Influenza H7N9 virus continues to cause infections in humans and represents a significant pandemic risk. During the most recent 5th epidemic wave in 2016/17 two distinct lineages with increased human infections and wider geographical spread emerged. In preparation for any future adaptations, broadly reactive antibodies against H7N9 are required for surveillance, therapy and prophylaxis. In this study we have isolated a panel of nanobodies (Nbs) with broad reactivity across H7 influenza strains, including H7N9 strains between 2013 and 2017. We also describe Nbs capable of distinguishing between the most recent high and low pathogenicity Yangtze River Delta lineage H7N9 strains. Nanobodies were classified into 5 distinct groups based on their epitope footprint determined using yeast display and mutational scanning. The epitope footprint of Nbs capable of distinguishing high pathogenic (HP) A/Guangdong/17SF003/2016 from low pathogenic (LP) A/Hong Kong/125/2017 (H7N9) were correlated to natural sequence divergence in the head domain at lysine 164. Several Nbs binding to the head domain were capable of viral neutralisation. The potency of one nanobody NB7-14 could be increased over 1000-fold to 113 pM by linking two Nbs together. Nbs specific for distinct epitopes on H7N9 may be useful for surveillance or therapy in human or veterinary settings. In the last century, four pandemics have led to many human deaths caused by three subtypes, H1N1 in 1918 and 2009, H2N2 in 1957 and H3N2 in 19683. In more recent years a number of avian influenza viruses, for example, H5N1 and H7N9, have crossed into the human population, but fortunately these zoonotic outbreaks have not yet become pandemics due to their low human-to-human transmissibility4. However, a more recent outbreak of H7N9 in China in 2016–2017 resulted in substantial geographical spread with the emergence of H7N9 strains carrying a polybasic cleavage site which can facilitate systemic spread beyond the lungs6. As such, H7N9 may be considered a serious pandemic threat and there is a pressing need to prepare for robust global responses which can be rapidly implemented should this virus ever become highly transmissible within the human population. Vaccines remain the main prophylactic treatment option and in March 2017 the WHO recommended new candidate vaccine viruses be made from the Yangtze River Delta lineage of the low pathogenic A/Hong Kong/125/2017 (H7N9) [(LP)HK/2017(H7N9)] and the high pathogenic A/Guangdong/17SF003/2016 (H7N9) [(HP)GD/2016(H7N9)] viruses, which are currently being evaluated in clinical trials8.Influenza A virus (IAV) remains a persistent threat to public health resulting in 200,000–500,000 deaths worldwide annually which can be even higher in a pandemic emergency9. Passive transfer of serum from convalescent patients is an option which was deployed, with some success, in the 1918 pandemic10 and more recently to treat a severely ill H5N1 patient11. However, this approach cannot be implemented on a global scale due to limited supply, high risk of toxicity, uncertain dosing, and difficulties in administration. A more promising approach is passive immunotherapy with recombinant monoclonal antibodies with broad reactivity against H7N9 which can be manufactured with consistent quality, stockpiled, and delivered immediately in a pandemic emergency14.Although vaccines remain the mainstay of infection control for influenza, their timely implementation, poor immunogenicity in certain patient groups and limited worldwide production capacity remain a considerable technical challenge. Anti-viral drugs are available although resistant viral strains are emerging15. HA mediates virus entry into host cells through the receptor binding site on the globular head interacting with sialic acid receptors on the cell surface. This in turn leads to virus internalisation, and then membrane fusion mediated by the HA stem region15. The pre-dominant host immune response is directed against the HA16 and this selective pressure drives the continuous antigenic changes in HA17. The high mutation rate and transmissibility means the discovery of new therapeutics is an active area of research14 and a number of broad spectrum human monoclonal antibodies (mAbs) specific for H7N9 have been described including CT14918, H7.16719, M82620, HNIgGD521 HNIgGA622 L4A-1423 and P52EO324. These monoclonal antibodies have been identified from human donors after either natural IAV infection or H7 subunit vaccination19 and unusually have limited somatic hypermutation. This suggests that they are the result of an early and immediate response to H7N9, likely biased by previous immune history of exposure to influenza23. In addition, recent reports have suggested that an effective protective immune response to H7N9 infection is extremely rare in humans23. Alternative approaches to generating mAbs to H7N9 which have been extensively optimised, could be expected to yield molecules of higher affinity and to potentially different epitopes to those targeted by the human immune system.As a typical member of the IAV family, H7N9 is classified through the major viral envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA is a homo-trimeric molecule with each monomer consisting of two chains, HA1 and HA2 which fold into a structure comprising a highly variable globular head and a more conserved proximal stem domain15. Several cross-reactive human antibodies have been isolated against the highly conserved HA stem26 or the receptor binding site of the head domain27. In several cases the route to cross-reactivity is achieved through using only their heavy chain for binding or through using a smaller binding footprint27. Conceptually a much smaller antibody epitope footprint may give a variable HA antigen less opportunities to escape binding and in effect there will need to be a mutational ‘bullseye’ for the virus to evade binding. Guided by these observations we have used nanobodies (Nbs) from alpacas as a potential route to cross-reactive antibodies30 as they are naturally devoid of a paired light chain and have a propensity to bind small pockets on protein surfaces31. In addition, given that camelid species are not thought to represent a zoonotic reservoir for influenza A, they may not be subject to the same immune constraints as human donors with prior exposure to IAV32. The unique properties of Nbs which also include small size, simple engineering into multi-domain antibodies and high stability are being exploited for a wide range of applications in biotechnology including diagnostics and immunotherapy33, with the first nanobody, Caplicizumab, having been approved for the treatment of a blood clotting disorder in August 201834.Although HA is continually changing, there are certain parts of the HA that are highly conserved, principally because they are essential for the infection process. Two functionally conserved sites lie within the receptor binding site and the HA stem which are required for viral uptake and membrane fusion, respectivelyIn this study we have isolated Nbs with both cross-reactive and strain specific binding to the prototype low pathogenicity (LP) and high pathogenicity (HP) A(H7N9) strains which have emerged in the most recent 5th epidemic wave. We correlate their specificity and functional activity with their epitopes which have been mapped using yeast display and mutational scanning. This technique allows high throughput epitope mapping and identifies mutations that directly interfere with antibody binding. Their potential as tools for surveillance or emergency therapeutics in both human and veterinary applications is discussed.7 was constructed using purified peripheral blood mononuclear cells. Two phage display library selection strategies were used, (i) sequential rounds of selection on the same antigen to yield all Nbs specific for H7-HA (H7N9 or H7N7) or (ii) alternating selections between H7-HA and H3-HA to bias towards cross-subtype reactive Nbs. Primary screening involved picking random clones and testing for binding to recombinant HA and whole virus inactivated antigen reagents of subtypes H3 and H7 sub-types as non-purified Nbs. Clones that were positive in ELISA were then sequenced and grouped into clonally related families based on their VHH-CDR3 sequences  of 113 pM  (Table 39. H7-specific Nbs (NB7-01 to 17) showed high affinity binding to both the HA1 domain and full length HA0  was sub-cloned into a yeast display vector in frame with an SV5 epitope tag. The use of a C-terminal epitope tag allows the detection and sorting of correctly folded HA displayed on the yeast surface independently of Nb binding using flow cytometry  were picked, sequenced and aligned to wild-type HA0 to identify HA mutations which were enriched compared to the unselected library. These selectively enriched mutations were predicted to form part of the physical