndoFree Plasmid Maxi Kit (Qiagen) or standard phenol-chloroform DNA extraction, respectively. Samples were sent to the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics, University of Oxford, for MVA genome sequencing.6 pfu of rMVA. For the heterologous prime boost regimen, mice were immunized with 100 μg of DNA, given i.m. (50μL per mouse) followed eight weeks later with 107 pfu, given intraperitoneal (i.p.) of rMVA. Procedures were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 under granted project licenses 30/2414 or 30/2889. Splenocytes were harvested seven, or 56 days post-MVA immunization for analysis by IFN-γ ELISpot or intracellular cytokine staining (ICS) and flow cytometry as previously described [+ or CD8+ T cell-specific 85A peptide pools for ICS. Two 85A-specific individual peptides were used for ELISpot; CD8+ T cell-specific p11 peptide, EWYDQSGLSVVMPVGGQSSF, and CD4+ T cell-specific p15 peptide, TFLTSELPGWLQANRHVKPT.Female BALB/c mice aged 6 to 8 weeks  were immunized intramuscularly (i.m.) in the tibialis muscles (50μL per mouse) or intradermally (i.d.) with a total of 10escribed , 31, usiescribed , 1μg/mL GraphPad Prism (GraphPad software) was used for statistical analysis and to plot data.


Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.  •Effect of mutation on affinity and efficacy of biased ligands mapped onto 3D models•Biased agonists form distinct interactions with the GLP-1R extracellular surface•Engagement of unique elements of the extracellular surface promotes biased agonism•Insights into class B GPCR activation/biased agonism can aid rational drug design Understanding how different ligands engage and activate signaling pathways downstream of the GLP-1 receptor, a key type II diabetes target, suggests strategies for shaping signaling outcomes to favor physiologically beneficial effects. G protein-coupled receptors (GPCRs) are critical for the transmission of extracellular signals across the cell membrane to initiate intracellular responses  and are Class B peptide hormone receptors are a subfamily of GPCRs that are major targets for the treatment of chronic disease, including type 2 diabetes, obesity, and dis-regulated bone metabolism . They inStructural data for class B receptors are limited to partial domains, including several NMR and crystal structures of peptide-bound N-terminal domains (reviewed in 2)  . The phe2) . Intrigu2) . This hi2+ mobilization, and phosphorylation of ERK1/2 (pERK1/2)  , each ofpERK1/2) . The conpERK1/2) .Using a combination of alanine-scanning mutagenesis, followed by pharmacological quantification of the effects of mutation on peptide agonist affinity and three distinct signaling pathways, we have identified critical regions within the extracellular face of the receptor core both for peptide agonist affinity and for driving receptor coupling to distinct signaling pathways, extending our initial work on ECL2 . We usedTo understand the functional interface at the GLP-1R extracellular surface, we completed alanine-scanning mutagenesis of the ECLs and adjacent TM residues, coupled with analysis of ligand affinity and signaling for three key pathways that are involved in GLP-1R function and rely on different effector engagement . We asseThe predicted ECL1 and adjacent TM boundary comprises 23 residues from L201 to S223, ECL2, 23 residues from G285 to L307 and ECL3, 16 residues from D372 to E387. The results for the pharmacology of ECL2 mutants have been published previously  and are All mutant receptors, with the exception of W306A, were expressed at the cell surface, and most were expressed at levels equivalent to wild-type  Koole e. Of the 125I-exendin-4 (9–39)) (Effects of mutations on agonist affinity were established by heterologous competition with the antagonist radioligand ( (9–39))  (Koole e (9–39)) . The aff (9–39)) . These wOverall, there was a high degree of overlap in the impact of alanine mutation on binding of GLP-1, exendin-4, and oxyntomodulin. In 3D space, there is a continuum of residues from K288, E292, D293, R299, N300 within the proximal part of ECL2 that link to TM6/ECL3 membrane-proximal residues D372, E373 and L379, K383, L384 in ECL3 that are globally important for binding affinity, along with most residues in the distal segment of ECL2  that are important for GLP-1 and exendin-4 affinity but have little role in oxyntomodulin binding  that is independent of both affinity and cell-surface expression levels  also had the same effect for exendin-4  lying outside of the predicted peptide-binding groove enhances the ability of GLP-1 to promote Caxendin-4  and 6.2+ mobilization. Whereas these latter aspects of receptor function required a large area of the protein’s extracellular surface for transmission of signal, residues important for pERK1/2 were localized mainly to membrane-proximal residues of ECL3  for GLP-1; L201 (ECL1) and F385 (ECL3) for oxyntomodulin; and W203 (ECL1) for exendin-4.Ligands binding to GPCRs modify the conformational landscape and thus stabilize a subset of conformational ensembles, providing the basis for both differential efficacy and biased agonism. There is limited information linking the dynamic events of receptor activation to engagement of specific effector proteins, and