In addition, hypoxia downregulated antigen presenting proteins like TAP 1/2 and LMP7 that are known to have a dominant role in surface display of peptide-MHC complexes. Corroborating oxygen-dependent regulation of MHC antigen presentation, hyperoxia (60% oxygen) transcriptionally upregulated MHC expression and increased levels of TAP2, LMP2 and 7. In conclusion, this study reveals a novel mechanism by which intra-tumoral hypoxia and HIF can potentiate immune escape. It also suggests the use of hyperoxia to improve tumor cell-based cancer vaccines and for mining novel immune epitopes. Furthermore, this study highlights the advantage of 3D cell cultures in reproducing hypoxia-dependent changes observed in the TME.Human cancers are known to downregulate Major Histocompatibility Complex (MHC) class I expression thereby escaping recognition and rejection by anti-tumor T cells. Here we report that oxygen tension in the tumor microenvironment (TME) serves as an extrinsic cue that regulates antigen presentation by MHC class I molecules. In support of this view, hypoxia is shown to negatively regulate MHC expression in a HIF-dependent manner as evidenced by (i) lower MHC expression in the hypoxic TME  The tumor microenvironment (TME) has been identified as a critical factor in controlling the progression and metastasis of cancer, along with dictating responses to conventional anti-tumor therapies –3. In paImmunogenicity is critical in the establishment of an effective adaptive immune response , 12 and MHC class I expression is primarily regulated transcriptionally by integration of tissue-specific intrinsic signals as well as extrinsic signals from the microenvironment , 25. In In this study, we focused on the regulation of MHC class I expression on tumors and found a positive correlation between local tissue oxygen tension and levels of MHC. Mechanistic studies showed a HIF-dependent MHC downregulation in hypoxic environments. In addition, oxygen may affect the density of peptide-MHC complexes at the cell surface by regulating TAPs and LMPs.2) or normoxia (21% O2) for 48h, followed by examination of changes in MHC expression. We show that, independent of anatomical location, both pulmonary tumors , whereas 16% of the population of cells from spheroid cultures was hypoxic, even under normoxic conditions , further confirming our observations of hypoxia-based downregulation of antigen presentation  enhanced MHC class I expression . This re T cells , 34. Thein vitro platform, we next explored the mechanisms by which oxygen tension affected MHC expression. Using MCA205 spheroids cultured in vitro under 1%, 21%, or 60% O2 for 48h and MCA205 pulmonary tumors from mice exposed to 10%, 21%, or 60% O2 for 48h, MHC class I mRNA levels were analyzed by RT-qPCR. We found that hypoxia downregulated while hyperoxia upregulated MHC class I mRNA expression both in vivo  cell lines that are extensively characterized and are known to have constitutively activated HIF expression due to inactivation of VHL gene . We compin vivo and in vitro. These components of the antigen processing machinery have a central role in determining the surface density of peptide-MHC complexes [in vitro [We next explored the effects of oxygen tension on constitutive expression of TAP1, TAP2, LMP2 and LMP7 proteins, both omplexes , 28 and in vitro .2 for 48h and MCA205 spheroids cultured in vitro under 1%, 21% or 60% O2 for 48h were analyzed by Western blot. Hypoxia significantly downregulated TAP1, TAP2 and LMP7 both in vitro  and functional VHL protein , along with siRNA of HIF, allowed us to firmly implicate the role of HIF in downregulating MHC class I expression. Interestingly, there is a noticeable delay from the induction of HIF and the downregulation of MHC class I. This observation, in conjunction with (i) an undefined hypoxia-response element in the MHC promoter (ii) evidence in literature that HIF-dependent gene suppression is typically due to indirect mechanisms rather than direct promoter binding [Mechanistically, we show that oxygen tension affects transcription of MHC class I heavy chains, with hypoxia downregulating and hyperoxia upregulating mRNA levels of MHC class I . Further binding , is indi binding , 42 are in vivo and in vitro. Since exposure to hyperoxic atmospheres can reverse tissue hypoxia and HIF-1α expression in tumors [in vitro data suggests the presence of a second mechanism. In vitro cell culture at 21% oxygen, especially as conventional 2D cultures, is not deeply hypoxic , 4T.1 (ATCC #CRL-2539), P815 (ATCC #TIB-64), RMA , B16 transfected with ovalbumin , MCA205 transfected with ovalbumin , EL4 (ATCC #TIB-39), renal cell carcinoma cells RCC4, UMRC2, Caki-2, A-498 and 786–0 with or without the lentiviral vector (pcDNA3) for VHL gene , were cCells were cultured as monolayers using conventional 96-well or 35mm culture plates . For spheroid culture, cells were grown in Scivax plates, either the 35mm dishes or 96-well plates . Cells were cultured for 24-48h to allow for the formation of spheroids before using in experiments, with the exception of RNAi experiments. Images of cells in 2D and 3D systems were captured using an inverted microscope fitted with Olympus camera.Female C57BL/6J (B6) mice, 11 weeks old, were purchased from Jackson Laboratory. All animal experiments were conducted in accordance with IACUC guidelines of Northeastern University .5 MCA205 cells suspended in 200μl of HBSS. For establishment of subcutaneous tumors, B6 mice were injected in the flank with 3×105 MCA205 cells suspended in 100μl of HBSS. When tumors reached 200-250mm3, day 11 (pulmonary tumors) or day 15 (subcutaneous tumors), mice were randomly divided into three groups and exposed to normoxia (21% O2), hypoxia (10% O2) or hyperoxia (60% O2) for 48h. Hypoxic exposure was achieved using the Biospherix Oxycycler (#A84XOV). Hyperoxic (60% oxygen) exposure was achieved using chambers as described previously [For establishment of pulmonary tumors, B6 mice were injected intravenously with 1×10eviously . Mice we2, 5% CO2), normoxia  or hyperoxia  for 48h. Hypoxyprobe™-1 Kit  was used to detect hypoxia. Briefly, cells in 2D and 3D systems were incubated with 150 μM of Hypoxyprobe for 1h. The cells were then extensively washed and levels of MHC class I and Hypoxyprobe were assessed using flow cytometry.Cells grown in 2D and 3D culture systems were exposed to hypoxia , human HLA-ABC , Hypoxyprobe . Dead cells were excluded by using the live/dead fixable dead cell stain . Tumor cells were analyzed using FlowJo (Tree Star) software.Pulmonary or solid tumors were excised and subjected to gentle mechanical dissociation by chopping the tumors into small sections and teasing apart tissue with needles. Enzymatic dissociation of tumors was intentionally avoided to prevent damage to cell surface markers. Cells cultured in 2D cultures were harvested by using cell dissociation buffer . 3D spheroids were harvested and dissociated by gentle pipetting. The cells were then incubated with mAbs, and acquired on a FACSCalibur. Monoclonal antibodies used were mouse MHC class I , mouse H-2KRNA was extracted from mice with 11-day established pulmonary tumors or spheroids treated with different oxygen tensions for 48h using the RNeasy Kit . RT-PCR was performed using the RT2 SYBR green PCR master mix  on an Applied Biosciences 7300. Primers were purchased from SABiosciences . Ribosomal protein L32  was included as the internal control.2. Efficacy of gene knockdown was assessed using Western blot.MCA205 cells were reverse transfected with scrambled control  or siRNA specific to HIF-1α gene  or HIF-2α gene , using Lipofectamine reagent per manufacturer’s instructions . The cells were then grown on Scivax plates for 48h under 21% or 1% Oin vivo tumor samples were lysed in RIPA buffer  with protease and phosphatase inhibitors . 40μg of protein was loaded per sample and fractionation with SDS–PAGE was followed by wet transfer to PVDF membrane. Antibodies used were HIF-1α ; HIF-2α ; TAP1 ; TAP2 ; LMP2 ; LMP7 ; β-actin .Spheroids and in vitro with 100 ug/mL OVA for 48h. CD8 T cells were then isolated  and used in the cytotoxicity assay.Splenocytes from OT-1 mice were activated 2 post-spheroid formation. Spheroids were dissociated using the spheroid dispersion solution  before use in cytotoxicity assays. To assess cytotoxicity, 10,000 target cells were incubated for 4h at 37°C, 21% O2, with different ratios of effector T cells as indicated. At the end of 4h, propidium iodide was used to determine the proportion of dead tumor cells .Ovalbumin-transfected MCA205 tumor cells were stained with Cell Tracker Deep Red  and grown in 3D cultures for 24h in 21% or 1% OP values are listed within the figures and figure legends.The significance of differences was analyzed by the Student’s t-test (two-sided). S1 Fig, P815 mastocytoma , RMA T lymphoma  and EL4 thymoma  were cultured as 2D monolayers (indicated as 2D) or as 3D spheroids (indicated as 3D) and cultured under 21% O2 or 1% O2 for 48h. Levels of MHC class I expression was determined using flow cytometry. Representative histograms of 4 independent experiments are shown. Grey filled: unstained control; red: hypoxia; blue: normoxia. MFI: mean fluorescence intensity. Inset of S1 G,H: 40X magnification of El4 cells grown at 1% O2 in 2D culture (G) or in 3D culture (H) representing the growth of these cells in clumps irrespective of the culture format.4T.1 breast carcinoma (TIF)Click here for additional data file.S2 Fig(S2A) or flat monolayer (S2B) in normoxic conditions (21% oxygen) and levels of hypoxia in each culture system assessed using hypoxyprobe. About 16% of the population was hypoxic in the 3D spheroids whereas there was no detectable levels of hypoxia in the 2D cultured cells. In 3D spheroids of EL4, about 20% of the population was hypoxic (S2D). Representative contour plots of three independent experiments shown.  Mean fluorescent intensity (MFI) of MHC class I expression on 3D spheroids  from less hypoxic (HP low) and more hypoxic (HP high) regions showed inverse correlation between hypoxia and MHC class I expression. Each point on graph represents an independent experiment with the average represented as a dash.MCA-205 fibrosarcoma cells were grown as spheroids (TIF)Click here for additional data file.S3 Fig, P815 mastocytoma , RMA T lymphoma  and EL4 thymoma  were cultured as 2D monolayers (indicated as 2D) or as 3D spheroids (indicated as 3D) and cultured under 21% O2 or 60% O2 for 48h. Levels of MHC class I expression was determined using flow cytometry. Representative histograms of 4 independent experiments are shown. Grey filled: unstained control; blue: normoxia; green: hyperoxia. MFI: mean fluorescence intensity.4T.1 breast carcinoma (TIF)Click here for additional data file.S4 Fig: siRNA mediated knockdown of HIF-1α reversed hypoxic downregulation of MHC class I expression as compared with the scrambled, non-targeting (NT) siRNA control. MCA205 tumor cells were reverse transfected with scrambled siRNA (NT) or with HIF-1α specific siRNA and cultured as 3D spheroids under 1% or 21% oxygen for 48h. Levels of MHC class I surface expression was determined using flow cytometry; quantitative analysis of representative histograms shown in (A). Levels of MHC class I transcripts were assessed using RT-qPCR (B). Average data of 3 independent experiments are shown.: Flow cytometry assessment of surface expression of HLA-ABC (S4C) and RT-qPCR analysis of HLA-ABC transcript levels (S4D) on paired isogenic renal cell carcinoma cell lines RCC4, UMRC2 and CAKI2. Each pair had the parental cell line that lacked endogenous wild-type VHL  and one with vector stably expressing functional VHL (VHL restored). Restoring VHL function and thereby reducing HIF expression, significantly increased HLA-ABC surface expression and transcript levels in the cells. Average data of 4 independent experiments are shown.