Lats1 in GH3 rat mammosomatotropinoma cells suppressed Prl and Gh promoter activity following an increase in YAP/TAZ levels. In conclusion, we have demonstrated activity of the Hippo kinase cascade in the human pituitary and association of high YAP/TAZ with repression of the differentiated state both in vitro and in vivo. Characterisation of this pathway in pituitary tumours is of potential prognostic value, opening up putative avenues for treatments.Tumours of the anterior pituitary can manifest from all endocrine cell types but the mechanisms for determining their specification are not known. The Hippo kinase cascade is a crucial signalling pathway regulating growth and cell fate in numerous organs. There is mounting evidence implicating this in tumour formation, where it is emerging as an anti-cancer target. We previously demonstrated activity of the Hippo kinase cascade in the mouse pituitary and nuclear association of its effectors YAP/TAZ with SOX2-expressing pituitary stem cells. Here, we sought to investigate whether these components are expressed in the human pituitary and if they are deregulated in human pituitary tumours. Analysis of pathway components by immunofluorescence reveals pathway activity during normal human pituitary development and in the adult gland. Poorly differentiated pituitary tumours  and papillary craniopharyngiomas (PCPs)), displayed enhanced expression of pathway effectors YAP/TAZ. In contrast, differentiated adenomas displayed lower or absent levels. Knockdown of the kinase-encoding  Drosophila Hippo) that activate large tumour suppressor homologs 1 and 2 (LATS1/2), which in turn phosphorylate and inhibit the activation of transcriptional co-activators Yes-associated protein (YAP), and WW domain-containing transcription factor (TAZ/WWTR1), the major effectors of the cascade. YAP/TAZ act as co-activators to TEAD transcription factors (TEA domain family members 1–4)  present with hyperplasia of the anterior pituitary lobe, but reduced secretion of hormones such as LH, PRL and GH , but not in differentiated tumours. Activating this pathway All procedures performed were in accordance with the ethical standards of the institutional research committee  and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Anonymised archival FFPE specimens of five ACP  twice for 10 min, followed by rehydration through a descending ethanol series. Antigen retrieval was carried out in citrate-based Declere unmasking solution (Cell Marque) in a Decloaking chamber NXGEN (Menarini Diagnostics) using the 110°C antigen retrieval protocol. Following blocking for 1 h in TNB-blocking buffer ), samples were incubated overnight in primary antibodies at 4°C in TNB at the following dilutions: YAP , pYAP (S127) , TAZ  and SOX2 . The following day, slides were washed in Tris–NaCl–Tween (TNT) buffer  and incubated in species-specific biotinylated secondary antibodies  diluted in TNB for 1 h at room temperature. Following washes in TNT, slides were incubated in ABC solution (Vector Laboratories PK-6100) for 30 min in the dark and in TSA-Cy3 diluted in TSA Stock Solution (Perkin Elmer NEL760001) for 10 min at room temperature. Subsequently, slides were washed and incubated in Hoechst labelling solution for 30 min at room temperature. After a final wash, they were mounted with soft-set mounting medium . Immunofluorescence staining was assessed as follows: Type A: high levels of both YAP/TAZ, frequent nuclear staining; Type B: robust levels of TAZ with frequent nuclear staining and moderate levels of YAP with occasional nuclear staining; Type C: moderate levels of YAP/TAZ, predominantly cytoplasmic, in over 50% of the tumour; Type D: low levels of YAP/TAZ, predominantly cytoplasmic, between 20 and 50% of tumour; Type E: absent YAP/TAZ staining or low levels in under 20% of the tumour.3, 10 mM HEPES, 2 nM Glutamine and 105 U/L penicillin-streptomycin. Cell culture materials were from Life Technologies, Nunc  and Sigma-Aldrich. Cells were transfected with SuperFect (Qiagen) following the manufacturer’s instructions. siRNA were against rat Lats1 (OriGene and Santa Cruz Biotechnology); a mix of scrambled non-specific siRNA was used as control. The Gh and Prl promoter reporter vectors have the proximal (_593) rat Gh promoter and rat Prl promoter respectively upstream to the luciferase gene . After transfection, cells were left for 48 h in low serum (2% FCS) DMEM, before being treated and/or assayed. The transfection efficacy was determined by cotransfection with the RSV-β-gal construct and results are presented as luciferase:β-galactosidase activity ratio. Each experiment was done in triplicate.GH3 cells (American Type Culture Collection) were cultured in 10% foetal calf serum Dulbecco’s modified Eagle medium (DMEM) supplemented with 2.2 g/L NaHCOCell proliferation was determined 48 h after transfection using the WST-1 colorimetric assay  following the manufacturer’s instructions.GH3 cells were lysed in ice-cold RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktail (Roche). Proteins were separated by polyacrylamide gel electrophoresis and blotted using standard procedures (BioRad). Primary antibodies were against LATS1 , YAP/TAZ  and phosphorylated pYAP(S127)   and β-actin . Anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies were used  and signal was developed with enhanced chemiluminescent solution (Roche). Each experiment was carried out in duplicate.n = 4)  that highly express the stem cell marker SOX2; the main body of the anterior lobe presented with moderate YAP and TAZ immunoreactivity . Both prn = 4) . Strong CTNNB1 mutations (encoding β-catenin)  has been well documented in both the papillary type (PCP), harbouring MAPK pathway mutations as well as the adamantinomatous type (ACP) that harbour catenin) , describcatenin) . Strong catenin)  as well catenin) . Null-cecatenin) . YAP andcatenin) , Table 1catenin) .Figure 2Altogether our data from normal pituitaries and pituitary tumours suggest that high levels of nuclear YAP/TAZ are associated with a more uncommitted state in the anterior pituitary, consistent with previous mouse data . In the Lats1 in rat mammosomatotropinoma GH3 cells reduced YAP phosphorylation at S127 and increased YAP and TAZ protein levels  and a Lister Institute Research Prize to C L A. E J L, A S, S R B, M T and C L A were supported by the Deutsche Forschungsgemeinschaft (DFG) within the CRC/Transregio 205/1 (Project A06 to C L A and S R B and B17 to M T) and CRC/Transregio 127/2, as well as GRK 2251.Concept and design of experiments: P X, M T, C L A. Provision of samples: J M, J R A, A S, T S J, V P, R L, J F. Acquisition of data: P X, E J L, M T, J M, A S. Analysis and interpretation of data: P X, E J L, J M, M T, C L A. Writing and review of the manuscript: P X, E J L, M T, C L A. Supervision of the work: S A, S R B, M T, C L A.


A successful HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs) that target the envelope glycoprotein (Env) spike on the virus. Native-like recombinant Env trimers of the SOSIP design now serve as a platform for achieving this challenging goal. However, SOSIP trimers usually do not bind efficiently to the inferred germline precursors of bNAbs (gl-bNAbs). We hypothesized that the inherent flexibilities of the V1 and V2 variable loops in the Env trimer contribute to the poor recognition of gl-bNAb epitopes at the trimer apex that extensively involve V2 residues. To reduce local V2 flexibility and improve the binding of V2-dependent bNAbs and gl-bNAbs, we designed BG505 SOSIP.664 trimer variants containing newly created disulfide bonds intended to stabilize the V2 loop in an optimally antigenic configuration. The first variant, I184C/E190C, contained a new disulfide bond within the V2 loop, whereas the second variant, E153C/R178C, had a new disulfide bond that cross-linked V2 and V1. The resulting engineered native-like trimer variants were both more reactive with and were neutralized by V2 bNAbs and gl-bNAbs, a finding that may be valuable in the design of germline targeting and boosting trimer immunogens to create an antigenic conformation optimal for HIV vaccine development. There remains an urgent need to create an effective vaccine to reduce the further spread of HIV-1. Many vaccine design strategies are now based on the elicitation of broadly neutralizing antibodies (bNAbs)N-linked glycans. Three or four additional glycans are present on ectodomain of gp41  –25. In g–26–26–36–39–As noted above, the BG505 and other genotypes of the SOSIP.664 native-like trimer now serve as platforms for structure-guided immunogen design as part of bNAb-development programs , 42–45. 42–48–52–The availability of high-resolution structural information on the architecture of BG505 and other SOSIP trimers is a critical guide to design improvements, including those to the V2 bNAb epitopes. The V1V2 domain folds into a four stranded anti-parallel β-sheet Greek key motif in which strands A and B are connected by the flexible V1 loop and strands C and D by the flexible V2 loop , 26, 43.43–Here, we sought to stabilize the V1 and V2 domains by introducing disulfide bonds either within the V2 loop or between the V1 and V2 loops. We show that both strategies can improve the presentation of V2 bNAb epitopes. Thus, the resulting SOSIP trimer variants have improved reactivity with V2 bNAbs and their inferred germline (gl) precursors. Moreover, the corresponding virus mutants are more sensitive to neutralization by V2 bNAbs. These trimer-improvement methods may therefore play a role in bNAb-induction strategies.A–C). To stabilize both domains simultaneously, we constructed three mutants, K155C/F176C, I154C/Y177C, and E153C/R178C, in which the C-terminal part of V1 is now linked to the N-terminal part of V2 by an engineered interloop disulfide bond (C). This category of mutant is hereafter referred to by the term V1-V2 disulfide bond. The positions for the Cys substitutions were chosen because of the close proximity of residues Lys155 and Phe176 (5.5 Å distance between the β-carbons (Cβ) atoms; PDB code 5CEZ), Ile154 and Tyr177 (5.4 Å), and Glu153 and Arg178 (4.1 Å); at such distances, it seemed feasible that a disulfide bond could be formed (C).We designed disulfide bond mutants to stabilize the flexible V1 and V2 domains on BG505 SOSIP.664 trimers , A–C. Toide bond C. This ce formed  Fig. 1CCA–C. To A–C). This category of mutant is hereafter referred to by the term V2-internal disulfide bond. The positions for the Cys substitutions were chosen because a twin cysteine motif is present at the corresponding positions in the V2 loops of SIV and HIV-2 Env and has been suggested to lead to the formation of a loop-stabilizing disulfide bond (D) (C).We also elected to stabilize the V2 loop by creating a new disulfide bond within it. Accordingly, we designed two mutants: Q183C/E190C and I184C/E190C , A–C. Thide bond D (18). Abond (D) C.A–C), and the resulting proteins were expressed transiently in 293T cells. We then assessed the yield, trimer formation, and antigenicity of each mutant, using the unpurified culture supernatants. All three V1-V2 disulfide mutants were expressed at acceptable yields , but a BN-PAGE gel showed that trimer formation was severely impaired (E). The implication is that the disulfide bond–linked V1V2 domains are no longer able to form the intersubunit interactions at trimer apex that are important for trimer formation. The conformationally nonselective 2G12 and VRC01 bNAbs bound the various V1-V2 disulfide bond mutant proteins efficiently . However, the conformationally sensitive (trimer-specific or trimer-preferring) bNAbs PG9, PG16, and PGT145 failed to bind to the K155C/F176C and I154C/Y177C mutants and reacted only poorly with the E153C/R178C mutant. These data patterns are consistent with the poor formation of trimers seen in the gel-based analysis .The five disulfide bond mutants described above were introduced into a D7324-tagged BG505 SOSIP.664 construct , A–C, animpaired E. The imAFig. S1). The Q183C/E190C mutant failed to form trimers, but a moderate level of trimer formation was observed for the I184C/E190C mutant (E). In the antigenicity studies, the Q183C/E190C mutant bound 2G12 and VRC01 somewhat less well than the SOSIP.664 comparator and did not bind PG9, PG16, and PGT145 at all . In contrast, the I184C/E190C mutant bound comparably to 2G12 and VRC01 and was also reactive with PG9, PG16, and PGT145, albeit to a lesser extent than SOSIP.664 . We judged that the extent of binding of these three V2 bNAbs was sufficient to pursue further studies of this V2-internal disulfide bond mutant (see below).The V2-internal disulfide bond mutants were both slightly less efficiently expressed than the parental BG505 SOSIP.664 construct . Each mutant virus was therefore passaged for a prolonged period in two independent SupT1 T cell cultures to see whether compensatory mutations arose that increased replication efficiency. The K155C/F176C and I154C/Y177C mutants failed to replicate detectably during the 3-month experiment, indicating that their defects were beyond repair. However, the E153C/K178C mutant acquired replication competence in both cultures after 3 weeks (note that position 178 in the LAI clone is a lysine and not the arginine present at the corresponding position in BG505 Env). Proviral DNA was therefore sequenced after 1, 2, and 3 months of culture. In both cultures, a point substitution at position 152 was found to have emerged within 1 month (B). Specifically, glycine 152 had changed to either glutamic acid  or valine . The significance of these observations is that residue 152 is directly adjacent to the cysteine substitution at position 153. The changes at position 152 persisted to the end of the 3-month culture period, but we also found that a second change, S128R, arose in the virus in culture 1 (B).As expected from the studies on the BG505 trimers, none of the three V1-V2 disulfide bond mutant LAI viruses was infectious, as measured in a single round infection assay in TZM-bl cells A. Each m 1 month B. Specifulture 1 B.C). To confirm that the G152E, G152V, and S128R compensatory mutations identified by sequencing the env genes were in fact responsible for the restored infectivity, we introduced these changes individually or in combination into an LAI clone that also contained the Cys153–Cys178 V1-V2 disulfide bond. In all three cases, the resulting virus mutants and the quasispecies present in the evolution cultures at month 3 had comparable infectivities for TZM-bl cells (C). The substitutions at position 152 had the strongest effect, whereas the S128R change modestly increased the infectivity of the G152E mutant. Taken together, the evolutionary repair experiments show that a compensatory substitution at position 152, either G152E or G152V, partially restores the infectivity of an otherwise replication-defective E153C/K178C V1-V2 disulfide bond mutant LAI virus.We next assessed the ability of the E153C/K178C virus quasispecies from the SupT1 cultures to infect TZM-bl cells and found that there was a gradual gain of infectivity during the 3 months of evolutionary rescue. By the end of the culture period, the E153C/K178C mutant virus in SupT1 cultures 1 and 2 showed infectivity levels of ∼50 and ∼100% compared with the parental LAI-PG9 virus, respectively C. To conbl cells C. The suA and BFig. S2). However, the G152V mutation was not beneficial .To assess whether the LAI compensatory mutations at residue 152 would translate to the BG505 SOSIP.664 trimer context, we introduced either the G152E or G152V change into the SOSIP.664 E153C/R178C mutant and expressed the proteins by transiently transfecting 293T cells. The G152E compensatory mutation clearly improved both trimer formation, judged using a BN-PAGE gel and the binding of the conformationally sensitive V2 bNAbs , confirming the results of the HEK 293T transfection (A).We then expressed the BG505 SOSIP.664 WT, E153C/R178C, and E153C/R178C/G152E constructs in 293F cells, purified the resulting Env proteins via 2G12 affinity chromatography, and analyzed them with size-exclusion chromatography (SEC). 2G12 binds to all possible forms of Env , and SEC allowed us to determine the trimer formation efficiency for each Env protein. Compared with SOSIP.664, the SEC profile of the E153C/R178C mutant contained many more monomers and dimers and proportionately far fewer trimers, but trimer formation was clearly increased when the G152E change was also present B, confirsfection A.C). Whereas the trimer yield for the BG505 SOSIP.664 construct was ∼2.0 mg/liter, the corresponding values for the E153C/R178C and E153C/R178C/G152E variants were ∼0.2 and ∼0.6 mg/liter, respectively .The improvement described above was not sufficient to allow the BG505 SOSIP.664 E153C/R178C/G152E trimers to be purified by the 2G12/SEC method. However, PGT145-affinity chromatography is an alternative approach to isolate native-like trimers , 63. We Fig. 3ectively . The yieectively . As expeAFig. S3). Alternative (i.e. aberrantly formed) disulfide bonds were also detected in low abundance, but they were quantitatively and qualitatively similar to ones previously identified in the WT BG505 SOSIP.664 trimer .To confirm that the engineered disulfide bonds had actually formed, the loop-stabilized BG505 SOSIP.664 trimers were trypsin-digested into fragments that were analyzed by MS to identify peptides linked by disulfide bonds . The E154 trimer . We concE) (E) (E). The I184C/E190C V2-internal disulfide bond mutant had a very similar appearance to the SOSIP.664 parental trimer, being predominantly (86%) in the closed native-like conformation (E).Negative-stain (NS) EM showed that the PGT145-purified SOSIP.664 E153C/R178C, E153C/R178C/G152E, and I184C/E190C disulfide bond mutant trimers were all in a native-like conformation. The SOSIP.664 trimer was in a closed, fully native-like conformation (>95%), as reported previously E 42). I. IE) (42E) E (64). TE) (E) , 63. We E) (E) , 63. TheE) (E) E. The I1ormation E.Tm = 65.3 °C versus 66.6 °C), whereas the additional presence of the G152E compensatory mutation reverted the Tm to 66.7 °C (F). The corresponding Tm value of 66.3 °C for the I184C/E190C V2-internal disulfide bond mutant trimer was also almost identical to SOSIP.664 (G). Thus, in contrast to when disulfide bonds are introduced into the core regions of a gp120 monomer (I201C/A433C)  to quantify the thermostability of the modified SOSIP.664 trimers. The E153C/R178C V1-V2 disulfide bond mutant was slightly less thermostable than SOSIP.664   or SOSIPC/A433C) , 65, engThe goal of our studies was to investigate whether we could improve the antigenicity of SOSIP.664 trimers for V2 bNAbs. We therefore tested an extensive panel of bNAbs and non-NAbs for their abilities to bind the disulfide mutant trimers in an ELISA. The various trimers were purified by PGT145 affinity chromatography as described above.A). Taken together, these results imply that the more open appearance of these trimers observed in NS-EM studies is associated with an antigenically less preferred conformation. The introduction of the G152E compensatory mutation improved VRC01, PGT151, and 2G12 binding and was also extremely beneficial to the epitopes for the V3-glycan bNAb PGT121 and the V2 bNAbs PG9, PG16, and CH01; indeed, the latter four bNAbs bound better to the E153C/R178C/G152E mutant than to SOSIP.664 (A and B). An increased binding to CD4bs bNAb CH103 was also observed for the E153C/R178C/G152E mutant (B). However, several non-NAbs also bound more strongly to this mutant trimer, compared with SOSIP.664 (A and B).Compared with BG505 SOSIP.664, the E153C/R178C V1-V2 disulfide mutant bound the VRC01, PGT151, and 2G12 slightly less well, whereas the 19b, 14e, and 17b non-NAbs targeting V3 and CD4i epitopes bound more strongly A. Taken OSIP.664 , A and BE mutant B. HoweveOSIP.664 , A and BB). Although binding to VRC01 was similar between mutant and SOSIP.664 trimer, binding to CH103 was improved for the V2-internal disulfide bond mutant trimer (B). There were no, or only very minor, differences in how the 2G12, PGT121, and PGT151 bNAbs or the F105 and 17b non-NAbs bound to the mutant and parental SOSIP.664 trimers (B).The I184C/E190C V2-internal disulfide bond mutant trimer bound all the V2 bNAbs  better than SOSIP.664. The titration curves showed that the maximum binding levels were substantially higher for the mutant trimers compared with SOSIP.664 B. Althout trimer B. There  trimers B.B) (B). Based on half-maximal binding (EC50) values, gl-PG9 binding to the mutant was increased by 5-fold, and the maximum binding level was also higher (C). A similar pattern was seen for the gl-PG16 and gl-CH01 bNAbs, although EC50 values could not be reliably determined because the titration curves did not reach a plateau (B and C).We also assessed whether the gl-PG9, gl-PG16, and gl-CH01 V2 bNAbs were more reactive with the mutant trimers. As reported previously, all three gl-bNAbs bound detectably to the BG505 SOSIP.664 trimer, albeit with lower affinities than the corresponding mature bNAbs B (66). HB) B. Based o higher C. A simi plateau , B and CB). None of the trimer variants bound gl-PGT145 or gl-VRC26.09 detectably (C).The V1-V2 disulfide bond mutant trimer was slightly more reactive with gl-PG9, gl-PG16, and gl-CH01 compared with SOSIP.664, although the beneficial effects of this engineered disulfide were markedly less than those conferred by the V2-internal disulfide B. None otectably C.Fig. S4). The binding site for gl-PG9 also remained stable over time on the V1-V2 disulfide mutant trimers, although incubation at 37 °C reduced the binding strength by 40% .Binding of V2 bNAbs to the V1-V2 disulfide mutant and SOSIP.664 trimer was not affected by preincubation of up to 10 days at 37 °C, indicating the exceptional robustness of the disulfide mutant BG505 trimers . The data pattern implies that the V2 bNAb and gl-bNAb epitopes are not stabilized in the same way by the two disulfide mutational strategies tested here. Superposition of the Env trimer structure with a V1V2 scaffold bound by PG9 shows that the V2 loop can interfere with V2 bNAb binding (A). Whether the V1-V2 disulfide bond mutant improves binding of V2 bNAbs to the BG505 Env trimer obviously depends on the position of the stabilized V2 loop and might be different for the V1-V2 disulfide and V2-internal disulfide mutant trimers.To determine whether the engineered disulfide bonds increased the presentation of V2 bNAb and gl-bNAb epitopes on the native, virion-associated trimer, we made variants of the BG505.T332N Env-pseudotyped virus containing the E153C/R178C/G152E or I184C/E190C changes. The V2-internal disulfide mutant virus was more sensitive to neutralization by V2 bNAbs compared with BG505 , although there was no difference in the sensitivity of the two viruses to PGT145 and VRC26.09, and gl-PG9 neutralized neither virus . Whether the V2 loop interferes with how different bNAbs see their CD4bs epitopes is likely to be influenced by their angle of approach to the trimer. Because CH103 approaches at a 45° angle relative to the trimer axis, it comes into close proximity with the V2 region; hence any alteration in the orientation of V2 caused by an engineered disulfide could alter access of CH103 to its epitope (B). In contrast, VRC01 approaches the CD4bs at an angle parallel to the virus membrane and at 90° to the trimer axis, which means that its neutralization potency is less likely to be affected by the precise orientation of the V2 loop (B).We noted that both disulfide bond mutant viruses were ∼10-fold more sensitive to neutralization by CH103, whereas there was no difference in sensitivity to VRC01, which also recognizes a CD4bs epitope . However, there were also examples, such as gl-PG9, where stronger trimer binding did not translate into more potent neutralization. The correlation was not statistically