this is particularly true for class B GPCRs. The current study explores the molecular determinants for ligand affinity and engagement of signaling. Specifically, we highlight crucial surface residues within a class B GPCR that link initial peptide agonist interactions to distinct intracellular signaling pathways and biased agonism.Peptide interactions with the extracellular surface and TM domains of class B GPCRs promote conformational transitions required to allow the binding of signaling effectors at the intracellular surface of these receptors. The ability of receptor mutants to affect signaling at only a single pathway highlights that different elements of the extracellular face are required for coupling to different effectors. In addition, differential effects on signaling by the three peptide agonists following mutation of individual residues supports the notion that the extracellular face of the receptor is important for initiating a switch in the conformational landscape that the receptor explores, with different ligands capable of promoting/stabilizing alternative subsets of ensembles that lead to biased agonism.Large portions of ECL2, ECL3 and the juxtamembrane positions of TM2/ECL1, ECL2/TM5 and TM6/ECL3 were important for molecular recognition of all peptide agonists but not for binding of the N-terminally truncated antagonist exendin-4(9–39). Despite the separation in sequence of these residues, they are all located together in 3D space. In addition, there is a network of residues provided by all three loops lining the cavity entrance in the TM bundle, and these residues are important for GLP-1 affinity, extending the peptide-binding groove from the N-terminal ECD into the TM domain cavity. These residues include L201, M204 (TM2/ECL1), E294, W297, T298, R299, N300, Y305 (ECL2), R380, L384 (ECL3) . Interes2.60, N2403.43, E3646.53, and Q3947.49) form part of a hydrogen bond network in the inactive, unliganded receptor and are important for the binding and function of GLP-1 and exendin-4, though these residues also have roles in the biased agonism of these peptides  in oxyntomodulin affinity or cAMP formation (9 (position 3) of the peptide and R1902.60 of the receptor. However, oxyntomodulin contains a Q at this position that would not be expected to form a salt bridge with R1902.60. Modified GLP-1 and oxyntomodulin peptides where the residue at position 3 is swapped converts the behavior of these two peptides such that R1902.60 is required for cAMP production by the modified oxyntomodulin but not for the modified GLP-1  of the signal attributed to this latter mechanism NH of cAMP , it reta of cAMP . This suBiased agonism is likely to be a crucial element of the function of class B GPCRs, as many of them can be activated by multiple endogenous ligands and their receptors are capable of activating multiple intracellular signaling pathways. Therefore, understanding the molecular determinants linking ligand interactions to activation of distinct signaling pathways, in addition to the physiological benefit of activating individual pathways, could have significant ramifications for future drug development and may provide the potential to rationally design future drug therapies, and this is highlighted by the in vivo actions of the biased GLP-1 agonist, P5 .We used Quikchange (Stratagene) to introduce mutations into the GLP-1R cloned into the pEF5/Frt/V5-Dest vector.Stable FlpIn CHO cell lines were generated using Gateway technology. For all assays, cells were seeded in 96-well plates at a density of 30,000 cells per well.125I-exendin-4(9–39) as the tracer ligand and competing with increasing concentrations of unlabeled peptide ligands as described previously  before lysis. Detection of cAMP and pERK1/2 in the lysates was performed using Alphascreen technology as previously described . Ca2+ mo50, and Emax values. Efficacy was calculated by applying the operational model of agonism:Bottom represents the y value in the absence of ligand, Em is maximal system stimulation, KA is the agonist-receptor dissociation constant, [A] is the ligand concentration, and τ is the operational measure of efficacy in the system, which incorporates signaling efficacy and receptor density. Derived τ values were corrected to cell-surface expression (τc) measured by ELISA, and errors were propagated from both τ and cell-surface expression.Concentration response data were analyzed using a three-parameter logistic equation to determine affinity, ECEnergy-based conformational modeling of the GLP-1R complex with GLP-1 was performed with Modeler 9.15, and peptide docking and energy optimization were guided by published experimental data  and S6, Detailed procedures and analysis are reported in the Conceptualization, D.W., J.S., P.M.S.; Methodology, D.W., C.A.R., K.J.S., J.C.M., M.L., P.E.T., P.M.S.; Software, C.A.R., K.J.S., J.C.M.; Formal Analysis, D.W., C.K., A.C., P.M.S.; Investigation, D.W., C.A.R., K.J.S., J.C.M., C.K., E.E.S., K.P., M.L., J.S., R.S.; Writing – Original Draft, D.W., C.A.R., L.J.M., A.C., P.M.S.; Writing – Review and Editing, C.K., S.G.B.F.; Visualization, D.W., P.M.S.; Supervision, D.W., C.A.R., P.E.T., P.M.S.; Project Administration, D.W., P.M.S.; Funding Acquisition, D.W., C.A.R., A.C., P.M.S.