(TIF)Click here for additional data file.S5 Fig(S5A) or 21% (S5B) oxygen for 48h. Levels of MHC class I surface expression was determined using flow cytometry . RT-qPCR was used to analyze MHC class I transcript levels. Ribosomal protein L32 was used as internal control (S5C). Efficacy of gene knockdown was assessed using western blot (S5D). β-actin was used as the loading control. Representative data of 2 independent experiments are shown.siRNA mediated knockdown of HIF-1α reversed hypoxic downregulation of MHC class I expression as compared with the scrambled, non-targeting (NT) siRNA control. EL4 tumor cells were reverse transfected with scrambled siRNA  or with HIF-1α specific siRNA (blue histogram) and cultured as 3D spheroids under 1% (PNG)Click here for additional data file.S6 Fig(S6A-C): siRNA mediated knockdown of HIF-1α, HIF-2α or both reversed hypoxic downregulation of MHC class I expression as compared with the scrambled, non-targeting (NT) siRNA control. MCA205 tumor cells were reverse transfected with scrambled siRNA (NT) or with HIF-1α, HIF-2α or both HIF-1α and HIF-2α specific siRNA and cultured as 3D spheroids under 1% or 21% oxygen for 48h. Levels of MHC class I surface expression was determined using flow cytometry (S6A). Transcripts levels were determined by RT-qPCR, with Ribosomal protein L32 as internal control (S6B). Efficacy of gene knockdown was assessed using western blot (S6C). Greater than 90% knockdown of HIF-1α/ HIF-2α was achieved. β-actin was used as the loading control. Average data of 3 independent experiments are shown.(S6D-F): Flow cytometry assessment of surface expression of HLA-ABC on paired isogenic renal cell carcinoma cell lines A-498 (D) and 786–0 (E). Each pair had the parental cell line that lacked endogenous wild-type VHL  and one with vector stably expressing functional VHL (VHL restored). Restoring VHL function and thereby reducing HIF expression, significantly increased HLA-ABC expression on the cells  and transcript levels (S6F). Representative histograms of 4 independent experiments are shown. Grey filled: Unstained control; Red: VHL null phenotype; Blue: VHL restored phenotype. For RT-qPCR, average data from 4 independent experiments shown.(TIF)Click here for additional data file.S7 FigS7A,B) 60% O2 significantly upregulated expression of the immunodominant peptide of ovalbumin, SIINFEKL. B16 melanoma cells (S7A) or MCA205 fibrosarcoma cells (S7B) transfected with ovalbumin were cultured as 3D spheroids for 48h either under 21% or 60% O2. Levels of MHC class I-SIINFEKL surface expression was determined using flow cytometry, with overnight incubation at 4°C with the mAb to MHC-SIINFEKL. Representative histograms of 4 independent experiments are shown. Grey filled: unstained control; blue: normoxia; green: hyperoxia. MFI: mean fluorescent intensity.((TIF)Click here for additional data file.


TT16 and TT15 independently control proanthocyanidin biosynthesis in the inner integuments of the Arabidopsis seed testa and their function extends beyond this tissue. tt16 and tt15 mutants display a very similar flavonoid profiles and patterns of PA accumulation. By using a combination of genetic, molecular, biochemical, and histochemical methods, we showed that both TT16 and TT15 act upstream the PA biosynthetic pathway, but through two distinct genetic routes. We also demonstrated that the activity of TT16 in regulating cell fate determination and PA accumulation in the endothelium is required in the chalaza prior to the globular stage of embryo development. Finally this study provides new insight showing that TT16 and TT15 functions extend beyond PA biosynthesis in the inner integuments of the Arabidopsis seed coat.Flavonoids are secondary metabolites that fulfil a multitude of functions during the plant life cycle. In Arabidopsis proanthocyanidins (PAs) are flavonoids that specifically accumulate in the innermost integuments of the seed testa (i.e. endothelium), as well as in the chalaza and micropyle areas, and play a vital role in protecting the embryo against various biotic and abiotic stresses. PAs accumulation in the endothelium requires the activity of the MADS box transcription factor TRANSPARENT TESTA (TT) 16 (ARABIDOPSIS B-SISTER/AGAMOUS-LIKE 32) and the UDP-glycosyltransferase TT15 (UGT80B1). Interestingly  TTG2/DSynthesis . It has late PAs .TT16/ABS/AGL32 (ARABIDOPSIS B-SISTER/ AGAMOUS-LIKE 32) encodes another TF involved in the transcriptional regulation of PA biosynthesis that belongs to the MADS box family  double mutant  were performed as described in All PCRs were carried out using high-fidelity Phusion DNA polymerase, according to the manufacturer’s instructions (Thermo Scientific Finnzymes). PCR products were subsequently sequenced after recombination or cloning into their destination vectors. All the primers used in this study are described in tt16-1 . The double tt15 tt16 mutant was obtained by crossing the tt15-2 and tt16-1 alleles. Plants expressing β-glucuronidase (GUS) under the control of the TT8 and TT15 promoters are described in Arabidopsis accession Wassilewskija (WS) was used as wild-type control. The mutant lines BANYULS/ANR, At1g61720; CHS/TT4, At5g13930; TT2/MYB123, At5g35550; TT8/bHLH042, At4g09820; TT15/UGT80B1, At1g43620; TT16/AGL32/ABS, At5g23260.The Arabidopsis gene IDs were as follows: PA staining of 4-day-old seeds was carried out using vanilline reagent as described in TT16 (pTT16: 1597 bp prior to the ATG) and BAN (pBAN: 236 bp prior to the ATG) promoters to the GatewayTM recombination cassette were carried out as described for the promoter of TT8 (1.5 kb prior to the ATG) in pTT16 and pBAN were PCR-amplified from genomic DNA (WS) with the pTT16-5′-HindIII/pTT16-3′-XbaI and pBAN-5′-HindIII/pBAN-3′-XbaI primer pairs, respectively. The obtained DNA fragments were subsequently cloned into the pBIB-Hyg-GTW vector  were PCR-amplified from WS cDNAs using the cTT2-B1/cTT2-B2 and cTT15-B1/cTT15-B2 primer pairs, respectively. Genomic TT16 (gTT16), which corresponds to the DNA sequence between the ATG and stop codons, including both exons and introns, was PCR-amplified from genomic DNA (WS) using the cTT16-gATG-B1/cTT16-gSTOP-B2 primer pair. The same primers were also used to amplify from WS cDNA cTT16L/ABSI (long: 759 bp) and cTT16S/ABSII (short: 744 bp) CDSs corresponding to the two splice variants of TT16. The obtained DNA fragments were then BP-recombined into the pDONR207 vector (GatewayTM).TT2 and TT15 CDSs were recombined into the pMDC32 vector .2 vector  for overcTT16L/ABSI and cTT16S/ABSII CDSs were LR-recombined into the pTT16:GTW vector.gTT16 was LR-recombined into pTT16:GTW, pTT8:GTW, pBAN:GTW, and pMDC32 vectors.TT2 promoter (2 kb prior to the ATG) for GUS analysis was cloned into pDONR207 and then LR-recombined into pGWB3  and incubated for 1 h at 4 °C in the fixation buffer (1× PBS (Eurobio), 2% formaldehyde, and 0.1% triton X-100) after vacuum treatment (three times). Samples were then dehydrated using a series of increasing ethanol concentration in PBS  at 4 °C (2 h each). Siliques were then stained using toluidine blue (0.01% in absolute ethanol), and transferred into a 2:1 followed by a 1:1 absolute ethanol–wax  solution for 2 h at 40 °C each, and finally transferred into a 1:2 solution (overnight at 40 °C). Samples were then incubated twice for 3 h in 100% wax solution at 40 °C before polymerization. Cross sections of 8 μm were finally cut using a Leica RM2165 microtome, and sample ribbons were placed on a drop of sterile water (Versol) on polyethylene slides, and left to dry overnight at 37 °C.Immunolabelling using the JIM4 and JIM8 monoclonal antibodies  was carried out as described in tt16 and tt15 share a very similar pattern of flavonoid accumulation. They both have lower content of the major accumulated flavonols (quercitrin and quercetin-3-rhamnoside) and very little PAs  showed that the tt15 had decreased PA accumulation strongly correlated with a decrease of BAN mRNA levels   was overexpressed in tt15 (using the p70S promoter). As a positive control for this complementation assay, overexpression of the TT15 CDS was also carried out using the same promoter. In both cases PA accumulation increased in immature and mature transgenic seeds, indicating that, similarly to TT16, TT15 functions upstream of the PA biosynthetic pathway (The two mutants ttle PAs  when compathway) , which w pathway .tt16, the endothelium cell shape of tt15 does not seem to be affected, suggesting that TT15 does not function upstream of TT16  and TT15 CDS were overexpressed (p70S promoter) in tt15 and tt16, respectively , confirming that TT15 and TT16 act through distinct genetic routes  and the ii1′ (parenchymatous cells) cell layers are derived from the ovular endothelium through periclinal divisions  expression starts prior to fertilization in the micropyle area and then propagates throughout the PA-accumulating cells upon fertilization  when compared with WT seeds (The main MBW complex involved in PA biosynthesis is composed of TT2 (R2R3-MYB), TT8 (bHLH) and TTG1 . TT2 andng cells . TT8 expTT16 for tt16 complementation experiments (TT8 (pTT8) and BAN (pBAN) were used, as the expression of the corresponding genes in PA-accumulating cells initiates at distinct developmental stages . We first successfully complemented the transparent testa (tt) phenotype of tt16 using the TT16 promoter , confirming that the genomic DNA fragment containing the TT16 CDS was functional  and pTT8:GUS activities  is fused to the uidA reporter gene, no GUS activity is observed in seeds, whereas the expression of TT16 in PA-accumulating cells requires the regulatory sequences that are present up to 3.4 kb prior to the TT16 start codon  and 744 bp (TT16S/ABSII) long (Brassica napus (canola), four homologues of the Arabidopsis TT16L variant (i.e. BnTT16.1 to BnTT16.4) displaying between 75 and 80% identity at the protein level were identified and characterized; each of them complements the tt16 mutation when ectopically expressed using the CaMV 35S promoter  seeds of WT, tt16, and tt16 transgenic lines were thus treated with vanilline reagent, which specifically stains PAs red . This strategy revealed that TT16 activity is required in the chalaza area prior to the globular stage of embryo development for proper endothelium development and PA accumulation  during seed development, we confirmed that a TT16 genomic sequence spanning the CDS region plays a key role in the control of PA accumulation in seeds (Brassica napus), we found that TT16L (as well as TT16S) was not sufficient to restore tt16 mutant phenotypes , a key transcriptional regulator of floral organ specification, for which most of the regulatory elements that control its expression are located in the second intron of the gene   . TT16 coTT16 and GOA/AGL63 (GORDITA) are paralogous genes  in response to TT16 activity. In addition a recent study has highlighted the importance of auxin production in the endosperm to initiate the development of the Arabidopsis seed coat, a process involving another MADS box TF, AGL62  protein complexes . Similarpression . Similar delayed . BecauseJXB online.Supplementary data are available at gTT16 overexpression are enhanced in tt15 mutants when compared with wild-type plants.Fig. S1. Growth defects in vegetative tissues (stunted plants with curly leaves and reduced flower size) due to tt16 and tt15 complementation experiments.Fig. S2. Arabidopsis seed immunohistolabelling using monoclonal antibodies targeting specific arabinogalactan-proteins present in the cell wall of the testa, in TT8 promoter activity in developing wild-type ovules and seeds revealed by the detection of GUS activity.Fig. S3. Pattern of Table S1. Primers used in this study.Supplementary_Table_S1Click here for additional data file.Supplementary_Figures_S1_S3Click here for additional data file.