epitope footprint and were chosen for further testing. Clones with mutations introducing/replacing cysteine or proline residues were discarded as they were predicted to have indirect or pleiotropic effects on binding28. Only HA mutations which were shown to interfere with Nb binding without affecting HA display level were classified as epitope residues. Selection for loss of binding to NB7-08 identified mutations D67G, D67V, L70S and D246N located in the HA1 domain,  that were not affected by the D67 mutations required further HA library selections to identify their respective epitopes. In selections with NB7-15, two separate mutations at residue 71 (E71G and E71V) were selected and placed NB7-15 in a separate epitope group (Gp2). Selections with NB7-03 gave mutations E111V, R163G and K164E, again placing it in a further separate epitope group, Gp3  with Gp 1 being divided into subgroups Fig. . Nbs belThe cross-reactive Nbs NB37X-01 and NB37X-05 were shown to bind only full length H7-HA0 with the absence of binding to the H3-HA1 head domain Fig.  suggesti40. This suggests that yeast display may be used as a tool to investigate the evolution of the HA molecule under different selective pressures without the need to use live virus and high containment facilities.Epitope group Gp3, comprising NB7-03, NB7-10 and NB7-13, were unique as these Nbs did not show any binding to the most recent highly pathogenic avian influenza A(H7N9) strain A/Guangdong/17SF003/2016 and the human H7N2 strain (A/NewYork/107/2003). The epitope footprint of these Nbs comprises E111, R163 and K164, and sequence comparison of the strains tested in ELISA identified this as a region of natural sequence divergence  with a 40% fatality rate. During the 5th wave in 2016–17, the emergence of highly pathogenic avian influenza (HP) A(H7N9) raised significant concerns as unlike (LP) A(H7N9) it maintained the capacity to infect via both human and avian receptors42. In preparation for any further adaptations in H7N9 which could threaten a pandemic, vaccines, anti-viral drugs and monoclonal antibodies which can be used for control, surveillance and as emergency prophylactics are essential.To date there have been 5 epidemic waves of zoonotic infections of influenza A(H7N9) with up to 1568 reported cases of human infection  A/Guangdong/17SF003/2016 and the low pathogenicity (LP) A/Hong King/125/2017 . Epitope group Gp1 was the largest group with 11 different Nbs which could be further divided into 4 sub-groups which, although sharing D67 as a key epitope residue  has been previously characterised as having 5 distinct antigenic sites e D Fig. . In anale D Fig. , we saw ion Fig.  may also51. The stem region is crucial in mediating viral infection and undergoes a large structural re-arrangement in the acidified environment of the endosomal compartment. It has been proposed that H106 and adjacent residues comprising M102 and E103 within the Gp5 epitope footprint function as a sensor for the enhanced protonation of HA at low pH15. The increase in binding we see at low pH is consistent with Nbs showing preferential binding to the HA stem after it has gone through the specific pH dependent conformational change. Non-neutralising human antibodies with a pH sensitive binding have been described which correlated with increased phagocytosis in vivo20. As Nbs NB37X-01 to 06 bind to a similar pH sensitive epitope they may be capable of similar mechanisms of viral neutralisation after appropriate reformatting as Fc-fusions to mediate interactions with phagocytic effector cells53.Nbs with H3/H7 cross-subtype reactivity and enhanced binding at low pH were located to the highly conserved HA stem and belonged to epitope Gp5 Fig.  Table S. Previou47 and may reflect a uniqueness to the immune response in alpacas that has not been shaped by prior exposure to IAV. This binding specificity could be correlated to natural sequence divergence at K164 between low (LP) and high pathogenicity (HP) H7N9 influenza viruses. This is a further example of how yeast display and mutational scanning is able to correlate Nb specificity with natural sequence change, which, if applied predictively, could be used to assess the suitability of stockpiled nanobody reagents as new viruses emerge30. To our knowledge we have described the first Nbs capable of distinguishing (HP) A/Guangdong/17SF003/2016 (H7N9) strain from (LP) A/Hong Kong/125/2017 (H7N9) and have been able to correlate this specificity to natural sequence divergence at K164 in the HA1 head domain. Since H7N9 presents in poultry with only mild symptoms, early warning signs of a pandemic may be extremely difficult to detect and the availability of specific Nbs formatted for rapid detection of this antigenic difference may be useful for influenza surveillance in the field using low cost biosensors54.Nanobodies binding to epitope group 3 had a unique specificity which doesn’t overlap with any of the previously described antigenic sites from human immune responses to H7N9 influenzaIn summary, we have isolated a panel of nanobodies to A(H7N9) which have been grouped on the basis of their epitopes and correlated this with their specificity and functional activity. They may be useful in monitoring the antigenic changes that occur in the HA of A(H7N9) during transmission in poultry, controlling the spread of virus in birds, or as emergency therapeutics in humans.Whole influenza virus inactivated antigen standards  used in this study were derived from strains: A/New York/107/2003 NIBRG-109 (H7N2) (08/362), A/Anhui/01/2013 NIBRG-268 (H7N9) (14/250), A/Mallard/Netherlands/12/2000 NIBRG-60 (H7N3) (07/336), A/Uruguay/716/2007 NYMC X-175C (H3N2) (08/278), A/Guangdong/17SF003/2016 NIBRG-375 (H7N9) (18/196), A/Texas/50/2012 (H3N2) (13/116) and A/HongKong/125/2017 (H7N9) ,. Purified recombinant hemagglutinins were full length HA0: A/Brisbane/10/2007 (H3N2), A/chicken/Netherlands/1/2003 (H7N7), A/Netherlands/219/2003 (H7N7), A/Shanghai/1/2013 (H7N9) (Protein Sciences) and HA1 (aa 26-344) A/chicken/Netherlands/1/2003 (H7N7), A/Hong Kong/125/2017 (H7N9) (e-ENZYME). Reverse genetic influenza strain A/Anhui/01/2013 (H7N9) NIBRG-268 was used.29. For antibody library construction approximately 10 ml samples of blood were collected from a immunised alpaca into heparinised tubes. Peripheral blood lymphocytes were purified using a ficol hypaque centrifugation procedure (Sigma-Aldrich) and RNA was extracted using a RiboPure RNA extraction kit (Novagen) according to manufacturer’s instructions. First strand cDNA synthesis was performed using Superscript III reverse transcriptase (Invitrogen) and oligo-dT primer with 200 ng of total RNA per reaction. A phage displayed library was constructed in pNIBS-1 vector as described previously29. This vector allows both phage display and production of soluble VHH nanobody fragments appended with a Histidine purification tag and a c-Myc epitope tag for detection of binding in ELISA.All methods performed on animals were carried out in accordance with relevant guidelines and regulations. All experimental protocols were reviewed and approved by the National Institute for Biological Standards (NIBSC) ethics committee and performed under United Kingdom Home Office License (PBF57622). A juvenile male alpaca was purchased through the Royal Veterinary College, Hertfordshire, UK. Primary immunisation and each subsequent boost was with 50 µg of purified H7-HA from A/Netherlands /219/2003 (Protein Sciences) and sample collection was performed as in29 using immunotubes (Thermo Fisher Scientific) coated overnight at 4 °C with 1 ml of 10 µg/ml recombinant H7-HA (Protein Sciences) or whole influenza virus inactivated antigen standards (NIBSC) reconstituted in PBS. Bound phage antibodies were eluted by adding 1 ml of 100 mM triethylamine followed by incubation for 10 min on a rotating platform at room temperature. The eluted phage were neutralised with 0.5 ml 1 M Tris-HCl pH 7.5. To amplify the selected phage for subsequent rounds of selection 1 ml of eluted phage were mixed with 5 ml of an Escherichia coli ER2738 (Agilent Technologies) culture grown to an OD600 of 0.5 and 4 ml of 2 × TY media followed by incubation in a water bath at 37 °C for 30 min. This was then spread onto 22 cm bioassay dishes containing 2 × TY agar supplemented with 100 µg/ml (w/v) carbenicillin and 2% (w/v) glucose. Plates were grown overnight at 37 °C and harvested and the phage titres before and after selection were determined29.Phage antibody library selections were performed essentially as inSfi1_YRec (gggcggaggatctggtggcggaggttctgcggcccagccggccATGGCACAGKTGCAGCTCGTGGAGTCNGGNGG) and Alp_FR4back_Not1_YRec (tccaaacccaacaatgggtttgggattggcttaccagcggccgc TGAGGAGACGGTGACCTG), where uppercase sequences anneal with antibody sequence extended lowercase sequences are included for subsequent yeast recombination and Sfi1/Not1 restriction