In synthesizing a double-stranded DNA from viral RNA, HIV-1 reverse transcriptase (RT) generates an RNA/DNA intermediate. RT also degrades the RNA strand and synthesizes the second DNA strand. The RNase H active site of RT functions as a nuclease to cleave the RNA strand; however, the structural basis for endonucleolytic cleavage of the RNA strand remains elusive. Here we report crystal structures of RT-RNA/DNA-dATP and RT-RNA/DNA-nevirapine (NVP) ternary complexes at 2.5 and 2.9 Å resolution, respectively. The polymerase region of RT-RNA/DNA-dATP complex resembles DNA/DNA ternary complexes apart from additional interactions of 2′-OH groups of the RNA strand. The conformation and binding of RNA/DNA deviates significantly after the seventh nucleotide versus a DNA/DNA substrate. Binding of NVP slides the RNA/DNA non-uniformly over RT, and the RNA strand moves closer to the RNase H active site. Two additional structures, one containing a gapped RNA and another a bulged RNA, reveal that conformational changes of an RNA/DNA and increased interactions with the RNase H domain, including the interaction of a 2′-OH with N474, help to position the RNA nearer to the active site. The structures and existing biochemical data suggest a nucleic acid conformation-induced mechanism for guiding cleavage of the RNA strand. Lys,3, complementing the primer-binding sequence of the viral ssRNA, forms a double-stranded nucleic acid segment that binds RT to initiate RNA-dependent DNA polymerization; (ii) RT adds nucleotides complementing the (+)RNA strand to synthesize (−)DNA strand starting from the 3′-end of the annealed tRNA; (iii) RNase H activity of RT degrades RNA strand from RNA/DNA hybrid leaving short A-T rich segments, known as polypurine tracts (PPTs), attached to the (−)DNA strand and (iv) the (+)DNA strand synthesis is initiated from the 3′-end of a PPT segment. A non-PPT RNA is usually cleaved in a non-sequence-specific manner. However, the presence of specific nucleotides at positions adjacent to the RNase H site and distal sites in the nucleic acid-binding cleft of RT can enhance RNase H cleavage efficiency (HIV-1 reverse transcriptase (RT) is a central enzyme responsible for copying the viral single-stranded RNA (ssRNA) into a double-stranded DNA (dsDNA) in the cytoplasm of an infected cell . This evficiency . The ratficiency ,7. Unlikficiency , i.e. HIficiency ; see revficiency  and Beilficiency . The prificiency ,15 mightEscherichia coli RNase H is a functionally independent enzyme, whereas the HIV-1 RNase H domain requires additional elements from the polymerase domain for RNase H cleavage activity  are approved for treating HIV-1 infection. In contrast, the RNase H activity has not yet been successfully targeted for blocking viral replication. HIV-1 RNase H has a two cation-dependent nuclease activity , and theactivity . The RNaactivity ,30. TakeStructurally, RT is a heterodimer of p66 and p51 polypeptide chains. The p66 subunit contains both the polymerase and RNase H active sites. The p51 subunit is proteolytically derived from a p66 chain by HIV-1 protease cleaving off the C-terminal RNase H domain; p51 plays a structural role. RT is a highly flexible enzyme that undergoes conformational changes to carry out its activity. Several conformational and functional states of RT have been structurally characterized . While mHere we report four structures of HIV-1 RT-RNA/DNA complexes in the presence of either a dATP substrate or an NNRTI drug, nevirapine (NVP); the RNA/DNA sequences used in the current study are shown in Figure HIV-1 RT construct RT127A containing an RNase H active site mutation D498N and a thumb mutation Q258C for RT-RNA/DNA cross-linking was cloned, expressed and purified following a previously reported protocol . The RNA2. The crystals were taken out of the drop and transferred to a stabilization solution containing 1.5 μl of 50 mM bis-tris propane pH 7.0, 14% PEG 8000 (w/v), 20 mM MgCl2, 5% sucrose (w/v), 5% glycerol (v/v) and 100 mM ammonium sulfate for 10 min. The crystals were then treated for 15 min in a solution containing 5 mM dATP, 14% PEG 8000 (w/v) and 1 mM MgCl2 for forming the RT-RNA/DNA-dATP complex in crystals. The crystals were cryoprotected by dipping for 1 min into the above solution with glycerol concentration increased to 20% (v/v), and flash frozen in liquid N2.The RT-RNA/DNA binary complex was concentrated to 9 mg/ml in 75 mM NaCl and 10 mM Tris pH 8.0. Crystals were grown at 4°C by hanging-drop vapor diffusion against well solution containing 50 mM bis-tris propane-HCl pH 7.0, 10% PEG 8000 (w/v), 100 mM ammonium sulfate, 5% glycerol (v/v), 5% sucrose (w/v) and 20 mM MgCl2.The RNA/DNA, gapRNA/DNA and bulgeRNA/DNA complexes were buffer exchanged and concentrated to 