significant for the V1-V2 disulfide bond mutant trimer and virus . Thus, stabilization of the V2 domain either by a disulfide bond within the V2 loop or by one that links the V1 and V2 domains can improve the presentation of V2 bNAb and/or gl-bNAb epitopes at the trimer apex. However, the beneficial effects of the engineered disulfide bond(s) are not universal for all the V2-targeted antibodies, and the two strategies do not lead to identical outcomes.Overall, the virus neutralization data and SOSIP.664 trimer-binding data were generally concordant. Compared with their unmodified counterparts, the V1-V2 and V2-internal disulfide bond mutant trimers generally interact more strongly with V2 bNAbs and gl-bNAbs compared with SOSIP.664 trimers, and this is also true for neutralization of the corresponding Env-pseudotyped viruses. Thus, increases in Ab binding to the V2-internal disulfide bond mutant trimer and neutralization of the corresponding mutant virus by the same Ab were correlated , but only two of them (E153C/R178C and I184C/E190C) yielded trimers with reasonable efficiency. Even for these mutants, there was a marked reduction in trimer yield compared with the SOSIP.664 trimer. We assume that the introduced cysteine residues interfere with the intricate process of gp120 folding and trimer assembly, perhaps by forming aberrant disulfide bonds leading to dead-end folding pathways or by distorting the gp120 subunit enough to impede trimer formation  arose in an evolutionary rescue culture of an HxB2 virus clone that was crippled by the deletion of a key glycan at position 262. In that case, the S128N change compensated for a substantial folding defect in the gp120 subunit –70. We h–The third step that was required to isolate native-like disulfide mutant trimers was affinity purification using the trimer-specific PGT145 bNAb. This positive-selection procedure allowed the isolation of stable, native-like V1-V2 and V2-internal disulfide mutant trimers. Nevertheless, the eventual yields of the V1-V2 and V2-internal disulfide trimers described here were still substantially  lower than that of the parental BG505 SOSIP.664 trimer. If these mutant trimers prove to be useful components of a bNAb-induction strategy, additional steps will need to be taken to address this limitation. For example, we have found that expressing a mutant trimer in a stable CHO cell line can sometimes improve the yield by severalfold, and modifying key residues at the gp120-gp41 interface can also be beneficial.The V2-internal disulfide bond did improve the trimer binding of V2 bNAbs. One explanation may be that the flexible V2 loop assumes a conformation in which V2 bNAb epitopes are transiently or permanently occluded on a subpopulation of BG505 SOSIP.664 trimers. Such masking could be mediated by the protein or the glycan components of the V2 loop itself or of the nearby V1 loop. Whatever the explanation, several V2 and one CD4bs bNAb epitopes were better presented on the V2-internal disulfide mutant trimer, perhaps because the additional cross-link stabilizes the loop and reduces its propensity to adopt a conformation in which certain epitopes are at least partially occluded. The disulfide bond connecting the V1 and V2 loops also improved the presentation of V2 bNAb epitopes, although less so than the V2-internal disulfide. We also intend to determine whether the V2-internal and V1-V2 disulfides can be incorporated into SOSIP trimers of other genotypes. The length of the V2 loop may be a variable that affects, in particular, the stabilizing and/or other effects of the V2-internal disulfide. Indeed, removal of 7 amino acids from the BG505 V2 contributed to the engagement of V2 gl-bNAbs .For an HIV-1 Env immunogen to induce bNAbs, it is likely to be necessary that it can first activate naïve B-cells that express appropriate gl-bNAb epitopes as their BCRs. A complex evolutionary process may then drive the emergence of a true bNAb. In this context, we found that the affinity of three inferred V2 gl-bNAb precursors for the V2-internal disulfide mutant was increased compared with SOSIP.664. Such an affinity increase may enhance the probability that naïve B-cells expressing similar gl-NAb epitopes will be found and activated when the modified trimers are used as immunogens. Whether improved antigenicity of the V2-internal disulfide mutant trimer correlates with an improved immunogenicity has to be addressed in future immunization studies. Such studies remain to be initiated, for example using knockin mice that express V2 gl-bNAbs as BCRs , 73. The183–Cys191) of the clade C ZM109F Env-pseudotyped virus has recently been described  motif in V2. However, in contrast to the effect of the disulfide that we introduced into the BG505.T332N virus, the ZM109F mutant virus was not more sensitive to neutralization by bNAb PG9 bNAb epitopes was improved when the V1 and V2 loops were stabilized by the V1-V2 disulfide and more so by the V2-internal disulfide.ECTO residue 664 (improvement of homogeneity and solubility) (ECTO and were constructed by adding the amino acid sequence GSAPTKAKRRVVQREKR after residue 664 in gp41ECTO (The BG505 SOSIP.664 construct has been described elsewhere . It was ubility) . SOSIP.6gp41ECTO . Point mThe various SOSIP Env constructs were expressed by polyethyleneimine-mediated transient transfection (together with the furin gene) of adherent HEK 293T cells or of HEK 293F cells adapted for serum-free suspension cultures, as previously described , 47. ForB, which included 2G12-purified trimers, all other experiments were performed with PGT145-purified trimers.For screening purposes, unpurified 293T-expressed trimers were analyzed directly using the culture supernatants obtained 6 days post-transfection. For extensive analysis of V2 bNAb reactivity, Env trimers were purified from 293F transfection supernatants by affinity chromatography using a PGT145 column or a 2G12 column followed by SEC, as described , 47, 63.Fig. 3Env proteins were analyzed using SDS-PAGE and BN-PAGE, followed by Western blotting or Coomassie Blue dye staining as previously described , 78.m NaHCO3 (pH 8.6) overnight. Serially diluted Abs were added for 2 h, washed, and then detected using horseradish peroxidase–labeled goat anti-human immunoglobulin (Jackson Immunoresearch). We also used a modified D7324-capture ELISA protocol that was optimized for detecting gl-bNAb binding , followed by blocking of the plates with casein blocking buffer. Then purified trimers were transferred to the plates in a concentration of 2.5 μg/ml and incubated at room temperature for 2 h. Next, binding of several antibodies to the BG505 trimers was tested using the following Ab concentrations: 2G12, 0.75 μg/ml; PGT145, 0.75 μg/ml; glPG9, 5.0 μg/ml; glPG16, 5.0 μg/ml; glCH01, 5.0 μg/ml; PG9, 0.75 μg/ml; PG16, 0.75 μg/ml; and CH01, 0.75 μg/ml. After incubating the antibodies for 1 h, similar steps were followed as for the D7324 ELISA to detect antibody binding.Purified BG505 trimers were incubated in PBS at room temperature or at 37 °C for 0, 60, 120, 180, or 240 h, and subsequently antigenicity was assessed with a lectin ELISA. Greiner half-well ELISA plates were coated with m, and association and dissociation were measured for 300 s. Trimer binding to a protein A sensor not loaded by antibody was set as background.Antibody binding to purified BG505 trimers was studied using a ForteBio Octet K2. All assays were performed at 30 °C and with agitation set at 1000 rpm. Trimer and antibody dilutions were made in running buffer  in a final volume of 200 μl/well. Antibody was loaded on protein A sensors (ForteBio) at 1.0 μg/ml in running buffer until a binding threshold of 0.5 nm was reached. Trimers were diluted in running buffer at 80 nPGT145-purified BG505 trimers were analyzed by negative stain EM as previously described , 63, 83.Thermal denaturation of purified Env proteins was studied using a MicroCal VP-Capillary DSC calorimeter  or a nano-DSC calorimeter (TA Instruments) as described previously . All EnvN-glycosidase F solution  in 100 mm ammonium citrate buffer (pH 6.5) for 1 week at 37 °C. The fully deglycosylated, and alkylated samples were digested overnight with trypsin (protein to enzyme ratio of 30:1) at 37 °C and were then analyzed by LC-MS using an Orbitrap Velos ProTM hybrid  mass spectrometer equipped with electron transfer dissociation module and coupled to an Acquity ultra performance liquid chromatography system . Approximately 5 μl  of the tryptic digest was injected onto a C18 PepMapTM 300 column , and the peptides were separated using a linear gradient starting from 3% B to 40% B gradient in 50 min and then 90% B for 10 min followed by re-equilibration at 97% A for 10 min. LC-MS runs were performed with a flow rate of 5 μl/min using mobile phases consisting of solvent A: 99.9% HPLC-grade H2O + 0.1% formic acid and solvent B: 99.9% HPLC-grade CH3CN + 0.1% formic acid. Data were collected using the data-dependent mode. To determine the disulfide connectivity profiles, the five most intense ions in a high-resolution scan in the Orbitrap were subjected to alternating collision-induced dissociation and electron transfer dissociation in the linear ion trap. The resulting data were analyzed using the Mascot search engine to identify peptides containing free cysteine residues, and the disulfide bond patterns were then determined manually, as described previously  and cloned into pLAI as SalI–BamHI fragments. The integrity of all plasmids was verified by sequencing. To create a PG9-sensitive LAI variant, four substitutions  were introduced into β-strand C of the V1V2 domain; the substitutions were based on the corresponding residues in the BG505 isolate, which is sensitive to PG9 . The resulting LAI-PG9 virus was then used as the basis for the disulfide mutant viruses described in the results.The pRS1 plasmid expressing the full-length LAI molecular clone (pLAI) was the basis for the WT and mutant viruses used in these studies . Env gene to PG9 . The out6 SupT1 cells were transfected with 5 or 20 μg of LAI-PG9. Virus replication in the cultures was monitored twice a week by visual inspection for the appearance of syncytia and by using a p24-antigen ELISA. The cultures were maintained for 3 months, during which period cell-free supernatants were passaged onto uninfected cells when virus replication was apparent. Smaller quantities of supernatant were used for passaging when the extent of virus replication was sufficient to cause significant levels of cell destruction in the cultures. At regular intervals, cells and filtered supernatant were collected for storage at −80 °C until needed for genotypic and phenotypic analyses. When a substantial increase in virus replication was identified, DNA was extracted from the infected cells using the QIAamp DNA mini kit (Qiagen). The complete proviral env genes were PCR-amplified using primers 1 (5′-ATAAGCTTAGCAGAAGACAGTGGCAATG-3′) and 2 (5′-GCAAAATCCTTTCCAAGCCC-3′) and then sequenced.Evolution experiments were performed as described before , 59, 61.50) was calculated using a sigmoid function in Prism software version 5.0.LAI viruses and BG505 Env-pseudotyped viruses were generated by transfection of 293T cells as described previously , 47, 59.S. W. d. T., I. A. W., J. P. M., and R. W. S. conceptualization; S. W. d. T., M. M.-R., and R. W. S. resources; S. W. d. T., E. P. G., K. S., A. T. d. l. P., and K. B. data curation; S. W. d. T., E. P. G., K. S., A. T. d. l. P., K. B., M. M.-R., W.-H. L., H. D., and R. W. S. formal analysis; S. W. d. T. and R. W. S. investigation; S. W. d. T. visualization; S. W. d. T. methodology; S. W. d. T. and R. W. S. writing-original draft; S. W. d. T. and R. W. S. project administration; S. W. d. T., K. S., H. D., I. A. W., J. P. M., A.B.W., and R. W. S. writing-review and editing; H. D., J. P. M., A.B.W., and R. W. S. supervision; J. P. M., A.B.W., and R. W. S. funding acquisition; A.B.W. software; R. W. S. validation.