sites are underlined. For each library, 20 µg of amplified PCR product was co-transformed with 20 µg of Sfi1/Not1 digested pNIBS-5 vector28 into S. cerevisiae EBY100 competent cells. pNIBS-5 is a yeast display vector which allows the display on yeast of proteins appended with a N-terminal SV5 epitope tag for detection. The final library size was determined through serial dilutions on selective plates as 2.25 × 107 clones. Standard procedures and recipes for growth, induction, yeast cell labelling, media and buffer preparation were used28. For yeast cell analysis and sorting we used 70 nM recombinant HA (eENZYME) followed by primary reagents monoclonal mouse anti-SV5  and biotinylated monoclonal rabbit anti-His  and by secondary reagents monoclonal goat anti-mouse IgG AlexaFluor488  and streptavidin AlexaFluor647 . For sorting ~ 108 cells were labelled in the first round and 50,000 events were sorted decreasing to 5,000 events for subsequent rounds. Yeast plasmid DNA was purified from the final outputs and transformed into TG1 electro-competent Escherichia coli cells and 24 single bacterial colonies were sequenced to identify there VHH-CDR3 sequence. All yeast cell analysis and sorting was performed on BD Canto II or BDAria III flow cytometers (Becton Dickinson) respectively. Data was analysed using FlowJo 10.4 software28.Yeast antibody libraries were generated using the same cDNA used for the construction of the phage libraries as template. Antibody genes were amplified with oligonucleotides: Alp_FR1_29. Influenza virus antigen standards  were reconstituted in 1 ml sterile water or PBS and then diluted 1/200 in 0.5 M bicarbonate buffer pH 9.6 prior to incubation overnight at 4 °C in a 96 well plate (Thermo Fisher Scientific). For primary screening soluble VHH antibodies were harvested from culture supernatants in a 96 well format. In short individual colonies from each round of selection were inoculated into 100 µl of 2 × TY medium supplemented with 100 µg/ml (w/v) carbenicillin and 2% (w/v) glucose in a 96 well flat bottom plate (Corning) using sterile toothpick and grown overnight in a shaking incubator at 30 °C. From this master plate a new 96 well round bottom plate containing 100 µg/ml (w/v) carbenicillin and 0.1% (w/v) glucose was grown at 37 °C for 6 h until OD 600 of approximately 0.9 was reached after which time 30 µl of 2 × TY supplemented with 100 µg /ml (w/v) carbenicillin plus 5 mM IPTG  was added and incubation continued overnight. The plates were then centrifuged at 600 × g for two minutes and supernatants containing the soluble VHH nanobodies were harvested and tested in ELISA. After addition of 100 µl of nanobody to individual wells and incubation for 1.5 h at room temperature, wells were washed with PBS and PBS-tween 0.1% (v/v) before 100 µl of anti c-myc 9E10-HRP (1/1000 dilution) (Roche) in 2% (w/v) milk powder was added and binding detected at OD 450 nM using TMB after 15 min incubation at room temperature. In all ELISA’s a negative control nanobody with no known specificity was used. For their large-scale production, cloned VHH gene fragments were transformed into the non-suppressor Escherichia coli strain WK6, expressed and purified29. Nanobodies were purified from periplasmic preparations using immobilised metal chelate chromatography from the periplasmic fraction using Talon resin (Clontech). Eluted antibodies were dialysed against PBS using dialysis cassettes of molecular weight cut-off of 3 kDa (Thermo Fisher Scientific)29.Primary screening was carried out in a 96 well ELISA plate (Nunc) coated with recombinant HA at 1 µg/ml overnight in PBS at 4 °CEscherichia coli strain WK6. Bivalent nanobodies were expressed and purified as above.Nbs were converted into bivalent molecules using a (G4S)6 linker to fuse two Nb binding domains ‘head to tail’. Sequences of each nanobody unit were optimised to limit the percentage of GC content and to reduce internal homology within the construct. Constructs were assembled by PCR from overlapping oligonucleotides, sub-cloned into pNIBS-1 and transformed into the 6 per well, in a total volume of 50 µl. Plates were then incubated in a humidified incubator at 37 °C, 5% CO2 for one hour, after which 1.5 × 104 HEK293T/17 cells were added per well in a total volume of 50 µl. After 48 h, supernatants were removed and a 50:50 mix of PBS and Bright-Glo (Promega Corporation) was added to each well. Plates were incubated at room temperature for five minutes and then luminescence was read using a Glomax luminometer (Promega Corporation). Signals were normalised to cell and virus only controls, representing 100% and 0% neutralisation respectively. IC50 values were calculated by non-linear regression using GraphPad Prism.Lentiviral pseudotypes were produced by transient co-transfection of HEK293T/17 cells using polyethylenimine. Plasmid p8.91 encodes the structural  genes and pCSFLW represents the genome incorporated into the pseudotypes bearing the firefly luciferase reporter. Influenza A hemagglutinin genes, in the expression plasmid pI.18 were also added to this mix alongside the Human Airway Trypsin (HAT) expression plasmid, pCAGGS-HAT, to allow for HA0 to HA1/2 maturation. The pseudotype based micro-neutralisation assay (pMN) was carried out in Nunc F96 microplates (Thermo Fisher Scientific). 1:2 serial dilutions of nanobodies were performed across the 96-well plate in a total of 50 µl DMEM + 10% (w/v) fetal bovine serum and 1% (w/v) penicillin/streptomycin. HIV-1 derived lentiviral pseudotypes bearing influenza HA were then added to yield a relative luminescence unit (RLU) input of 1.5 × 1039. In brief, HA was immobilised onto a Biacore CM5 chip in 10 mM sodium acetate pH 5.5 using an amine coupling kit  to create surface densities of between 1000 and 3000 RU. A concentration series of purified nanobody were sequentially run over the different antigen surfaces ranging from 1 to 25 nM. A reference surface was subtracted prior to evaluation of the sensograms using the single cycle kinetics procedure of the Biacore T200 evaluation 3.1 software  in combination with a 1:1 fitting model.For binding and affinity ranking against different full length recombinant HA0 and HA1 head domains we used single cycle kinetics on a Biacore T100 machine SfiI-NotI restriction sites (Integrated DNA Technologies), cloned into the yeast display vector pNIBS-5 and transformed into S. cerevisiae EBY10028. Standard procedures and recipes for growth, induction, yeast cell labelling, media and buffer preparation were used. Staining with purified Nbs was performed by incubating cells with 100 nM of Nbs followed by monoclonal mouse anti-SV5  and polyclonal chicken anti-cMyc  followed by secondary reagents monoclonal goat anti-mouse IgG AlexaFluor488  and goat anti-chicken IgG AlexaFluor647 . Anti-influenza control H3 and H7 specific antibodies MIA-H7-334 and MIA-H3-501 were used (eEnzyme). Labelled yeast cells were analysed on BD Canto II or BDAria III flow cytometers.Full length HA0, HA1 and HA2  respectively, were codon optimized for yeast expression and synthesised, including Sfi1/Not1 digested pNIBS-5 yeast display vector28 into S. cerevisiae EBY100 competent cells (Invitrogen). The final library size was determined through serial dilutions on selective plates, and yeast plasmid minipreps of the library was transformed into TG1 electro-competent Escherichia coli cells (Agilent Technologies) for single colony sequencing. Individual colonies were picked and sequenced to assess mutation frequency. The yeast library was grown in selective medium for induction of HA display. 108 cells were co-stained with 100 nM Nb, followed by anti-SV5/anti-cMyc antibodies to detect Nb binding and HA display using fluorescent secondary reagents as described above. Flow cytometric cell sorting was performed using BDAria III. For the first round of a sorting a gate was chosen to sort cells displaying HA  but absence of nanobody binding (lower right quadrant of a FACS dot plot). A second round was performed using the same sorting conditions. A third round of ‘positive’ sorting was done using 200 nM of a non-competing nanobody of NB37X-01 for epitope mapping of HA1 specific nanobodies  and NB7-14 for epitope mapping of the stem specific nanobodies . Yeast plasmid DNA was purified from the final outputs and transformed into TG1 electro-competent Escherichia coli cells and single bacterial colonies were sequenced. Sequence reads were assembled and aligned to the wild-type HA0 or HA1 genes to identify candidate mutations. Yeast clones with defined mutations were separately labelled for HA display and Nb binding. The Nb binding mean fluorescence intensity (MFI) of each Nb-mutant HA pair was divided by the MFI value of the wild-type H7-HA interaction, and the resulting ratio normalised to percentage values. Binding was categorized as follows, ≤ 15% no binding, between 15 and 40% intermediate binding and ≥ 40% strong binding (green).Error prone PCR amplification of the HA gene was carried out using a GeneMorph mutagenesis kit according to manufacturer’s instruction (Agilent Technologies) and 20 µg of mutated PCR product was co-transformed with 20 µg of Supplementary Information.Supplementary Figure S5.