Serping1 or C1 inhibitor, which is known to inhibit the initiation of the complement cascade. The complement cascade is composed of three pathways; the classical, lectin, and the alternative pathway; the first two are inhibited by C1 inhibitor, and all three converge at the level of C3. Knockdown or knockout of Serping1 affected neuronal stem cell proliferation and impaired neuronal migration in mice. Knockdown of Serping1 by in utero electroporation resulted in a migration delay of the electroporated cells as well as their neighboring cells demonstrating a non-cell autonomous effect. Cellular polarity was also affected. Most importantly, expression of protein components mimicking cleaved C3 rescued the knockdown of Serping1, indicating complement pathway functionality. Furthermore, we propose that this activity is mediated mainly via the complement peptide C5a receptors. Whereas addition of a selective C3a receptor agonist was minimally effective, the addition of a dual C3aR/C5a receptor agonist significantly rescued Serping1 knockdown-mediated neuronal migration defects. Our findings suggest that modulating Serping1 levels in the developing brain may affect the complement pathway in a complex way. Collectively, our findings demonstrate an unorthodox activity for the complement pathway during brain development.Current knowledge regarding regulation of radial neuronal migration is mainly focused on intracellular molecules. Our unbiased screen aimed at identification of non-cell autonomous mechanisms involved in this process detected differential expression of  Serping1  encoding for the C1 inhibitor protein. C1 inhibitor is a member of the serpin family of protease inhibitors  with an estimated prevalence of 1:50,000, where about 25% of the patients exhibit Serping1 in the developing brain. A study examining single cell RNA expression in the E14 developing mouse brain revealed that Serping1 is expressed in subventricular zone (SVZ) basal progenitors  in an unbiased screen aimed at identifying molecules which may affect neuronal migration in a non-cell autonomous way  or a multipolar appearance (following Dclk shRNA treatment). The results of the screen showed that the levels of Serping1 were 2.10-fold higher in Dclk shRNA vs. Dcx shRNA. The differences at the mRNA level were verified by realtime qPCR  demonstrated that Serping1 mRNA is expressed in the developing cortex, where the highest expression levels are seen in the SVZ as previously reported , as well as SERPING1 protein in the developing brain . Neuronal stem cell proliferation was tested by application of a short IdU pulse, which is incorporated during S phase following by immunostaining of embryonic brain sections using the respective antibodies. Embryos were in utero electroporated at E13 and analyzed at E14. The analysis was targeted at the IdU/GFP positive cells, which were likely to receive the respective shRNA plasmids. There was a statistically significant difference between the IdU/GFP double positive cells, where the introduction of Serping1 shRNA reduced the number of cells in S-phase  or IdU/EdU/GFP triple positive cells  thus suggesting that the cell cycle length was not affected. The proportion of IdU/GFP double positive cells was reduced in Serping1 shRNA consistent with previous experiments . In addition significantly more GFP only positive cells were noted in case of Serping1 shRNA treatment, suggesting that these cells were not actively in S-phase during the tested period . Collectively, our results suggest that knockdown or knockout of Serping1 results in a reduction in the number of cycling cells of both ventricular zone  and intermediate  progenitors during cortical development.To investigate the role of s Figure . SimilarSerping1 in regulation of radial migration was investigated by knockdown of the gene using in utero electroporation of Serping1 shRNA and by studying knockout embryos generated by CRISPR/Cas9 gene editing. Serping1 shRNA significantly impaired radial neuronal migration   and significantly improved the position of the cells in three out of four bins  Figure . The poss Figure . Additios Figure . Neurona Figures . Neurons Figures . Next, t Figures . Althoug Figures .Serping1 may affect neuronal migration in a non-cell autonomous way in addition to the cell autonomous effect observed above was examined  successfully migrated through the layer of previously born cells (green) treated with control shRNA . However, it also covalently binds and inhibits the activity of the closely related MASP1 and MASP2 proteases, which initiate the lectin pathway. It can also physically bind and functionally affect the interaction between complement factor C3b and complement factor B, and thus to interfere also with the alternative pathway is important for cortical development at several stages. At early stages of cortical development Serping1 is important for the proliferation of neuronal stem cells. A decrease was noted in the number of cells in S phase that include both radial glia and intermediate progenitors. The effect may be transient since the total number of TBR2 positive basal progenitors, cells was not affected. Serping1 has been reported to be expressed in the SVZ basal progenitors  and knockdown of Masp2 resulted in a statistically significant increase in the number labeled progenitors (IdU positive)  rescued the migration impairment neurons. Moreover, our data indicate that signaling is likely to be also transmitted through the complement C5a receptors, which when activated, completely rescued neuronal migration deficits observed in the case of Serping1 knockdown. It should be noted that this study could not distinguish between a role for C5a receptor 1 or C5a receptor 2 (C5L2). However, it has been previously demonstrated that perturbations in C5a receptor 1 signaling during rodent brain development can result in select neuronal defects   . The control shRNA plasmid used is pLKO.1-TRC control (Addgene) containing non-hairpin 18 bp sequence (5′- CCGCAGGTATGCAACGCG).The following shRNA constructs were cloned into pSuper vector  was purchased from OpenBiosystems (ThermoScientific) and subcloned into pCAGGS plasmid. This plasmid was used as a basis for creating shRNA resistant plasmids. 4 mismatches with the shRNA sequence were inserted in the original sequences by PCR using the following primer: 5′- AAGCTCGAGCTGTCCAAATTCCTGCCCACTTACCTACCA TGCCACACATAAAGT (Serping1).A plasmid containing the complete coding sequences of C3 (BC043338.1) was purchased from OpenBiosystems (ThermoScientific). C3a was subcloned from the C3 plasmid with following primers: 5′- ATATGGCTAGCTCAGTACAGTTGATGGAAA and 5′- ATAGCGGCCGCTCACCTGGCCAGGCCCAGCACG. C3b beta was subcloned from the C3 plasmid with following primers: 5′- ATATGGCTAGCATCCCCATGTATTCCATCATT and 5′- ATAGCGGCCGCTCAGGCTGCTGGCTTGGTGCACTC. C3b alpha was subcloned from the C3 plasmid with following primers: 5′- ATATGGCTAGCATCCCCATGTATTCCATCATT and 5′- ATAGCGG CCGCTCAGTTGGGACAACCATAAAC. The C3 fragments were subcloned into the pCAGGS plasmid that contained C3 signal peptide (5′- ATGGGGGACCAGCTTCAGGGTCCCAGCTACTAGTGCTACTGCTGCTGTTGGCCAGCTCCCCATTAGCTCTGGGG).A plasmid containing the complete coding sequences of Serping1 the following primers were used: 5′- GCCCAATTCGATGACCATAC and 5′- AAGTTGGTGCTTTGGGAACA; 5′- GCCCAATTCGATGACCATAC and 5'-AGTGGGGTTGAGAGCCTTTT; 5′ –TTCCCTGAAAGAGATGACTCCTGGA and 5′- CGTTGGCTACTTTACCCATGGTGTC; 5′- TGGAGTCCCCCAGAGCCTACA and 5′- GAGGAGGCTGGCAATGCTGA. 29rps primers were used as a reference: 5′- GTATTTGCGGATCAGACCGT and 5′- CTGAAGGCAAGATGGGTCA.For Real-Time PCR of Serping1 cDNA the following primers were used: 5′- AGAGAGCTTCCCTGAAAGAGATG and 5'- TGAGGAGGCTGGCAATGCTGA.For PCR of Rabbit anti SERPING1/C1INH , rabbit anti EMERIN , mouse anti-MAP2 , rat anti C3b antibodies  were used for western blotting.The following antibodies were used for immunostainings: chicken anti TBR2 , rabbit anti CUX1 , chicken anti TBR1 , mouse anti IdU-B44 , chicken anti GFP . Mouse CTR433 antibodies ;Cas9 plasmid and plasmids encoding guide RNAs were purchased from the University of Utah Mutation Generation lab. The following oligonucleotides were used for construction of gRNA vectors:In vitro transcribed Cas9 RNA(100 ng/ul), and sg RNA(50 ng/ul), were injected into one cell fertilized embryos isolated from superovulated CB6F1 hybrid mice mated with CB6F1 males Harlan Biotech Israel Ltd. . Injected embryos were transferred into the oviducts of pseudopregnant ICR females as previously described  was injected intraperitoneally (0.01 ml of 5 mg/ml IdU solution per gram body weight) into pregnant mice at the indicated time points.4, and 100 mM ascorbic acid. This reaction was followed by a click reaction with a non-fluorescent molecule . After treatment with 10 mM ascorbic acid and 4 mM CuSO4, followed by incubation with 20 mM EDTA, the relevant immunostainings were performed.For labeling cells in S phase, pregnant mice (E14) were injected with (50 mg/gr body weight) 5-ethynyl-2′-deoxyuridine (EdU) solution and were scarified 30 min post-injection. The brains were removed and fixed in 2.5% PFA-PBS overnight, washed and cryoporotected by immersion in 30% sucrose-PBS solution. The cryosections (10 μm) were pretreated in boiling sodium citrate buffer  for 30 min. The click reaction was performed with Cy3 azide (2.5 μM) in the PBS-based buffer containing 100 mM Tris-HCl, 1 mM CuSOin utero electroporation using previously described methods  were used for the experiments. For proliferation experiments embryos were in utero electroporated on E13.5 (E13) with 7 mm electrodes (39 mV pulses). IdU was injected in 24 h after electroporation for 30 min. Post-fixed brains were cryopreserved in sucrose and cryosections (10 μm) were used for proliferation analysis. For double electroporation the first in utero electroporation was performed at E13.5 with 7 mm electrodes (39 mV pulses). The second electroporation was performed 24 h later as described above.Plasmids were transfected by Floating vibratome sections (60 μm) were permeabilized using 0.1% Triton X-100 and blocked in blocking solution  for 60 min. Antibodies were incubated in blocking solution over night at 4°C. After washing, appropriate secondary antibodies (Jackson ImmunoResearch) were diluted in blocking solution, and incubated for 2 h at room temperature. Slices were mounted onto glass slides using Aqua Polymount (Polysciences). For IdU immunostainings (E18) the brain slices were pretreated with HCl (30') followed by neutralization with borate buffer. For IdU immunostainings (E13 and E14) the cryosections (10 μm) were used. Antigen retrieval procedure was performed by boiling slides in sodium citrate buffer  for 30 min.Images were taken using confocal microscopy , equipped with Axio Observer Z1 microscope, and imaged with either Plan-apochromat 20x/0.8, or Plan-apochromat 40x/1.2, or Plan-apochromat 63x/1.4 oil objectives. The scaling data are 0.624X0.624 μm per pixel for 20X magnification, 0.312X0.312 μm per pixel for 40X magnification, and 0.198X0.198X0.51 μm per voxol for 60X magnification. The images were processed by ZEN software and/or Imaris software.t-tests or one-way or two-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison analysis, using PRISM 7 for Mac (GraphPad software). Error bars represent standard error. For the measurement of leading edge length high resolution z-stack images were collected of the relevant slices. The analysis was performed with the ImageJ program.Cell count, positioning and colocalization analyses were performed using Imaris software . At least three brains were analyzed for each treatment. Four representative slices from each brain were chosen for analysis. The size of the area of interest was determined and preserved per each experiment. For each slice the area