Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the development of innovative translational strategies against challenging human-tropic viruses.Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and  Several virus-mediated molecular processes remain poorly described, which dampen the development of potent anti-viral therapies. Hence, new experimental strategies need to be undertaken to improve and accelerate our understanding of these processes. Here, as a proof of concept, we employ amino-acid coevolution as a tool to gain insights into the structural rearrangements of Hepatitis C Virus (HCV) envelope glycoproteins E1 and E2 during virus fusion with the cell membrane, and provide a basis for the inhibition of this process. Our coevolution analysis predicted that a specific domain of E2, the Back Layer (BL) is involved into significant conformational changes with E1 during the fusion of the HCV membrane with the cellular membrane. Consistently, a recombinant, soluble form of the BL was able to inhibit E1E2 fusogenic rearrangements and HCV infection. Moreover, predicted coevolution networks involving E1 and BL residues, as well as E1 and BL-adjacent residues, were found to modulate virus fusion. Our data shows that coevolution analysis is a powerful and underused approach that can provide significant insights into the functions and structural rearrangements of viral proteins. Importantly, this approach can also provide structural and molecular basis for the design of effective anti-viral drugs, and opens new perspectives to rapidly identify effective antiviral strategies against emerging and re-emerging viral pathogens. Flaviviridae such as Hepatitis C Virus (HCV), Dengue Virus (DENV), Zika Virus (ZIKV) or West Nile Virus (WNV) are cause of several acute and chronic diseases worldwide. The continuous investigation of the molecular processes by which these RNA viruses infect and replicate into their host is critical to develop innovative anti-viral strategies and anticipate viral resistance to pre-existing drugs.The high potency of viral genomes to mutate is often considered as a major limitation for the development of effective anti-viral strategies. Nevertheless, the high-mutation rate of RNA viruses represents a unique opportunity to decrypt viral protein functions and structures. Highly-evolving viral genomes are shaped by important evolutionary constraints to maintain genetic structure and proper protein folding. Amino-acid coevolution, which refers to mutations of different residues at a similar time frame, mirrors such constraints. Hence, the identification and characterization of coevolution signals imprinted within viral protein sequences can provide unique insights into viral protein functions and conformational changes, and ultimately guide the design of original anti-viral strategies.Virus entry is a conserved, critical step during the viral life cycle and represents a valuable target for the development of antivirals and vaccines. The entry process of HCV is orchestrated by two envelope glycoproteins, E1 and E2, which are incorporated onto the virion surface. During entry, E1E2 mediate viral particle attachment to cell surface receptors and induce the merging  of endosomal and virus membranes at acidic pH, thus leading to release of viral RNA into the cytosol . In contThe structure of a large region of the E2 ectodomain (E2 core) ,7 exhibiMutagenesis studies have shown that both E1 and E2 domains, as well as E1-E2 dialogs, are involved in the HCV fusion process ,14,19–21Here, using HCV fusion as a model of study, we aimed to provide a proof of concept that amino-acid coevolution and protein evolutionary constraints can shed light on viral protein functions and rearrangements. We hypothesized that detection of E1-E2 coevolution patterns can uncover their functional interplays as well as critical features of the HCV fusion mechanism.Blocks In Sequences (BIS), that can robustly detect coevolution signals within conserved cellular and viral proteins using a limited number of protein sequences [in vitro. Beyond providing novel insights into the HCV fusion mechanism, our work also demonstrates that coevolution analysis can shed light on viral-mediated processes and can open avenues for the accelerated design of innovative anti-viral compounds against challenging human tropic-viruses.We recently developed an original computational method, equences –24. TakiS1 Fig), can accurately detect coevolution signals within a wide range of well-characterized cellular and viral proteins [S1 Table). Among those, three clusters  displayed a strong statistical significance  and involved coevolving blocks between E and Pr. When E is assembled as dimer at low pH condition, cluster 2 coevolving block positions supported the close proximity of E DIII and Pr . Similarly, cluster 7 also recapitulated the close proximity between Pr and E domain DII on a trimeric E-Pr structure that form at neutral pH in the endoplasmic reticulum . On a trimeric Pr-E structure (one dimer + one monomer of PrM-E) found at low pH when particles mature in the trans-Golgi, cluster 9 also supported a close proximity between Pr and E DII, but also between E dimers . Two other clusters (cluster 3 and 8) of strong statistical significance  were identified by BIS but did not involve coevolving residues located within E protein (S1 Table). Although cluster 8 was composed of two coevolving blocks located within M protein only, clusters 3 involved coevolving residues located within both M and Pr. As cluster 3, cluster 2 and 9 also supported the existence of interactions between Pr and M . As M and Pr are the two cleavage products of a single PrM protein, these three clusters hence represent additional evidence of BIS ability to recapitulate biologically significant protein interactions. Taken together, our results demonstrate that BIS has the ability to accurately predict the tridimensional assembly of two viral proteins within different conformational states.We have previously reported that BIS, a combinatorial-based coevolution analysis method , and determined whether coevolution signals can also predict E fusogenic rearrangements. Using 17 different DENV E serotype 2 sequences, BIS identified 12 clusters (S2 Table). Among them, nine clusters  displayed associated p-values ranging between 7e-3 to 2e-7 and two clusters (clusters 9 and 10) exhibited associated p-values of 0.058 (S2 Table). Cluster 1 and 2 were either conserved  or too large respectively to be considered. Several clusters  displayed small blocks located within a single region of E both in the linear protein sequence and on the tridimensional structure, suggesting that coevolution signals might contribute to the structural organization of secondary protein sub-domains  . DENV E DI and DII are composed of two or three sub-domains that are distant on the linear protein sequence but form single structured domains in the protein tertiary structure. Cluster 8 blocks were mostly located within the two sub-domains of DII and were consistent with the tridimensional organization of this protein domain . Cluster 8 blocks located within the second sub-domain of DII (DII-2) of each E monomer were in close contact on the dimeric E structure , hence suggesting that coevolution signals can predict point of contacts between E monomers once organized as dimer . Despite lower statistical significance (p<0.06), cluster 9 and 10 coevolving blocks also supported E structural organization as these blocks were distant on the linear structure but close on the E dimer structure . Finally, three clusters  displayed coevolving blocks that were both distant on the linear and tridimensional E structure. During fusion, E DIII folds-over toward DII, and DII becomes at close proximity with the E transmembrane domain [Fig 2C). Cluster 5 and 11 coevolving blocks organization were consistent with these structural rearrangements , suggesting that BIS can recapitulate viral glycoprotein fusogenic conformational changes.As DENV E has been demonstrated to mediate DENV viral fusion, we then aimed to study the intra-protein coevolution signals within DENV E only (S3 Table). Interestingly, most of the identified clusters involved residues in both E1 and in E2 suggesting the existence of conserved tight dialogs between E1 and E2 proteins . Only a few number of statistically significant clusters were found among genotype 4a to 6a sequences, due to the low number of sequences available and to their very low genetic divergence.Following validation of BIS ability to model viral envelope glycoprotein structural rearrangements, we then applied the BIS methodology to HCV E1E2. We analyzed independently using BIS ten groups of E1E2 sequences from different genotypes (gt) or sub-types   for which only a fraction of its/their conformations have been characterized (which is the case for HCV E1 and E2), BIS coevolution clusters can thus only suggest, but not contradict a given conformational hypothesis, this unless the full panel of a protein conformations is known. Consequently, in vitro experiments.Given this particular experimental context and in order to identify putative E1E2 rearrangements during HCV fusion, we thus adopted the following approach. First, we aimed to assign to each E1E2 cluster a given function by mapping clusters blocks with residues previously identified in the literature to impact E1E2 folding/heterodimerization, binding or fusion. Second, we sought to identify among BIS clusters classified as “fusion clusters” a putative protein rearrangement supported by several fusion clusters across multiple genotypes, prior experimental challenge through S3 Fig; note that BIS numbered E1 and E2 residues by considering the first amino acid of E1 as residue #1) showed E1 as coevolving systematically with all the E2 domains. Plotting the gt1a coevolving blocks onto reference sequences  revealed a strong correlation between blocks and residues previously identified in the literature to be important for heterodimer folding or viral binding site conformation  or fusion. This correlation allowed us to propose functions  for most gt1a clusters . When plotted on the E2core structure , most fusion clusters involved blocks located within the E2 BL . Interestingly, some of the fusion clusters involved distant blocks in both E1 and E2 , highlighting that E1 terminal regions and the E2 BL could be in close proximity during fusogenic rearrangements. In addition, BIS also suggested an association between fusogenic rearrangements and a potential packing of E2 domains . Thus, BIS proposed that interdependent rearrangements of E1 and BL could represent a hallmark of E1E2 fusogenic conformational changes.We first focused our efforts on analyzing gt1a clusters. Detailed analysis of these clusters . Statistically significant coevolution networks between