Ccr1l1 (Ccr1-like 1) encodes an orphan G-protein-coupled receptor (GPCR) with the highest homology to the inflammatory and highly promiscuous chemokine receptors Ccr1 and Ccr3 . Ccr1l1 was first cloned in 1995, yet current knowledge of this putative chemokine receptor is limited to its gene organization and chromosomal localization. Here we report that Ccr1l1 is a Rodentia-specific gene selectively expressed in eosinophils. However, eosinophil phenotypes, development, and responsiveness to chemokines were all normal in naïve Ccr1l1 knockout mice. We demonstrate for the first time that recombinant Ccr1l1 is expressed on the plasma membrane of transfected cells and contains an extracellular N terminus and an intracellular C terminus, consistent with GPCR topology. Using receptor internalization, β-arrestin recruitment, calcium flux, and chemotaxis assays, we excluded all 37 available mouse chemokines, including Ccr1 ligands, and two viral chemokines as Ccr1l1 ligands, and demonstrated that mouse Ccr1, but not Ccr1l1, exhibits constitutive signaling activity. However, sequence analysis and structural modeling revealed that Ccr1l1 is well equipped to act as a classical signaling GPCR, with N-terminal sulfotyrosines as the only signaling and chemokine-binding determinant absent in Ccr1l1. Hereof, we show that a sulfatable N-terminal Ccr1 Y18 residue is essential for chemotaxis and calcium responses induced by Ccl3 and Ccl9/10, but substituting the corresponding Ccr1l1 F19 residue with tyrosine failed to confer responsiveness to Ccr1 ligands. Although Ccr1l1 remains an extreme outlier in the chemokine receptor family, our study supports that it might respond to unidentified mouse chemokine ligands in eosinophil-driven immune responses.Mouse  Chemokines are a large family of chemotactic cytokines that regulate immune cell development and migration by signaling through 7-transmembrane domain (7TM) receptors. Chemokines are divided into four subfamilies, termed CC, CXC, XC, and CX3C, based on the number and arrangement of their N-terminal cysteine residues . Two typChemokine receptors are integral plasma membrane proteins, consisting of an extracellular N terminus, an intracellular C terminus, and 7 transmembrane domains (TMI–VII) connected by three intracellular loops (ICL) and three extracellular loops (ECL) . In addiChemokine GPCR intracellular signaling is transduced by heterotrimeric G proteins and/or β-arrestins, resulting in the release of intracellular stores of ionized calcium, actin polymerization and cell migration, or receptor internalization, respectively , 24. AftChemokine receptors can also be classified as homeostatic or inflammatory depending on their functional roles , 38. MosA). Ccr1l1 maps to chromosomal region 9qF4 flanked by Ccr1 and Ccr3. This region is syntenic to human 3p21, which contains CCR1 and CCR3 but lacks a Ccr1l1 ortholog. Ccr1 and Ccr1l1 are separated by approximately 14 kb and have the same transcription orientation (A).Given its extremely strong homology with Ccr1 and Ccr3, we framed our initial analyses of Ccr1l1 within the CC chemokine receptor subfamily. Ccr1l1 is located on mouse chromosome 9, together with seven other CC chemokine receptors  A. Ccr1l1entation A.Figure B). Notably, no Ccr1l1 orthologs were found in any other mammalian families, indicating that Ccr1l1 is a highly conserved gene restricted to Rodentia. Ccr1l1 amino acid sequence is 70% and 53% identical to mouse Ccr1 and Ccr3, respectively, whereas it has slightly lower amino acid identity to human CCR1 (66%) and CCR3 (51%), and substantially lower homology with other CC chemokine receptors (C). To understand the evolutionary relationship of Ccr1l1 with other CC chemokine receptors, we performed a phylogenetic analysis of all CC chemokine receptors found in these 11 rodent species and humans. The different chemokine receptors were correctly clustered by a maximum likelihood analysis that established evolutionary relationships—common ancestors shared by Ccr6, Ccr7, Ccr9 and Ccr10; by Ccr4 and Ccr8; and by Ccr2 and Ccr5—similar to a previously published phylogenetic analysis (D) (D). In conclusion, mouse Ccr1l1 and Ccr1 are two highly related receptors with similar chromosomal location, transcription orientation, amino acid sequence, and evolution.A BLAST-P search using the predicted mouse full-length Ccr1l1 protein sequence as the query revealed orthologs in at least ten other rodent species with amino acid identities >78% B. Notableceptors C. To undanalysis D 2). We. WeB). Nysis (D) D. In conA). Then, we sorted naïve splenocytes into T, B, and non-T/non-B cell (non B/T) populations and found that Ccr1l1 expression was significantly higher in non-B/T cells (B). Consistent with this, a search in the RNA-seq Gene Skyline databrowser of the Immunological Genome Project or ImmGen (http://rstats.immgen.org/Skyline/skyline.html) (C). To confirm this, we analyzed the expression of Ccr1l1 in bone-marrow-derived macrophages (BMDM) and eosinophils (BMDE) generated in vitro from wild-type Ccr1l1+/+ C57BL/6NTac mice or Ccr1l1−/− mice as a control. We found that while both BMDM and BMDE expressed Ccr1l1, the relative expression was 2 logs higher in BMDE (D). As expected, Ccr1l1 was undetectable or near the detection limit in cells derived from Ccr1l1−/− animals or in nonretrotranscribed controls (D). We concluded that Ccr1l1 is expressed in most lymphoid tissues and in some myeloid cells, with the highest constitutive expression in eosinophils.Next, we investigated the tissue and cellular expression of Ccr1l1. In the absence of an available antibody reagent to detect Ccr1l1 protein, we analyzed Ccr1l1 mRNA expression in multiple tissues and cell types by qPCR. Relative transcript abundance of Ccr1l1 was particularly high in the bone marrow and the spleen of naïve C57BL/6NTac mice compared with other tissues such as the brain, kidney, liver, or heart A. Then, /T cells B. Consisne.html)  revealedne.html) C. To con in BMDE D. As expcontrols D. We conA). In addition, this model supported that the β-hairpin fold typically observed in the ECL2 of chemokine receptors and the two conserved extracellular disulfide bridges (TMVII-N-terminus and TMIII-ECL2) are preserved in Ccr1l1 (B). Importantly, the model predicted that Ccr1l1 conserves the G-protein-coupling DRY motif at the boundary between TMIII and ICL2 (C). Alignment of the amino acid sequences of 11 different rodent Ccr1l1 species with mouse Ccr1 confirmed that Ccr1l1 preserves the canonical DRYLAIV sequence of the DRY motif, with most rodent Ccr1l1 orthologues encoding a DRYLAVV sequence (D). This exact sequence is also found in the corresponding location of human CCR8 and CCR5 , and it has been proposed to play only a minor role in signaling by chemokine GPCRs  of Ccr1l1 TMV, and S131 is substituted for a highly homologous T129 in Ccr1 and all Ccr1l1 species (D). In addition, Y45, W94, Y116, and E288 located at the bottom of the chemokine binding pocket of CXCR4 were shown to be critical signal initiators . Finally, we found that a TMVI hydrophobic bridge involved in the signal propagation from the signal initiators on the extracellular side to the intracellular activation determinants of GPCRs (D). Together, these observations support that Ccr1l1 is well equipped to act as a canonical signaling GPCR.Recently, a comprehensive mutagenesis analysis of CXCR4 revealed other amino acids required for G protein coupling, activation, and signal transmission . For ins species D. In additiators . As showof GPCRs , 44, is of GPCRs D. TogethA). Protein expression was detected by fluorescence microscopy without membrane permeabilization (-Triton) after transfection with pNT2, whereas detection of the C-terminally tagged pNT1-encoded protein required permeabilization . We confirmed and quantified these results by fluorescence-activated cell sorting (FACS) (C and D). As shown in Figure 4D, comparable levels of pNT1-and pNT2-encoded proteins were detected in permeabilized cells, but only the N-terminally tagged pNT2-encoded protein was detectable in nonpermeabilized cells. Thus, Ccr1l1 can be expressed on the plasma membrane and contains an extracellular N terminus and an intracellular C terminus, consistent with a functional chemokine receptor.Ccr1l1 conserves major structural and signaling features of bona fide chemokine GPCRs, and therefore, it is likely to bind chemokines and signal through G proteins if it can be expressed on the cell plasma membrane. To test this, we transiently transfected HEK293 cells with two different