of interest was positioned so that the center of the electroporated area is in the center of the area of interest. For the cell count and positioning the relevant channel of an area of interest was analyzed with “Spots” module of Imaris, every spot labeling approximate center of the cell body. The “y” position of all the dots was analyzed by Microsoft Excel Histogram tool. The data were presented in percentages out of total analyzed cells per bin. For Figure n = 6 for each time point) were dissected in cold PBS and fast-freeze in liquid nitrogen. RNA preparation and Real-time RT-PCR were performed as described above.For confirmation of shRNA efficiency neurospheres from E13.5 were grown in Neurobasal medium (Gibco) supplemented with B27, glutamax, gentamicine, EGF (20 ng/ml), bFGF (20 ng/ml), and heparin for 2 days. The cells were transfected by NEPA21 electroporator (Nepagene) according to manufacturer's instructions. The cells were grown for additional 48 h and collected for RNA isolation . After Dnase treatment (Sigma), first-strand cDNA synthesis was done using M-MLV RT (Promega). Relative levels of Serping1 expression were normalized to the 29rps gene. Real-time PCR with SYBR FAST ABI qPCR kit (Kapa Biosystems) was performed using StepOnePlus Real-Time PCR System (Applied Biosystems). E13, E14, E16, E18 cortices (in utero electroporated with control shRNA or Serping1 shRNA together with GFP (E14-E17). The electroporated areas were dissected under fluorescent binocular. The dissected areas were homogenized in lysis buffer  supplemented with protease inhibitor cocktail (Sigma). Fifty microgram of total protein was mixed with SDS sample buffer, separated by SDS-PAGE and subjected to western blot analysis with the indicated antibodies.Brains were AG and TS planned, conducted experiments, analyzed data, and wrote the manuscript. TW contributed receptor agonists, planned experiments, and wrote the manuscript. OR planned experiments, analyzed the data, and wrote the manuscript.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.


To evaluate the effects of HCV NS5B amino acid substitutions on treatment outcome in Ledipasvir (LDV)/Sofosbuvir (SOF) for Japanese patients with genotype 1b HCV infection, NS5B sequences were examined in i) seven patients experiencing virologic failure after LDV/SOF in real-world practice, ii) 109 SOF-naïve patients, iii) 165 patients enrolled in Phase-3 LDV/SOF trial. A218S and C316N were detected in all patients with viral relapse; the percentages of these substitutions in SOF-naïve patients were 64.2% and 55.0%, respectively. Genotype 1b HCV strains with NS5B-C316N mutation were located in the leaves different from those in which HCV strains without such substitutions were present on the phylogenetic tree. Structural modeling revealed that amino acid 218 was located on the surface of the NTP tunnel. Free energy analysis based on molecular dynamics simulations demonstrated that the free energy required to pass through the tunnel was larger for triphosphate SOF than for UTP in NS5B polymerase carrying A218S, but not in wild-type. However, no susceptibility change was observed for these substitutions to SOF in replicon assay. Furthermore, the SVR rate was 100% in patients enrolled the Phase-3 trial. In conclusion, NS5B A218S and C316N were detected in all patients who relapsed following LDV/SOF in real-world practice. These substitutions did not impact the overall SVR rate after LDV/SOF, however, further studies are needed to elucidate the impact of these substitutions. In DAA therapies including NS5A inhibitors, a baseline resistance-associated substitution (RAS), especially the NS5A-Y93H mutation, was shown to be associated with virologic failure1. Thus, we developed a simple assay to quantify the percentages of HCV-RNA levels of NS5A-Y93H mutant HCV strains and NS5A-Y93 wild-type HCV strains relative to the total HCV-RNA levels using cycling-probe real-time polymerase chain reaction (PCR)2 and established a diagnostic system in combination with direct sequencing to evaluate NS5A-RAS, including the NS5A-L31M/V mutation3. Also, Yoshimi et al. established an assay system for NS5A-RAS using the INVADER method4. These diagnostic systems contributed to an improvement in the therapeutic efficacies of dual oral therapies with DCV plus ASV in real-world practice through the exclusion of patients with HCV strains carrying NS5A-RAS from the potential candidates for treatment5.Combination therapies using 2 or 3 direct-acting antiviral agents (DAAs) have improved sustained viral response (SVR) rates in patients with genotype 1b HCV. DAAs are classified into 3 categories: nonstructural (NS) 3/4 A protease inhibitors, NS5A inhibitors and NS5B polymerase inhibitors. In Japan, dual oral therapy with daclatasvir (DCV), an NS5A inhibitor, and asunaprevir (ASV), a second-generation NS3/4 A protease inhibitor, was approved for patients with genotype 1b HCV as the first interferon-free regimen in July 20146 belongs to the former classification, while beclabuvir (BEC)7 and dasabuvir (DSB)8 belong to the latter. In Japan, SOF was approved for clinical use for patients with genotype 1b HCV in July 2015 as a combination tablet with ledipasvir (LDV), an NS5A inhibitor. The therapeutic efficacy of DAAs, including SOF-based treatments, was shown to be excellent;10 especially in a Japanese trial10, all the patients with genotype 1 HCV who received LDV/SOF without ribavirin (RBV) achieved an SVR. In real-world practice, however, virologic failure can occur in patients with genotype 1b HCV even among those without previous DAA therapies including NS5A inhibitors11. For SOF, NS5B-S282T confers resistance to SOF with an EC50 value relative to that of NS5B-S282 wild-type HCV strains of 9.512. Also, Donaldson et al reported that a baseline NS5B-C316N mutation was potentially associated with a reduced response to SOF in patients with genotype 1b HCV based on bioinformatics characterization13, whereas HCV strains carrying this mutation showed no resistance against SOF in an in vitro replicon system14.Another type of DAA, known as NS5B polymerase inhibitors, is classified into nucleotide and non-nucleotide inhibitors; sofosbuvir (SOF)16. In current science, in silico assay is applied to verify the interaction, in which in vitro and in vivo experiments failed to clarify the mechanisms18. Molecular dynamics simulation is also used to sample conformations of molecules for free energy calculations and has been applied to various analyses such as protein-protein interactions, protein ligand interactions, protein folding, and so on26.Bioinformatics approach based on structural modeling and molecular dynamics simulations, was established by Karplus, Levitt and Warshel in 1976 as a multiscale model for analyzing complicated chemical reactionsin silico by bioinformatics analysis as well as in vitro by replicon system.Thus, in the present study, we examined prevalence of HCV NS5B amino acid substitutions in patients failing treatment with LDV/SOF, and evaluated the impact of these substitutions at baseline on response to LDV/SOF treatment in patients with HCV genotype 1b infection in Japan. Moreover, the significance of these substitutions were assessed Amino acid substitutions in the NS5A and NS5B regions were evaluated in HCV sequences obtained from the 7 patients who experienced virologic relapse after LDV/SOF treatment . As shown in Fig. -7), who had previously experienced virologic failure after DCV plus ASV therapy. In this patient, triple RAS in the NS5A region  was detected at the baseline of LDV/SOF therapy. The quadruple amino acid substitutions in the NS5B region  were also observed at baseline in this patient.Among these 7 patients, the baseline serum sample was available only in one patient (Patient: 40 patients (36.7%) with HCV harboring A207, A218, C316 and Q464 wild-type in the NS5B region, 47 patients (43.1%) with HCV harboring A207T, A218S, C316N and Q464E, and 22 patients (20.2%) with HCV harboring A218S with wild-type phenotypes at either aa207, aa316, or aa464. Phylogenetic trees based on the 115 amino acid sequences in the NS5B region are shown in Fig. -type phenotypes, 97.2% (35/36) in those with HCV virus harboring A207T, A218S, C316N and Q464E substitutions.To further understand the pre-treatment prevalence of these NS5B amino acid substitutions in genotype 1b Japanese patients especially in our institutes, baseline samples from 109 SOF-naïve patients were assessed. The demographic and clinical features of the patients are shown in Table ).Among 165 genotype 1b patients who received LDV/SOF as a part of a phase 3b clinical trial in Japan, the overall SVR12 rate was 100% (165/165), of which HCV in 32% (53/165) of patients harbored A207T, A218S, C316N and Q464E at baseline (Table 2P < 0.05). Consequently, HCV with NS5B-C316N substitution were detected in 61.0% of the patients without previous RBV administration; the ratios were significantly higher than in patients with previous RBV administration (37.5%).As shown in Table The three-dimensional structure of an NS5B polymerase in HC-J4 strain was shown as a cartoon model in Fig. -type polymerase , one of the popular methods for free energy analysis based on molecular dynamics simulations, were performed using 4WTA and 4WTG, in which amino acid residues were substituted by those of the wild-type genotype 1b HCV (HCV-K1-S2) strains and those of the mutant HCV strains in a patient failing to achieve SVR (LC216929). The former polymerase showed an A207/A218/C316/Q464 wild-type phenotype, while the latter polymerase showed an A207T/A218S/C316N/Q464E mutation. In the case with the wild-type NS5B polymerase, the free energies were not different between activated triphosphate SOF and UTP, while the deviation was large in the simulations using activated triphosphate SOF Fig. . This prase Fig. , while tase Fig. . A similase Fig. , whereasase Fig. . In contUTP Fig. , but thiype Fig. .Figure 7The ratios of EC50 values of each mutant HCV strain relative to that of wild-type genotype 1b HCV strain are shown in Table 29, and NS5A-RASs such as L31M/V and Y93H substitutions developing at the time of virologic failure during and after DCV + ASV therapy were shown to be responsible for virologic failure after retreatment with LDV/SOF. Also, the HCV strains carrying NS5A-L28M and/or R30Q with Y93H substitutions shows high level of resistance to NS5A inhibitor30. In our hospital, the amino acid sequences of HCV strains at virologic failure after LDV/SOF therapy were evaluated in 7 patients, and 2 patients had prior DCV + ASV therapy. Various combinations of NS5A-RASs, showing diverse extent of susceptibility to NS5A-inhibitors, were observed, while NS5B-S282T mutation was absent in all the patients. Considering that SOF retains antiviral activity for patients with HCV strains harboring these NS5A-RASs, NS5B amino acid substitutions different from NS5B-S282T mutation might contribute to virologic relapse in patients receiving unsuccessful LDV/SOF therapy.In Japan, virologic failure occurs in some patients with genotype 1b HCV following LDV/SOF therapy in real-world practice. Most patients with virologic failure had a history of previous DCV plus ASV therapies7 × 100). This suggests that these substitutions may be enriched among patients failing LDV/SOF treatment, either due to an impact on treatment outcome or due to emergence during therapy due to resistance selection. These observations prompted us to postulate a possible decreased sensitivity of HCV harboring NS5B-A218S substitution to SOF compared to that of wild-type HCV strains. However, no change in susceptibility was observed in the in-vitro replicon assay. To further understand this relationship, samples from the 155 genotype 1b patients treated in the LDV/SOF phase 