E1 and BL were also found among genotype 3 sequences . Similar networks were also found among sequences of genotype 4 to 6, but displayed poor statistical significance for the reason described above. Genotype 3 to 6a cluster positions are available through the webpages indicated in the data availability statement.Analysis of clusters from another HCV genotype (gt2) provided similar findings as fusion clusters also involved the BL  as well as distant blocks on E1  and E2  (S10 Table), hence strengthening the functional significance of BIS analysis.HCV E1 and E2 transmembrane were previously shown to be critical for E1E2 heterodimerization and correct E1E2 functions –28. Cons), reinforcing BIS as a relevant method to model viral protein conformations. The detailed analysis of all the clusters of gt1a and 2, regardless of their attributed function, can be found in  respectively.In parallel, BIS also revealed several gt1a and gt2 structural and multifunctional clusters as supportive of the E2core central scaffold structure , through dialogs with E1, could mediate the evolution from a potential stretched E2 pre-fusion structure toward a domain-packed E2 post-fusion structure .Altogether, BIS coevolution analysis of E1E2 sequences suggested that E2 may adopt a pre-fusion structure distinct from E2core as well as yet unreported molecular rearrangements that could occur during fusion. We hence hypothesized that a movement of the BL  from the H77 gt1a strain, detectable through Coomassie blue staining and Western immunoblotting . Non-reducing SDS-Page electrophoresis and Dynamic Light Scattering (DLS) analysis ( confirmed the homogeneity of the peptide in solution and suggested that BLd-H77 fold as a monomer. The far UV circular dichroism (CD) spectrum of BLd-H77 eluted from size exclusion chromatography displays the molar ellipticity per residue expected for a protein folded mainly in α-helix  harboring envelope glycoproteins of gt1a (H77/JFH-1) but also gt2a (JC1) . Although BLd-H77 was able to slightly inhibit HCVcc infection when pre-incubated with cells prior infection, it showed a strong potency to inhibit infection when present during the first four hours of infection  thus suggesting that BLd-H77 might likely act on early steps of the virus life cycle. Consistently, BLd-H77 was able to efficiently inhibit infection of Huh7.5 by non-replicative retroviral pseudoparticles harboring HCV E1E2 (HCVpp) from different genotypes . This inhibition was specific as BLd-H77 was not able to inhibit infection by pseudoparticles harboring VSVG envelope (VSVGpp). Time-course experiments using BLd-H77  but not on cells. No effect of BLd-H77 on HCV cell entry receptors expression could be observed . Moreover, BLd-H77 was also able to inhibit cell-to-cell transmission  in addition to cell-free infection . Altogether, these results highlighted that BLd-H77 likely inhibits a conserved mechanism during HCV entry without affecting cell susceptibility for infection. Importantly, BLd-H77 was able to inhibit HCVcc and primary human hepatocytes-derived HCVcc virus (or HCVpc) infection of primary human hepatocytes (PHH) and Huh7.5 respectively . Finally, we assessed the ability of BLd-H77 to inhibit infection in vivo. BLd-H77 showed a potency to inhibit HCVcc JC1 infection over time in humanized liver mice treated with 150 μg of BLd-H77 under a prophylactic protocol . Despite their uneven infectivity [S11G and S11H Fig). BLd-H77 had no impact on human hepatocyte viability in mice as assessed by serum albumin concentration over the course of infection . Altogether, our results indicate that BLd-H77 is able to inhibit entry of different types of HCV particles in vitro and in vivo and thus target a strongly conserved virus entry mechanism.To challenge the potential role of the BL in E1E2 rearrangements, we generated a soluble 9kDa 6His-tagged BL domain  and an fication ), demonsectivity , our resFig 4F and 4H), we showed that BLd-H77 could irreversibly neutralize HCV particles regardless of mix dilution, hence suggesting that BLd-H77 can bind native viral particles prior viral entry . To assess the presence of an interaction between HCV particles and the BLd-H77, we constructed a transmembrane form of BLd-H77  . Following lentiviral transduction, BLd-tm expression was detectable at Huh7.5-BLd-tm surface , and did not impact HCV receptor expression . BLd-tm expression at Huh7.5 surface, but not expression of a similar construct encoding for an anchored HIV-1 fusion inhibitor (namely C46), inhibited HCVcc propagation both in a cell-free and cell-to-cell transmission manner . HCVpp entry, but not VSVpp entry,  was inhibited following infection of Huh7.5-BLd-tm, hence highlighting that BLd-H77 specifically inhibits HCV entry likely through binding of E1E2 glycoproteins. Consistently, more HCVpp were detected at Huh7.5-BLd-tm cell surface 4h post infection in comparison to Huh7.5 , suggesting a potential containment of HCV particles by BLd-tm at the cell surface. The ability of recombinant soluble E2 (sE2) to bind more efficiently Huh7.5-BLd-tm cells than Huh7.5 cells in a dose dependent manner  further suggested that virus entry is inhibited through an interaction between E2 and BLd-tm. In order to explore more precisely a putative interaction between E2 and BLd-H77, we designed an ELISA assay to quantify the ability of sE2 to be captured by coated BLd-H77. Our result showed a significant ability of sE2 to bind coated-BLd-H77 and coated-anti-E2 antibody AR3B [S12E Fig). Altogether, these results strong suggest that BLd-H77 inhibit HCV entry by binding to E2 glycoprotein.We then sought to elucidate how BLd-H77 blocks HCV entry. By pre-incubating viral particles with BLd-H77 and diluting the mix prior to infection to reach a BLd-H77 concentration below its efficient neutralizing activity , HCVpp-H77 particles or soluble E2  on Huh7.5 cells. Using a highly specific and previously established HCVpp binding assay [S13B–S13D Fig). Moreover, BLd-H77 neutralizing activity was not competing with the activity of a neutralizing anti-E2 antibody known to inhibit viral particle binding [S13E Fig). Consistently, BLd-H77 could bind viral particles following their binding at the cell surface, and was shown to have its more potent neutralizing activity during post-binding steps . Using a cell-cell fusion assay, we showed that BLd-H77 could strongly inhibit cell-to-cell fusion in comparison to control envelope glycoproteins . Importantly, cell-to-cell fusion was only inhibited when cells were incubated with BLd-H77 before low pH exposure that activate membrane fusion , underlining BLd-H77 ability to specifically binds E1E2 pre-fusion conformations. Finally, using a HCVpp fusion assay with liposomes, which are devoid of any receptors or cell factors, the BLd-H77 inhibited fusion in a dose-dependent manner . Altogether, these results suggest that BLd-H77 specifically blocks E1E2 fusogenic rearrangements and the formation of post-fusion structures through binding to E2 protein, in accordance with BIS predictions.Next, we explored which step of HCV entry is inhibited by BLd-H77. BLd-H77 had no significant effect on attachment of HCVcc  as an interesting candidate for supporting E1 and E2 dialogs, BL movements and transition from E1E2 pre-fusion to post-fusion states. This cluster, similar to gt1a fusion cluster 5, linked two central blocks in E1  and one block in E2 BL . In order to challenge its potential role during fusion, we used cluster 5 blocks to guide the rational design of E1E2 chimeric constructs. The E1 region containing two cluster 5 blocks  and E2 BL regions containing the other cluster 5 block  or not  were swapped between two E1E2 heterodimers from different gt2 strains, one allowing efficient HCVpp entry (J6) and another one mediating sub-optimal HCVpp entry (2b1) . All chimeras were similarly expressed and incorporated . Although J6 chimera carrying 2b1 cluster 5 regions (J6-1/2) displayed a poor entry efficiency, similar to 2b1 parental glycoproteins, a 2b1 chimera carrying J6 cluster 5 regions (2b1-1/2) exhibited >10-fold improved entry ability . In contrast, 2b1 chimera carrying J6 Region 1 and 3  were not optimal for entry, hence suggesting that the E1 cluster 5 blocks and the N-terminal half of the BL domain are involved in a dialog regulating virus entry. Production and titration of HCVcc particles harboring these different chimeric envelopes confirmed these results . Interestingly, 2b1-1/2 also displayed improved ability to mediate cell-to-cell fusion  as well as higher fusion efficiency at neutral pH than at acidic pH , suggesting that this chimera exhibited an E1E2 conformation already primed for fusion at neutral pH. In contrast, J6-1/2 chimera did not increase J6 fusion efficiency and abrogated E1E2 sensitivity to low pH at levels similar to those of 2b1 . Altogether, consistently with BIS predictions, these results suggest that conserved interplays between the central region of E1 and the N-amino-terminus region of the BL likely govern E1E2 fusogenic conformational states. Our results also support the maintenance of such dialogs through coevolution as they appeared to be mediated by genotype-specific regions of E1 and the BL.Beside highlighting potential E1E2 rearrangements during fusion, BIS can identify pairs of residues that need to mutate in concert to guarantee structural compensations and proper viral fitness. Indeed, we have previously demonstrated how HCV entry depends on strain-specific dialogs between particular E1 and E2 domains . Thus, w), which involved four residues within E1 central region (position 78 and 113–115) and a domain of 10 amino acids (452–461) within the Stem region. We employed two poorly divergent genotype 1a E1E2 sequences, H77 and A40, that displayed two E1  and one E2  amino acid differences located at the borders of the gt1a fusion cluster 5 blocks . Unlike J6 and 2b1, the level of functionality of H77 and A40 were relatively close (2.4x104 and 1.6x104 GFP i.u. per ml respectively) despite being significantly different , hence making it challenging to predict the influence of residue swaps on envelope functionality. H77 chimera harboring both swapped E1 and E2 A40 residues significantly impacted HCVpp infectivity, although swapped E1 or E2 residues alone did not impact H77 functionality  despite similar E1E2 expression and incorporation . Importantly, H77 chimeras harboring only the E1 or E2 A40 residues showed defect for cell-cell fusion compared to H77, although fusion ability of the H77 chimera harboring both the E1 and E2 mutations were enhanced . Altogether, consistently with BIS predictions, our results suggest that these E1 and E2 residues −and to larger extend the E1 central region and the E2 stem− are part of a coevolving network that regulates the fusogenic properties of gt1a viral envelope.To extend