plasmid constructs, termed pNT1 and pNT2, containing the coding sequence of Ccr1l1 cloned from mouse splenic mRNA in frame with a C-terminal or N-terminal HA-tag, respectively A. Protei+Triton, B. We cong (FACS) , C and DA, both cell lines expressed high levels of the corresponding myc-tagged receptor on the plasma membrane and as expected, Ccl3 and Ccl9/10 but not Ccl24 (negative control) induced downregulation of cell surface Ccr1. However, none of these ligands affected surface expression of Ccr1l1 (A). To confirm this, we analyzed by bioluminescence resonance energy transfer (BRET) the β-arrestin recruitment induced by these chemokines in 293T cells transiently transfected with Ccr1l1 or Ccr1. We found that Ccl3 and Ccl9/10 but not Ccl22 (negative control) increased the net BRET signal over time in Ccr1-transfected cells (B). However, the net BRET signal after the addition of these chemokines did not diverge from that of the buffer in Ccr1l1-transfected cells (B). Interestingly, we observed that regardless of chemokine treatment, the baseline net BRET signal was higher in Ccr1-than in Ccr1l1-tranfected cells (B), suggesting constitutive β-arrestin recruitment by Ccr1. To address this more directly, we analyzed the net BRET signal in the absence of chemokine treatment after transfection with 100 or 50 ng of plasmids encoding Ccr1, Ccr1l1, or a control plasmid encoding only the Rluc8 reporter. While the net BRET recorded in Ccr1l1-transfected cells did not surpass that of cells transfected with the control plasmid, the signal was markedly higher in Ccr1-tranfected cells (C), indicating that mouse Ccr1 constitutively recruits β-arrestin in this system. It has been previously reported that human CCR1 is a constitutively active receptor  or induced a detectable migration or calcium flux response in these cells (E). As expected, Ccl3 and Ccl9/10 induced chemotaxis and calcium mobilization in myc-Ccr1-expressing cells (E). Importantly, Cxcl12  induced calcium mobilization in untransfected as well as myc-Ccr1-and myc-Ccr1l1-expressing L1.2 cells , confirming that all three cell lines were capable of responding in this type of assay. Of note, all chemokines used in the internalization assays of Figure 5D were also tested in chemotaxis and calcium flux assays, but only selected chemokines are presented in Figure 5E for clarity purposes. Therefore, we concluded that there is no conventional chemokine ligand for Ccr1l1 among the 37 known and commercially available mouse chemokines.Since the Ccr1 ligands we tested induced neither internalization nor β-arrestin recruitment in Ccr1l1-expressing cells, we next tested all 37 commercially available mouse chemokines on the myc-Ccr1l1 cell line using receptor internalization, calcium flux, and chemotaxis assays. As shown in ell line D or induse cells E. As expng cells E. Import.2 cells E, bottomHerpesviridae family, encode chemokine homologs to hijack the chemokine antiviral response of their hosts . Also, although the efficacy was low, only Ccr3-expressing cells showed detectable and reproducible migration in response to vCCL2, with the migration peak at 10 nM of chemokine . The inability of vCCL2 to activate mouse Ccr8 in our experiments may indicate that this viral chemokine is specific for the human counterpart of this receptor. However, it is important to note that, unlike its CCR3-mediated activity, which has been independently confirmed by several groups and now also by us with mouse Ccr3, the vCCL2 action at CCR8 remains unclear . In stark contrast, we noticed that the N-terminal domain of mouse Ccr1l1 does not present any tyrosine adjacent to acidic residues . In fact, a Ccr1 DY motif (D17 and Y18 of Ccr1) is replaced most commonly with DF in seven of 11 Ccr1l1 rodent species, including mouse Ccr1l1, whereas it is conserved in the other four species . The phenylalanine replacement in mouse Ccr1l1 would preserve most physical properties of a tyrosine, but it cannot be sulfated since it lacks the necessary hydroxyl group. To test the significance of this anomaly in mouse Ccr1l1, we generated stable L1.2 cell lines expressing an F19Y variant of mouse Ccr1l1 (myc-Ccr1l1-19Y) and golden hamster (Mesocricetus auratus) Ccr1l1 (myc-ghCcr1l1), one of the four rodent Ccrl1l species that conserves the N-terminal DY motif . Also, both as a control and to investigate the role of Y18 in ligand binding by Ccr1, we generated a stable cell line for a Y18F Ccr1 mutant (myc-Ccr1-18F). As shown in Figure 6C, all cell lines expressed high levels of the corresponding receptors on the cell surface. Consistent with the experiments in Fig. 6C). In contrast, internalization of myc-Ccr1-18F was reduced compared with myc-Ccr1 in response to both Ccr1 ligands, pointing to the functional importance of Ccr1 Y18 in ligand interaction, possibly due to tyrosine sulfation of the site (C and D). Importantly, myc-Ccr1-18F was more resistant to Ccl3-mediated internalization (35% cell surface receptor) than to Ccl9/10-mediated internalization (20% cell surface receptor) , suggesting that Y18 plays a more decisive role in the Ccr1-Ccl3 complex than in the Ccr1-Ccl9/10 complex. However, F19Y substitution in mouse Ccr1l1 failed to confer any internalization of the receptor in response to Ccr1 ligands (C and D). Similarly, cell surface ghCcr1l1, despite its conserved N-terminal DY motif, was not downregulated by Ccl3 or Ccl9/10 (C and D). Therefore, we concluded that the absence of a sulfatable tyrosine in the N terminus of Ccr1l1 does not fully explain its inability to interact with Ccr1 ligands.To understand the structural basis for why Ccr1 ligands fail to interact with Ccr1l1 despite >70% overall amino acid identity for the receptors, we focused on differences in the N-terminal domain, which, structural studies have shown, forms an extended ligand-binding interface for chemokine receptors , 17, 19.Fig. 618 in receptor signaling, we performed chemotaxis and calcium flux assays using these N-terminal tyrosine mutant receptor-expressing cell lines. As expected, Ccl3 and Ccl9/10 induced strong chemotactic and calcium responses in Ccr1-expressing cells, whereas, consistent with the internalization data of Figure 6D, no response was detected in Ccr1l1-or Ccr1l1-19Y-expressing cells (E). However, with the only exception of a small calcium flux bump induced by Ccl9/10 , the Y18F substitution nearly abolished Ccr1 chemotactic and calcium responses (E). Therefore, although Ccr1-18F was still capable of chemokine binding to some extent, as indicated by the internalization experiments (D), we concluded that Y18 is essential for Ccr1 signaling.To evaluate the role of Ccr1 Yng cells E. Howeve Ccl9/10 E, bottomesponses E. Thereferiments D, we conD), we compared the chemotactic and calcium flux responses to Ccl3 (Ccr1 ligand) and Ccl11 (Ccr3 ligand) in BMDE obtained from Ccr1l1+/+ and Ccr1l1−/− mice. Importantly, naïve Ccr1l1−/− mice displayed normal tissue eosinophil phenotypes (A) and overall unaltered immune cell populations, with the exception of slightly elevated T cells in the liver (B). Furthermore, absence of Ccr1l1 did not affect the development of BMDE, identified as Ccr3+ SiglecF+ cells (C), from bone marrow cells stimulated with IL-5. Both Ccr1l1+/+ and Ccr1l1−/− BMDE gave strong calcium responses after stimulation with Ccl3 and Ccl11 (D). In chemotaxis experiments, no differences in the efficacy and potency of Ccl3 and Ccl11 to attract Ccr1l1+/+ or Ccr1l1−/− BMDE were observed (E). To explore the possibility that Ccr1l1 may interfere with other eosinophil chemokine receptors, we extended the calcium flux analysis of BMDE to 20 different chemokines, including at least one ligand for every known mouse chemokine receptor and the Ccr1/Ccr3 ligands Ccl3, Ccl9/10, Ccl11, and Ccl24 as controls. Both Ccr1l1+/+ and Ccr1l1−/− BMDE displayed the same chemokine response profile (F). In addition to the Ccr1 and Ccr3 agonists, Cxcl12 (ligand for the ubiquitous Cxcr4 receptor), Cxcl5, and Cxcl2 induced comparable calcium flux responses in both types of BMDE (F). Cxcl2 is a specific ligand for Cxcr2, whereas Cxcl5 activates Cxcr1 and Cxcr2; both receptors have been reported to be expressed in IL-5 primed eosinophils  and housed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals under the protocol LMI-8E approved on 12/31/2015 and annually renewed by the Animal Care and Use Committee of NIAID.2, and 100% humidity. Recombinant mouse chemokines were from R&D Systems or PeproTech and reconstituted and stored per the manufacturer's recommendations. Recombinant viral chemokines vCCL1 and vCCL2 were purchased from R&D Systems.L1.2 cells  were grown in RPMI-GlutaMAX medium supplemented with 10% FBS, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, and 100 U/ml penicillin-streptomycin . HEK293 and 293T cells (ATCC) were grown in DMEM-GlutaMax medium (Life Technologies) supplemented