The observed data on mutated sequences in clonally-related B cells with a MZ-B cell or follicular B (FO-B) cell phenotype indicates that mutated IgM+ MZ-B and FO-B cells have a common origin. To further investigate the origin of mutated IgM+ MZ-B cells we determined whether mutations occurred in rearranged IGHV-Cμ transcripts using IGHV4 and IGHV5 genes from neonatal rat MZ-B cells and FO-B cells. We were not able to detect mutations in any of the IGHV4 and IGHV5 genes expressed by MZ-B cells or FO-B cells obtained from neonatal rat spleens. Germinal centres (GCs) are absent from neonatal rat spleen in the first few weeks of their life, and no mutations were found in any of the neonatal sequences, not even in the IGHV4 gene family which accumulates the highest number of mutated sequences (66%) in the adult rat. Therefore, these data do not support the notion that MZ-B cells in rats mutate their IGHV genes as part of their developmental program, but are consistent with the notion that mutated rat MZ-B cells require GCs for their generation. Our findings support that the splenic MZ of rats harbors a significant number of memory type IgM+ MZ-B cells with mutated IGHV genes and propose that these memory MZ-B cells are probably generated as a result of an antigen driven immune response in GCs, which still remains to be proven.Previous studies in rodents have indicated that only a minor fraction of the immunoglobulin heavy chain variable region (IGHV-Cμ) transcripts carry somatic mutations and are considered memory B cells. This is in marked contrast to humans where nearly all marginal zone B (MZ-B) cells are mutated. Here we show in rats that the proportion of mutated IgM This contrasts with the dominant population of mature (naïve) follicular B (FO-B) cells located in the follicles of peripheral lymphoid organs, which express low levels of IgM and high levels of IgD (IgMlowIgDhigh). MZ-B cells appear to be in a “pre-activated” state, which is illustrated for example by their high expression of CD80/CD86 and complement receptor 2 (CD21) on their membrane surface in comparison with FO-B cells [+ MZ-B cells carry mutated IgM-encoding IGHV genes [+IgD+CD27+ B cells [+IgD+CD27+ MZ-B cells are not memory cells, but cells that develop in the absence of antigen, in a TI fashion, outside the GCs, along a pathway that differs from classical memory B cells. This conclusion was initially based on the finding that patients with CD40L or CD40 deficiency harbor mutated IgM+IgD+CD27+ MZ-B cells in the blood [+IgD+CD27+ B cells are mutated, and these authors hypothesized that somatic hypermutation (SHM) in this population occurs mainly during foetal development and in very young children. MZ-B cells are already found early during ontogeny in the rat spleen [+ mutated MZ-B cells are memory B cells that can be considered to be formed in GCs.The splenic marginal zone (MZ) is a distinct anatomical compartment dominated by a unique population of B (MZ-B) lymphocytes, in addition to macrophages, dendritic cells in rodents and in humans also CD4 T cells –3. This -B cells , 6. Anot-B cells . MZ-B ce-B cells . The maj-B cells . Autoant-B cells , 12. In -B cells –18. A ha-B cells . Indeed,HV genes , 20. Expptor 2 CD on theirptor 2 CD on theirHV genes , 23. PheHV genes . Human M B cells . The rea B cells . Methods B cells , 23 and  B cells , 20 muta B cells . On the  B cells . Weller  B cells , 26, 30.he blood , 30. Thehe blood , 26. Schhe blood  also obst spleen , 32. At t spleen , 34. WheAdult male at 4.5 months of age and pregnant BN/SsNOlaHsd rats were obtained from Harlan . Rats were housed under clean, conventional conditions at the Central Animal Facility of the University Medical Center Groningen. The adult male rats were housed until the age of 9 months. Two-day-old neonatal rats of both sexes were killed by decapitation. All experiments were approved by the Animal Ethics Committee of the University of Groningen.- and CD90- cells) were sorted. Herewith, we were able to exclude immature B cells (i.e. CD90+ B-cells: [-IgDhighIgMlow) and MZ-B cells (CD90-IgMhighIgDlow) were collected in sterile FACS tubes  containing 500 μl newborn calf serum . At least one million cells per B cell subset were sorted. B cell subsets were obtained with > 95% purity. FlowJo software  was used for flow cytometry data analysis.Rat B-lymphocytes were isolated and purified from splenic tissue as described previously , 35. BriB-cells: , 35, T cRCCTGGAAGATCCT-3' and IGHV5'-CTTAGTGCAGCCTGGAAGGT-3'IGHV3:5'-TGAAACCCTCACAGTCACTC-3', IGHV4:5’-GGTGCA [5’-CAACACTGAAGTCATCCAGGG-3’. To assess the amount and quality of the cDNA, PCR was also performed for β-actin, using β-actin-specific primers as described by Stoel et al. [Total RNA was extracted from sorted cells using the Absolutely RNA Miniprep kit  according to instructions of the manufacturer. Briefly, sorted cells were pelleted by 300xg centrifugation for 10 min at 4°C and then resuspended in a total volume of 350μl lysis buffer containing β-mercaptoethanol (Stratagene). First strand cDNA was synthesized using an oligo-(dT)12-18 primer  and SuperScriptTMII reverse transcriptase  as described in the manufacturer’s protocol. Rearranged IGHV3-Cμ, IGHV4-Cμ IGHV5-Cμ transcripts were amplified in a 50μl reaction mixture, containing 2μl cDNA of either IGHV3-Cμ, IGHV4-Cμ or IGHV5-Cμ family-specific primer, plus 0.6 pmol/μl universal Cμ constant region primer and 2.5U Taq DNA Polymerase (Invitrogen). The IGHV gene family specific primers were: l et al. . The PCRTM PCR Cloning Kit (Fermentas Life Sciences). TOP10F E. coli competent cells (Invitrogen) were transformed with plasmids containing the PCR product. Plasmid DNA was isolated from randomly picked colonies with the Nucleospin Plasmid QuickPure kit . Size of the inserts was determined by digestion of a DNA sample of the plasmids with the appropriate restriction enzymes followed by agarose gel electrophoresis. Plasmids containing an insert of approximately 500 bp were sequenced in both directions by ServiceXS . Sequence processing was performed using EMBL/Genbank Data Libraries and Chromas software . IGHV-Cμ sequences displaying 100% identity and obtained from the same PCR amplification, might be derived from a single B cell and were therefore counted only once in our subsequent analysis. The presence of SHM in IGHV genes are another hallmark of memory B cells and therefore mutated IGHV-Cμ transcripts identified with >2 number of mutations in the VDJ region were considered to be IgM+ memory B cells. Mutational analysis was performed using IMGT/V QUEST database (www.imgt.org/IMGT_vquest/share/textes/). We have calculated the mutations (>2 in the VDJ region) of H-CDR and H-FR regions from IGHV-Cμ transcripts as previously reported by Hendricks et al. [PCR products were cloned into the pJET1/blunt vectors using the GeneJETs et al. .Statistical analysis of the sequence data was performed using SPSS 16 software . IGHV-Cμ sequences displaying 100% identity were considered to be derived from a single B cell and counted only once for statistical analysis. Since Taq DNA polymerase errors might be responsible for 1–2 mutations per sequence, we considered only sequences with more than 2 mutations as truly mutated [- cells) were further analyzed. FO-B cells and MZ-B cells were subsequently defined as CD90-IgMlowIgDhigh and CD90-IgMhighIgDlow. The post sort purity for FO-B cells and MZ-B cells was >95%.Recently, we have constructed and annotated the complete genomic repertoire of the IGH locus of the BN rat . The comExpressed IGHV3, IGHV4 and IGHV5 genes from adult rats were amplified, cloned and sequenced . These IThree of the four IGHV3 germline genes were expressed as productive genes in both B cell subsets, i.e. IGHV3S1, IGHV3S3 and IGHV3S5 . From thWe subsequently analyzed the obtained IGHV-Cμ transcripts that were amplified from both B cell subpopulations. The number of mutations within each rearranged IGHV gene was assessed on the basis of the nucleotide identity to the closest corresponding germline of the IGHV gene counterparts. Sequences with only 1 or 2 mutations were considered to be germline because we cannot exclude the possibility that these differences compared to germline IGHV genes were due to PCR artifacts . We firsWe subsequently analyzed whether this difference in percentage of mutated sequences between FO-B cells and MZ-B cells was present in all three IGHV families tested. There was no statistical difference in percentage mutated sequences between the two B cell subsets among sequences of the IGHV4 and IGHV5 gene families, albeit that there was a strong trend with a higher percentage of MZ-B cells expressing mutated IGHV4 genes present compared to the percentage of mutated IGHV4 genes of FO-B cells  . In contWe analyzed whether there was a difference in the percentage of mutated IGHV-Cμ transcripts between the three IGHV gene families  in the two B cell subsets. As we show in We next compared the number of mutations among the mutated IGHV-Cμ transcripts (i.e. > 2 mutations per transcript) IGHV genes in FO-B cells and MZ-B cells. When taking all mutated sequences from the three families together, MZ-B cells have a significantly  higher number of mutations than FO-B cells . In MZ-BH-CDR3 regions can be used to assess clonal relationships between B cells because the H-CDR3 region is virtually unique for each different IGHV rearrangement. A total of 10 independent clone sets (designated as C#1-C#10) were found among the two B cell subsets. Seven clone sets (2–3 members per clone) had members found exclusively found within the MZ-B cell subset and the remaining three clone sets had shared members between the MZ-B cell subset and the FO-B cell subset see . The seq-IgMhighIgDlow cells and CD90-IgMlowIgDhigh respectively. By including CD90 in our staining combination, we excluded immature MZ-B and FO-B cells from our analysis.Weller et al and others postulated that mutations in MZ-B cells are not the consequence of an antigen-driven response, but are an intrinsic property of these B cells, introduced during their development , 26. In + MZ-B cells varied significantly when different IGHV gene families were analyzed. This proportion of mutated MZ-B cell-derived sequences occurred up to 28% for IGHV5 transcripts and up to 66% for IGHV4 transcripts in adult rats. For this reason, we chose to analyze IGHV-Cμ transcripts encoding for IGHV5 and IGHV4 family genes. IGHV-Cμ mRNA transcripts from both neonatal FO-B cells and MZ-B cells were amplified by RT-PCR, cloned, sequenced and analyzed for the presence of SHM. The IGHV5 gene family is the second largest IGHV gene family in the rat and consists of 27 potentially functional IGHV genes [+ memory B cells are absent in neonatal animals supports the hypothesis that IgM+ memory B cells are generated in the GCs. The absence of mutation profiles of IGHV genes in neonatal rats therefore revealed that mutated IgM MZ-B cells are possibly generated in an antigen GC-dependent process in rats as opposed to the GC-independent process proposed by Weller et al. for humans [In the analysis of adult IGHV genes, we showed that the proportion of mutated IgMHV genes . In totaHV genes , of whicHV genes , 38, ther humans .