12 mg/ml in 75 mM NaCl and 10 mM Tris-HCl pH 8.0. The crystals were grown at 4°C in sitting drops, by vapor diffusion against well solution containing 50 mM bis-tris propane pH 7.2 – 7.4, 9 – 11% PEG 8000 (w/v), 100 mM ammonium sulfate, 5% glycerol (v/v), 5% sucrose (w/v) and 20 mM MgCl2+ ions, then into 50 μl of solution II [solution I containing 2 mM NVP and 4% DMSO (v/v)] for 3 h 30 min and finally into 10 μl solution III [solution II with glycerol concentration increased to 20%] for 1.5 min. The RT-RNA/DNA-NVP crystals harvested from solution III were flash frozen in liquid N2. A similar procedure was followed to obtain the crystals of RT-bulgeRNA/DNA-NVP or RT-gapRNA/DNA-NVP complexes , 10% (v/v) glycerol and 12% (w/v) PEG 8000] for 30 min to remove Mgs Figure –C. Two m3 crystal of RT-RNA/DNA-dATP, a 200 × 200 × 60 μm3 crystal of RT-DNA/RNA-NVP, a 280 × 120 × 30 μm3 crystal of RT-gapRNA/DNA-NVP and a 240 × 120 × 40 μm3 crystal of RT-bulgeRNA/DNA-NVP complex. Individual datasets were processed and scaled using HKL2000 (http://www.pymol.org/) or ChemBioDraw (http://www.cambridgesoft.com/).Flash-frozen crystals were transported to the F1 beam line at the Cornell High Energy Synchrotron Source (CHESS). X-ray diffraction data were collected from a 280 × 120 × 30 μm HKL2000   compared to the other (Mol-2). Differences in subdomain arrangements are also observed from comparison of multiple crystal structures of RT/NVP binary complex; e.g. PDB IDs 3HVT  versus 1In structures of HIV-1 RT in complexes with RNase H inhibitors, the inhibitors chelate both RNase H active site cations . We aligProtein–nucleic acid interaction and crystallographic B-factor analyses of dATP and NVP complexes suggest that the binding of RNA/DNA to RT is primarily defined by its interactions with palm, thumb and finger subdomains in the polymerase domain, and with the RNase H primer grip in and nearby the RNase H domain . The parThe RNase H primer grip is important for degradation of RNA by RT, and mutations of the primer grip residues cause significant loss of RNase H cleavage activity . When coAn RNA strand of an RNA/DNA duplex is cleaved periodically. The template RNA strand does not consistently interact with RT near the RNase H domain, and thereby no structural element of RT continuously threads the RNA to the RNase H active site for the cleavage of phosphodiester bonds. It seems likely that an RNA/DNA duplex constantly undergoes conformational changes while RT slides over it ,15, and To artificially introduce conformational variations and investigate the effects on the proximity of the RNA strand with respect to the RNase H active site, we introduced (i) a single-nucleotide bulge and (ii) a single-nucleotide gap in the RNA strand of the RNA/DNA duplex Figure  and C. FThe structures of gapRNA-NVP and bulgeRNA-NVP complexes were determined at 3.2 and 3.0 Å resolution, respectively Table . As desi2+ ions are the cofactors for RNase H activity in vivo. However, structures of inhibitors chelated at the RNase H active site of RT could only be obtained in the presence of Mn2+ ions  is observed bound at position A at the RNase H active site, and no binding was observed at position B  might be preventing the formation of a catalytic complex in the current structures. Another structural constraint in nearly all crystal structures of RT is the formation of an extended β-sheet involving two RNase H domains of adjacent molecules. This extended sheet may also restrict the flexibility of RT to attain an RNase H catalytic complex in crystals.2+ (or Mn2+) ion present at the RNase H active site in bulgeRNA or gapRNA structures (with a minimum distance of ∼4 Å between the oxygen and ion)  increased by the binding of an NNRTI and (ii) decreased by the binding of a dNTP . All str) Figure  and D. C) Figure  and B. C) Figure  and D.A recently reported nicked RNA/DNA-RT-efavirenz structure shows a significant deviation in the binding of the RNA/DNA compared to the current structures  and all RT-nucleic acid complexes previously described in the literature; the authors proposed that enhanced interaction between the p51 C-terminus and RNA strand might be critical for RNase H degradation . Our anaThe current series of structures provide insights into how the non-uniform sliding of RNA/DNA, and conformational changes of the duplex near to and distal from the RNase H active site assist the RNA strand in accessing the active site for cleavage ,52,54. FThe atomic coordinates and structure factors for the structures of RT-RNA/DNA-dATP, RT-RNA/DNA-NVP, RT-gapRNA/DNA-NVP and RT-bulgeRNA/DNA-NVP complexes are deposited in Protein Data Bank (PDB) under accession numbers 4PQU, 4PUO, 4Q0B and 4PWD, respectively.Supplementary Data are available at NAR Online.