with 10% FBS. All cells were maintained at 37 °C, 5% COCcr1l1−/− lines, and all experiments were reproduced with at least two independent knockout lines.Ccr1l1-deficient mice were generated from Taconic C57BL/6NTac mice using the CRISPR-Cas9 genome-editing method and the small RNA guides Ccr1l1-sgRNA1 (gctttgctattaaacacgtaggg) and Ccr1l1-sgRNA3 (ggtatctatcaatcgtaagcagg). Five of 31 pups were confirmed to be homozygous for the mutation by PCR using the primers molMR4316 (gacccaggtaatgatgctactgatg), molMR4317 (cagggtttagtaaactctaggc), and molMR4318 (gacaatgacctttatatttgc). By DNA sequencing, we found that four Ccr1l1 knockout founder mice had the same deletion from position –58 to +387 relative to the ATG initiation codon, and one mouse had a deletion between –61 and +428. Therefore, in these five mice, our CRISPR-Cas9 editing of Ccr1l1 resulted in the elimination of the initiation codon and at least the first 129 residues of the protein. All five mice were bred as separate Ccr1l1−/− and Ccr1l1+/+ mice were analyzed by FACS. Tissues of interest were harvested and placed in phosphate-buffered saline (PBS) on ice. Blood was collected by cardiac puncture and red blood cells were immediately lysed using ammonium-chloride-potassium (ACK) lysing buffer. Single-cell suspensions from spleen, lymph nodes, and thymus were generated by pressing the tissues through 70 μm cell strainers. Bone marrow cells were collected from femurs and tibias by flushing with 5 ml of cold PBS. Lungs and liver were collected, minced, resuspended in digestion media (FBS-free DMEM-GlutaMax media containing 0.1 mg/ml Liberase TL and 20 μg/ml DNAseI [enzymes from Sigma]), and incubated for 1 h at 37 °C. Cells from the small intestine were isolated essentially as previously described from the colon , T cells , CD4 cells , CD8 cells , macrophages , dendritic cells , and neutrophils .Single-cell suspensions were centrifuged, red blood cells were lysed using ACK buffer, and cells were resuspended in PBS-staining buffer (1X PBS supplemented with 1% FBS and 1% BSA). Approximately one million cells/sample were stained for FACS analysis. Before antibody staining, cellular Fc receptors were blocked using TruStain FcX (Biolegend). Cells were stained in PBS-staining buffer with APC-Cy7-conjugated rat anti-mouse CD45 , PerCP-eFluor710-conjugated rat anti-mouse SiglecF , BUV395-conjugated rat anti-mouse CD11b , PE-conjugated rat anti-mouse Ccr3 , APC-conjugated rat anti-mouse F4/80 , BV785-conjugated rat anti-mouse Ly6G , FITC-conjugated hamster anti-mouse CD11c , BV650-conjugated hamster anti-mouse CD3ε , FITC-conjugated rat anti-mouse CD4 , PE-conjugated rat anti-mouse CD8α , BV785-conjugated rat anti-mouse B220 , and Fixable Viability Dye eFluor450 (eBioscience). In total, 100,000 events were collected on a BD Fortessa cytometer, and data were analyzed using FlowJo (both from BD Biosciences). Cell duplets and aggregates were excluded by SSC-H Ccr1l1+/+ or Ccr1l1−/− mice. Bone marrow was harvested from tibias and femurs by flushing and straining the opened bones with PBS. For the generation of BMDM, five million bone marrow cells were cultured in a 100 mm petri dish in RPMI-GlutaMax media supplemented with 20% FBS and 30% L929-conditioned media. Fresh media was added 3 days after isolation and cells were cultured for four additional days before experiments. For the generation of BMDE, after lysis of red blood cells with ACK buffer, the leukocyte fraction was separated by centrifugation  of bone marrow cells in Lympholyte-M separation media (Cedarlane). Cells at the interface were collected, counted, and cultured at 107 cells/ml in BMDE media  supplemented with 100 ng/ml stem cell factor and 100 ng/ml Flt-3 ligand (both from Peprotech). Two days later, an equal volume of fresh BMDE media supplemented with stem cell factor and Flt-3 ligand was layered on top of the cells. On day 4, cells were moved to new flasks at 3 to 5 × 106 cells/ml in BMDE media supplemented with 10 ng/ml of recombinant mouse IL-5 . On day 7, the cells were moved again to new flasks at 1 × 106/ml in BMDE media containing mIL-5. On days 9 and 11, half of the media was replaced with fresh BMDE media supplemented with mIL-5. On day 14, cells were collected for experiments. To confirm the purity of our BMDE cultures, 106 cells were washed with PBS-staining buffer  and stained with PerCP-eFluor710-conjugated rat anti-mouse SiglecF  and PE-conjugated rat anti-mouse Ccr3 . Data were collected on a BD Fortessa cytometer and analyzed using FlowJo (both from BD Biosciences).BMDM and BMDE were differentiated from bone marrow cells of (Cq[Ccr1l1]-Cq[Gapdh]).Mouse tissues were homogenized in 1 ml of PBS using a tissue homogenizer , and 100 μl of the homogenate was rapidly mixed with 1 ml of Trizol (Life Technologies). Splenocytes were sorted into B, T, and non-T/non-B cells using mouse CD19 and CD90.2 microbeads in an autoMACS Pro separator . Cell pellets of sorted splenocyte populations as well as those of BMDE and BMDM were directly lysed with 1 ml Trizol. After chloroform extraction, the RNA-containing clear phase was diluted with an equal volume of 70% ethanol, and total RNA was purified using the Isolate II RNA mini kit (Bioline). Contaminant genomic DNA was eliminated from RNA samples using the Turbo DNA-free kit (Life Technologies), and cDNA was obtained from 100 to 500 ng of RNA using the SensiFast cDNA synthesis kit (Bioline). Where indicated, separate RNA samples were mock-treated without reverse transcriptase. The expression of Ccr1l1 was calculated relative to the expression of Gapdh by qPCR. The Cq values for gene amplification were obtained in a CFX96 Real-Time System (Bio-Rad) using the SensiFast SYBR kit (Bioline) and the following primer pairs: Gapdh F (aactttggcattgtggaagg) and Gapdh R (acacattgggggtaggaaca); Ccr1l1qPCR F2 (acacacacttcagagacgtga) and Ccr1l1qPCR R2 (tgtgacagctggaatctccatt). Ccr1l1 relative expression was calculated as 2The complete ORF of mouse Ccr1l1 was obtained by RT-PCR from mRNA isolated from splenocytes of C57BL/6NTac mice (Taconic). For transient expression in HEK293 cells, Ccr1l1 was amplified from splenic cDNA by PCR to incorporate a Kozak sequence and a C-terminal or N-terminal HA tag using the primer pairs Ccr1l1 F1/R1 (ccgcttaagccaccatggagattccagctgtc/ccgtctagattaagcataatctggaacatcatatggatataagtcggcagaggtctc) or Ccr1l1 F2/R2 (gcccttaagccaccatggcgtatccatatgatgttccagattatgctgagattccagctgtcacag/ccgtctagattataagtcggcagaggtctc), respectively. PCR fragments were cloned into a pcDNA3.1 plasmid resulting in the plasmids pNT1 (HA tag at the C terminus) and pNT2 (HA tag at the N terminus). For the generation of stable L1.2 cell lines, mouse Ccr1l1, golden hamster Ccr1l1 (ghCcr1l1), and mouse Ccr1 were cloned in frame with a myc tag at the N terminus. For this, Ccr1l1 was amplified by PCR from pNT2 using the primers Ccr1l1 F5 (ccgcttaagccaccatggaacaaaaactcatctcagaagaggatctgg) and Ccr1l1 R2; a synthetic ghCcr1l1 was obtained from GeneArt (Life Technologies) and amplified using the primers ghCcr1l1 F2 (ccgcttaagccaccatggaacaaaaactcatctcagaagaggatctggagatttcagccatcacag) and ghCcr1l1 R1 (cggtctagattataagccagcagaaagc); and mouse Ccr1 was amplified by PCR from an existing plasmid in our laboratory using the primers Ccr1 F5 (ggccttaagccaccatggaacaaaaactcatctcagaagaggatctggagatttcagatttcacag) and Ccr1 R1 (ccgtctagattagaagccagcagagagctc). PCR fragments were cloned in a pcDNA3.1 plasmid, and the resulting plasmids were termed pAT20 (mouse Ccr1l1), pAT17 (golden hamster Ccr1l1), and pAT15 (mouse Ccr1). Plasmids for the expression of the Ccr1 Y18F and Ccr1l1 F19Y mutants were obtained by site-directed mutagenesis using the QuikChange Lightning kit  with the primer pairs Y18F F1/R1 (gtggagtccccaaagtcaaattctgtagttgtgggg/ccccacaactacagaatttgactttggggactccac) and F19Y F1/R1 (ctgaagcataagaatccagacatatagtcattctttggcaactgtgttg/caacacagttgccaagaatgactatatgtctggattcttatgcttcag) on the templates pAT15 and pAT20, respectively. The correct incorporation of the desired mutations was confirmed by DNA sequencing, and the resulting plasmids were termed pAT15mut and pAT20mut, respectively.For BRET assays, mouse Ccr1 and Ccr1l1 were cloned in a pcDNA-Rluc8 plasmid for their expression in frame with a C-terminal renilla luciferase variant 8 (Rluc8) reporter gene. A 6-amino acid linker (GGGSGG) was introduced between the receptor and the reporter gene to allow the proper folding and functioning of both domains. For this, first, Ccr1 and Ccr1l1 were amplified by PCR from pAT15 and pAT20, respectively, using the primers Rluc8pJK1/2F (ctatagggagacccaagctgggccaccatggaacaaaaactc) and Rluc8pJK1R (gtcgtacaccttggaagccatgccgccgctgccgccgccgaagccagcagagagctcatg), or Rluc8pJK1/2F and Rluc8pJK2R (gtcgtacaccttggaagccatgccgccgctgccgccgcctaagtcggcagaggtctc), respectively. Then, PCR fragments were cloned into a pcDNA-Rluc8.6-535 plasmid using the NEBuilder HiFi DNA Assembly Kit  