3b clinical study conducted in Japan were assessed. Although the baseline prevalence of these substitutions was similarly high in this group as compared to the other cohort, all patients achieved SVR, further suggesting no impact of these substitutions on treatment outcome. Recently, however, the study group in Kagoshima prefecture showed that NS5B-A218S/N316N mutations were significant factors associated with virologic failure after LDV/SOF therapy by multivariate analysis in about 500 patients with genotype 1b HCV in real-world practice . Thus, the significance of these substitutions should be further investigated in future.Amino acid mutations in the NS5B region of genotype 1b HCV were assessed in reference to Con1 and/or HCV-K1-S2 strains, and four variants , were detected in all 7 patients experiencing virologic failure after SOF/LDV therapy. For one patient, with available baseline sample, these substitutions were also present at baseline. Across 109 SOF-naïve patients linkage disequilibrium was found among these mutations. The prevalence of patients with HCV harboring all 4 substitutions was 43.1%, and the probability of this result accidentally occurring in 7 consecutive patients was 0.28%  of 2.4 angstroms, and the overall fold is quite similar. Thus, we propose that 4WTA/4WTG structures are appropriate for analyzing the effect of amino acid substitutions on the binding of UTP/SOF to NS5B complexed with RNA. Next, amino acid residues of 4WTA/4WTG were substituted with those of the wild-type genotype 1b HCV (HCV-K1-S2) strains and mutant HCV strains from the serum in other patient with virologic failure after therapy (LC216929). The former polymerase manifested A207, A218, C316 and Q464 wild-type phenotypes, while the latter polymerase carried A207T, A218S, C316N and Q464E substitutions. The movement of activated triphosphate SOF and UTP can be visualized; they pass through the NTP tunnel leading to linkage with an extending RNA at the activity region. The distance of travel was calculated to be about 20 Å. The free energy differences required for activated triphosphate SOF and UTP to pass through the NTP tunnel were evaluated under various situations, and we found that the phenotype of aa218 was a crucial factor determining the difference in energies between activated SOF triphosphate and UTP. Free energy analysis based on molecular dynamics simulations demonstrated that activated triphosphate SOF was inferior to UTP in terms of passage through the UTP tunnel, especially in the case of NS5B polymerase manifesting the A218S substitution, suggesting that the therapeutic efficacy of SOF may be deranged in patients with genotype 1b HCV carrying the NS5B-A218S substitution. NS5B-A218S substitution was less common among patients with previous RBV administration, compared with RBV-naïve patients. Since RBV is an analog of guanine that manifests a hydrophilic character similar to that of UTP, the therapeutic efficacy of SOF may be theoretically improved when RBV is administered together with SOF in patients with HCV strains carrying the NS5B-A218S substitution.The weighted histogram analysis method (WHAM) is one of the most popular method based on molecular dynamics simulations to analyze free energy profile along with a defined reaction coordinateIn the present study, we focused on the interaction between activated-SOF and NS5B polymerase, which may be involved in therapeutic efficacy of LDV/SOF therapy in patients with genotype-1b HCV. Thus, deep sequencing analysis was not done in the present study. To clarify the significance of HCV strains carrying NS5B-A218S and C316N mutations, however, deep sequencing analysis should be done especially focusing on dynamics of quasispecies in patients with NS5B-A218S and C316N mutant HCV strains. These matters should be investigated in future.In conclusion, genotype 1b HCV strains were classified into 2 groups by a phylogenetic analysis based on amino acid sequences of NS5B; HCV strains with and those without NS5B-C316N mutations, which showed high linkage disequilibrium with NS5B-A207T, NS5B-A218S and NS5B-Q464E mutations. The 7 patients experiencing virologic failure harbored these 4 substitutions. Structural modeling and free energy analyses revealed that the NS5B-A218S substitution may have an effect on the passage of activated triphosphate SOF through the NTP tunnel. Overall, the presence of these substitutions individually or in combination with NS5A-RASs did not impact the treatment outcome to LDV/SOF, however, further studies are needed to elucidate possible effects in patients with advanced disease.Three patient groups were assessed. The first group included 6 patients who were referred to Saitama Medical University Hospital after experiencing virologic relapse to LDV/SOF. The second group included 109 SOF-naïve patients seen at our hospital between September 2015 and September 2016. 92 of whom received LDV/SOF, and 17 of whom received ombitasvir (OBV), paritaprevir (PTV) with booster ritonavir (r). Baseline serum samples were obtained from all 109 patients and serum samples were also obtained at the time of virologic failure for the 6 patients in the first group and for the 1 subject who failed treatment with LDV/SOF in the second group. The third group included165 Japanese patients with genotype 1b chronic HCV infection enrolled in a Phase 3b clinical trial in Japan evaluating LDV/SOF .Written informed consent was obtained from all the patients prior to the collection of blood samples. The study conformed to the ethical guidelines of the Declaration of Helsinki and was conducted with the approval of the Institutional Review Board of Saitama Medical University Hospital.HCV-RNA was purified from sera and subjected to nested PCR followed by direct sequencing. The amino acid substitutions were assessed by referring to the sequences of the Con1 strain (GenBank accession number AJ238799.1) and the HCV-K1-S2 strain (D50485.1). HCV strains with or without mutations were classified according to a phylogenetic tree analysis based on amino acid sequences in the NS5B regions. Also, the 3-dimensional structure of NS5B polymerase was evaluated using a bioinformatics analysis, and the significance of the mutations was assessed using molecular dynamics. Furthermore, the ratios of EC50 values of each mutant HCV strain for SOF relative to that of wild-type genotype 1b HCV strain were evaluated using genotype 1b HCV replicon assay.® Terminator v3.1 Cycle Sequence Kit  using the PCR primers. Direct sequencing was performed using a 3730xl DNA Analyzer (Applied Biosystems), and the resulting nucleotide sequences were assembled using ATGC for Windows version 8 software . The nucleotide mixture detection during sequencing was determined automatically by the software. All working processes were performed according to the manufacturer’s protocol.HCV-RNA was purified from sera using the QIAamp MinElute Virus Spin Kit  and was subjected to nested PCR followed by direct sequencing of NS5A and NS5B regions. Reverse transcription and the first PCR amplification were performed for purified RNA samples using the PrimeScript™ II High Fidelity One Step RT-PCR Kit  with primer sets: set of NS5A_L/NS5A_R for aa12–125 in the NS5A region, and HCV-16L/HCV-19R for full-length NS5B region Table . For dirThe amino acid sequences in the NS5A and NS5B regions of the HCV strains were determined using GENETYX for Windows version 13 (GENETYX Corp.), and the mutations were assessed by referring to the sequences of the Con1 strain (GenBank accession number AJ238799.1) and the HCV-K1-S2 strain (D50485.1). HCV strains with or without mutations were classified according to a phylogenetic tree analysis based on amino acid sequences in the NS5B regions. Also, the 3-dimensional structure of NS5B polymerase was evaluated using a bioinformatics analysis, and the significance of the mutations was assessed using molecular dynamics.The nucleotide sequences of the HCV strains in patients before LDV/SOF therapies and in those at viral relapse after the therapies were submitted to the DDBJ/EMBL/GenBank databases under accession numbers LC210145 to LC210252 and LC216928 to LC216934, respectively.For the patients in the LDV/SOF clinical trial, the HCV NS5B coding regions were amplified by DDL Diagnostic Laboratory  using standard reverse transcription polymerase chain reaction (RT PCR) technology, in available plasma/serum samples with HCV RNA was > 1000 IU/mL. Deep sequencing using MiSeq platform  was performed by DDL or WuXi AppTec .The amino acid sequences of the NS5B regions obtained by direct sequencing were multiple-aligned using MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets, and the phylogenetic trees were constructed using the maximum likelihood method adapted with best fit model (Kimura 2-parameter plus Gamma distributed with Invariant sites). To confirm the reliability of the phylogenetic analysis, bootstrap resampling was performed 1,000 times.http://pymol.org). Mutant amino acids were labeled as various colors and their locations were evaluated using a cartoon model or a model showing the surface of each amino acid.The three-dimensional structure of NS5B polymerase in genotype 1b HCV was constructed based on nucleotide sequences of the HC-J4 strain (1NB7) using the software PyMOLTM 1.8.x ) and 4WTG (with activated triphosphate SOF) substituting the amino acid residues using SCWRL 441 with the AMBER ff14SB force field42 for proteins and parameters generated by R.E.D. Server36 for UTP and activated triphosphate SOF. To determine the potential of the mean force (PMF) of UTP/SOF through the tunnel, the weighted histogram analysis method (WHAM)42 with 25 2-ns windows  was used. This method is one of the popular methods to obtain free energy profile along a defined reaction coordinate. The distance from the O3 atom in U3 (chain ID: P) and the C5 atom in UTP/SOF was chosen as the reaction coordinate (10–22 Å).All the simulations were executed using GROMACS ver.4.6.5Each of the PMF simulations was repeated 3 times per model. The models subjected to the analysis were as follows, 4WTA and 4WTG in which amino acid residues were substituted with those of the wild-type genotype 1b HCV (HCV-K1-S2) strains, 4WTA and 4WTG adding either of C316N, A218S, A218S/C316N, A207T/C316N/Q464E or A207T/A218S/C316N/Q464E substitutions to the wild-type HCV strain, 4WTA and 4WTG substituted with amino acid residues of the mutant type genotype 1b HCV strain obtained from a patients failing to achieve SVR (LC216929) and 4WTA and 4WTG in which an amino acid residue at aa218 was reverted from A218S mutation to A218 wild-type in the mutant HCV strain.43. Replicon RNAs were transcribed in vitro from replicon-encoding plasmids using a MEGAscript kit . RNA was transfected into Huh-lunet cells using the method of Lohmann et al44. Compound were diluted in 100 percent DMSO and added to cells. Cells were treated for 3 days, after which culture media were removed, cells were lysed, and Renilla luciferase activity was quantified using a commercially available assay  and a Top Count instrument . EC50 values were calculated as the compound concentration at which a 50 percent reduction in the level of Renilla reporter activity was observed when compared with control samples with DMSO.NS5B substitutions were introduced into the genotype 1b replicon by site-directed mutagenesis and tested in transient transfections as previously describedCategorical data were compared using the Fisher exact test. Distributions of continuous variables were analyzed using the Mann-Whitney U-test to identify factors associated with NS5B-RAS. All the tests of significance were two-tailed, and P values of less than 0.05 were considered statistically significant. SPSS Statistics version 22  was used for the analyses.