+ expressing, memory MZ-B cells in the rat [highIgDlow) in comparison with FO-B cells (IgMlowIgDhigh) obtained from the adult BN rat spleen. The analysis was confined to three different IGHV gene families, which differ in size: IGHV3, IGHV4 and IGHV5. These three IGHV gene families have 4, 2 and 26 functional IGHV genes, respectively. The IGHV3 and IGHV4 gene families were chosen to determine whether there is a difference in mutation frequencies among members of the IGHV gene families that are relatively small and to compare this frequency to the second largest IGHV gene family (IGHV5) in the rat, which had been analyzed previously in the PVG rat [+ memory B cells among IGHV5 and IGHV6 gene families were all mutated which shows that the high frequency of mutations is not limited due to individual IGHV genes. Further, Colombo et al. [highCD27+), GC B cells and class-switched B cells were mutated, albeit with a lower average number of mutations than both GC and class-switched B cells. However, the average number of mutations in human MZ-B cells (11.8) [+ MZ-B cells (8.8) and IgG+ MZ-B cells (7) [is due to rats requiring fewer mutations to diversify their antibody repertoire after immunization than humans, because rats can encode a larger pool of different antibodies from their primary repertoire. In addition, it is possible that differences in life span and environmental conditions also contribute to differences in the average mutation frequency per IGHV gene. During their long lives, humans may encounter a greater variety of antigens than laboratory rats that live in well-controlled laboratory conditions.Previous studies provided evidence supporting the existence of mutated, IgM the rat . Dammers the rat  demonstr the rat , 41, 42. the rat  it becam the rat . Here we PVG rat . The BN  PVG rat , 44, we  PVG rat . This di PVG rat , 20. Our PVG rat , 41, 42. PVG rat  analyzedells (7)  found inhighIgDlow MZ-B cells.Several pathways for the development of IgM and IgG B cells have been proposed. As previously suggested , shorter+ MZ-B cells are derived from GCs or whether they represent a GC-independent B cell population. Among the mutated sequences, we observed groups of mutated sequences that were derived from clonally related B cells, i.e. these sequences had identical H-CDR3 regions, used the same IGHV gene, expressed shared mutations and were from the same rat. Most (70%) of groups of cells had members that were confined to the MZ-B cell compartment. However, importantly, some clonally related groups of cells had members that were found among both MZ-B cells and FO-B cells. This clonal relationship strongly suggests that mutated IgM+ MZ-B cells and mutated IgM+ FO-B cells have a common origin. This is consistent with our previous finding that clonally related class-switched, mutated B cells co-exist with members in both the MZ-B and FO-B cell compartment [+ FO-B cells and MZ-B cells are probably GC derived. In contrast to mice, mutated IgM+ B cells can still be found in humans with CD40 or CD40L deficiency , that lack classical CD40L mediated T cell help and lack GC formation [+IgD+CD27+ cells, also called natural effector cells, that correspond to splenic MZ-B cells [+ B cell populations could not be formed in CD40/CD40L deficient HIGM patients. Berkowska et al. [+IgD+CD27+ cells have a relatively low replication history. Furthermore they are already present in very young (< 2 years) children and in human foetuses [+IgD+CD27+ MZ-B (-like) cells are not derived from GCs and are generated in the absence of T cell help. Weill and colleagues [+ MZ-B cells can be derived from GCs. Recently, Aranburu and co-workers [+ MZ-B cells are not GC-derived memory B cells. Instead, these authors postulated that the mutations in human MZ-B cells are acquired during their development in order to diversity their primary repertoire, in a GC and T-cell independent fashion. To test this hypothesis in rats we investigated the possible presence of mutated IgM+ MZ-B cells in neonatal rats. Neonatal rats do not develop GCs in the first weeks of life [+ MZ-B cells seen in adult animals are bona fide memory cells which are most probably generated under the influence of external antigenic stimuli in the GC.Memory cells are generally believed to be generated in GCs. It is still controversial, however, whether mutated (memory) IgMpartment . Possiblpartment , 51. Thuormation , 52–54. a et al.  observedfoetuses , 31, 54.lleagues  speculatlleagues . Our obslleagues  observed-workers  proposed-workers , 54, 56  of life , 38. Thu of life , 26, 56,


WO-Seq: a bisulfite-free and base-resolution sequencing method based on peroxotungstate oxidation is presented for the identification of hm5C sites in the transcriptome. Combining the peroxotungstate oxidation with TET enzyme oxidation, m5C can also be detected in a procedure termed TET-Assisted WO-Seq (TAWO-Seq). 5C) and 5-hydroxymethylcytidine (hm5C) have also been identified in RNA, their distribution and biological function in RNA remain largely unexplored, due to the lack of suitable sequencing methods. Here, we report a base-resolution sequencing method for hm5C in RNA. We applied the selective oxidation of hm5C to trihydroxylated-thymine (thT) mediated by peroxotungstate. thT was subsequently converted to T during cDNA synthesis using a thermostable group II intron reverse transcriptase (TGIRT). Base-resolution analysis of the hm5C sites in RNA was performed using Sanger sequencing. Furthermore, in combination with the TET enzyme oxidation of m5C to hm5C in RNA, we expand the use of peroxotungstate oxidation to detect m5C in RNA at base-resolution. By using this method, we confirmed three known m5C sites in human tRNA, demonstrating the applicability of our method in analyzing real RNA samples.5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), two of the best-studied DNA modifications, play crucial roles in normal development and disease in mammals. Although 5-methylcytidine (m M5C is abundant in noncoding RNA, and has the ability to stabilize tRNA secondary structure,5C has been shown to be enriched in Drosophila melanogaster mRNA, increase mRNA translation and play a central role in Drosophila brain development.5C also exists in mammalian RNA, albeit at low levels,5C in the mammalian transcriptome remain unknown due to the lack of sensitive and robust sequencing methods.Epitranscriptome, which refers to the multitude of RNA chemical modifications, has vital roles in post-transcriptional gene regulation.5C in RNA is to adopt bisulfite sequencing, which is widely used to sequence 5mC in DNA. Bisulfite treatment deaminates unmethylated cytosine to uracil in single-strand RNA, while leaving m5C unconverted. Therefore, bisulfite sequencing provides base-resolution information of m5C. Using bisulfite sequencing, widespread m5C sites were identified in both coding and non-coding RNAs.5C in RNA are immunoprecipitation-based that use m5C-specific antibodies or methyltransferases to pull down m5C-containing RNA.5C. Mapping hm5C in RNA is even more challenging. To date, there is no base-resolution sequencing method for hm5C. The only reported method is the antibody-based immunoprecipitation approach.5C and m5C based on peroxotungstate oxidation.The most common way to sequence mthT). thT is a thymine derivative, and will induce C-to-T transition in DNA after PCR. However, the peroxotungstate oxidation reaction requires single-strand DNA. The reaction is strongly inhibited in double-strand DNA with a conversion rate of less than 10%, which severely limits its application.5C in RNA, which is mostly single-stand . To enable us to monitor the efficiency of the hm5C-to-T conversion, one hm5C was positioned so that, upon successful hm5C-to-T conversion, a TaqαI restriction enzyme recognition site in the resulting RT-PCR product was destroyed 5C sites, while Superscript III and Bst 3.0 DNA polymerase induced truncations at the reacted hm5C sites   was also tested for the TAWO-Seq, which gives similar results with NgTET1  . The resAWO-Seq) . RestricAWO-Seq) . Sanger g. S7 ESI, 33.3% oAsp(GUC) in 293T cells. The tRNAAsp(GUC) contains three known m5C sites at structural positions 38, 47 and 48  was cloned and sequenced. As shown in Fig. S10b and c , we found that m5C at position 48 has the highest C-to-T conversion rate . According to the tRNAAsp(GUC) structure, this site is in a double-stranded CpG context, which is an ideal substrate of TET enzyme.5C-to-T conversion rates of three m5C sites are caused by the sequence preference of the TET proteins.To further demonstrate the utility of TAWO-Seq on real RNA sample, we applied it to the endogenous tRNAand c ESI, 35.2% o5C in RNA. We demonstrate the specific hm5C-to-T transition using peroxotungstate to oxidize the RNA followed by cDNA synthesis with the TGIRT enzyme, and Sanger sequencing results have proved the base-resolution sequencing ability of this method. We further demonstrate the ability of TAWO-Seq to detect m5C by combining WO-Seq with the prior NgTET1 or mTet1 oxidation of m5C to hm5C. The successful detection of m5C sites in human tRNA demonstrates our method is applicable for real RNA samples. Both WO-Seq and TAWO-Seq could potentially solve the false positive issue of bisulfite sequencing since they directly detect modified cytosine without affecting unmodified cytosine. Further improvement of both methods to increase the conversion rate and apply to mRNA samples using next-generation sequencing technology are underway in the lab. We believe this method could be highly useful for the identification of unexplored m5C/hm5C distribution and function in the transcriptome.In conclusion, we have described WO-Seq as an RNA friendly, chemical oxidation-based, base-resolution method to sequence hmWe would like to acknowledge P. Spingardi, G. Berridge and B. Kessler for helping with the HPLC-MS/MS; C. He for the mTet1; F. Howe for editing the manuscript. This work was supported by the Ludwig Institute for Cancer Research. Work in the C.-X. Song lab is also supported by Cancer Research UK (C63763/A26394 and C63763/A27122), NIHR Oxford Biomedical Research Centre, and Conrad N. Hilton Foundation. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. F. Yuan and Y. Bi are supported by China Scholarship Council.There are no conflicts to declare.Supplementary informationClick here for additional data file.