L) and heavy strand (OH) replication within the major arc. However, under certain disease conditions, rarer, minor arc deletions are detected. Currently, there are few techniques which would allow the detection and quantification of both types of mtDNA deletions in single muscle fibres. We have designed a novel triplex real-time PCR assay which simultaneously amplifies the MT-ND4 gene in the major arc, the MT-ND1 gene in the minor arc, and the non-coding D-Loop region. We demonstrate that this assay is a highly sensitive and reliable tool for the detection and quantification of a broad range of major and minor arc mtDNA deletions with the potential to investigate the molecular pathogenesis in both research and diagnostic settings.Mitochondrial DNA (mtDNA) mutations are commonly found in the skeletal muscle of patients with mitochondrial disease, inflammatory myopathies and sarcopenia. The majority of these mutations are mtDNA deletions, which accumulate to high levels in individual muscle fibres causing a respiratory defect. Most mtDNA deletions are major arc deletions with breakpoints located between the origin of light strand (O Each mitochondrion harbours multiple copies of mtDNA and each cell contains multiple mitochondria. Point mutations and large-scale rearrangements (deletions or duplications) of the mitochondrial genome are associated with a wide-range of human clinical presentationsL) into the minor arc910H and OL) are essential for mtDNA duplicationL is dispensable14MtDNA deletions are frequently reported in disease and ageing2346transmitochondrial cybrid cellsMT-ND1 that is rarely deleted and MT-ND4 which is often deleted as the majority of deletions appear within the major arc1819MT-ND1 indicates the total amount of mtDNA present in the sample, whilst the relative level of MT-ND4 allows quantification of the proportion of the deleted species. Finally, an additional TaqMan real-time PCR multiplex assay, which allows quantification of deletions as well as mtDNA copy number by measuring two mitochondrial targets (MT-ND4 and D-Loop region) and a nuclear gene (β2M) has recently been describedThe gold standard technique for the detection and quantitative assessment of mtDNA deletions is Southern blotting; however, this requires large quantities of DNA and is therefore not suitable for small samples such as single cell preparationsMT-ND1 and MT-ND4. In order to facilitate analysis of a much broader spectrum of mtDNA deletions we have developed a new assay which simultaneously amplifies part of the D-Loop, MT-ND1 and MT-ND4. The D-Loop target is positioned within the non-coding region (NCR) of mtDNA, which has been shown to be conserved in 98.8% of mtDNA molecules reported to harbour a mtDNA deletionAlthough our duplex real-time PCR assay is a useful tool for quantifying major arc deletions of the mitochondrial genome, it fails to detect and quantify rarer mtDNA deletions which extend into the minor arc deleting or partially deleting both DNA samples from a variety of different tissues were used in this study including whole blood from healthy, young individuals (n = 3), muscle homogenate from a young control (n = 1) and single large-scale mtDNA deletion patients SD; n = 2), and single muscle fibres from sporadic inclusion body myositis patients (sIBM) (n = 4) and SD patients (n = 2). We used SD samples as they had well characterised major arc deletions and sIBM samples as they encompass unusual mtDNA rearrangements, and sinEthical approval was granted from Newcastle and North Tyneside LREC. The experiments were carried out in accordance with the approved guidelines. An informed consent was obtained from all subjects taking part in this study.MT-ND4 gene (605 bp) was amplified with the forward primer 5′-TGTAAAACTGCGGCCGCTCTCCCTCTACATATTTACCAC-3′ and the reverse primer 5′-CATGCGGCCGCTATGACCGTGGCTCAGTGTCAGTTCG-3′. The amplicon was sequentially digested with BsrBI and NotI and the resulting 580 bp fragment was inserted into the EcoRV and NotI restriction sites of the pcDNA3.1( + ) vector (Life Technologies) multiple cloning site. The forward and reverse primers 5′-GCAATGGCATTCCTAATGCTT-3′ and 5′-CAGGAATGCATCATGACCAGAGTGCGTCATATGTTGTTC-3′ were used to amplify a region of the MT-ND1 gene (724 bp). This PCR product was cut with BsmI and PsiI (642 bp) and inserted into the MT-ND4 pcDNA3.1( + ) vector, digested with BsmI and BstZ17I. Finally, a 634 bp sequence of the mitochondrial D-Loop was amplified with the forward primer 5′-TATCGAAGCTTCAACCCTCAACTATCACA-3′ and reverse primer 5′GTATCGGGTACCTGTGCAGACATTCAATTGTTA-3′. After digestion with KpnI and HindIII, the 621 bp product was inserted into the KpnI and HindIII restriction sites of the MT-ND4/MT-ND1 pcDNA3.1( + ) vector multiple cloning site (In order to obtain a template for standard curve generation a plasmid construct was engineered as follows. A portion of the ing site . The resMT-ND1 and MT-ND4 have been previously reportedMT-ND1 VIC-5′-CCATCACCCTCTACATCACCGCCC-3′-MGB (np 3506-3529), MT-ND4 FAM-5′-CCGACATCATTACCGGGTTTTCCTCTTG-3′-MGB (np 12111-12138), D-Loop NED-5′-ACATCACGATGGATCAC-3′-MGB (np 16559–6)  . All priMT-ND1 or MT-ND4) was amplified (singleplex), and in a reaction where the D-Loop, MT-ND1 and MT-ND4 were amplified simultaneously (triplex). All reactions were conducted on the same plate. The amplification curves were examined for consistency, the amplification efficiencies calculated using the curves’ slope values for each target [E = (10^(−1/slope)−1)*100] and compared between singleplex