The standard of treatment for botulism, equine antitoxin, is a foreign protein with associated safety issues and a short serum half-life which excludes its use as a prophylactic antitoxin and makes it a less-than-optimal therapeutic. Due to these limitations, a recombinant monoclonal antibody (mAb) product is preferable. It has been shown that combining three mAbs that bind non-overlapping epitopes leads to highly potent botulinum neurotoxin (BoNT) neutralization. Recently, a triple human antibody combination for BoNT/A has demonstrated potent toxin neutralization in mouse models with no serious adverse events when tested in a Phase I clinical trial. However, a triple antibody therapeutic poses unique development and manufacturing challenges. Thus, potentially to streamline development of BoNT antitoxins, we sought to achieve the potency of multiple mAb combinations in a single IgG-based molecule that has a long serum half-life. The design, production, and testing of a single tri-epitopic IgG1-based mAb (TeAb) containing the binding sites of each of the three parental BoNT/A mAbs yielded an antibody of nearly equal potency to the combination. The approach taken here could be applied to the design and creation of other multivalent antibodies that could be used for a variety of applications, including toxin elimination. The mainstay of clinically proven treatment for botulism is therapeutically administered antitoxin, made by the immunization of human volunteers or by hyperimmunizing horses. Equine antitoxin ,2 and hureatment ,6. An alreatment .We have generated a series of combinations of three mAbs that bind all toxin subtypes tested of BoNT/A, /B, /C, /D, /E, /F or /HA and that result in highly potent BoNT neutralization in mice ,8,9. TheHere, we show that such single bi-epitopic mAbs (BeAb) and tri-epitopic mAbs (TeAb) can be generated that have similar BoNT binding and neutralization as the combination of two or three BoNT/A mAbs. Design of the TeAb was informed by first designing and testing a bi-epitopic antibody (BeAb) based on dual-variable domain (DVD) mAbs . The BeAN) and CR2 bind an epitope on the N-terminal subdomain (HCN) of the BoNT/A binding domain (HC) [CC) of the BoNT/A binding domain (HC) [The bi-epitopic antibody BeAb, A was desain (HC) . The triain (HC)  variableain (HC) .H) ran at the expected sizes . The BeAb was expressed from stably transfected CHO cells and purified by Protein G affinity chromatography with yields of approximately 10 mg/L of culture supernatant. Under non-reducing conditions, the BeAb ran at the expected size of approximately 200 kDa, larger than the predicted size of the 150 kDa 2G11 and CR2 IgG, and smaller than the TeAb . Under rD) of 2G11 and CR2 binding sites of the BeAb for antigen were measured using flow fluorimetry in a KinExA  and specific recombinant LC-HN and HCN domains, respectively  than the CR2 IgG. The lower affinity was largely the result of a 10-fold slower association rate (onk), likely due to steric hindrance from the 2G11 binding site  ofing site . Use of D approximately the same as the 2G11 IgG (KD 2G11 IgG = 1.14 × 10−11 M and KD BeAb 2G11 binding site = 1.28 × 10−11 M), as shown in D of the BeAb for holotoxin compared to CR2 IgG (higher affinity) than the CR2 IgG (off)k, likely reflecting avid binding from the 2G11 binding site. Differences in the binding of antibodies to the HCN domain vs. holotoxin reflect the fact that the recombinant domain is not a perfect mimic of the CR2 epitope in the holotoxin [Using holotoxin, the BeAb bound the 2G11 binding site with a K CR2 IgG  results C epitope that blocks the ability of BoNT/A to bind to its receptor [To ensure that the BeAb recapitulated the functional activity of CR2 and 2G11 in vitro, the ability of the BeAb, CR2 IgG, 2G11 IgG and the combination of CR2 + 2G11 IgG to prevent uptake of BoNT/A into neurons and cleavage of SNAP25 was compared . CR2 IgGreceptor .H, the CR2 VH and the CH1, CH2 and CH3 domains, and two representing the 2G11 Vk, the CR2 Vk, the Ck and the VH and Vk of RAZ1.The TVD, TeAb-K and TeAb-H were expressed from stably transfected CHO cells and purified by Protein G affinity chromatography with yields of approximately 5–10 mg/L of culture supernatant. The TVD and TeAb-H could not be purified to greater than 80% purity and the TVD also had a tendency to aggregate. These constructs were not studied further. Under non-reducing conditions, the TeAb-K ran close to its predicted size of 250 kDa (the 200 kDa of the BeAb component plus two 25 kDa scFv) . Under rD) of the individual RAZ1, 2G11, and CR2 binding sites of the TeAb-K for antigen were measured using flow fluorimetry in a KinExA and specific recombinant HCC, LC-HN, and HCN domains, respectively  than the RAZ1 KD  was compared to the potency of individual mAbs 2G11, CR2 and RAZ1, the combination of 2G11 and CR2 IgG and the combination of 2G11 + CR2 + RAZ1 IgG. Fifty μg of each of individual mAbs completely neutralized between 20 and 100 mouse LD the MNA   were sequentially inserted into the CR2-IgG expression vector, which was created using the IgG1 expression vector N5KG1 as previously reported [H domains fused to the C-terminus of the CR2 Vk and VH domains respectively separated by a 14-amino acid linker1 of amino acid sequence TVAAPSVFIFPPSD for Vk, or linker2 ASTKGPSVFPLAPS for VH. Briefly, the Vk genes plus a linker were PCR- amplified from the mAb 2G11-IgG1 expression vector N5KG1 [BglIIinDraIIIoutVk5′primer: (5′-ATCACAGATCTCTCACCATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCCGATG-3′)DraIIIinBsiWoutVklinker3′primer: (5′-ACTGCTCATCACATCGTGAGAAGATGAAGACAGATGGTGCAGCCACGGTACGTTTGATTTCC-3′)The BeAb and three different TeAb expression-vector plasmids were constructed using the 2G11, CR2 or RAZ1- IgG1 N5KG1 expression vectors or their scFv leads in pYD2 vectors  similar to those previously reported . For BeAreported , with seor N5KG1  using thBglII and DraIII restriction enzymes , purified and ligated into the mAb CR2-IgG1 expression vector backbone which was digested and purified similarly. The ligation product was used to transform E. coli DH5α competent cells, and vector plasmids containing the insertion were purified from the transformant for DNA sequencing. After sequence verification of the correct 2G11 Vk insertion into the mAb CR2-IgG1 plasmid from one of the transformants, the resulting vector was used for 2G11 VH insertion to create the BeAb expression plasmid. The VH genes with a second linker2 were polymerase chain reaction (PCR) amplified from the same mAb 2G11-IgG1 expression vector using a different pair of primers for the VH + linker2 gene fragment: SalIinMluIoutVH5′primer: (5′-GACCCGTCGACATGGGTTGGAGCCTCATCTTGCTCTTCCTTGTCGCTGTTGCTACCCGTGTCTTGTCCC-3′)MluIinNheIout VH3′ tail primer: (5′-GGAGGAGGGTGCCAGACGCGTGACCGATGGGCCCTTGGTGCTTGCTGAGGAGACGG-3′)The PCR-amplified Vk + linker1 fragment was double cut with H + linker2 gene fragment was double digested with SalI and MluI restriction enzymes, purified, and subcloned into the plasmid from the previous step. The resulting plasmid was digested with same enzyme pairs before ligation and transformation. The ligation products were used to transform E. coli DH5α competent cells, and new plasmids were prepared from those transformants for sequencing. Both V-genes of mAb 2G11 and CR2 were confirmed to be in the BeAb expression vectors, and they were in frame with the leader sequence, and also in frame with the constant domain gene plus the designed linkers. This confirmation was achieved via sequencing the plasmid from upstream of the leader sequence and downstream of the constant domain genes using VHfor, VHback, Vkfor and Vkback primers as previously published [The PCR-amplified Vublished  and addiThree triepitopic antibodies (TeAbs) were created based on the BeAb construct with the 2G11 and CR2 V-domain at the N-terminus of the IgG. The third binding site (RAZ1 V-domains) was inserted as either the innermost tandem V-domains fused to the C-terminus of CR2 V-domains and the N-terminus of the Ck and CH1 domains (TVD) or as a scFv at the C terminus of the Ck (TeAb-K) or CH3 (TeAb-H) as illustrated in H linker4 is ASTKGPSVTRVLS. Linker3 and linker4 genes were added to the RAZ1 V gene via PCR by using the IgG expression plasmid and primer pairs RAZBsiWIlinkerVk5′primer and Vk3′primer, or RAZNheIlinkerVH5′ primer and VH3′ primer  linker5 sequence added for splicing. After splicing PCR amplification, the entire fragment was double digested with BsiWI and EcoRI, then ligated into the BeAb expression plasmid backbone which had been digested with the same enzymes and purified. After ligation, the product was used to transform E. coli DH5α, and plasmid purified from individual transformants for sequence confirmation using primer TeAbKscfvSeqprimer. For TeAb-K expression-vector construction, the RAZ1 scFv gene was first PCR amplified from RAZ1-scFv pYD2 yeast expression vector , using per pairs  of TeAbKSmaI restriction site at the 5′ end. The splicing product was used to transform E. coli DH5α, and plasmid prepared from individual transformants for sequence confirmation using sequencing primer ForTeAbscfvseq and primer4scfvconfirm.For TeAb-H expression, vector construction was also performed using a three-step PCR and splicing strategy. The RAZ1 scFv gene was first PCR amplified from RAZ1-scFv pYD2 yeast expression vector, using primer pairs  LinkerHs280 measurement and used immediately or stored at −80 °C prior to characterization. All V-genes and each linker gene sequence in the BeAb or TeAb expression vectors were confirmed to be as designed, and in frame with the leader sequence and with the constant domain gene. Sequencing of the plasmid was performed from upstream of the leader sequence and downstream of the constant domain genes as described above. Stably-transfected CHO DG44 cells were selected using the G418  resistant marker as previously described . The traD) and kinetic association and dissociation rate constants  of BeAb and TeAb for holotoxin and for specific recombinant BoNT domains were measured using a KinExA 3200 flow fluorimeter  as previously described [D. The exponential decrease in the concentration of free antigen as a function of time was fit to a standard bimolecular rate equation using the KinExA Pro software to determine the kon. The koff was calculated from the product of onk × KD.Equilibrium dissociation constants  for 30 min at room temperature. Each mixture was then added to individual wells of 12- to 14-day-old primary neurons incubated at 37 °C for 30 min before phosphate-buffered saline (PBS) washing and 10% goat serum blocking of non-specific binding. BoNT/A1 without antibody was used as a positive uptake and cleavage control. After fixing the treated neurons with freshly prepared 4% paraformaldehyde, permeabilization of fixed cells was done by incubation with 0.2% Triton X-100. The neurons were sequentially incubated with 1 μg/mL of mAb 4E17.1 