Measles virus (MeV) is a highly contagious, re-emerging, major human pathogen. Replication requires a viral RNA-dependent RNA polymerase (RdRP) consisting of the large (L) polymerase protein complexed with the homo-tetrameric phosphoprotein (P). In addition, P mediates interaction with the nucleoprotein (N)-encapsidated viral RNA genome. The nature of the P:L interface and RdRP negotiation of the ribonucleoprotein template are poorly understood. Based on biochemical interface mapping, swapping of the central P tetramerization domain (OD) for yeast GCN4, and functional assays, we demonstrate that the MeV P-to-L interface is bipartite, comprising a coiled-coil microdomain proximal to the OD and an unoccupied face of the triangular prism-shaped C-terminal P X-domain (P-XD), which is distinct from the known P-XD face that binds N-tail. Mixed null-mutant P tetramers regained L-binding competence in a ratio-dependent manner and fully reclaimed bioactivity in minireplicon assays and recombinant MeV, demonstrating that the individual L-binding interface elements are physically and mechanistically distinct. P-XD binding competence to L and N was likewise trans-complementable, which, combined with mathematical modeling, enabled the mechanistic characterization of P through two- and stoichiometrically-controlled three-way complementations. Only one each of the four XDs per P tetramer must be L or N binding-competent for bioactivity, but interaction of the same P-XD with L and N was mutually exclusive, and L binding superseded engaging N. Mixed P tetramers with a single, designated L binding-competent P-XD caused significant RdRP hyperactivity, outlining a model of iterative resolution and reformation of the P-XD:L interface regulating polymerase mobility. Paramyxoviridae recommend the P-XD:L protein-protein interface as premier target for directed drug discovery against emerging and re-emerging paramyxoviruses.MeV belongs to the order of non-segmented negative polarity RNA viruses, which includes devastating human pathogens. While all feature encapsidated RNA genomes and P-L type polymerase complexes, insight into the intermolecular interactions within the polymerase hetero-oligomer and between the polymerase complex and the RNA-encapsidating N protein is rudimentary. Our mapping of the MeV P-to-L interaction revealed a bipartite interface with physically and mechanistically distinct contact zones, which provided a unique experimental platform to dissect the stoichiometry and dynamics of P interactions with L and N through functional trans-complementation assays in minireplicon settings and, ensuring physiological significance, recombinant virions. The identification and functional characterization of a novel L-binding face on the P-XD triangular prism, distinct from the side contacting N-tail, places P-XD at the center of a regulatory mechanism that controls the dynamics of polymerase advancement along the encapsidated genome through iterative separation and restoration of P-XD interaction with L. These observations and the high structural homology of polymerase components within the  Non-segmented negative polarity RNA viruses comprise the etiological agents of some of the most devastating acute viral infections, such as Ebola virus, rabies virus, and, of the paramyxovirus family, Nipah virus and MeV. Common to all viruses in this order, the single-stranded RNA genomes are wrapped by nucleocapsid (N) proteins into helical ribonucleoprotein (RNP) complexes, and encapsidated RNAs are exclusively recognized as templates for transcription and replication by the viral RNA-dependent RNA-polymerases (RdRPs) complexes .0-P complexes with newly synthesized free N, preventing premature N multimerization and ensuring delivery of N to the replication site for co-transcriptional encapsidation of nascent genomic and antigenomic viral RNAs [The core components of these RdRPs are the viral large (L) and phospho- (P) proteins –4. L conral RNAs –18. Althral RNAs , little 0-P complexes [We have addressed this knowledge gap in the present study by example of the polymerase complexes of an archetype of the paramyxovirus family, MeV. As is characteristic for all paramyxovirus P proteins, MeV P has a modular organization in which three defined functional modules, an N-terminal domain, the central OD (residues 303–377), and the C-terminal P-XD (residues 459–507) are separated by large stretches of intrinsically disordered residues , 20 Fig. The N-tomplexes . The OD omplexes –24. In tP-XD does not tetramerize but stabilizes RdRP interaction with the RNP template through interaction with a molecular recognition element (MoRE) that is located in the largely unstructured C-terminal tail domain of the N protein , 26. Co-Large stretches of the C-terminal half of MeV P (PCT) including the OD have been implicated in contributing to P interaction with L , 31–33. In this study, we have combined biochemical interface mapping with mechanistic assays to better understand the dynamic nature of the MeV P interface with L, probe a role of this interface in ensuring polymerase mobility along the RNP template, and explore determinants mediating spatiotemporal control of the process. Having identified a bipartite P to L contact zone consisting of a microdomain immediately downstream of the OD and an uncharacterized side of the P-XD prism that was previously considered unoccupied, we have examined the individual contributions of these microdomains to physical P interaction with L, interaction dynamics, and RdRP bioactivity in a groundbreaking trans-complementation assay of the distinct contact zones. Supported by mathematical models of experimentally observed trans-complementation profiles, our results reveal a multiplexed and highly dynamic interaction between P, L, and the RNA encapsidating N moieties that attributes a central regulatory role in orchestrating polymerase negotiation of the encapsidated template to P-XD. These data provide a framework for a revised mechanistic model of paramyxovirus RNA synthesis.1708 lacking the L methyltransferase domain, that we have previously shown to be fully folding competent and capable of forming bioactive polymerase complexes when post-translationally combined with the corresponding C-terminal L fragment [1708 or full-length L with P are indistinguishable  or replfragment . Co-IP euishable , but L17aneously .303-360). Further extension of the GCN4-substituted area up to P residue 377 eliminated any appreciable interaction with L. Native-PAGE analysis confirmed that both GCN4-substituted P mutants tetramerized efficiently, whereas PΔOD predictably remained monomeric  for a linear combination model in which only tetramers consisting of four wild type (4×wt) or three wild type and one mutant (3×wt/1×P (361–364)Ala) P monomers contribute to RdRP bioactivity, while all other P tetramer species are biologically inactive Ala mutation that we consider to be due to interference with coiled-coil extension.We tested a series of mathematical models for the best description of the experimental competition data. Assigning all mutant and wild type P proteins co-expressed in the same cell equal probability to tetramerize, we considered mutant occupation of one P monomer to be independent of the other three monomers. Goodness of fit was excellent titution . Even atModeling attempts revealed that these data could not be described with a weighted linear combination function. Therefore, we explored non-linear models, but the complex biology of P-XD prevented a satisfying mathematical representation of the experimental results without overfitting the data set. While our results thus reveal that partially reducing the number of L and MoRE binding-competent XDs within a P tetramer boosts overall RdRP bioactivity, meaningful mathematical modeling of the phenotype requires a better-targeted approach than removal of P-XD entirely. We therefore explored mapping of the P-XD interface contacting L.1708 fragment impacted co-IP results, we re-examined two P mutations abolishing interaction, P (361–364)Ala and P-V463R, against full-length L. Neither P mutant precipitated L efficiently, validating the L1708-based results   . We concAla, respectively Ala was recovered successfully, replicated as efficiently as the genetic parent virus  P and L. Both viruses share considerable protein sequence homology and identity, and heterotypic co-IPs confirmed that MeV P associates with MeV or CDV L with equal efficiency . HeterotAla mutant. However, all mutants tested with alanine substitutions in the connector domain, including those capable of physical interaction with L, significantly reduced P bioactivity in minireplicon assays Ala (Ala was likewise successful with P-V463R but not with P (361–364)Ala Ala . Trans-c–364)Ala , placing+ MoRE−) and L-binding defective (L−MoRE+) P-XDs Ala monomer can be present in a bioactive P tetramer, this mutant enabled us to probe the stoichiometric requirements of the different P-XD functionalities. In contrast to our successful earlier trans-complementation of (L−MoRE+) P-XDs with P (361–364)Ala, the combination of (L+ MoRE−) P-XDs with P (361–364)Ala did not restore RdRP activity. These results indicated that the presence of one MoRE binding-competent or one L binding-competent XD per P tetramer is sufficient for RdRP activity, but these competencies must not reside on the same P-XD if the other three P-XDs are unable to interact with MoRE  P-XDs . Albeit +) P-XDs . We thenith MoRE .Ala double mutant, while the combination of P-V463R-D493K with P (361–364)Ala was bio-inactive inactive . The preThis study demonstrates that distinct microdomains in MeV P are involved in efficient P hetero-oligomerization with L. We favor the view that these microdomains engage L through direct protein-protein contacts, although more indirect associations involving, for instance, host-derived adapter proteins cannot be excluded. Here, the term P:L interface will be used to refer to both direct and multi-component interactions between the P and L proteins. Based on a combination of biochemical and functional assays, we have pioneered a trans-complementation system to probe the stoichiometric requirements for productive P:L interactions. While trans-complementations were carried out in minireplicon assays, a recombinant MeV carrying a P trans-complementation pair in two tandem open reading frames could be recovered readily, efficiently proliferated with standard MeV-like growth profile, and was genetically stable over multiple passages. The absence of compensatory or reversion mutations, in particular, serves as a compelling indicator that the mutant recMeV does not experience strong selective pressure. These results confirm that trans-complementation restored all RdRP activities required for successful paramyxovirus replication. Thus assured of the physiological relevance of the strategy, the assay has advanced our mechanistic understanding of how paramyxovirus RdRp negotiates the encapsidated template in four areas:Ala with wild-type P. It furthermore is consistent with a very recent study demonstrating that the coiled-coil OD of MeV P is required for viral gene expression [Ala and wild type P, only mixed P tetramers harboring precisely one copy of this mutant are bioactive. We have thus shown that three wild type P monomers are able to structurally control one mutant and form proper tertiary assemblies, but two or more P (361–364)Ala subunits in a P tetramer cannot be contained. The P:L interface is bipartite. P monomers were unable to interact with L, but replacement of P-OD with yeast GCN4 was tolerated, demonstrating that P tetramerization is essential for L binding. In contrast to previous suggestions , howeverpression . MathemaDownstream of the P OD proximal section, we identified a conserved second microdomain centered around P residues 404–410 that was a major determinant for the efficiency of P-to-L binding. Changes in this connector region likewise had a dominant-negative effect on P bioactivity, suggesting that the section between P OD and P-XD very likely does not exist monomeric in P complexes, but engages in a tertiary assembly. Trans-complementation revealed that the P-OD proximal and connector sections reside in the same functional groups, raising the possibility of an extended interface with L or, if only one of these regions engages in physical contact with L, interference of mutations in the other microdomain with proper assembly of the actual contact zone.Remarkably, we identified specific residues located on two different α-helices of P-XD that are essential for P-to-L binding. These residues map to a microdomain on a previously unappreciated face of the P-XD triangular prism that is distinct from