our transfer between the bioinformatics identification of coevolving amino acid clusters to the functional linkage of these domains, we tested the ability of BIS to pinpoint specific amino acids located in other regions than BL and furthermore, in the context of another genotype than genotype 2 (studied above). For this purpose, we sought to challenge a gt1a fusion cluster identified by BIS . Importantly, the E2 BL emerged as a key element of these rearrangements that could mediate the transition of E1E2 complex from a pre-fusion to a post-fusion conformation. We propose that during this transition, the endogenous E2 BL packs with the front sheet of E2. Thus, a recombinant soluble BLd-H77 could compete with the endogenous E2 BL and block HCV fusogenic rearrangements, then leading to inhibition of membrane fusion . Such competition is consistent with the idea that E2 may harbor a stretched pre-fusion structure exposing internal epitopes. The existence of other E2 structures is also supported by the ability of neutralizing antibodies to target an epitope that is not exposed in the E2core structure .Coevolution signals detected within DENV E and Pr recapitulated several structural features of DENV E/E-Pr protein complexes in different conformational states, hence highlighting the structural accuracy of BIS predictions. Coevolution analysis of HCV E1E2 sequences from several genotypes and sub-types led to the identification of several coevolution signals in HCV E1E2 and suggested that E1 and E2 are strong coevolving partners that refold interdependently during fusion (a305-324 ). Whethea305-324 , suggestBy employing an original multi-disciplinary approach combining computational analysis and experimental assays, our work sheds light on potentially important structural and functional features of the HCV fusion mechanism. Although the clear roles of E1 and E2 during the HCV fusion process still remain to be better defined through the structural resolution of the E1E2 heterodimer at different pH, our work initiates a path toward an experimentally-supported model for HCV-cell fusion.Fig 8).In this putative model, E1 would play the role of a fusion protein protruding onto the virus surface whereas E2 would be a receptor binding protein and a fusion chaperone concealing E1 epitopes. Although E2core is a truncated E2 protein and may not exhibit full E2 properties, E2core does not respond to pH variation . This, cThe Ig-fold β-sandwich structure of E2core has been proposed to display similarities with domain III or B from class II fusion proteins . By chapA primary limitation of current coevolution analysis approaches relies on the availability of a large number of sufficiently divergent evolutionarily-related sequences . Such seIn strong contrast to our previous work  that wasBeyond uncovering important features of the HCV fusion mechanism, our study provides altogether a proof of concept that coevolution can be successfully harnessed to decrypt viral protein rearrangements and interactions, as well as to expand our knowledge of viral proteins-mediated biological processes. Virus entry mechanisms and envelope glycoproteins conserved epitopes represent valuable targets for the development of drugs and innovative vaccine strategies against challenging human pathogens –46. By uExperiments were performed in accordance with the EU guidelines (Directive 2010/63/EU) on approval of the protocols by the local ethical committee , Lyon, France.5 cells/well) into Gentest seeding medium (BD Biosciences) completed with 5% FCS. The next morning, PHH were washed and cultured with the culture medium for PHH William’s E medium  supplemented with 7,5% BSA, 1% ITS , 10-7M of Dexamethasone (Sigma), 1% of non-essential amino acids (Gibco), 1% of Glutamax (Gibco) and 1% Penicillin-Streptomycin solution (Gibco). Sera containing HCV particles were obtained from a gt1b infected patient  and were subsequently amplified in uPA-SCID humanized liver mice. Viral loads (RNA copies number/ml) were determined by RT-qPCR using a clinical diagnostic kit (Abbot).Human Huh-7.5 , BRL3A rat hepatoma (ATCC CRL-1442), and 293T kidney (ATCC CRL-1573) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Invitrogen). Primary human hepatocytes  were centrifuged using a F-12 HAM medium (Sigma) and seeded overnight in collagen-coated 48 well plate  that coevolve with another region of the BL (block 7–2), forming altogether a fusion cluster (cluster 7). Hence, in order to rigorously challenge BIS predictions that suggested a potential redefinition of the BL, we constructed a soluble BLd peptide from residue 390 to 460. Hence, DNA sequence of HCV E2 coding for the residues 390 to 640 was amplified from a genotype 1a envelope H77 (AF009606) cDNA sequence. This sequence was subcloned into a phCMV plasmid to fuse the last 18 amino acids from the C-term part of HCV core that act as a signal peptide, and a CH3-terminal 6 Histidine-tag was added. The resulting peptide of 77 amino acids (71+6) was then named as BLd-H77.In order to construct a relevant BLd soluble peptide derived from a genotype 1a sequence, we took into account the BL borders previously suggested (eference ), but aleference  between BLd-H77 was expressed in 293T following transient transfection and purified from cell culture supernatant (OptiMEM) by fast protein liquid chromatography on a Superdex G-75 gel filtration column . BLd-H77 was re-suspended in 1XPBS. The concentration of purified BLd-H77 peptides was determined by absorption at 205 nm. The mass of BLd-H77 peptide was measured by ESI mass spectrometry using a Finnigan LCQ ion trap mass spectrometer (Thermo Electron Corporation). Analysis by SDS-PAGE electrophoresis was performed using a standard Tris–glycine system and 11% acrylamide gels, in reducing or non-reducing condition. Electrophoresis was followed by either coomasie blue staining or western immunoblotting with an anti His tag antibody.O6-benzylguanine and BCNU. BLd-tm expression in Huh-7 cells was quantified by FACS analysis using a mouse anti human IgG2 and an anti-mouse APC antibody.An anchored form of BLd-H77 was engineered by adding a hinge region (human IgG2) and the transmembrane domain of the CD34 protein to the E2 BLd. The construct, named BLd-tm, was then inserted into Gae-SFFV-IRES backbone harboring selectable marker gene P140K MGMT. A similar construct, but encoding for a HIV-1 gp41 fusion inhibitor peptide , namely BRL cells expressing CD81 or SRBI were washed and stained for 1h at 4°C with a mouse anti-human IgG2 antibody (for detection of C46 and BLd-tm), an anti-CD81 (JS81) and with an anti-human SR-BI (CLA-1) respectively. Cells were then washed and incubated with a secondary anti-mouse or rat APC antibody for 1h at 4°C. Cell surface expression levels were then quantified by flow cytometry (FACS CANTO II–BD Biosciences). For HCV receptor detection on Huh7.5 and Huh7.5-BLd-tm cells, cells were fixed with 2% formaldehyde for 20 min at room temperature and washed. For Occludin staining, cells were permeabilized with Perm/Wash Buffer (BD Biosciences) for 15 min at 4°C prior staining. Human CD81 was stained with JS81 mAb, human SRBI with CLA-1 mAb, human Claudin-1 with the MAB4618 and human Occludin with an anti-Occludin mAb targeting the human Occludin C-terminal region. To characterize the effect of BLd-H77 on HCV receptor expression, Huh7.5 were incubated overnight with BLd-H77 (50μg/ml) prior staining.HCVpp were produced as previously described ,51 from Transfected 293T cells were lysed and nuclei were removed by centrifugation at 12 000 rpm for 10 min. HCVpps were purified and concentrated from the cell culture medium by ultracentrifugation at 82,000xg for 1h 45 min through a 20% sucrose cushion. Cell lysates and viral pellets were subjected to western blot analysis using 3/11 anti-E2 antibody and an anti-MLV-CA antibody as described previously .MultiCode-RTx Real-Time PCR (Luminex) according to manufacturer’instructions and run on a Step One Plus quantitative PCR machine (Life Technologies). Data were analyzed using the MultiCode Analysis Software v1.6.5 (Luminex). The following primers were used for the detection of HCV RNA: GCTCACGGACCTTTCA (sense) and GGCTCCATCTTAGCCC (antisense).Plasmid pFK H77/JFH1/HQL (kind gift of R. Bartenschlager), termed as H77/JFH-1, displaying HCV genome with adaptive mutations  and harboring H77 sequence derived from the BLd-H77peptide, as well as plasmid pFKi389-Venus-Jc1, termed as Jc1, (an intra-genotypic recombinant between J6-CF sequence (AF177036) and JFH1 sequence) were used to produce and electroporate into Huh7.5 the respective H77/JFH-1 and JC1 viral RNAs as described previously . Huh7.5 H77/JFH1 virus was used to infect Huh7.5 and Huh7.5-BLd-tm . At day 1, 3 and 5 post infection, cells were fixed with 2% formaldehyde for 20 min at room temperature, washed and permeabilized with Perm/Wash Buffer (BD Biosciences) for 15 min at 4°C. NS5A expression levels were then quantified using anti-NS5A antibody 9E10 by flow cytometry (FACS CANTO II–BD Biosciences). In parallel, cell supernatants were harvested at each time point, filtered and used to infect naïve Huh7.5. 72h post infections, infectious titers (FFU/ml) of cell supernatant were determined as described above.HCVpp H77 and H77/JFH-1 HCVcc particles were preincubated for 1h at room temperature with PBS or BLd-H77 (50 μg/ml or 35 μg/ml respectively). Then, BLd-H77 and concentrated viral particles were diluted (1/5) with cell culture media or not prior infection of Huh7.5. 4 days post infection, percentages of GFP-positive cells were determined by flow cytometry and FFUs/ml were determined by NS5A immunostaining as described above.H77/JFH-1 were used to infect Huh7.5 for 4h at 37°C . After washing, cells were incubated with anti-E2 antibody AP33 (25μg/ml) alone, or mixed with BLd-H77 (35μg/ml), or with PBS. 72h post infection, cells were fixed and numbers of cell per foci for each condition were quantified through NS5A immunostaining as described above.PHH were washed and infected by JC1 HCVcc virus at different m.o.i. . 4 days post infection, cell culture supernatants were harvested and used to infect naïve Huh7.5. Infectious titers were revealed through NS5A immunostaining 4 days post infection as described above. Indirect titrations were performed as infected PHH were poorly detectable through NS5A immunostaining, making the indirect titration the only accurate method to quantify the amount of infectious viral particles  in a PHH-cell culture supernatant. For neutralization assay, JC1 virus or JC1-derived HCVpc were pre-incubated with BLd-H77  or with PBS for 1h prior PHH  or Huh7.5 infection respectively . 