and triplex conditions. Similarly, serial dilutions of plasmid p1D7 (concentration of 50 ng/μl) were also validated on a separate plate in a triplex reaction, and amplification efficiencies were calculated in the same way.In order to validate the assay, five serial dilutions of young control DNA (concentration of 62.5 ng/μl based on Nanodrop measurement) extracted from a muscle homogenate (which we refer to as “standard”) were tested both in a single real-time PCR reaction, where just one target  were mixed in different ratios  in sextuplicate to test precision and accuracy of the assay. Finally, a set of single cell DNA samples (n = 40) from two patients with single mtDNA deletions and four sIBM patients (n = 60) were tested for mtDNA deletion load to verify whether this assay was sensitive enough for single cell analysis. Ratios are presented as a percentage. For example, an MT-ND4/MT-ND1 ratio of 90% signifies that MT-ND4 levels are 90% of MT-ND1 levels, in other words 10% of mtDNA molecules tested are harbouring an MT-ND4 deletion.Serial dilutions of the standard DNA were analysed in triplicate in eight separate experiments and amplification efficiencies were calculated in order to assess reproducibility across independent plates. In addition, three normal control DNA samples were quantified in a minimum of six independent experiments and DNA samples from selected single cells used in the triplex assay were amplified using a long range PCR protocol and 130 F (np 130–161) and 16382 R (np 16361–16382) oligonucleotide primers. PCR reactions were carried out using Prime Star GXL polymerase kit (Clontech) and the cycling conditions were: 35 cycles of 98 °C for 10 sec and 68 °C for 11 min. Amplified products were separated through 0.7% agarose gels and imaged using ChemiDoc (Biorad).We used deep sequencing to characterise mtDNA deletion breakpoints. Briefly, selected amplicons ( ≤ 9 kb), were purified using Agencourt AMPure XP reagent and assessed for concentration and quality using an Agilent 12000 DNA kit on a Bioanalyzer . 100 ng of each DNA sample processed using the Ion Xpress Plus Fragment Library Kit (Life Technologies) according to manufacturer’s instructions. The samples were fragmented using Ion Shear Plus Enzyme, then ligated to barcoded adapters. Fragments ~330 bp in length were recovered with E-gel Size Select 2% gels (Life Technologies). The libraries were then amplified and assessed on a Bioanalyzer using the Agilent High Sensitivity DNA Kit. The libraries were pooled in equimolar ratios , then clonally amplified onto Ion Sphere Particles using the Ion OneTouch system and the Ion OneTouch 200 Template kit v2. Subsequently, the Ion OneTouch ES Instrument allowed enrichment of template-positive Spheres using magnetic bead technology. The enriched samples were loaded onto an Ion 316v2 Chip for deep sequencing on an Ion Torrent system. Data were analysed using the Torrent Browser (v4.0.2) and alignments to the human mtDNA reference sequence were interpreted using IGV .For each ratio under study, the distribution of the ratio values for control samples was both examined visually and tested for normality using three common tests MT-ND1, MT-ND4 and D-Loop, using serially-diluted standard DNA. The amplification curves for each mtDNA target from the singleplex and triplex reactions overlapped ; 106.4 ± 9.7, 113.3 ± 7.8, 106.8 ± 5.7 (case 2); 102.8 ± 6.4, 106.5 ± 6.3, 103.7 ± 2.4 (case 3)  . The ratle range . HoweverMT-ND1/D-Loop and MT-ND4/D-Loop ratios in control muscle homogenate and whole blood DNA. Normalisation of the data is achieved by dividing each ratio by the average from all normal muscle homogenate and blood DNA tested, to achieve a normalised MT-ND1/D-Loop and MT-ND4/D-Loop ratio of 100%.To correct for the extra D-Loop binding, we established the normal ranges of the Results from 29 independent experiments, obtained from 4 samples from blood and muscle homogenate were analysed using the standard DNA standard curve, and 31 independent experiments from the same samples using plasmid p7D1. Both distributions were Gaussian, thus we were able to use the mean for normalisation, and the standard deviation to define a tolerance ( ± 1.96 x standard deviations).MT-ND1/D-Loop and MT-ND4/D-Loop ratios respectively; for the plasmid p7D1 standard curve the normalised tolerances were ± 14.5% and ± 10.6% respectively , whereas the MT-ND1 site was entirely preserved (around 100% MT-ND1/D-Loop ratio) . Both patients had previously characterised major arc deletions: SD1 harbouring a 8704 bp mtDNA deletion (breakpoints: np 7175 and np 15879) and SD2 a 4113 bp deletion (breakpoints: np 11262 and np 15375). p ratio) . Over 40MT-ND1 or MT-ND4, mimicking the results of respiratory-normal counterparts. A range of mtDNA deletions were detected in other COX-deficient single fibres from sIBM patients. The majority of cells had mtDNA deletions encompassing MT-ND4 only, but we also identified cells with reduced levels of either MT-ND1 or both MT-ND1 and MT-ND4 (We then investigated mtDNA deletions in single respiratory-deficient (COX-deficient) cells and respiratory-normal (COX + ve) cells (serving as endogenous control) from four sIBM cases. The data were analysed in 60 cells using standard DNA and/or plasmid p7D1 standard curves and normalised. Representative results are shown in d MT-ND4 . These uMT-ND1 deletion and combined MT-ND1 + MT-ND4 deletion) and subjected them to long range PCR amplification. The primers were chosen to amplify almost the entire wild type