the side binding MoRE. Importantly, this microdomain resides in a separate functional group from the P OD proximal and connector regions, and mutations destroying this newly discovered L-binding face of P-XD were recessive in competition studies with wild type P. These results indicate that the P-XD interaction with L is mediated by a single XD subunit of the P tetramer, and that at least two distinct binding sites for different P microdomains exist on L, the P oligomerization domain-proximal tetrameric regions (tetrameric feet) and the P-XD (monomeric head) . Neither Only one each of the four XDs per P tetramer must be L or MoRE binding-competent . By intrAla competition profile with wild type P, that at most one P (361–364)Ala monomer can be present in bioactive mixed P tetramers. Employing the P (361–364)Ala mutant as a tool to control the stoichiometry of trans-complemented P tetramers, we noted that although only one each of the four P-XDs must be L or MoRE binding-competent, combining these competencies on only one P-XD per tetramer completely abrogates polymerase activity (+ MoRE+) P-XD and three (L−MoRE+) P-XDs in a mixed P tetramer was sufficient to restore wild-type P like polymerase activity (+ MoRE+) P-XD and three (L+ MoRE−) P-XDs) was entirely bio-inactive (+ MoRE−) P-XDs happens to contact L) did not remain bioactive. P-XD interaction with L and MoRE is mutually exclusive, and L-binding of a given P-XD supersedes MoRE-binding . These cactivity . Presumaactivity , but theinactive . This coAla monomer to otherwise wild type P tetramers reduced bioactivity by approximately 40% according to our regression model. Consequently, wild type P-like 100% bioactivity seen in the Ala monomer. Our competition studies of the P-V463R mutant with wild type P returned even higher peak RdRP values reaching 250% of reference bioactivity. During RNA synthesis, P-XD iteratively separates from and reengages with L . In all + MoRE+) P-XD monomer present in every RdRP complex that can potentially initiate RNA synthesis in this constellation will eventually engage L. Because no other MoRE binding-competent P-XDs are present in that P tetramer, this event appears to create a dead-end situation, presumably resembling that experienced by the trans-complementation pair represented by What could be the mechanistic basis for a boost in RdRP activity when only one P-XD subunit is able to interact with L? A previous study considered that negotiation of the encapsidated template by paramyxovirus polymerases might depend on continuous structural rearrangements within the RdRP complex, possibly including the repetitive dissolution and reassembly of P:L interaction . Based oWe propose a 4-step model to describe the dynamic interplay between P, L, and the RNP . The tet2. The stable cell lines were incubated in the presence of G418 (Thermo-Fisher) (100 μg/ml) at every fifth passage. Cells were transiently transfected using GeneJuice (Novagen) according to the manufacturer’s instructions.Baby hamster kidney cells  stably expressing T7 polymerase (BSR-T7/5), African green monkey kidney epithelial (Vero) cells , and Vero cells stably expressing human signaling lymphocytic activation molecule (Vero-hSLAM) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 7.5% fetal bovine serum at 37°C and 5% CO1708, and P were synthesized in vitro (GeneWiz). The PΔOD variant lacking residues 303–360 was generated through PCR amplification and religation at an added HindIII site. Yeast-derived GCN4 tetramerization domain was PCR amplified from a previously generated template [Ala in tandem was generated based on non-codon optimized mutant versions of IC-B P using the NEBuilder HiFi DNA Assembly kit, introducing and artificial intergenic sequence (IGS) between the two P copies. The assembled recMeV IC-B P-V463R-N-IGS-P (361–364)Ala cassette was transferred to the full-length genomic plasmid between the N-P and P-M intergenic junctions using existing XbaI and SalI restriction sites. Mutagenesis success and the integrity of all PCR-amplified nucleic acids was confirmed through Sanger sequencing.Codon-optimized open reading frames encoding MeV IC-B-derived L, Ltemplate  and insetemplate . A full-5 cells/well) with 1 μg of plasmid DNA encoding codon-optimized MeV L1708 with a C-terminal FLAG epitope tag and 1.8 μg of plasmid DNA encoding MeV P or P mutants, each with a C-terminal HA epitope tag. After 36 hours, cells were washed two times with phosphate buffered saline (PBS) and lysed chemically (50 mM HEPES (pH 7.2), 300 mM NaCl, 1.0 mM EDTA, 1% Triton X-100, protease inhibitors (Roche)). Cleared  lysates were mixed with 5×urea buffer . Samples were incubated for 30 minutes at 50°C and separated on 8% SDS-PAGE gels, tank-blotted on polyvinylidene difluoride (PVDF) membranes (Millipore), and subjected to chemiluminescence detection using specific antibodies directed against the FLAG  or HA  epitopes, MeV N , or against cellular glyceraldehyde-3-phosphate dehydrogenase  as specified. Immunoblots were developed using a ChemiDoc digital imaging system (Bio-Rad) for image visualization. Densitometry was carried out on non-saturated images with global background correction. A full set of positive (wild type P) and negative  controls were included on each immunoblot, and no normalizations across different blots were conducted.Cells were transfected in a 6-well plate format (5 x 10Cells transfected with 1.0 μg MeV P or P mutant-encoding plasmid DNA were harvested after 36 hours as specified above. Cleared lysates were mixed with 1 μl G-250 sample additive (Invitrogen) and 4×Native-PAGE sample buffer (Invitrogen). Electrophoretic gel fractionation was carried out using 3–12% Bis-Tris gradient gels (Invitrogen) and NativePAGE running buffer. Immunoblotting and detection were performed as outlined above.5 cells/well) were transfected with MeV L1708 and P or P mutant-encoding plasmid DNA as detailed above. Cells were lysed 24 hours after transfection and cleared lysates incubated with specific antibodies directed against HA or HIS epitopes / × 100, with signalmax corresponding to cells having received wild type P and signalmin corresponding to cells having received equal amounts of pUC-19 in place of P-encoding plasmid. Results calculated for each biological repeat represent the means of the nine technical repeats, and each condition  was assessed in at least three biological repeats.BSR-T7/5 cells  were transfected in nine technical replicates per condition assessed with plasmids encoding MeV IC-B L (0.02 μg), IC-B N (0.016 μg), the MeV luciferase replicon reporter (0.044 μg), and either IC-B P or P mutants as specified . In all experiments that involved transfecting variable amounts of mutant and wild type P-encoding plasmid DNA ratios, empty vector (pUC-19) DNA was added in the appropriate amounts to ensure that all transfection reactions received the same total amount of DNA. Firefly luciferase activities were determined 24 hours post-transfection in a Synergy H1 microplate reader (BioTek), using Bright-Glo luciferase substrate (Promega) directly added to the wells and signal detection after a 1 to 2-minute stabilization period. Relative RdRP activities, expressed as percentages of that observed in the presence of wild type P, were determined according to the formula % rel. activity =  and the plasmids encoding IC-B N (0.42 μg), IC-B-P (0.54 μg), and IC-B-L (0.55 μg). Transfected cells were overlaid after 48 hours onto Vero-hSLAM cells. Emerging infectious centers were individually transferred to and then passaged twice on Vero-hSLAM cells, followed by whole RNA extraction from infected cells , first strand synthesis using random hexamer primers and Superscript III reverse transcriptase (Invitrogen), PCR amplification of synthesized cDNAs using appropriate primers, and Sanger sequencing. Titers of MeV stocks were determined through TCIDescribed .5 cells/ well) and infected with recovered viruses, either recMeV IC-B or recMeV IC-B P-V463R-P (361–364)Ala at a multiplicity of infection (MOI) 0.01 TCID50 units per cell for 1-hour, followed by replacing the inoculum with growth media. To ensure accurate inoculum concentrations, virus stocks were pre-diluted to approximately 5,000 TCID50/ml and inoculum titers validated through TCID50 titration. Cell-associated viral particles were harvested in 12-hours intervals through scraping of cells in serum-free DMEM, two consecutive freeze/thaw cycles, and clearance centrifugation . Viral titers in cleared samples were determined through TCID50 titration.Vero-hSLAM cells were plated in 12-well format (1.0×105 cells/well) and infected with either recMeV IC-B or recMeV IC-B P-V463R-P (361–364)Ala at a MOI of 0.01 TCID50/cell for one hour, then inoculum was replaced with growth media. Infected cells were imaged in 2-hour increments over a period of 36 hours post-infection with a Cytation5 automated high content imager with phase contrast microscopy capacity (BioTek). The increase in μm2 area size covered by individual infectious centers first detected at 24 hours after infection was quantified over time using the Gen5 microplate software program (Ver. 3.05.11) (BioTek). A maximum of four syncytia/well were followed, quantitations are based on 10 distinct syncytia/virus analyzed. CPE kinetics are expressed as fold-change of area size relative to the 24-hour after infection timepoint.Vero-hSLAM cells were plated in a 12-well format (1.0×10x) and wild type (wt) P was denoted as ρ. Adapting a previous approach to a comparable problem [Based on behavior in native-PAGE, mutant and wild type P co-expressed in the same cell were given equal probability to tetramerize and the mutant occupation of one P monomer was treated independent of the other three monomers. The ratio between mutant  are bio-inactive  are linearly additive:ive Figs , S4 Fig,0wt4M) are bio-inactive Ala and P-V463R were assigned a default bioactivity data point A = 1 when ρ = 0.Experimental measurements of relative RdRP bioactivities were fitted to the above models using Isqcurvefit, a nonlinear regression model in Optimization Toolbox of MATLAB R2017a (MathWorks). Goodness of fit was calculated using ROne or two-way ANOVA with Tukey’s post-hoc multiple comparisons tests or T-tests were used to assess statistical difference between samples, in all cases using the Prism 8 software package (Graph Pad). The nature of individual statistical tests applied is specified in each Figure legend, p value-intervals are shown. Wherever possible, graphical representations of experimental results show both the means of all biological replicates ± standard deviations (SDs) and individual biological repeats. All experiments were carried out in at least three biological repeats.S1 FigCartoons provide a schematic overview of the L constructs. Numbers refer to amino acids. Immunoblots of input and co-precipitated material assessing P interaction with full-length and truncated L. Detection and quantitative analysis as in (TIF)Click here for additional data file.S2 FigSDS-PAGE shows immunoblots of the identical samples after denaturation and reduction.(TIF)Click here for additional data file.S3 FigAmounts of plasmids encoding L and N were kept constant. Symbols show means of experimentally observed biological repeats ± SD (n = 3). Statistical analyses through one-way ANOVA with Dunnett’s multiple comparison test, relative to starting conditions (0.1 μg); .(TIF)Click here for additional data file.S4 FigColumns represent mean relative RdRP activities, symbols show individual biological repeats ± SD (n = 3). Statistical analyses through one-way ANOVA and Tukey’s multiple comparison test .(TIF)Click here for additional data file.S5 Fig1708 and full-length L as co-IP targets.Interaction analysis was carried out as specified in (TIF)Click here for additional data file.S6 FigAlignment with Clustal Omega algorithm (MeV P (NP_056919.1); CDV P (AIN44014.1); RV P (AAB23268.1); hendra virus (HeV) P (APT69525.1); nipah virus (P) P (QBQ56717.1); SeV P (AAB06279.1); HPIV-1 P (AAL89402.1); HPIV-3 P (BAA00921.1); mumps virus (MuV) P (BAA00260.1), HPIV-2 P (ART66806.1), and PIV-5 (YP_138512.1). α-helical regions are highlighted above the sequences and heptad repeats in P-OD indicated; numbering refers to MeV. P residues 361–364 are highlighted in green, color-coding of individual residues in P-XD according to the scheme used in (TIF)Click here for additional data file.S7 FigInteraction analysis, immuno-detection and signal quantitation in (TIF)Click here for additional data file.S8 FigColumns show means ± SD, symbols represent individual biological repeats (n = 3). Statistical analysis through one-way ANOVA and Tukey’s multiple comparison test .(TIF)Click here for additional data file.