for two hours and 5 μg/mL of goat anti-human IgG secondary Ab for one hour at room temperature. A polyclonal guinea pig antibody for vesicular glutamate transporter 1 , a synaptic vesicle marker, was also used to show the location of presynaptic terminals when necessary. After washing, the samples were mounted and sent for imaging analysis.For microscopy, internalized BoNT/A1 toxin inside the neuron was detected using mAb 4E17.1  and DyLig for 20 min, the supernatant was then collected and protein concentration was determined by the absorbance at the 280 nm. The extracted protein from the cell lysate was loaded onto a 4–20% SDS-PAGE gradient gel for electrophoresis and the separated proteins were then transferred onto a polyvinylidene difluoride membrane . The membrane was blocked with 5% fat-free milk, then immunoblotted overnight with a primary antibody against SNAP-25  at 4 °C, followed by incubation with horseradish peroxidase-conjugated anti-mouse secondary antibody  and visualization using SuperSignal West Femto kit  and autoradiography using Kodak film . The scanned images were also analyzed by using NIH Image J software for semi-quantitation followed by Western blotting  at the concentration of 0–4 nM mixed with 1 nM of BoNT/A1 in the Opti-MEM media were monitored. Cells were scraped off the dishes and protein was extracted by using radio immunoprecipitation assay (RIPA) lysis and extraction buffer with proteinase inhibitors (Thermo Scientific). After centrifugation at 13,400 × blotting B.The United States Army Medical Research Institute of Infectious Diseases (USAMRIID) Institutional Animal Care and Use Committee (IACUC) approved the animal care and use protocol to conduct the animal studies described in this sub-section and in All procedures involving animals performed at the United States Department of Agriculture (USDA) were reviewed and approved by the Institutional Animal Care and Use Committee of the USDA, Western Regional Research Center. Animal use protocol for BoNT mouse bioassays (Protocol #15-8) was approved by the Western Regional Research Center Institutional Animal Care and Use Committee (WRRC-IACUC) on 27 July 2015.50 of BoNT/A1 holotoxin  in gelatin phosphate buffer (pH 6.5) and incubated at room temperature for 30 min. For the mAb pairs, antibody was added in a 1:1 ratio; for triple mAb combinations, antibody was added in a 1:1:1 ratio. The mixtures were injected intraperitoneally (i.p.) into groups of 10 female . Mice were observed at 4–8 h intervals after toxin challenge. Mouse survival was recorded for 5 days after injection.BeAb, TeAb, individual IgGs of 2G11, CR2 or RAZ1, or a combination of two or three IgGs  were added to an indicated number of mouse LDw/v) uranylformate, and aspirated to dryness as reported [Purified antibody as individual IgG or BeAb or TeAb, ciBoNT/A, or antibody-toxin complexes were applied to glow-discharged 400 mesh carbon-coated copper grids, washed, stained with 0.75%  were injected intravenously (iv) via the lateral tail vein with 5000 pg ciBoNT/A and 10 µg of mAb  per mouse at indicated times. MAbs were diluted in PBS (pH 7.2). Blood was collected from the submandibular site into serum collection tubes with gel separators (BD Biosciences). Samples were incubated on ice for at least 30 min before centrifugation at 3000× The concentration of ciBoNT/A in sera was determined by an electrochemiluminescence (ECL) ELISA using a SECTOR Imager 2400 . ECL was performed as described previously with slight modification . Brieflyonk. Such a decrease in affinity due to a reduction in onk for the inner-variable domains has been previously reported for a DVD binding TNFα [onk for the CR2 binding site for BoNT/A was 4.6-fold slower than the CR2 IgG, this was more than compensated by a 8.6-fold slower offk for the CR2 site in the BeAb compared to CR2 IgG. These findings suggest that the BeAb avidly bound to BoNT/A, either simultaneously binding a single BoNT/A at both the 2G11 and CR2 epitopes or cross-linking multiple BoNT/A molecules. This is likely due to the flexibility of the 2G11 and CR2 binding sites in the DVD format [50 between 20,000 and 40,000 LD50 with a 50 µg/mouse total antibody dose. The potency of the BeAb, like the mAb pair combination, was approximately 100 times greater than the individual mAbs.We have partially reproduced within four-fold the increased BoNT neutralization potency of double or triple mAb combinations in a single IgG-based molecule by creating biepitopic (BeAb) and triepitopic (TeAb) antibodies. The BeAb was designed based on the previously described dual variable domain (DVD) IgG . Using eing TNFα  and coulD format . The BeA50 for a 40,000 LD50 dose of BoNT/A of approximately 2.5 μg, or about 40-fold more potent than the BeAb and 20-fold more potent than the combination of mAbs 2G11 and CR2. The TeAb, however, was approximately four-fold less potent in BoNT/A neutralization than the combination of 2G11 + CR2 + RAZ1 IgG.Three TeAb constructs were designed based on the successful BeAb structure. Of these, only the TeAb-K, with the RAZ1 scFv fused to the C-terminus of the kappa light-chain constant domain was produced with sufficient purity and quantity for detailed characterization. The TeAb-K had comparable affinity for the 2G11 and CR2 epitopes as the BeAb as measured by the epitope specific domains. The affinity of the TeAb for the RAZ1 epitope was approximately 19-fold lower than RAZ1 IgG, likely due to conversion to the scFv format. Affinity for BoNT/A at the 2G11 and CR2 epitopes were 5.4 and 3.4 times higher than for the 2G11 and CR2 IgG and the reduction of affinity of the RAZ1 scFv was only 8.8-fold. These data, like for the BeAb, suggest avid binding of the TeAb to BoNT/A. The TeAb-K had an EDmAb combinations can synergize by a number of different mechanisms to neutralize BoNT. First, binding of multiple mAbs can interfere with multiple steps in the intoxication pathway, such as receptor binding and cell entry, endosomal translocation or intracellular catalysis . Second,Multifunctional antibodies have entered preclinical as well as clinical development. Despite the significant progress on bi-specific antibody constructs ,36,37,38In summary, we have described how the synergy observed by combining three BoNT/A mAbs can almost be reproduced in a single tri-epitopic IgG-like molecule. Potency is increased more than 2000-fold compared to single mAbs. While it is unproven as to how easily such triepitopic antibodies can be developed as therapeutics, they may provide an alternative to mAb combinations for the treatment and prevention of botulism.


Reverse transcriptase (RT) is the target for the majority of anti-HIV-1 drugs. As with all anti-AIDS treatments, continued success of RT inhibitors is persistently disrupted by the occurrence of resistance mutations. To explore latent resistance mechanisms potentially accessible to therapeutically challenged HIV-1 viruses, we examined RT from the related feline immunodeficiency virus (FIV). FIV closely parallels HIV-1 in its replication and pathogenicity, however, is resistant to all non-nucleoside inhibitors (NNRTI). The intrinsic resistance of FIV RT is particularly interesting since FIV harbors the Y181 and Y188 sensitivity residues absent in both HIV-2 and SIV. Unlike RT from HIV-2 or SIV, previous efforts have failed to make FIV RT susceptible to NNRTIs concluding that the structure or flexibility of the feline enzyme must be profoundly different. We report the first crystal structure of FIV RT and, being the first structure of an RT from a non-primate lentivirus, enrich the structural and species repertoires available for RT. The structure demonstrates that while the NNRTI binding pocket is conserved, minor subtleties at the entryway can render the FIV RT pocket more restricted and unfavorable for effective NNRTI binding. Measuring NNRTI binding affinity to FIV RT shows that the “closed” pocket configuration inhibits NNRTI binding. Mutating the loop residues rimming the entryway of FIV RT pocket allows for NNRTI binding, however, it does not confer sensitivity to these inhibitors. This reveals a further layer of resistance caused by inherent FIV RT variances that could have enhanced the dissociation of bound inhibitors, or, perhaps, modulated protein plasticity to overcome inhibitory effects of bound NNRTIs. The more “closed” conformation of FIV RT pocket can provide a template for the development of innovative drugs that could unlock the constrained pocket, and the resilient mutant version of the enzyme can offer a fresh model for the study of NNRTI-resistance mechanisms overlooked in HIV-1. The majority of anti-AIDS drugs target the reverse transcriptase (RT) enzyme of the HIV-1 virus. RT catalyzes the central step in the virus replication cycle converting the viral RNA genome into DNA for subsequent integration into the host genome. As with all anti-AIDS treatments, continued success of RT inhibitors is persistently disrupted by the occurrence of resistance mutations. To explore latent resistance mechanisms potentially accessible to therapeutically challenged HIV-1 viruses, we examined RT from the related feline immunodeficiency virus (FIV). FIV closely parallels HIV-1 in its replication and pathogenicity however is resistant to all non-nucleoside inhibitors of HIV-1 RT. We resolved the crystal structure of FIV RT, and using mutational and biochemical analyses, we show that specific differences in the FIV RT structure inhibit the binding of non-nucleoside inhibitors. We further show that mutating the protein to facilitate binding of the inhibitors does not confer sensitivity to these inhibitors, suggesting that other variances inherent in FIV RT modulate a second layer of resistance. These insights can help in the development of novel drugs against evolving HIV-1 RT resistance. Reverse transcriptase (RT) is the most common target for anti-AIDS drugs being the enzyme that catalyzes the central step in the HIV-1 replication cycle converting the viral RNA genome into DNA for subsequent integration into the host genome . AlthougAs with all anti-AIDS treatments, resistance mutations persistently disrupt the continued success of RT inhibitors. While the mechanistic bases are not yet clearly defined, the prevailing view establishes that NNRTI-resistance mutations mainly alter the association and dissociation kinetics of NNRTIs (reviewed in ). ResistCurrent NNRTIs are HIV-1 specific. RT enzymes from HIV-2, simian (SIV) and feline (FIV) immunodeficiency viruses are intrinsically resistant to HIV-1 NNRTIs. The intrinsic resistance of FIV RT (fRT) is particularly interesting since FIV harbors the Y181 and Y188 NNBP sensitivity residues absent in both HIV-2 and SIV  14]. Un. Un14]. https://hivdb.stanford.edu/). While the exact role and significance of each of these variances, separately and combined, remains to be determined, fRT presents a pertinent model for probing the NNRTI-resistance