4 days post infection, infectious titers of PHH cell culture supernatants were determined by infecting Huh7.5 as described above. In parallel, infectious titers of JC1-derived HCVpc following Huh7.5 infection were quantified as described above. Sera containing HCV particles were used to infect PHH at a m.o.i. of 0,1 following BLd-H77 (30 μg/ml) or PBS preincubation for 1h at room temperature. 4 days post infection, cell culture supernatants were harvested and viral loads (RNA copies number/ml) were determined by RT-qPCR using a clinical diagnostic kit  with a limit of quantification (LOQ) of 12 IU/mL (i.e. 51.6 HCV RNA copies/ml). Given a serum dilution of 1:100 in PBS, LOQ = 5160 HCV RNA copies/ml.Retroviral vectors expressing human CD81 (NM_004356) and SR-BI (Z22555) were described previously . RetroviBinding assays were performed as described previously . Briefly5 or 5x105 cells) in presence of 0.1% sodium azide for 1h at 37°C. After washing, bound soluble E2 were detected using anti-E2 H53 antibody as described above. Levels of binding enhancement were determined relatively to the basal E2 binding on naïve Huh7.5. For HCVpp binding enhancement assay, 1x105 Huh7.5 cells or Huh7.5-BLd-tm were infected with HCVpp for 4 hours. Cells were then trypsinized and washed with PBFA . Bound viruses were quantified by flow cytometry  following cell staining with the mouse H53 anti-HCV E2 and an APC goat anti-mouse immunoglobulin-G.Concentrated soluble E2  were mixed with equivalent number of Huh7.5 or Huh7.5-BLd-tm (2x104 i.u.) were pre-incubated with BLd-H77 (35 μg/ml), Heparine (250 μg/ml) or with PBS for one hour at 37°C. Viral particles were then mixed with 1x105 Huh7.5 cells for 2h at 4°C. After 3 washings, cells were lysed using the RLT Buffer (QIAGEN) complemented with β-mercaptoethanol. Total RNAs were then extracted using the RNeasy mini kit (QIAGEN) as recommended by the manufacturer. Extracted RNAs were reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad) and HCV viral RNA (5-CTTCACGCAGAAAGCGCCTA and 5-CAAGCGCCCTATCAGGCAGT) and human GAPDH (5- GAAGGTGAAGGTCGGAGTC and 5- GAAGATGGTGATGGGATTTC) were then quantified by qPCR using the FastStart Universal SYBR Green Master kit (Roche Applied Science) on an Step One Plus quantitative PCR machine (Life Technologies). Cell-associated viral RNA copies were normalized on human GAPDH expression for each sample.JC1 HCVcc particles (5x1096-well plates (Corning) were coated overnight with different amounts of a mouse IgG isotype , anti-E2 antibody AR3B  (10 and –/–Rag2–/–Il2rg–/–) mice (mixed background: C57BL/6 and 129Sv) were housed in our animal facility . Because of their lethal phenotype, mice are maintained on 8 mg/l of NTBC (nitro-4-trifluoro-methylbenzoylcyclohexanedione) in the drinking water. 48h prior the engraftment, adult (6–10 weeks old) mice were injected intravenously with 2x109 p.f.u. of an adenoviral vector encoding the uPA transgene. 7x105 to 1x106 PHH (BD Biosciences) were injected intrasplenically as previously described [FRG .5 i.u.) or with patient-derived HCV particles (5x104 i.u.) via intraperitoneal route. Mice sera were collected at day 7 and day 14 post-infection by retro-orbital bleeding. At day 21, mice were sacrificed, sera were collected and levels of human albumin were determined . JC1 infectious titers in mice sera were determined through infection of Huh7.5 with different dilutions of sera as described above. In parallel, for HCV particle containing sera, viral load was determined by RT-qPCR using a clinical diagnostic kit  with a limit of quantification (LOQ) of 12 IU/mL (i.e 51.6 HCV RNA copies/ml). Given a serum dilution of 1:100 in PBS, LOQ = 5160 HCV RNA copies/ml.Highly reconstituted mice (HSA >15mg/ml) were infected with JC1 HCVcc particles inocula . 7 mice were treated under a prophylactic protocol with PBS (4 mice) or with 30 μg (3 mice) of BLd-H77. Mice were treated via intra-peritoneal route one day prior infection, and at day 1, 7 and/or 14 post-infection infection. For all the mice, sera were harvested and viral titers were quantified at day 7, 14 and during sacrifice 21 post infection. A second independent cohort of 7 mice was attributed for another in vivo inhibition assay of JC1 infection (5 mice + 2 negative control). Here, JC1 infection was challenged with a dose of 150μg of BLd-H77 (2 mice) or with PBS (3 mice) under a prophylactic protocol similar as described above. A cohort of 14 mice (12 mice + 2 negative control) was attributed for an in vivo inhibition assay of serum-derived HCV particles infection, challenged with one dose of BLd-H77  or with PBS (7 mice) under a prophylactic protocol similarly to what has been described above. Serum-derived HCV particles viral load were determined as described above. Following sacrifice, the level of human Albumin was quantified for each untreated and treated mice in order to ensure that HCV infection inhibition was not due to a decrease of liver humanization caused by BLd-H77.A first cohort of 11 mice was attributed for an 5 cells/well seeded in six-well tissue culture dishes 24 h before transfection) were co-transfected using calcium phosphate reagent with a HCV  or VSV-G envelope encoding-plasmids and with an HIV-1 LTR  luciferase reporter plasmid. After 12h, transfected HEK-293T cells were detached with versene  and co-cultured (5x104 cells/well) with Huh-7-Tat indicator cells (5.104cells/well) in a 24-well plate. After 24 h, the cells were washed with serum free DMEM, incubated for 3 min in either pH 7 or pH 5 buffer  and then washed three times with serum free DMEM. The luciferase activity was measured 72 h later using a luciferase assay kit according to the manufacturer’s instructions (Promega). For fusion neutralization assay, coculture were washed, pre-incubated with BLd-H77 (50μg/ml) or with PBS for 1h at 37°C prior a second washing and exposure to pH buffer. Alternatively, co-cultured cells pre-treated by BLd-H77 or PBS were incubated for an extension of 24h following pH shock by BLd-H77 (50μg/ml) or PBS.Cell-cell fusion assays were performed as described previously ,19. BrieHCVpp/liposome lipid mixing was performed as previously described ,56. R18-Structural analysis of Dengue E pre-fusion structure (PDB 1K4R), E-Pr complex structure (3C6E), E post-fusion structure (1OK8) and E2core structure (4MWF) were realized using Chimera software  (UCSF).p<0.05, **p<0.01, ***p<0.001).GraphPad Prism software (version 6) was used for statistical analysis. Statistics were calculated using Student’ t test and/or two-way ANOVA when appropriate. Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S1 TableS1 Fig as an example).17 amino acids sequences of DENV E and PrM serotype 2 were aligned and 14 Clusters were identified by BIS. Clusters are computed with the BIS coevolution analysis method –24 and t(DOCX)Click here for additional data file.S2 TableS1 Fig as an example).Clusters are computed with the BIS coevolution analysis method –24 and t(DOCX)Click here for additional data file.S3 TableS4 Table, S7 Table and in the related HCV webserver (http://www.lcqb.upmc.fr/HCVenv/HCVenv.html) are specific to each genotype and set of patient sequences analyzed.Ten groups of sequences were assembled and analyzed independently with the BIS method. Groups were constituted of E1E2 sequences from HCV types and sub-types from genotype 1a to 6a. Groups of sequences from genotypes 1 and 2 were constituted by pools of sequences from subtypes 1a and 1b (50 sequences) and sequences of genotypes 2a and 2b (30 sequences). Total numbers of detected clusters for each genotype and sub-type is reported, as well as the number of statistically significant clusters among them (when p<0.05). For each group of sequence, we also report the number of statistically significant clusters only involving E1 positions (“intra-E1”), the number of clusters only involving E2 positions (“intra-E2”), and the number of clusters across E1 and E2 (“inter-E1-E2”). The assignment of a given cluster block to E1 or E2 was determined by mapping the reference genome sequence of genotype 1b (accession: AJ238799) to the multiple sequence alignment, for each genotype. E1 and E2 were identified on AJ238799 at positions 192–383 and 384–746, respectively. Note however that residue positions displayed in (DOCX)Click here for additional data file.S4 TableS1 Table. Note that residue positions displayed in this table are specific to the set of patient sequences analyzed. Hence, nucleotide gaps generated during the analysis of the patient sequences by BIS were taken into account when plotting gt1a clusters into a gt1a reference E1E2 reference  and into gt1a E2core structure .Clusters are computed with the BIS analysis method similarly to (DOCX)Click here for additional data file.S5 TableS4 Fig, where each block is referenced as follow: “Cluster ID-Block N°”.For each block, the initial and final position of the block predicted by BIS and the name of the cluster it belongs to are given. Blocks from each cluster are numerated from 1 to x (Block N°) to easily identify their position on the E1E2 sequences in (DOCX)Click here for additional data file.S6 TableS4 Table). Genotype 1a clusters harboring blocks that mapped residues previously reported in the literature to have a specific function  are classified. The known role(s) are indicated: Folding or heterodimerization (blue), viral binding site conformation (green) or fusion mechanism (red). According to these roles, clusters are categorized into three different categories: structural , fusion or multifunctional cluster. Clusters harboring blocks that did not map any residues with a previously reported function were classified as clusters with “undefined role.We aligned 25 E1E2 amino acids sequences of HCV genotype 1a and identified using BIS method 16 clusters ((DOCX)Click here for additional data file.S7 TableS1 Table. Note that residue positions displayed in this table are specific to the set of patient sequences analyzed. Hence, nucleotide gaps generated during the analysis of the patient sequences by BIS, as well as gaps between gt1a and gt2 sequences, were taken into account when plotting gt2 clusters into a gt2 reference E1E2 reference  and into gt1a E2core structure .Clusters are computed with the BIS analysis method similarly to (DOCX)Click here for additional data file.S8 TableS7 Fig, where each block is referenced as follow: “Cluster ID-Block N°”.For each block, the initial and final position of the block predicted by BIS and the name of the cluster it belongs to are given. Blocks from each cluster are numerated from 1 to x (Block N°) to easily identify their position on the E1E2 sequences in (DOCX)Click here for additional data file.S9 TableS7 Table). Genotype 2 clusters harboring blocks that mapped residues previously reported in the literature to have a specific function are classified. The known role(s) of the residues in each cluster are indicated: Folding or heterodimerization (blue), viral binding site conformation (green) or fusion mechanism (red). According to these roles, clusters are categorized into three different categories: structural , fusion or multifunctional cluster. Clusters harboring blocks that did not map any residues with a previously reported function were classified as clusters with “undefined role”.We aligned 30 E1E2 amino acid sequences of HCV genotype 2 (2a and 2b) and, using the BIS method, we identified 21 clusters ((DOCX)Click here for additional data file.S10 TableList of clusters identified by BIS that connect the transmembrane of E1 and E2. Six clusters were identified across the two major HCV genotypes. Location of the transmembrane domains within E1 and E2 are indicated for each genotype and sub-types. Position of the coevolving blocks located within the transmembrane of E1 and E2 are indicated for each cluster. TMD, Transmembrane Domain. Only significant clusters are reported .