During the tests, chlorobenzene (CB) was used as a model reagent since the dioxins are poisonous. The results showed that introducing GO to Ce-V-Ti catalysts can improve the specific surface area and promote the CB adsorption on the surface of catalysts. Simultaneously, the Ce-V-Ti with 0.7 wt % GO support showed the high activity with the conversion of 60% at 100 °C and 80% at 150 °C. The adsorb ability of catalysts is strengthened by the electron interaction between GO and CB through π-π bond. In the case of Ce-V-Ti catalysts, Ce played a major catalytic role and V acted as a co-catalytic composition. After GO modification, the concentration of Ce3+ and V4+ were enlarged. The synergy between Ce3+ and V3+ played the critical role on the low-temperature performance of catalysts under sintering flue gas.Ce-V-Ti and Ce-V-Ti/GO catalysts synthesized by the sol-gel method were used for the catalytic combustion of dioxins at a low temperature under simulating sintering flue gas in this paper. The catalytic mechanism of Ce-V-Ti catalysts modified with graphene oxides (GO) at a low temperature was revealed through X-ray diffractometer (XRD), Brunauer–Emmett–Teller (BET), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), H Dioxin (polychlorinated dibenzofurans and polychlorinated dibenzodioxins) can cause a series of environmental problems such as photochemical smog, atmospheric ozone depletion and ground-level ozone generation, which are considered to be extremely hazardous contaminants due to serious carcinogenicity and different discharges from the body . It is mCatalysts are crucial for the catalytic combustion of dioxins at low temperature. During the past few decades there are a number of catalysts developed for dioxin catalytic combustion, most of which can be classified into three types: noble metals ,6, transIt is reported that graphene oxide with oxygen functional groups is an excellent material for the functionalization of graphene and tunability of its optical properties due to its ultrathin two-dimensional (2D) sheets with 98% optical transmittance . The graIn past research, V-Ti , Ce-Ti , V-Ce 1 and grap3)3·6H2O ethanol solution was added to 34.10 g Ti[O(CH2)3CH3]4 ethanol solution under stirring. Then, 0.07 g commercial industrial graphene oxide  with diameter of 3–10 μm was added without further treatment. The solution of NH4VO3 (0.64 g) and oxalic acid  was added under stirring. The solution was slowly gelled after finishing the reaction between Ti[O(CH2)3CH3]4 and H2O at room temperature. The gel was dried at 110 °C for 12 h and then calcined at 450 °C for 3 h. The obtained samples were labeled as Ce-V-Ti and Ce-V-Ti/GO(x) catalysts, where x represented the weight percent of graphene oxide. TiO2 was the carrier, CeO2 was the active component, VOX was the co-active component, and GO was the modified component in Ce-V-Ti/GO catalysts.Ce-V-Ti and Ce-V-Ti/GO catalysts were prepared by sol-gel method. Firstly, 3.78 g Ce with Cu Kα radiation . The surface area and distribution of pore size were carried out using an ASAP-2020 surface analyzer from Micromeritics, Norcross, GA, USA. Transmission electron microscopy (TEM) was performed with JEM-2100. X-ray photoelectron spectroscopy (XPS) was used to analyze the oxidation states on the surface of the CeO2-VOx-TiO2/GO catalysts with AXIS ULTRP, which used Al Kα (1486.6 eV) radiation as the excitation source (powered at 10 mA and 15 kV). The temperature-programmed reduction (H2-TPR) test was carried out by an N-3000 dual-channel chromatography workstation device. FT-IR spectra were recorded at room temperature on a Fourier Transform Infrared Spectrometer  in the 500–4000 cm−1 range and a resolution of 4 cm−1.Characterization by XRD, BET, transmission electron microscope/high-resolution transmission electron microscopy (TEM/HRTEM), XPS, FTIR and H−1. Feed stream to the reactor was prepared by delivering liquid CB with a syringe pump, and the injection position was cooled with ice water bath to ensure the complete evaporation of the liquid reactor feeds. The reaction flue gas was composed of 16% O2 and 84% N2, and the CB concentration was set to 100 ppm to simulate iron ore sintering flue gas. The reaction temperature was controlled with a thermocouple. The recent gases were analyzed under a given temperature by using an online gas chromatograph (GC) equipped with flame ionization detector (FID1) and SE-54 capillary column for the quantitative analysis the inlet and outlet of CB. During the CB analysis the temperature of the column and detector of GC with nitrogen as carrier gas were set up as 100 °C and 150 °C, respectively.Catalytic conversion was tested in a fixed-bed flow reactor as shown in η) was calculated as follows:inCB and outCB were the inlet and outlet concentration of CB in the system at steady-state, respectively.The conversion ratio of CB .Research about catalytic combustion of dioxins over metal oxide catalysts is shown in 2 produced during catalytic reaction [2 in off-gas were analyzed by online gas chromatograph (GC) equipped with a flame ionization detector (FID). Because of the sufficient oxygen in the reaction gas, no CO was detected. It indicated that CB was complete combustion during the Ce-V-Ti and Ce-V-Ti/GO catalysts combustion reaction. HCl/Cl2 were detected by ion chromatography (LC-2010 PLUS) after adsorption by NaOH solution. The result was analyzed strongly with HCl and Cl2 in − and H+ can combine to produce HCl, and Cl2 is attributed to Deacon Reaction (2HCl + 1/2O22 + H2O), which inevitably generates Cl2. These indicated that CB underwent a completely catalytic oxidation into CO2, H2O and HCl/Cl2 by the detection of CO2 and Cl species.For industrial applications, the stability in catalytic activity is essential for long-time operation. reaction . In orde2 adsorption-desorption isotherms revealed the textural properties of the samples. The BET surface area, pore volume, and pore size of the Ce-V-Ti and Ce-V-Ti/GO catalysts are summarized in 2/g and increased to 123.8 m2/g after adding GO. Meanwhile, the pore volume decreased from 0.29 to 0.15 cm3/g after adding GO, and the pore diameter remained nearly similar. The results indicated that Ce-V-Ti/GO catalysts had porous structures. Studies [The N Studies ,31 have 2 in the in 10–80° range  [2 (JCPDF # 43-1002) [2 by hindering the forming of a Ce-O-Ti solid solution. No diffraction peaks characteristic of carbon species was observed owing to the low carbon loading and the relatively low diffraction intensity of GO. Thus, the presence of GO was further determined by XPS.XRD characterization was performed to study the internal crystal structure of the samples . All sam21-1272) . No obvi43-1002) . These r2 particles were found on the Ti-based structures. The lattice spacing (0.312 nm) matched that of the <111> crystal plane of a standard CeO2 sample (JCPDF #43-1002). Along with CeO2 particles, the TiO2 particles showed an exposed <101> crystal plane with a lattice spacing of 0.352 nm .The morphology of the catalysts was characterized by TEM. As shown in 2 in Ce-V-Ti/GO(0.7) catalysts.The TEM images also showed that the catalysts were homogeneous with no crystalline vanadium oxide phases. To further characterize the distribution of vanadium species in the catalysts, elemental mapping was conducted on Ce-V-Ti/GO(0.7) by scanning electron microscopy (SEM). As shown in 4+ species, while u′ and v′ were assigned to Ce3+ species [3+ species than the rest of the catalysts.The atomic concentration and element chemical state on the catalyst surface was investigated by XPS.  species ,34. Ther3+ ions can be stabilized by decreasing the energy of the Ce 4f levels. The reducibility of ceria was not infected by the fraction Ce3+ ions that were already present. As the additional electrons in Ce3+ occupy localized cerium 4f- states, Ce3+ ions did not interact with the reducibility of catalysts [3+ species could create vacancies with one-charge imbalances and unsaturated chemical bonds on the catalyst surface, which could increase the amount of surface chemisorbed oxygen [3+ ions increased. It indicated that GO changed the chemical environment and the oxidation state of Ce [The fraction of Ced oxygen . After ate of Ce .β) and the other BE at 531.0-531.4 eV were assigned to surface-adsorbed oxygen species (Oα) such as O22− or O− [α shifted to lower values due to “Ce←O” electron-transfer processes through the formation of Ce-O-Ti. Moreover, the amount of Oα (estimated by peak area) increased with the amount of Ce species (α. The Ce-V-Ti/GO(0.7) catalysts showed the highest concentration of Ce3+ and a considerable amount of active surface oxygen chemically adsorbed on the vacancies (O−). Thus, Ce-V-Ti/GO(0.7) showed the highest activity for the catalytic combustion of CB.The O 1s spectra b were de2− or O− . The pea species . Thus, C3/2 level at about 516 eV was ascribed to V4+ species [4+  increased the amount of Lewis acid on the surface [4+ was oxidizable and can assist in the reduction of Ce4+ to accelerate the reaction catalytic cycle. species . As repo species , cerium, species . Higher  surface . V4+ was−1 were attributed to O-H stretching vibrations of hydroxyl, carboxylic, and phenolic groups [−1 could be attributed to C=C stretching vibrations in carboxylic or pyridine-like structures [−1 could be attributed to the stretching and vibration bending vibrations of aliphatic C-H groups, respectively [−1 were ascribed to the bending vibrations of C-O groups [−1), C-H , C=O (1064 cm−1), and C=C (1631 cm−1) existed in Ce-V-Ti and Ce-V-Ti/GO catalysts.The samples were analyzed by FTIR to study the effects of GO doping on Ce-V-Ti catalysts . The absc groups . The adsructures . The banectively . The banO groups . Figure −1 peaks were more intense in the Ce-V-Ti/GO catalysts than in the GO-free catalysts. Ce-V-Ti/GO(0.7) catalysts showed a new peak at 2917 cm−1. The peaks at 2917 and 3417 cm−1 were attributed to aliphatic C-H groups and O-H stretching vibrations in hydroxyl, carboxylic, and phenolic groups, respectively. Hence, the addition of GO strengthened the intensity of aliphatic C-H groups and O-H stretching vibrations, increased the number of hydroxyls, carboxylic, and phenolic acids and favored new aliphatic C-H groups (2917 cm−1). As a consequence, these groups favored the adsorption of CB on Ce-V-Ti/GO catalysts, facilitating the desorption of CB [The 3417 cmon of CB .2-TPR was used to investigate the reducibility of surface oxygen on Ce-V-Ti and Ce-V-Ti/GO catalysts within 200–700 °C  [2 was increased with GO addition. Further reduction reaction was facilitated with the increasing temperature, which resulted in a higher number of Ce3+ ions [4+ to Ce3+ on the surface of nano-crystallites with different sizes was observed in the H2-TPR plots of Ce-V-Ti/GO(0.7) [4+ was promoted after adding GO [H0–700 °C . The H2- species . The peao Ce2O3) . After te3+ ions . Interes/GO(0.7) . It indidding GO . The int2 content of Ce-V-Ti catalysts efficiently increased the specific surface area, which could result in a high distortion extent. After being modified by GO, the low temperature catalytic activity of Ce-V-Ti catalysts was further improved.The catalytic activity test showed that CB combustion over Ce-V-Ti catalysts was higher than most normal catalysts at low temperature. It was because the nano-TiO3+/Ce4+ and V3+). Chlorine was adsorbed by nucleophilic oxygen (basic lattice oxide ions or hydroxyl groups) to form a phenolate intermediate. The catalysts adsorbed the gas-phase oxygen on the active sites to replenish the consumed oxygen. The active surface oxygen species attacked the aromatic ring, and finally oxidized CB into CO2 and H2O. To obtain deeper insight into this reaction at the molecular level, the mechanism of CB combustion over Ce-V-Ti catalysts was proposed based on the above analyses.Generally, CB combustion over the Ce-V-Ti catalyst was the Deacon reaction and followed the MVK mechanism . The chl2 and VOx. Here, O* includes O2−, O−, and O2−.First, the oxidation and reduction reactions could proceed between CeO2/VOx catalysts. During the oxidation process, oxygen could transfer from Ce-OH to VOx. It may promote the rate of cyclic catalytic reactions by accelerating electron transfer between Ce and V. The presence of V3+ improved the catalytic activity and ensured faster electron transfer during the mutual transformation of V3+ and Ce4+.V acted as a co-catalyst in the CeO2, and H2O on the active sites of the catalyst. The CB molecules were then adsorbed on the surface of the catalysts. The C-Cl molecular bond of CB break on the surface of the catalyst and degraded into an intermediate, which in turn degraded into CO2 and H2O and simultaneously produced HCl.In the third step, according to the MVK mechanism, O* attacked CB and oxidized CB into HCl, CO2O3 captured O from oxygen on the surface of the catalysts and was re-oxidized to CeO2.Fourth, CeBET is 500–1000 m2/g). It was consistent with the catalytic activity of Ce-V-Ti/GO catalysts.Hence, catalytic combustion was a process of catalytic combustion and adsorption. Improving the rate of catalytic combustion and the adsorption capacity of the catalyst could effectively improve catalytic activity. Consequently, adding GO into Ce-V-Ti catalysts can increase catalytic activity by enhancing adsorb ability and changing the adsorption mode. GO possessed a similar layered structure to graphene, and was heavily decorated with oxygen-containing groups on the planes or carbon atoms . Studies3+ ions. The chemical interaction between Ce and Ti could improve the amount of oxygen vacancies and Ce3+ ions, both of which can effectively improve the adsorption of acidic sites on the catalysts surface. In addition, a certain amount of low-priced vanadium oxides appeared in Ce-V-Ti/GO catalysts, which could help promote the rate of catalytic combustion through accelerating the recycle reaction of Ce.After adding GO, the adsorption mode could be changed from vertical adsorption mode to parallel adsorption mode, as shown in (1)The catalytic activity of Ce-V-Ti catalysts was improved significantly after modification with GO. The CB conversion over Ce-V-Ti/GO catalysts achieved 60% at 100 °C and 80% at 150 °C, which showed excellent catalytic activity at low temperature.(2)3+ and V4+ on the surface was enlarged, which corresponded with oxygen vacancies. Moreover, the adsorption mode could be changed from a vertical adsorption mode to a parallel adsorption mode by interaction of π-π bonds between CB and GO.After being modified with GO, the specific surface area and adsorb ability of CB were both enhanced. The concentration of Ce(3)3+ ions. V3+ could help to promote the reduction reaction.Ce played a major catalytic role and V acted as a co-catalytic composition during catalytic combustion. The chemical interaction between Ce and Ti could improve the amount of oxygen vacancies and Ce