following the manufacturer's instructions. pcDNA-Rluc8.6-535 was a gift from Sanjiv Sam Gambhir (Addgene plasmid #87125). The resulting plasmids were termed pJK1 (Ccr1-Rluc8) and pJK2 (Ccr1l1-Rluc8). The sequence of all constructs was confirmed by DNA sequencing.2 fragment conjugated with AlexaFluor 488 . Both antibodies were used at a 1:800 dilution. In total, 20,000 events were collected in a BD Fortessa bioanalyzer and analyzed using FlowJo (both from BD Biosciences). Only single cells (identified by SSC-H versus SSC-A gating) negative for the viability staining (live cells) were included in the analysis. For fluorescence microscopy analysis, cells were plated as indicated above in six-well plates containing microscopy coverslips. After transfection, coverslips were fixed with 2% methanol-free formaldehyde (Sigma) in PBS for 15 min at room temperature, then stained with 5 μg/ml of an anti-HA tag mAb (Sigma) and an anti-mouse-AlexaFluor488 secondary antibody (Life Technologies) in PBS containing 5% FBS with or without 0.1% of Triton X-100. Samples were mounted using Prolong Gold mountant with DAPI and imaged in an AxioVert 200 inverted fluorescence microscope (Zeiss).To confirm that the Ccr1l1 ORF encodes a plasma membrane protein, HEK293 cells were transiently transfected with the HA-tagged Ccr1l1-expressing plasmids pNT1 and pNT2. For this, cells plated in six-well plates at one million cells/well were transfected using Fugene HD (Promega) with 2 μg of pNT1, pNT2, or pcDNA3.1 as control. Ccr1l1 expression was analyzed 48 h after transfection by FACS or fluorescence microscopy. For FACS, cells were collected with 1 mM EDTA in PBS and washed twice with PBS-staining buffer . Then, 400,000 cells were stained with Zombie Violet viability dye (Biolegend) for 10 min at room temperature and a rabbit anti-HA tag mAb  on ice for 20 min followed by an anti-rabbit F(ab′)L1.2 cells were used for the generation of stable lines for the expression of Ccr1 and Ccr1l1. For this, two million cells were transfected with 2 μg of pAT15, pAT15mut, pAT20, pAT20mut or pAT17 using SG Cell Line kit in a 4D-Nucleofector X (both from Lonza) following the manufacturer's instructions. Two days after transfection, cells were plated in 24-well plates in pools containing 2400 cells/well in selection media . Media was replaced with fresh selection media on day 7, and cells were cultured for seven additional days. Then, surviving pools were screened for the expression of myc-tagged receptors by FACS. For this, Fc receptors were blocked using mouse TruStain FcX (Biolegend), and receptor surface expression was detected by staining with a PE-conjugated anti-Myc antibody  in PBS-staining buffer . Data were acquired in a BD LSR II and analyzed using FlowJo (both from BD Biosciences). Pools with the highest expression levels were further selected by single cell limiting dilution in selection media. Clones were screened for receptor expression by FACS as explained before. Selected clones were expanded and frozen in geneticin-free selection media. For experiments, clones were grown and maintained in selection media containing 1 mg/ml geneticin at all times.6 cells/well in internalization buffer (RPMI-Glutamax supplemented with 0.5% BSA and 10 mM HEPES). Cells were incubated in the presence or absence of 100 nM chemokine in internalization buffer for 45 min at 37 °C and 5% CO2. Subsequently, cell surface receptor levels were analyzed by FACS as explained above.L1.2 stable cell lines expressing myc-tagged chemokine receptors were seeded into a 96-well plate at 1 × 105 cells/well, and cultured for an additional 24 h. Then, media was removed and 30 μl/well of assay buffer  was carefully added on top of cells. Subsequently, coelenterazine h  and 100 nM of the indicated chemokines in assay buffer or buffer alone were added after obscuring the bottom of the plate with opaque adhesive, and the luminescence (480 nm) and fluorescence (540 nm) signals were recorded over time (up to 80 min) using a Mithras LB 940 Multimode Microplate Reader (Berthold Technologies). BRET was calculated as the YFP/Rluc8 emission ratio. The BRET signal recorded in wells transfected only with an Rluc8-containing plasmid was subtracted from the BRET in sample wells to calculate the net BRET in each condition.The capacity of Ccr1 and Ccr1l1 to recruit β-arrestin in a ligand-dependent or -independent way was analyzed by BRET. For this, 293T cells were transiently cotransfected using Fugene HD (Promega) with a beta-arrestin2 mYFP plasmid and pJK1 (Ccr1-Rluc8), pJK2 (Ccr1l1-Rluc8) or empty pcDNA-Rluc8 as control. Beta-arrestin2 mYFP plasmid was a gift from Robert Lefkowitz (Addgene plasmid # 36917). Twenty-four hours after transfection, cells were collected, plated in clear-bottom 96-well white plates at 10L1.2 stable cell lines or BMDE were seeded into clear-bottom 96-well black plates at 400,000 or 200,000 cells/well in 100 μl of media, respectively. Cells were incubated for 20–30 min at 37 °C and then incubated with 100 μl of FLIPR Calcium 6 Assay Kit (Molecular Devices) for 2 h at 37 °C. Chemokine dilutions were prepared at ninefold of the indicated final concentration in a 1:1 vol:vol mix of cell culture media and calcium assay buffer (HBSS supplemented with 20 mM HEPES). Then, 25 μl/well of chemokine solution or buffer alone was added using the robotic pipettor of a FlexStation 3 microplate reader (Molecular Devices), and the relative fluorescence signal was recorded  for 180 s.Chemotaxis of BMDE and L1.2 cells was tested using ChemoTx 96-well plates (NeuroProbe) with 3 or 5 μm pore size membranes, respectively. To enhance the expression of the transfected receptors, L1.2 stable lines were incubated with 5 μM butyric acid (Sigma) for 18 h before the experiment. Recombinant chemokines at the indicated concentrations in assay buffer (RPMI-GlutaMax supplemented with 0.5% BSA and 10 mM HEPES) were placed in the bottom wells. In total, 200,000 cells in assay buffer were placed on the top membrane and allowed to migrate for 4 h or 30 min at 37 °C for L1.2 cells and BMDE, respectively. Then, the top membrane was removed, and the total number of migrated cells was calculated by interpolation in a cell standard curve after incubation with Cell Titer Aqueous One Solution Kit (Promega) and determination of the absorbance at 490 nm in a FlexStation 3 microplate reader (Molecular Devices).Mouse Ccr1l1 (NCBI reference sequence number NP_031744.3) protein was used as query in BLAST-P and I-TASSER analyses. No restrictions or template sequences were introduced in I-TASSER, which selected human CCR5 as the best structure existing in the databases to generate a homology model of Ccr1l1. Images of the predicted three-dimensional structure of Ccr1l1 were generated using Chimera. All the sequence alignments and phylogenetic analyses were generated and edited using the sequence analysis and tree/dendrogram building tools in the Geneious software platform.All data are contained within the article.The authors declare that they have no conflicts of interest with the contents of this article.


Silencing of DDX3X causes accumulation of rNMPs in the cellular genome. These results support the existence of alternative RER pathways conferring high flexibility to human cells in responding to the threat posed by rNMPs incorporation.Removal of ribonucleotides (rNMPs) incorporated into the genome by the ribonucleotide excision repair (RER) is essential to avoid genetic instability. In eukaryotes, the RNaseH2 is the only known enzyme able to incise 5′ of the rNMP, starting the RER process, which is subsequently carried out by replicative DNA polymerases (Pols) δ or ϵ, together with Flap endonuclease 1 (Fen-1) and DNA ligase 1. Here, we show that the DEAD-box RNA helicase DDX3X has RNaseH2-like activity and can support fully reconstituted  Compelling experimental evidence accumulating in the last years, led to the realization that the most abundant type of endogenous DNA damage in mammalian cells is represented by the >1 million ribonucleotides (rNMPs) erroneously incorporated into the genome at each round of replication. Unexpectedly, the number of erroneously incorporated rNMPs is even higher than the well-known oxidized bases or cyclobutane pyrimidine dimers . Indeed,in vitro thanks to yeast enzymes and no other Pol has been shown, up to now, to be able to act in RER reactions  of at least three-four residues. In contrast, RNaseH2 is the only enzyme able to process single rNMPs into a dsDNA context cleaving on the 5′ side of them and promoting an error-free and specialized repair pathway called ribonucleotide excision repair (RER) . After Reactions .In RNaseH2-deficient mammalian cells, there is the activation of an alternative and highly mutagenic pathway directed by Top-1 enzyme that is deleterious for genome stability ,8. MoreoThe DEAD-box RNA helicase DDX3X is a well-known