(DOCX)Click here for additional data file.S1 FigCoevolution analyses require the identification of sequence variability and they are usually realized on families of homologous protein sequences, typically very divergent and represented by a large number of sequences. In contrast, studying viral sequences requires methods that can analyze serotypes and genotypes, usually constituted by a limited number of conserved sequences. BIS allows to track small parallel changes likely corresponding to compensatory patterns preserving the structure and function of the protein. Given a sequence alignment, the coevolution analysis method Blocks In Sequences (BIS) –24 ident(TIF)Click here for additional data file.S2 FigS2 Table for cluster positions). Each cluster is identified by a distinct color. Cluster 1 and 2 were too large to be considered. A linear representation of Dengue E protein is also shown at the top of the figure. Starting and ending residue positions of each E domain are indicated. E domains are annotated by distinct colors: DI, domain I (red); DII, domain II (yellow); DIII, domain III (blue); Tmd, transmembrane (black).Mapping of the DENV E clusters 3 to 12 (illustrated by distinct colors) on the E dimeric structure (PDB 1K4R). For each cluster, positions of the coevolving blocks in E sequence are displayed within “strips” located above each structure (See (TIF)Click here for additional data file.S3 FigS4 Table for cluster positions). On the top of each coevolving block is indicated the corresponding protein or E2 domain the block belongs to: HVR1, Hyper Variable Region 1; FL, Front Layer; VR2, Variable Region 2; βS, β-Sandwich; BinL, CD81 Binding Loop; BL, Back Layer; Stem; Tmd, Transmembrane domain). Two small linear representations of HCV E1E2 are located at the top of each “strip” column, and can be used as reference for determining the position of each coevolving block within the E1E1 sequence. At the top of the figure is shown an enlarged linear representation of HCV E1E2, where the starting and ending residue positions of each protein and domain are indicated. E2 domains are highlighted by distinct colors .The 16 gt1a clusters (illustrated by distinct colors) are displayed within “strips” representing the E1E2 sequence. For each cluster, positions of the coevolving blocks in the E1E2 sequence are indicated within the corresponding “strip” (see (TIF)Click here for additional data file.S4 FigS5 Table. Residues (boxes) or domains  previously identified in the literature to have a function are indicated .Genotype 1a cluster blocks are represented as red horizontal bars positioned above the E1 and E2 H77 or JFH-1 aligned sequences. Each horizontal bar is numerated as follow: “Cluster ID-Block N°”. Respective positions of these blocks are referenced in (TIF)Click here for additional data file.S5 FigA), structural clusters 11 (orange), 2 (green) and 6 (red) (B) and undefined role clusters 3 (pink), 9 , 13 (brown), 14 (blue) and 15 (yellow) (C) are plotted on a vertical linear representation of E1 and on a tridimensional view of E2 core (PDB 4MWF). Each cluster is composed of blocks harboring a similar color, according to S3 Fig. The Stem region (Stem) is represented by a dotted line after the C-terminal part of the BL. The transmembrane domain (Tmd) is represented as a rectangle following the Stem region. In B and C, bold lines link E1 and E2 blocks that coevolve. For each cluster, block positions in E1 (at the left of the linear structure) and E2 (below boxes whose color match the color of the corresponding cluster) are indicated. In A, the E2 central scaffold is enlarged and red circles highlight regions where cluster 4 blocks are in close proximity. Rotation angles of the E2core structure are indicated. Viewing angle of E2core is indicated by a black cross . FL, Front Layer; BinL, CD81 Binding Loop; VR2, Variable Region 2; BL, Back Layer; βS, β-Sandwich.HCV E1E2 gt1a multifunctional clusters 4 (blue) and 16 (orange) ((TIF)Click here for additional data file.S6 FigS7 Table for cluster positions). On the top of each coevolving block is indicated the corresponding protein or E2 domain the block belongs to: HVR1, Hyper Variable Region 1; FL, Front Layer; VR2, Variable Region 2; βS, β-Sandwich; BinL, CD81 Binding Loop; BL, Back Layer; Stem; Tmd, Transmembrane domain). Two small linear representations of HCV E1E2 are located at the top of each “strip” column, and can be used as reference for determining the position of each coevolving block within the E1E1 sequence. At the top of the figure is shown an enlarged linear representation of HCV E1E2, where the starting and ending residue positions of each protein and domain are indicated. E2 domains are highlighted by distinct colors .The 21 gt2 clusters (illustrated by distinct colors) are displayed within “strips” representing the E1E2 sequence. For each cluster, positions of the coevolving blocks in the E1E2 sequence are indicated within the corresponding “strip” (see (TIF)Click here for additional data file.S7 FigS8 Table. Residues (boxes) or domains  previously identified in the literature to have a function are indicated (see Online Methods for references). A color code, reported below the sequence alignment, links residues (boxes) or domains (arrow) to specific functions according to the literature: blue for folding/heterodimerization, green for binding and red for fusion. Multi-colored code boxes represent residues with two identified functions. In E2, the AR3A antibody epitope is shown as three orange arrows. The putative function determined for each cluster is summarized in S9 Table. The addition of one amino acid on the left and on the right of each block is considered to take into account potential structural adjustments after mutation that are not considered in BIS calculation. Cluster blocks positions are numbered according to JFH-1 gt2 .Genotype 2 cluster blocks are represented as blue horizontal bars positioned above the E1 and E2 H77 or JFH-1 aligned sequences. Each horizontal bar is numerated as follow: “Cluster number-Block N°”. Respective positions of these blocks are referenced in (TIF)Click here for additional data file.S8 FigA) Plot of the gt2 structural cluster 19 on a tridimensional view of the E2core structure (PDB 4MWF). Gt2 structural (B), multifunctional (C), structural and multifunction (D), fusion (E) and undefined (F) clusters were plotted both on a vertical linear representation of E1 and onto the E2 core structure. Each cluster is composed of blocks harboring a similar color, according to S6 Fig. The Stem region (Stem) is represented by a dotted line after the C-terminal part of the BL. The transmembrane domain (Tmd) is represented as a rectangle following the Stem region. In (B) and , bold lines link E1 and E2 blocks that coevolved. For each cluster, block positions in E1 (at the left of the linear structure) and E2 (below boxes whose color match the color of the corresponding cluster) are indicated. Inter-E1E2 interactions mediated by gt2 structural clusters 7, 8, 11 and intra-E2 interactions mediated by gt2 structural cluster 18 and 21 are represented in B, while multifunctional clusters 5 and 12 are represented in (C). Areas of close proximity between blocks in cluster 19 (A) and between blocks in cluster 11 (D) and 12 (D) are enlarged and highlighted by red circles. Panel E shows gt2 fusion clusters 6, 16 and 17. Panel F shows gt2 undefined role clusters 3, 4, 9, 14, 15 and 20. Rotation angles of the E2core structure are indicated. Viewing angle of E2core is indicated by a black cross . HVR1, Hyper Variable Region 1, FL, Front Layer; BinL, CD81 Binding Loop; BL, Back Layer, βS, β-sandwich.((TIF)Click here for additional data file.S9 FigFig 3B). Genotype 3 cluster positions are available through the following webpage: http://www.lcqb.upmc.fr/HCVenv/HCVenv.html/.Genotype 3 cluster 4 (blue), cluster 8 (red) and cluster 9 (green) are plotted on a tridimensional view of the E2core structure (PDB 4MWF) and on a vertical linear representation of E1. Each cluster is composed of blocks harboring a similar color. The Stem region (Stem) is represented by a dotted line after the C-terminal part of the BL. The transmembrane domain (Tmd) is represented as a rectangle following the Stem region. The BL region is highlighted with a dotted circle on the E2core structure and enlarged alone at the right of the E1 linear representation. Bold lines link E1 and E2 blocks that coevolved. For each cluster, block positions in E1 (at the left of the linear structure) and E2 (below boxes whose color match the color of the corresponding cluster) are indicated. Rotation angles of the E2core structure are indicated. Viewing angle of E2core is indicated by a black cross (Reference: (TIF)Click here for additional data file.S10 FigA) Amino acid sequence of the soluble BLd-H77. Dotted lines represent internal disulfide bridges that might be involved in the functional folding of the soluble BLd-H77. (B) Detection of BLd-H77 in reducing and non-reducing condition following SDS-Page electrophoresis and coomassie blue staining. (C) Size distribution profiles as a function of the intensity and volume of soluble BLd-H77, by Dynamic Light Scattering. The graph shows the superposition of three successive measurements from 12 runs at each concentration, which are representative for at least three independent experiments. (D) Far UV circular dichroism spectrum of soluble BLd-H77 after purification by size exclusion chromatography.