mitochondrial genome (~16.5 kb). As expected, the largest PCR product was derived from a cell devoid of mtDNA deletions (cell A), a band of approximately 7 kb was amplified from a cell with MT-ND1 (cell B) deletion and the smallest (~5 kb) deletion was detected in a cell harbouring MT-ND1 + MT-ND4 deletion (cell C)  . Deep se(cell C) . A shortng sites .MT-ND1/MT-ND4 real-time PCR duplex assay developed by our group18L and extending far into the minor arc, it is very important to develop a tool to easily discern and quantify these deletions in cells and tissues. An in-depth review of mtDNA rearrangements by Damas et al. demonstrates that the most conserved region of mtDNA (preserved in 98.8% of mtDNA molecules in which deletions have been reported to date) is the non-coding control region, containing the D-loopMT-ND1 and MT-ND4. The D-Loop region has been previously used as a target in a multiplex assay in conjunction with MT-ND4 allowing detection of major arc deletions onlyThe purpose of this study was to design an assay allowing detection and quantification of the great majority of mtDNA deletions in single cells. The MT-ND1, MT-ND4 and D-Loop) as they all amplified with the same efficiency in singleplex and triplex reactions.One of the challenges of developing a multiplex real-time PCR assay is avoiding non-specific interactions between primers amplifying different DNA targets. Therefore it is essential to directly compare amplification efficiencies of each target tested both individually and in the multiplex reaction. There was no inhibitory effect of any of the three targets (H and terminates just after termination-associated sequence (TAS)Xenopus oocytes)The D-Loop target used in this assay binds to the non-coding region (NCR) of the mitochondrial genome. The NCR spans approximately 1.1 kb between nucleotide positions 16024 and 576Phe and LSP–the fragment of NCR not associated with RNA, RNA/DNA hybrid or 7S DNA. However, it was not feasible to find an optimal primer pair in this short sequence of approximately 130 nucleotides.Primers targeting the triple-stranded region will thus bind to both the H-strand and 7S DNA if it is present, which will perturb the ratio of the D-Loop binding compared to other mtDNA targets. To avoid this, we attempted to design primers binding to the region between tRNAMT-ND1/D-Loop and MT-ND4/D-Loop ratios, as the amount of D-Loop could potentially be influenced by disease. The latter would involve scaling the data by the mean amount of D-Loop in healthy cells  from several cases of the same disease.Nevertheless, we showed that by quantifying the variability in the D-Loop ratios from controls it was possible to normalise the data obtained using the D-Loop primers/probe. Our data demonstrate that such normalisation was sufficient to enable an accurate assessment of the deletion load in all samples. This step was not included in the previous study despite the D-Loop primers binding within the triple stranded region of the NCRMT-ND4/D-loop and MT-ND4/MT-ND1 ratios in SD1 (34.79% and 33.63% mtDNA deletion respectively) and SD2 (70.45% and 70.41% respectively). Single laser-microdissected COX-deficient muscle fibres did not differ by more than 0.6% between MT-ND4/D-loop and MT-ND4/MT-ND1. In COX-deficient cells from sIBM cases we detected some without mtDNA deletions, some with major arc deletions removing only the MT-ND4 gene, some with minor arc deletions removing both MT-ND1 and MT-ND4 genes, and some with deletions removing MT-ND1 but preserving MT-ND4. Long range PCR and deep sequencing of mtDNA obtained from a representative group of three single cells with isolated MT-ND1, combined MT-ND1 + MT-ND4 deletion and “no deletion” confirmed the expected locations of deletion. Interestingly, the cell, in which no deletion had been detected using the triplex assay, harboured a short mtDNA deletion which only encompassed a proportion of the MT-ND4 gene. The identification of such a variety of mtDNA deletions in sIBM is in agreement with previously published reports from inflammatory myopathiesMT-ND2 gene which will be missed using our assay. This highlights the importance of developing new multiplex PCR-based techniques to permit the detection of other, currently undetectable, mtDNA deletions in single cells. In summary, we have shown that the addition of a D-Loop target to the established MT-ND4/MT-ND1 assay allows quantification of minor arc mtDNA deletions, providing the opportunity for more comprehensive studies into the role of mtDNA deletions in disease. Unlike the previously published real-time PCR assay incorporating the D-Loop targetWe tested the triplex real-time assay on a range of samples (homogenate and single cell DNA) from single deletion patients and sIBM, and we obtained reliable and reproducible results. As confirmed before by sequencing of the mtDNA deletion breakpoints, both SD patients’ muscle homogenate tested in this study contained major arc deletions, which were detected with K.A.R. carried out the experimental work and wrote the majority of the manuscript with contributions from J.P.G. and H.A.L.T. J.P.G. performed the statistical analysis. H.A.L.T. constructed the p7D1 plasmid and advised on many aspects of the methodology. J.P.G., H.A.L.T., D.M.T. and R.W.T. read and reviewed the manuscript.How to cite this article: Rygiel, K. A. et al. Triplex Real-Time PCR - an improved method to detect a wide spectrum of mitochondrial DNA deletions in single cells. Sci. Rep.5, 9906; doi: 10.1038/srep09906 (2015).