mechanisms. The FIV model is the only non-primate model of a lentivirus that induces AIDS-like syndrome in its natural host [FIV RT inherently combines numerous substitutions frequently observed in HIV-1 resistance to NNRTIs including  M41L, V75M(I), K101E/Q(Q), V118I, I132L, E138K/A(A), Q145M/L(V), V179D, F227L/C(Y), K238T, L283I(M), N348I, I393L(T), T/A376S(R), and T369I/V(L)   and superimposable NNRTIs in a model of open fRT NNBP (The NNBP of unliganded RT is rimmed at the p51/p66 interface (residues 100–105 of p66 and E138-loop of p51) and largely filled by the primer grip (β9-β10) and the catalytic loop (β7-β8) . The pocfRT NNBP .p51 and I135Kp51 of p51 . Intriguingly, a group of FIV variances clusters at the rim of the NNBP entryway. fRT is altered to K101Q, D192N, D177Q and V179D in the p66 subunit, and E28N, T139G, E138A, I135K and S134R, in the p51. The prominent contributors to key physiochemical alterations at the entryway appear to be K101Q and V179D of p66, and E138Ap51 Figs  and 3A.p51 and potentially with I135Kp51. In HIV-1 RT, the negatively charged and bulky side chain of E138p51 may shield Y181 against these interactions , preventing their interaction. In another HIV-1 RT structure (PDB code: 1DLO) E138p51 is rotated away from Y181, allowing a 3.4 Å interaction between Y181 and N136p51, 0.5 Å farther, and consequently weaker, than the interaction in fRT (2.9 Å)  to carbonyl oxygen of three p66 residues . The bulkier side chain of S134Rp51 may further contribute to a tighter entryway by filling more space at the p51/p66 interface and interacting (3.6 Å) with the carbonyl oxygen of I180 (p51) and two hydrogen bonds   . Moreove of I180 . fRT als138Ap51) . Consequ138Ap51) . FIV var138Ap51) .Additionally, a cluster of substitutions in fRT creates extra interactions that could restrain the flexibility of the primer grip (β9-β10) rimming the NNBP. Particularly, Gln of H221Q in fRT forms an extra interaction to backbone amine of L228T. Also, OH of F227Y interacts with backbone amines of V106 or K223E . These iThe closed architecture of the NNBP entryway readily indicates that NNRTI binding to fRT would not be favorable. Previous studies documented the failure of NNRTIs to inhibit fRT activity but did not establish if this is a result of fRT inability to bind NNRTIs , 17. In To further validate the restricted-pocket configuration, we mutated the fRT p51-loop to match its HIV-1 RT equivalent  and found that the mutated fRT was able to bind EFZ and RVP with comparable affinities to HIV-1 RT  and bindNoteworthy, the fRT mutant had reduced thermal stability (~ 2.5°C), presumably as a result of the interactions lost through this mutation .The structure of the intrinsically resistant fRT provides a novel model of a constrained NNRTI pocket, which when combined with the numerous other NNRTI-resistance mutations inherent in fRT could result in a resilient enzyme. Although mutating the p51-loop opened the NNBP for drug binding, this was not sufficient to render fRT sensitive to these drugs. While potently inhibiting HIV-1 RT, none of the three inhibitors EFZ, DLV or RPV reduced the activity of the mutated fRT: the mutant retained the same activity as wild type fRT in both the presence and absence of these NNRTIs . AlthougVariances distinct to fRT clustering around the nucleotide-binding site may also add to the overall resilience of the feline enzyme. A cross-species study investigating FIV virus evolution revealed that the majority of positive-selection mutations mainly cluster in the nucleotide-binding pocket and sites contacting nucleic acid substrates . Modelinhttps://www.hiv.lanl.gov/) shows that distinct substitutions of fRT p51 can occur in HIV-1 p51: K101Q , I135K , E138A (1.88%), and S134R (3 occurrences combined with E138A or S134N). Of the 14 records of K101Q, one was combined with E138A and 4 with I135R. There was yet no record of I135K/R with E138A. The resistant mutant version of FIV RT, combining these mutations together, may now provide a model to predict novel resistance mechanisms that a challenged HIV-1 may exploit in escaping inhibition of bound NNRTIs. The resilient FIV mutant RT offers a model for NNRTI-resistance based on distinct variances inherent in FIV but overlooked in HIV-1, HIV-2 and SIV. These intrinsic species-specific differences, which apparently hinge on variations as subtle as few or single residue mutations, highlight the importance of cross-species analysis for discovering potential unknown alternative routes awaiting HIV-1 exploitation. However, while NNBP residues are not crucially conserved and recombinant RT mutants were found enzymatically active, future studies need to evaluate the replication competence of FIV and HIV-1 viruses containing the p51-loop mutations.Given its importance for productive virus infectivity, it is not surprising to find that RT accumulates the majority of positive-selection mutations during a cross-species study investigating virus evolution . NNBP reProminently, the wild type FIV RT offers a unique model to a more constrained pocket than has previously been observed in other lentiviruses and may, therefore, pave the way for the development of novel NNRTIs that need to disrupt such a locked pocket that may emerge in HIV-1. Whereas RPV appears to be one such inhibitor, further studies need to investigate whether the observed RPV binding to wild type fRT  is speciamp resistance, Novagen) and p51 subunit (residues 1–430) in pET28b  with a thrombin-cleavable His-tag at the N-terminus. FIV RT mutant (FIVm) was generated by introducing the specific mutations into the p51 subunit using QuikChange Kit (Agilent Technologies Genomics). HIV-1 RT construct, containing p66 and protease coding regions in pT5M plasmid, was a generous gift of Prof. Stephen H. Hughes .The two subunits of FIV RT  were subcloned into separate plasmids to facilitate site-specific mutagenesis. The p66 subunit (residues 1–554) was cloned into pET22b (FIV RT constructs were expressed in ArcticExpress cells (Agilent Technologies Genomics) with 0.5mM IPTG induction overnight in 16°C. HIV-1 RT was expressed in BL21(DE3) with 1mM IPTG induction for 4 hours in 37°C.RT proteins from FIV and HIV-1 were purified using HisPur Cobalt Resin (Thermo Fisher Scientific) in a buffer containing 500 mM NaCl, 20mM potassium phosphate pH 7.5, 5 mM β-mercaptoethanol and dialyzed into a final buffer containing 500mM NaCl, 20mM Hepes 6.0, 2 mM DTT. Thrombin  was used to cleave the His-tag of FIV RT , and was subsequently removed using benzamidine sepharose resin . FIV RT proteins were further purified using HiTrap SP HP cation exchange  eluting at 350 mM NaCl.For crystallization, FIV RT was further purified using Superdex-200 size exclusion column  equilibrated with 300 mM NaCl, 20mM Hepes pH 7.4 and 2mM DTT, and concentrated to 10 mg/ml. Protein crystals were grown using the hanging-drop vapor-diffusion method at 4°C. Initial crystals were obtained in C9 condition of crystals screen (Hampton Research) containing 4.0 M sodium formate. Diffraction quality crystals were obtained in 3.8 M sodium formate supplemented with 0.2 M ammonium formate. Cryoprotectant solutions contained an additional 20% glycerol.Diffraction data were collected at beamline I-24 of Diamond Light Source Ltd , and processed with XDSAPP . The strDNA-dependent DNA polymerization activity of RT enzymes was assessed using a DNA primer/template prepared by annealing of 70-mer ssDNA template with a complementary 25-mer ssDNA primer labeled with 6-FAM at 5’-end (Integrated DNA Technologies).Reaction mixtures contained 100 mM Tris-HCl pH 7.5, 75 mM KCl, 0.5 mM dNTP mixture, 60 mM MgCl2, 5 mM DTT and 0.15 μM RT, 0.5 μM DNA substrate. Reactions were incubated for 30 minutes at 37°C and stopped using urea sample loading buffer and heating at 95°C for 10 minutes. Reaction products were resolved using denaturing urea polyacrylamide gel electrophoresis . PercentRT proteins were labeled with Monolith NT protein labeling kit BLUE-NHS (NanoTemper Technologies) and eluted in microscale thermophoresis (MST) buffer . Labeled RT proteins were incubated with serial dilutions of Efavirenz or Delaviridine, and MST measurements were carried out using the Monolith NT.115 in standard capillaries with 40% LED and 20% MST power.RT proteins were diluted (into 20 mM Hepes pH 7.4 and 300 mM NaCl) to reach a final concentration of 0.15 mg/ml and subsequently filled into standard treated capillaries. Label-free differential scanning fluorimetry (nanoDSF) measurements were carried out using Prometheus NT.48 (NanoTemper Technologies) and thermal unfolding was monitored with temperature changing from 15°C to 95°C, with the rate of 1°C/min.Protein sequences were aligned using T-Coffee  and annoS1 FigSequences of RT from HIV-1, HIV-2, SIV and FIV are compared. Top numbering and secondary structure are shown for HIV-1 (PDB code: 5HBM) and are shown for FIV RT at the bottom. Identical residues are shown in bold red font and conserved equivalents in red font. NNRTI-resistance mutations known in HIV-1 and intrinsic in FIV are in blue. FIV substitutions in known HIV-1 NNRTI-resistance positions are in pink except for two well-investigated mutations K101 and E138 in cyan.(TIF)Click here for additional data file.S2 FigElectrostatic surfaces of RT from FIV (left) and HIV-1  are displayed over cartoon representation. Indicated are Y181 (red asterisk) and p51 loop (black asterisk), which is positively charged (blue) in fRT and negatively charged (red) in HIV-1 RT.(TIF)Click here for additional data file.S3 Figm) of wild type RT (45.8 ± 0.7°C) and mutant (43.1 ± 0.1°C) are mean values (with standard deviations) of two repeats (each with triplicate measures) and are presented with black and gray vertical lines, respectively. The difference in Tm (2.7°C) is statistically significant . For clarity, the Y-axis scale of FIVm is shifted downwards by 0.03 ratio points.A ratiometric measurement of the fluorescent signal (Ratio) is plotted against increasing temperature for wild type FIV RT  and the p51 mutant . The melting temperatures (T(TIF)Click here for additional data file.S4 Fig(A) DNA polymerization activity of wild type FIV RT as affected by EFZ (black marks) and DLV (clear marks). (B) Representative gels used in the quantification of NNRTI effect on activity. Gels show the resolution of the fluorescent-primer/template substrate (PT) and the polymerization product (DNA), which is fluorescently labeled only if the fluorescent-primer was elongated by RT activity.(TIF)Click here for additional data file.S5 FigWalleye stereo view of FIV RT (green cartoon) after being modeled bound to DNA (orange) and dNTP (magenta sticks) using HIV-1 RT complex structure . EFZ (red stick-spheres) and dislocated primer-grip (β9/10) and catalytic-loop (β7/8) (purple cartoon) are modeled using was modeled using 1FK9. Potential salt bridges are indicated with black dashed lines.(TIF)Click here for additional data file.