((TIF)Click here for additional data file.S11 FigA) Quantification of HCVcc infectious titers following dose-dependent neutralization of HCVcc H77/JFH-1 by BLd-H77. Four days following BLd-H77 dose-dependent neutralization of HCVcc H77/JFH-1 infection , Huh7.5 cell culture supernatants were harvested and used to infect naïve Huh7.5 cells. Four days post-secondary infection, viral titers were determined by NS5A immunostaining . (B) Average IC50 of BLd-H77 inhibition (μg/ml) for HCVpp or HCVcc particles of different genotypes or sub-types. ND, not determined. (C) Bafilomycin A1 and AR4A mediated-inhibition of HCV entry. Huh7.5 cells (cells) or HCVpp  were incubated 1h with Bafilomycin A1 (20nM) or AR4A (25μg/ml) prior infection (2), during the 4h infection (3) or following infection (4). As control, cells were incubated at each step with equivalent volume of PBS (1). Percentages of infection were calculated 72 post infection based on viral titer obtained from control conditions. . Statistical significances  were determined for each experimental condition versus control condition (100%). N.A., non applicable, as significant cell death was observed for this experimental condition. (D) HCV receptors expression is not impaired by BLd-H77. CD81, SR-BI, Claudin-1 and Occludin expression in Huh7.5 cells pre-incubated overnight with PBS (upper line) or BLd-H77 . Following PBS or BLd-H77 pre-incubation, cells were fixed, permeabilized (only for Occludin staining) and stained for the different HCV receptors with (white areas) or without (grey areas) receptor-specific antibody. Expression levels were then determined by flow cytometry. Data are representative of two independent experiments. (E) BLd-H77 inhibition of cell-to-cell transmission. After HCVcc H77/JFH-1 infection, cells were pre-incubated with PBS (control), with AP33 antibody alone (25μg/ml) or with AP33 mixed with BLd-H77 (35μg/ml). Average numbers of cells per foci were determined by immunostaining for each condition 4 days post infection. **p<0.01, ns non-significant. (F) Titration of primary human hepatocytes (PHH) cell culture media following HCVcc JC1 virus infection. PHH were infected with HCVcc JC1 virus at different m.o.i. 4 days post-infection, PHH cell culture supernatants were used to infect Huh7.5 cells. Viral titers were determined 4 days post-secondary infection via NS5A immunostaining . (G) BLd-H77 mediated-inhibition of patient-derived HCV particles in vivo. Patient-derived HCV particles were pre-incubated with PBS or BLd-H77 (30μg/ml) and were used to infect primary human hepatocytes (m.o.i. 0.1). Four days post-infection, cell culture supernatants were harvested and viral load from primary infection were determined using a clinical diagnostic kit. Limit of detection (l.o.d.) of the kit is indicated by a dotted line (n = 1 due to the limited amount of patient sera available). (H) Serum-derived HCV viral loads from a mouse cohort treated with 150μg of BLd-H77  or with PBS  under a “prophylactic” protocol. BLd-H77 or PBS was injected one day prior virus infection with patient serum as conducted in Fig 4L. Serum-derived HCV particle viral loads were determined as described in online methods. The limit of detection (l.o.d.) is indicated within the graph (dotted line). Viral loads below the dotted line represent non-quantifiable viral loads. One non-engrafted liver mice and one non-infected mice were used as negative controls . A statistical analysis was realized on all quantifiable viral loads for all time points (p = 0.02). (I) Serum albumin concentration in humanized liver FRG mice prior infection and (Pre-infection) at the time of sacrifice (Week 3). Albumin concentration were determined in the context of HCVcc JC1 (JC1) and serum-derived HCV infection (Serum), and with BLd-H77 treatment (BLd) or not (PBS). Medians are shown for each experimental group. (5–7 mice per group). ns non-significant.((TIF)Click here for additional data file.S12 FigA) Cell surface staining of C46 and BLd-tm following transduction of Huh7.5. Expression of C46 and BLd-tm (white) was measured by flow cytometry using an anti-hinge human IgG2 antibody and compared to the level of expression within non-transduced Huh7.5 (Grey). (B) CD81, SR-BI, Claudin-1 and Occludin expression within Huh7.5 (left column) and Huh7.5 expressing the transmembranous form of the BLd-H77 (Huh7.5-BLd-tm) (right column) cells. Cells were fixed, permeabilized (only for Occludin staining) and stained for the different HCV receptors with (white areas) or without (grey areas) receptor-specific antibody. Expression levels were then determined by flow cytometry. Data are representative of two independent experiments. (C) Effect of BLd-tm expression on cell-to-cell viral spread. Huh7.5, Huh7.5-C46, Huh7.5-BLd-tm cells were infected by H77/JFH-1 HCVcc. Four days post-infection, average number of cells per foci were determined for each cell type . ***p<0.001, ns non-significant. (D) Retention of HCVpp particles at the Huh7.5-BLd-tm cell surface. Huh7.5 (left) and Huh7.5-BLd-tm (right) were infected with H77 HCVpp particles. Presence of HCV E2 at the cell surface was quantified by flow cytometry using anti-E2 H53 antibody. Gating were set up on non-infected Huh7.5 (left) and Huh7.5-BLd-tm (right). Data are representative of two independent experiments. (E) Interaction between BLd-H77 and sE2 detected by ELISA. Different amounts of mouse IgG isotype, AR3B and BLd-H77 were coated overnight into 96-well plates. Coated peptides and antibodies were then incubated with 10ng of soluble E2 (sE2). After washing, soluble E2 was detected using the anti-E2 antibody 3/11 or a rat isotype IgG antibody. Following incubation with an anti-rat HRP antibody, 3/11 binding specificity to E2 was determined by calculating the ratio of O.D. between condition using 3/11 antibody and the rat isotype IgG antibody, for each coating condition . ****p<0.0001, ns non-significant.((TIF)Click here for additional data file.S13 FigA) BLd-H77 effect on E2 and HCVpp binding to Huh7.5 cells (Left). Huh7.5 cells, pre-incubated with PBS (top panels) or BLd-H77 (50μg/ml) (bottom panels), were mixed (white) or not (grey), at 37°C for 1h with soluble E2 or with concentrated H77-HCVpp. Bound soluble E2 or HCVpp were then stained with an anti-6his tag antibody or an anti-E2 antibody, respectively. Binding was then quantified by flow cytometry following incubation with appropriate APC antibody. Data are representative of two independent experiments. Ability of BLd-H77 to bind to Huh7.5 cell membrane (Right). BLd-H77  or PBS (grey) was incubated for 1h at 37°C with Huh7.5 cells. Absence of bound BLd-H77 at Huh7.5 cell surface was verified by flow cytometry following staining using an anti-6his antibody or an anti-E2 antibody as control. (B) Expression of CD81 and SR-BI at BRL cell surface and HCVpp binding. BRL cell lines were transduced by vectors encoding human CD81 or SR-BI (hSR-BI). After selection, naïve BRL (grey) or transduced-BRL (white) cells were stained by anti-CD81 or anti-hSR-BI and by the appropriate secondary APC antibody. Quantification of CD81 and hSR-BI expression was determined by flow cytometry. (C) BLd-H77 effect on E2 and HCVpp binding to CD81 and hSR-BI. Naïve BRL (grey) or HCV-receptor expressing BRL (white) cells were mixed with concentrated H77-HCVpp pre-incubated with PBS (top panels) or BLd-H77 (50μg/ml) (bottom panels). Bound HCVpp were then stained with an anti-E2 antibody and with an appropriate APC antibody. Binding was then quantified by flow cytometry following incubation. Data are representative of three independent experiments. (D) Relative quantification of HCVpp binding to BRL cells expressing exogenous hCD81 or hSR-BI. Binding of HCVpp is presented as percentage of binding, relatively to the binding efficiency of HCVpp pre-incubated with PBS. . Statistical significances  were determined for each experimental condition versus control condition (100%). (E) Competitive entry inhibition assay using BLd-H77 and a neutralizing anti-E1E2 antibody. HCVpp-H77 were incubated for 1h at room temperature with BLd-tm (35 or 50 μg/ml), AR4A antibody (2 or 17 μg/ml) or with both protein (35 and 2 μg/ml or 35 and 17 μg/ml), and used to infect Huh7.5. Percentage of infection were quantified 72h post infection and calculated according to viral titer obtained for control conditions . *p<0.05, **p<0.01.((TIF)Click here for additional data file.S14 FigA) LTRhiv-luciferase vector transduced 293T cells expressing H77 HCV (left panel) or VSV (right panel) envelope glycoproteins were co-cultured with Tat-expressing Huh7.5 cells. Co-culture were pre-incubated for 1h with 50μg/ml of BLd-H77 or PBS, washed and incubated for 3 min with a pH7 (red) or pH5 (orange) buffer. Luciferase activities were determined 72h post-exposure. Results are presented in relative light units (RLU) for each experimental condition . *p<0.05, **p<0.01. Statistical significances represent the significant differences between pH5 and pH7 condition, for each viral envelope and treatment (PBS or BLd-H77). (B) Inhibitory effect of the BLd-H77 prior and following pH shock. Co-cultures were either pre-incubated with BLd-H77  for 1h prior pH shock (pH5), for 24h following pH shock  or both (4). As a control, co-cultures were pre-incubated with PBS for 1h prior pH shock, followed by an additional 24h incubation (1). 24h later, cells were washed and incubated for 48h. Relative fusion efficiency at pH5 between control (pre- and post-treated by PBS) and the different BLd-H77 incubation periods are indicated . Statistical significances  were determined for each experimental condition versus control condition (100%). (C) Specificity of HCVpp-liposome fusion assay. No enveloped HCVpp, H77 HCVpp, fusion mutant E1E2 HCVpp particles and HANApp particles  were mixed with R18-labelled liposomes. Dequenching of R18 was quantified following sample acidification (pH5) over 5 minutes (300 seconds). Data are represented as non-linear polynomial fitted curves and display the evolution of the fusion rate from the time of acidification, relatively to the HCVpp-H77 fusion rate which has been normalized at 100% at 300s post acidification. Curves are representative of three independent experiments.((TIF)Click here for additional data file.S15 FigA) Protein sequence alignment of E1E2 J6 and 2b1. Level of conservation for each amino acid position is indicated. Position of the coevolving blocks that belong to gt2 fusion cluster 5 are indicated by red rectangles. Position of the interchanged amino acid regions between J6 and 2b1 for the construction of the J6/2b1 chimeric envelope are shown . (B) Structural location of the E2 gt2 cluster 5 block (orange) and of the five J6/2b1 amino acids differences  within the E2 BL. A dotted line symbolizes the rational experimental dissection of the BL into two distinct sub-domains, noted region 2 (non-β sheet region) and region 3 (β sheet region), based on the distinct rearrangement of these two sub-domains. The distinct structural contexts of the mutation 1–2  and of the mutation 3–5  are highlighted. (C) J6/2b1 E2 chimera expression and incorporation onto HCVpp. Expression in transfected 293T cells (Cell lysates) and incorporation onto concentrated pseudoparticles  of E2 from J6, 2b1 and J6/2b1 chimera. Detection of E2 on pseudoparticles harboring no envelope glycoproteins was used as negative control. MLV-Capsid (CA) was detected to control equivalent HCVpp production between chimera. (D) Quantification of HCV viral RNA in the supernatant of Huh7.5 cell electroporated with viral RNA coding for HCVcc harboring different J6/2b1 envelope chimera. Cell culture supernatant were harvested at 48h and 72h post electroporation and viral RNA copy numbers per ml were determined by RT-qPCR for each viral strain . *p<0.05, **p<0.01.((TIF)Click here for additional data file.S16 FigProtein sequence alignment of E1E2 H77 and A40. Level of conservation for each amino acid position is indicated. Position of the E1 and E2 block of interest belonging to the gt1a cluster 5 are indicated by red asterisks. Position of the three H77 amino acid residues that will be replaced by A40 residues for the construction of H77/A40 chimeric envelopes are indicated by red rectangles (two in E1 and one in E2).(TIF)Click here for additional data file.