Phlebovirus, family Bunyaviridae). Vaccination is the only known effective strategy to prevent the disease, but there are no licensed RVF vaccines available for humans. A live-attenuated vaccine candidate derived from the wild-type pathogenic Egyptian ZH548 strain, MP-12, has been conditionally licensed for veterinary use in the U.S. MP-12 displays a temperature-sensitive (ts) phenotype and does not replicate at 41°C. The ts mutation limits viral replication at a specific body temperature and may lead to an attenuation of the virus. Here we will review well-characterized ts mutations for RNA viruses, and further discuss the potential in designing novel live-attenuated vaccines for RVF.Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to the African continent. RVF is characterized by high rate of abortions in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. RVF is caused by the Rift Valley fever virus (RVFV: genus  Rift Valley fever virus has a tripartite negative-stranded RNA genome designated Small (S)-, Medium (M)-, and Large (L)-segments. The S-segment encodes two open reading frames (ORF) for a nucleoprotein (N) and a non-structural protein (NSs) in an ambi-sense manner. The M-segment encodes a single ORF for a polyprotein precursor. The precursor protein is co-translationally cleaved into four different proteins: Gn, Gc, 78-kD protein, and a non-structural protein (NSm). The L-segment encodes a single ORF for the RNA-dependent RNA polymerase (L) protein.DC-SIGN, dendritic cell specific intercellular adhesion molecule-3-grabbing non-integrin, is a receptor for RVFV and binds to oligosaccharides attached to virions . After vRift Valley fever virus encodes two non-structural proteins, NSs and NSm. Both proteins are dispensable for viral replication. However, NSs serves as a major virulence factor as it counteracts host antiviral responses. NSs suppresses host general transcription by interrupting the assembly of transcription factor (TF) IIH, which is essential for the function of cellular RNA polymerase I or II . RVFV NSRift Valley fever virus is an arbovirus and can replicate in both mosquito and mammalian hosts in nature. RVFV can replicate at 28°C in insect cells , and at Figure 1). The MP-12 vaccine does not replicate efficiently in vivo, though the S-segment encodes a functional NSs gene. Currently, little is known about ts mutations for RVF vaccine candidates. The RVFV MP-12 strain was developed by 12 serial plaque isolations in human lung diploid (MRC-5) cells in the presence of a chemical mutagen, 5-fluorouracil . As a re3 pfu, i.p)  . The stuCulex pipiens, which fed on hamsters infected with the pathogenic ZH501 strain. T1 strain displayed a ts phenotype at 41°C, and produced uniformly small plaques. The T1 strain is also highly attenuated in hamsters, and the LD50 is >6.3 × 105 pfu (i.p). On the other hand, the RVFV T46 strain, which was isolated from Aedes taeniorhynchus that fed on ZH501-infected gerbils, also predominantly produced small plaques, but was pathogenic in hamsters, without showing a ts phenotype. As the full genome sequences are available , we analyzed the mutations that occurred in the T1 and T46 strains compared to the parental ZH501 strain. The T1 strain encodes two mutations in the N gene: the G144U (G to V) mutation and a deletion of A at nt.640, which causes a frame-shift and a premature termination of N protein synthesis. T1 strain also encodes a mutation in the 5′-M-untranslated region (C3818U), and two silent mutations in the L-segment (C282U and A2691G). On the other hand, T46 strain encodes only one mutation in the M-segment (U1174A: M to K). The T1 strain has not been further evaluated for vaccine development.Meanwhile, in vivo and can be screened for using culture cells. Ts phenotypes have been characterized for many viruses . Non-segmented negative-stranded RNA viruses encode six conserved regions  in the RNA-dependent RNA polymerase  ts114 mutant encodes three amino changes  in the L-segment compared to the non-ts parental strain. The ts phenotype occurs from D575G, which is located between PreMotif A and Motif A in Region III . The ts1The respiratory syncytial virus (RSV) cold-adapted, temperature-sensitive (cpts) 248/404/1030 is a live-attenuated vaccine strain . It encoThe live-attenuated FluMist vaccine consists of master donor virus for influenza virus A (MDV-A) and influenza virus B (MDV-B). MDV-A has been developed by serial passages of the wt A/Ann Arbor/6/60 strain in primary chicken kidney tissue culture at successively low temperatures down to 25°C . MDV-A eTemperature-sensitive mutants have been successfully developed as licensed vaccines, or candidate vaccines, in particular, for respiratory diseases: e.g., FluMist (influenza A and B viruses), FluAvert (equine influenza virus) , and MED5 pfu) or intranasal vaccination (~1 × 104 pfu in 1.0 ml volume) of rhesus macaques with MP-12 induced serum neutralizing IgG (80), which was maintained for 6 years. The vaccinated rhesus macaques were protected from an aerosol challenge of the pathogenic ZH501 strain  or ts mutations, to fully attenuate the S-, M-, and L-segments, toward the development of highly safe and efficacious RVF vaccines .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.