However, it remains unclear whether one enhancer element could have been produced via cooperation among multiple, yet distinct, TEs during evolution. Here we show that an evolutionarily conserved genomic region named AS3_9 comprises three TEs , inserted side-by-side, and functions as a distal enhancer for wnt5a expression during morphogenesis of the mammalian secondary palate. Functional analysis of each TE revealed step-by-step retroposition/transposition and co-option together with acquisition of a binding site for Msx1 for its full enhancer function during mammalian evolution. The present study provides a new perspective suggesting that a huge variety of TEs, in combination, could have accelerated the diversity of cis-regulatory elements involved in morphological evolution.Acquisition of  cis-regulatory elements is a major driving force of evolution, but its whole evolutionary significance is poorly understood. Here, we found an unprecedented case in that three TEs cooperatively and synergistically act as a distal enhancer for wnt5a expression in mammalian frontonasal region, being involved in the evolution of the secondary palate formation. We elucidated the stepwise evolutionary history of getting the enhancer activity via (retro-)transposition of TEs during mammalian evolution. Taking various types of TEs constituting nearly half of mammalian genomes into consideration, this study provides a new perspective for the potentiality that almost infinite combinations of proximally-located different TEs could have generated a huge diversity of developmental enhancers and had a great impact on mammalian morphological evolution.Acquisition of  Genomic DNA (10 μg each) from F1 individuals was digested with Bln I and Bsp1407 I (for the 5’ probe), EcoR V (for the 3’ probe), or Sac I (for the Neo probe). Signals of 12.0, 6.2, and 6.3 kb for the mutant alleles were detected by Southern blotting using the 5’, 3’, and Neo probes, respectively  was used to identify proteins that could bind to the AS3_9 sequence. A SMART cDNA library was constructed using the kit from the frontonasal tissues of 21 mouse embryos at E14.5. The bait DNA (chr14:29028539–29029109 of GRCm38/mm10) consisted of AmnSINE1 and X6b_DNA regions because the sequence is responsible for the enhancer activity in maxillary processes, the origin of PS outgrowth. Yeast one-hybrid screening was conducted with two consecutive selection steps with 100 ng/ml (first step) and 500 ng/ml (second step) of the antibiotic aureobasidin A. Among 11.4 million clones of the library screened, 14 positive clones  were isoin silico screen of the human genome (hg38) to identify AmnSINE1-derived CNEs proximal to other TE-derived CNEs. By comparing a list of conserved elements (phastConsElements100way) from the UCSC genome database and the latest TE annotation list by RepeatMasker , all TEs overlapping >30 bp with the conserved elements (LOD score >100) were extracted. Among the 626 evolutionarily conserved AmnSINE1 sequences found, those proximal to (<600 bp) another TE-derived CNE were collected and listed in We performed an http://www2.clst.riken.jp/arg/mutant%20mice%20list.html (Accession No. CDB0941K).Details of the AS3_9-ko lines are available at S1 FigUpper jaws (A) and coronal sections (B) are illustrated for E13.5–15.5 embryos. Palatal shelves are shown in magenta.(TIF)Click here for additional data file.S2 FigThe top line of the alignment shows the consensus sequences of AmnSINE1.(TIF)Click here for additional data file.S3 FigEmbryos from the transient enhancer analysis (A) and the three stable lines α–γ (B) at E9.5–15.5 were stained. Scale bar: 2 mm.(TIF)Click here for additional data file.S4 Figwnt5a, (B) erc2, (C) lrtm1, (D) cacna2d3, (E) selk, (F) actr8, (G) il17rb, and (H) chdh. Red arrowheads denote wnt5a expression in palatal shelves.(A) (TIF)Click here for additional data file.S5 FiglacZ embryos , and ISH for lacZ  and wnt5a . Arrowheads: frontonasal region. Ventral view of the X-gal–stained upper jaw of AS3_9-lacZ , coronal sections of the X-gal–stained AS3_9-lacZ embryos , and ISH for wnt5a in coronal sections . Arrowheads: palatal shelves.X-gal–stained AS3_9-(TIF)Click here for additional data file.S6 FigBln I, Bs: Bsp1407 I, E: EcoR V, and S: Sac I sites. (B) Positions of the genotyping primers and PCR product lengths for wild-type and mutant alleles. (C–E) Southern hybridization of the F1 heterozygote AS3_9-ko mice with 5’ (C), 3’ (D), and Neo sequence (E) probes. Five and three individuals of the two independent lines  were used, respectively, as well as a wild-type mouse .(A) Schematic representation of wild-type and mutant alleles as well as the targeting vector. The 800-bp region containing the three TEs in AS3_9 was replaced with a Neo cassette (Pr-Neo-pA). Black bars: 5’ and 3’ probe regions used for Southern hybridization. Bl: (TIF)Click here for additional data file.S7 FigCoronal sections (10 μm) of E14.5 (A) and E15.5 (B) embryos of AS3_9-ko mice were stained with hematoxylin and eosin. The embryos were compared among two to five littermates from eight different litters. WT: Wild type. Scale bar: 0.5 mm.(TIF)Click here for additional data file.S8 FigSequence alignments of the X6b_DNA (A), and MER117 (B) regions of AS3_9. The top line of each alignment is the consensus sequences of the TE. The putative Msx1-binding site (reverse-complement of the TAATTG motif) is denoted by a red line in (A).(TIF)Click here for additional data file.S9 Fig(a–i) Embryos harboring the AS3_9 construct (a) and related deletion constructs (b–i), corresponding to those in (TIF)Click here for additional data file.S10 Fig(A–C) Binding of Msx1, Msx2, Gtf2ird1, and Zbtb7c to the AS3_9 sequence detected by yeast one-hybrid assay using SD/-Leu medium with (B) or without (C) antibiotic (aureobasidin A). Empty vector was used as the negative control. (D–F) Yeast one-hybrid assay was used to assess the viability of Msx1-binding-site mutants using SD/-Leu medium with (E) or without (F) aureobasidin A.(TIF)Click here for additional data file.S11 FigEpigenetic marks in AS3_9 from the ENCODE (A) and the Roadmap Epigenomics (B) projects. (A) The human AS3_9 region in the UCSC genome browser (hg19) showing the DNase-hypersensitive clusters and the transcription factor binding signals by the ChIP-seq analyses from the ENCODE project. (B) ChIP-seq or DNase-hypersensitive signals with scores >10 were heat-mapped for each tissues/cells. Maximum scores in each TE region were retrieved form the Roadmap Epigenomics genome browser.(TIF)Click here for additional data file.S1 TableIn total 14 positive clones contained 12 genes, some of which are included in the same clones.(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableThe Locus ID 12 corresponds to AS3_9 enhancer.(XLSX)Click here for additional data file.


The length of the G1 phase in the cell cycle shows significant variability in different cell types and tissue types. To gain insights into the control of G1 length, we generated an E2F activity reporter that captures free E2F activity after dissociation from Rb sequestration and followed its kinetics of activation at the single-cell level, in real time. Our results demonstrate that its activity is precisely coordinated with S phase progression. Quantitative analysis indicates that there is a pre-S phase delay between E2F transcriptional dynamic and activity dynamics. This delay is variable among different cell types and is strongly modulated by the cyclin D/CDK4/6 complex activity through Rb phosphorylation. Our findings suggest that the main function of this complex is to regulate the appropriate timing of G1 length. During the gap 1 (G1) phase of the cell cycle, cells grow in size and synthesize mRNAs and proteins in preparation for the S phase when DNA is replicated . During Despite our extensive knowledge of the cell cycle regulation, how the known molecular effectors control G1 length in single cells remains poorly understood. In the canonical model for cell cycle regulation, the control of the G1/S transition mediated by the Rb/E2F network is thought to be exerted by multiple G1 cyclin/cyclin-dependent kinase (CDK) complexes . FurtherCdh1 inactivation shortly before S phase entry and significantly later than E2F target gene expression, suggesting that more intricate regulation of E2F activity at multiple layers may exist at the temporal scale [Analysis of cell-cycle progress in cell populations is insufficient to address this issue, given the extensive cell-cell heterogeneity in cellular behavior and gene expression –16. In cal scale . MoreoveTo this end, we developed an E2F activity reporter that captures the dynamic regulation of free E2F activators following their release from the inhibitory sequestration by Rb. We used this reporter to monitor E2F activity dynamics at the single-cell level and in real time. Our results indicate that the E2F activity reporter serves as a reliable indicator of S phase entry at the temporal scale. Furthermore, our quantitative analysis of E2F activity dynamics linked to G1/S transition underscores the role of cyclin D/CDK4/6 activity as a main determinant of G1 length.A clone of REF52 cells expressing the transcriptional reporter pQCXIP-hE2F1p::4NLS-d4Venus was described in our previous publication . For the5 per ml and then seeded in μ-Slide I  channel slides by adding 1 ml volume of the cell suspension. After overnight culturing, REF52 cells were synchronized in quiescence by shifting into Minimum Essential Medium Alpha medium with 0.02% BGS (starvation medium) for 36 h. For perturbation experiments, PD0332991  was added into cells immediately after cells were released from serum starvation (by adding 10% BGS). hTert-HME1 was starved in basal medium (MEBM without additive) for 36 hours after seeding overnight.REF52  fibroblasts were routinely grown in Minimum Essential Medium Alpha Medium (Gibco/Invitrogen) supplemented with 10% bovine growth medium . hTert-HME1  cells were cultured in Mammary Epithelial Basal Medium  with addition of MEGM SingleQuots . For the preparation of E2F dynamics measurement by using time-lapse microscopy, cells were first trypsinized, resuspended at a density of 1×10For time-lapse microscopy, quiescent cells growing in μ-Slide I slides were released from starvation by shifting to serum containing medium and placed under Leica DMI 6000 B inverted fluorescence microscope (Leica). Images were taken using Leica N PLAN L 20×0.4 objective lens with phase contrast, Semrock BrightLine GFP filter set, YFP filter set or Texas Red filter set, and Hamamatsu ORCA AG digital camera (Hamamatsu) with uniform parameter setting: binning = 4, offset = 0, gain = 255 and exposure time = 0.01 s (phase channel) or 0.15 s (YFP channel). The microscope was enclosed with an environmental chamber with 37 C temperature, atmosphere (5% CO2) and humidity. Images were acquired every 30 or 60 min for 24~48 h. Time-series image acquisition was controlled by SimplePCI 6 Software (Hamamatsu).Phospho-Rb (Ser807/811) (D20B12) XP Rabbit mAb  following the manufacturer’s instruction. EdU staining was performed after E2F dynamics measurement in ibidi μ-Slide I slides by using Click-iT EdU Imaging Kits (Invitrogen) with Alexa Fluro 594 azide according the manufacturer’s instructions. The only modification of the protocols for immunostaining and EdU staining was to wash cells with PBS with 0.2% Tween three times after all the staining steps. Images were taken using Leica N PLAN L 20×0.4 objective lens with phase contrast or a Semrock BrightLine GFP filter set, YFP filter set, Texas Red filter set, and Hamamatsu ORCA AG digital camera with uniform parameter setting: binning = 4, offset = 0, gain = 255 and exposure time = 0.01 s (phase channel) or 0.8 s (Red channel).Immunostaining was performed after E2F dynamics measurement in ibidi μ-Slide I slides with The time-lapse microscopy resulted in two series of raw images of the cells in 30 or 60-min increments for 36–72 h, one each for the phase channel and the fluorescence channel(s). E2F signals were extracted from these images using ImageJ (NIH) software. The first time-point of each series of images was loaded side-by-side into the software. Using the ROI Manager Tool, a circular selective marker with a fixed area was placed around each cell nucleus in the phase channel image. The location of each marker was then copied to the fluorescence channel(s) image and the integrated grey value of the selected pixels was measured. In the case of cell division, the selective marker was applied to one of the daughter cells. This value was normalized to the background by subtracting the integrated grey value of the same area of pixels in an empty part of the image. This process of measuring normalized grey value of the nuclei was repeated for each time point by adjusting the location of the selective marker to account for the movement of cells, thus giving a time-series measurement of the fluorescence reporter on the E2F fluorescence. Throughout this process, data from cells that left the field of view at any time were discarded; and the time of cell division was also noted. The similar approach was applied to quantify the fluorescence signal from immunostaining and EdU staining.t1 and t2:t1,t2} that gives This time-series E2F dynamics trajectories were analyzed in Matlab (MathWorks) to determine various E2F parameters, e.g. time delay, amplitude, etc. The processed trajectories without spikes were then smoothened by using a 3-window Gaussian averaging algorithm. Each smoothed and processed trajectory was then fit to the following two-phase regression model to automatically derive optimal values for Relevant parameters represent:t1: initial delayt2: activation timey0: basal level ymax: peak level yE2F(t): E2F signal in dynamic trajectory at the moment ttΔ, we modify the decay constant of cyclin D, dCD, and cyclin E, dCE, respectively. In both cases, we change the decay constant between 1.5/h– 4.5/h. We demonstrate the effect of inhibiting either cyclin E/CDK2 or cyclin D/CDK4/6 under these conditions on tΔ, E2F activity, and E2F transcription  and perturbed (1 μM PD0332991) conditions. The statistics are reported in both box and beeswarm plots and the difference is estimated by Wilcoxon rank test.1/S transcriptional program leading to cell cycle entry. To measure the regulatory activity of free E2F protein (henceforth referred as “E2F activity dynamics”) in individual cells, we engineered an E2F activity reporter by placing a destabilized mCherry protein downstream of a truncated human E2F1 promoter. The truncated E2F1 promoter only includes the two putative E2F binding sites, but lacks the canonical Myc binding site and other potential regulatory sites located in the more distal region of the E2F1 promoter  and repressors (E2F3b-8). E2F activators are regulated at multiple levels, with a key regulation of their activity provided via their tight but reversible sequestration by the retinoblastoma (Rb) tumor suppressor protein ,21,22. Ipromoter . A similAmpact), the initial delay (tα-act), the activation time (tß-act), and, for cells that undergo division, a post-activation time (tγ-act). The area under the curve (AUC) of the activity tracing, a metric geometrically related to the previous variables, captures the integrated activity of E2F over time . To this end, we combined the measurements of E2F activity with that of 5-ethynyl-2’-deoxyuridine (EdU) incorporation into newly synthesized DNA, a surrogate for determining how much DNA has been synthesized over time . Our meaTo measure the onset time of E2F activity relative to the start of S phase, we constitutively expressed a GFP-PCNA fusion protein  in REF52tα-act) in E2F activity dynamics is a good predictor of the length of the G1 phase, and that the mean tα-act can vary substantially among different cell types .To further examine the temporal correlation between E2F activity dynamics and S phase entry, we relied on the property of the FUCCI reporter system . During tΔ) varies substantially among individual cells  that captured E2F transcriptional dynamics at the single-cell level . Since tal cells .tΔ.Given the Myc/Rb/E2F network structure , it is ntΔ and G1 lengthening. Experimentally we treated REF52 cells expressing both the transcriptional and activity reporter with the CDK4/6-specific inhibitor–PD0332991. In concert with our prediction, alignment of both dynamics trajectories in individual cells over time indicates a significant increase of tα-act under the inhibition of cyclin D/CDK4/6 activity  could be split into two periods, the first corresponding to an initial delay in the transcriptional dynamics (tα-trans) and the second being the delay between the initial increase in E2F activity and E2F transcriptional activation or tΔ , suggesting that tΔ is the predominant interval that influences the whole cell cycle duration  is constrained by E2F transcription but is modulated at the temporal scale through G1 cyclin/CDKs-mediated Rb phosphorylation. Taken together these studies provide a quantitative framework to describe the regulation of E2F activation during G1/S transition.The information on both E2F transcriptional and activity dynamics provides a novel interpretation about the function of G1 cyclin/CDKs that regulate cell cycle commitment and progression. The prevailing view claims that the cyclin D/CDK4/6 activity is the trigger of cell cycle entry, whereas our results suggest that the cyclin D/CDK4/6 complex only control the timing of the onset of the E2F1 activity, even after E2F1 has been transcribed. This timing control reconciles the diverse, sometimes conflicting roles of the cyclin D/CDK4/6 activity in cell cycle regulation. First, cyclin D/CDK4/6 activity has been implicated as the driver of G1/S transition, because microinjection of anti-cyclin D1 antibody into normal diploid human fibroblasts prevented cells from entering S phase . Accordiin vitro or in vivo with cyclin D/CDK4/6 inhibitors results in cellular senescence and tumor suppression [Our findings have implications for the understanding of cellular senescence, which was described as a state of “permanent cell cycle arrest” but was poorly distinguished from other non-proliferative states such as quiescence and terminal differentiation at the mechanistic level. From the model supported by our data, senescence is likely to be a state in which cells are trapped within a permanent cell cycle progression due to the potent inhibition of G1 cyclin/CDK activities. Indeed, evidence has been provided in several studies that treatment of epithelial cells pression ,31. TogeCell cycle lengthening, particularly the extension of G1 phase, is a hallmark of cell cycle alteration during lineage specification in different physiological contexts ,7. UnravS1 FigREF52 cells (a) and hTert-HME1 cells (b) released from serum starvation. The dot in each trajectory corresponds to the cell division time point. Grey trajectories represent cells that did not divide during the observation window.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigRepresentative views of hTert-HME1 cells imaged in phase, mCherry and Alexa Fluo 488 (phosphorylated Rb) channels. Scale bar, 50 μm.(TIF)Click here for additional data file.S4 FigRepresentative views of hTert-HME1 cells imaged in phase, mCherry and Alexa Fluo 488 (EdU staining) channels. Scale bar, 50 μm.(TIF)Click here for additional data file.S5 Fig0). Cells were imaged every 30 min from t0 to t60 in the GFP and RFP channels . (a) Time series of images (from time t26 to t36) showing the nuclear pattern of GFP-PCNA in a single cell. (b) The signal from the E2F activity reporter in cell shown in (a) was quantified. Activity values (relative fluorescent units) are reported from t25  to t36 (one frame before nuclear envelope breakdown). Arrow indicates time (t28) of first recorded increase in reporter activity over base line. (c) 47 single cells were scored for the delay between the onset of E2F activity and GFP-PCNA fine puncta formation. Delays fell into 5 categories . Counts indicate the number of cells in each time delay category.REF52 cells expressing the E2F activity reporter and a GFP-PCNA fusion protein were starved for 48h and released into the cell cycle with 10%BGS (t(TIFF)Click here for additional data file.S6 FigtΔ between the two dynamics trajectories measured over ~50 cells under the condition of (a).(a) Example trajectories of the E2F transcriptional dynamics and activity dynamics in cells released from serum starvation back into the cell cycle after growth stimuli. (b) Statistics of (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 Movie(AVI)Click here for additional data file.S2 Movie(AVI)Click here for additional data file.