enzyme for its involvement and therapeutic relevance in viral infections and cancer . It operDifferent studies utilized specifically mutated polymerases to increase the level of misincorporated rNMPs in the genome. Comparing cells with wild type- and deficient- RNaseH2 enzyme, it appears clear that it is not just the absence of RNaseH2, but also of the ribonucleotide processing that causes deleterious consequences. If the level of rNMPs exceeds a specific threshold, there is the insurgence of cellular stresses also in the presence of a completely functional RNaseH2 . Moreovein vitro characterization and preliminary in vivo evidence, here we show that the RNA helicase DDX3X has RNaseH2-like activity able to initiate RER in in vitro reconstituted systems and that its silencing causes accumulation of rNMPs in cellular genome. Moreover, we demonstrate that the specialized DNA repair Pols β and λ can fully support in vitro reconstituted RER reactions, with either RNaseH2 or DDX3X. These results support the existence of alternative RER pathways in human cells, suggesting a hitherto unappreciated flexibility of cells in responding to the threat posed by rNMPs incorporation.Thanks to biochemical Unlabelled rNMPs and dNTPs were from Roche Molecular Biochemicals . All other reagents were of analytical grade and were purchased from Merck  or Fluka .Oligonucleotides were purchased from Biomers.net GmbH  or Metabion AG . The sequences of the substrates used are (bold letters indicate ribonucleotides):39R1D15: D55Dds 55rC/55 mer Substrate *CCAACACACAACACCGTGTGAATTCGGCACTGGCCGTCGTATGCTCTTGGTTGTA-3′5′-GGTTGTGTGTTGTGGCACACTTAAGCCGTGACCGGCAGCATACGAGAACCAACAT-5′FAM3′-19R1D4: D24Dds 24rC/24mer Substrate *CTACGGCTCACACTATCTCACACT-3′5′-GATGCCGAGTGTGATAGAGTGTGA-5′FAM3′-18R1D5: D24Dds 24rC/24mer Substrate *CTACGGCTCACACTATCTCACACT-3′5′-GATGCCGAGTGTGATAGAGTGTGA-5′FAM3′-19R4D18: D41Dds 41rCGUA/41 mer Substrate *TATCCACCAATACTACCCTACGCACACCTCCACTCAAACTC-3′5′-ATAGGTGGTTATGATGGGAUGCGTGTGGAGGTGAGTTTGAG-5′FAM3′-18: R38*Rds 18/38 mer RNA Substrate AUGAAGGUUUGAGUUGAGUGGAGAUAGUGGAGGGUAGU-3′5′-UACUUCCAAACUCAACUC-5′ FAM3′-54: *D48 + D30DFEN-1 Flap control Substrate 54mer:TACCAACAACTTTCAACCCCAATCTACCCCTCTTCCACCCAATTCCATTATACC-3′5′-30mer:GGTATAATGGAATTGGGTGGAAGAGGGGTA-3′5′-48mer(flap):GATTGGGGTTGAAAGTTGTTGGTATACCTTAAAACATATTCTCTAACT-3′FAM 5′-Single- and double- underlined sequences indicate the hybridization positions of the respective oligonucleotides, to generate a dsDNA with a 24 nucleotides (nt) flap.39R1D15: D398oxoG1D15Dds 55rC/55oxoG Substrate *CCAACACACAACACCGTGTGAATTCGGCACTGGCCGTC8TATGCTCTTGGTTGTA- 3′5′ -GGTTGTGTGTTGTGGCACACTTAAGCCGTGACCGGCAGCATACGAGAACCAACAT-5′FAM3′ -2 and 250 mM NH4Ac, heated at 95°C for 5 min and then slowly cooled down at room temperature.Each 6-carboxyfluorescein (FAM) 5′-labeled primer was mixed to the complementary template oligonucleotide at 1:1 (M/M) ratio in the presence of 150 mM HEPES–KOH pH 7.4, 500 mM KCl, 10 mM MgClE. coli expression vector pET-30a(+). ShuffleT7 E. coli cells were transformed with the plasmid and grown at 37°C up to OD600 = 0.7. DDX3X expression was induced with 0.5 mM IPTG at 15°C O/N. Cells were harvested by centrifugation, lysed and the crude extract centrifuged at 100 000×g for 60 min at 4°C in a Beckman centrifuge before being loaded onto a FPLC Ni-NTA column from GE Healthcare . Column was equilibrated in Buffer A . After extensive washing in Buffer A, the column was eluted with a linear gradient in Buffer A from 25 to 250 mM Imidazole. Proteins in the eluted fractions were visualized on SDS-PAGE and tested for the presence of DDX3X by Western Blot with anti-DDX3X A300-A474 from BETHYL  at 1:4000 dilution in 5% milk. Fractions containing the purest DDX3X protein were pooled and dialyzed using Slide-A-Lyzer® MINI Dialysis Devices 20K MWCO from Thermo Scientific  for 3 h  in order to remove the salt excess. Finally, proteins were stored at –80°C.Human recombinant full length DDX3X was cloned in the Escherichia coli expression vector pET-30a(+) and BL21(DE3) cells were transformed with the plasmids. Proteins induction and purification were the same as for the full-length protein, with the only difference that proteins were not dialyzed to avoid further loss of material due to the dialysis procedure.Human recombinant DDX3X mutants here specified were induced and purified as the full-length protein . E256A and R262A mutants were cloned in the E. coli expression vector pTrchisA. BL21 E. coli cells were transformed with the plasmid and grown at 37°C up to OD600 = 1. DDX3X expression was induced with 0.5 mM IPTG at 15°C O/N. Purification procedure was the same of full-length protein.Human recombinant DDX3XΔins (1–248/260–662) was cloned in the E. coli expression vector pET-30a(+). BL21(DE3) E. coli cells were transformed with the plasmid and grown at 37°C up to OD600 = 0.7. DDX5 expression was induced with 0.5 mM IPTG at 37°C for 4 h. Cells were harvested by centrifugation and lysed and the crude extract centrifuged at 13 000×g for 60 min at 4°C in a Beckman centrifuge before being loaded onto Dyanabeads® His-Tag Isolation & Pulldown beads from Life Technologies . Protein was eluted according to the manual.Human recombinant DDX3X (132–607) truncated mutant was expressed and purified as previously described . Human r2-D electrophoresis (2-DE) and mass spectrometry. The first electrophoresis run  was performed on 18 cm long, non-linear pH 3–10 gradient Immobiline-polyacrylamide DryStrip gels  using the Ettan™ IPGphor system . For analytical and MS-preparative gels, strips were rehydrated with 20 and 60 μg of protein, respectively, denatured in 350 μl of 2-DE buffer  and 0.2% or 2% (v/v) IPG-buffer , for 1 h at 0 V and overnight at 30 V, at 16°C. IEF was performed at 16°C and complete protein focusing was reached when a total of 100 000 Vh was applied. The focused strips were equilibrated for 12 min in 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.05 M Tris–HCl pH 6.8, 2% (w/v) DTE; and for further 5 min in 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.05 M Tris–HCl pH 6.8, 2.5% (w/v) iodoacetamide, and trace of bromophenol blue. The second dimension (SDS-PAGE) was run on house-made 9–16% polyacrylamide linear gradient gels. at 10°C, in Protean II Multi Cell  applying 40 mA/gel constant current. The run was stopped when the bromophenol blue dye reached the end of the gel. Analytical and MS-preparative gels were stained with ammoniacal silver nitrate and with a MS-compatible silver staining, respectively. 2-DE stained gels were scanned using the ImageScanner III , and the acquired images were analyzed by ImageMaster 2D Platinum v6.0 software .http://www.matrixscience.com). The following database search criteria were set: E. coli or Homo sapiens as taxonomy, 100 ppm as peptide tolerance, trypsin as the digestion enzyme with one allowed missed cleavage, cysteine carbamidomethylation as fixed modification, and methionine oxidation as variable modification. For protein identifications the number of matched peptides, the extent of sequence coverage, and the probabilistic score were considered.Protein spots cut from MS-preparative gels, were destained and acetonitrile dehydrated. Spots were rehydrated in trypsin solution  and in-gel trypsin digested overnight at 37°C. 1.25 μl of each tryptic peptides sample was directly spotted onto the MALDI target and air-dried. Then 0.75 μl of matrix solution  acetonitrile and 0.5% (v/v) trifluoroacetic acid) was added to the dried samples and allowed to dry again. Protein identification was carried out by peptide mass fingerprinting (PMF) using an Ultraflex III MALDI-TOF/TOF mass spectrometer . Mass spectra were acquired with the mass spectrometer in reflector positive mode with a laser frequency of 100 Hz and analyzed by Flex Analysis software v.3.0. PMF database searching was carried out in SwissProt database  using the on-line available Mascot software  to 40% solvent B  over 30 min and from 40% to 100% solvent B in 2 min at a constant flow rate of 0.25 μL/min, with a single run time of 33 min in case of single bands, or with a linear gradient from 95% solvent A to 40% solvent B over 55 min and from 40% to 100% solvent B in 5 min at a constant flow rate of 0.25 μl/min for peptides obtained with STAGE-diging.m/z.MS data were acquired using a data-dependent top 12 method, the survey full scan MS spectra (300–1750 Th) were acquired in the Orbitrap with 70 000 resolution, AGC target 1e6, IT 120 ms. For HCD spectra resolution was set to 35000, AGC target 1e5, IT 120 ms; normalized collision energy 25% and isolation width of 3.0 E. coli, unknown version, 23014 entries), with the following parameters: enzyme Trypsin, maximum missed cleavage 2, fixed modification carbamidomethylation (C), variable modification oxidation (M) and protein N-terminal acetylation, peptide tolerance 10 ppm, MS/MS tolerance 20 mmu. Peptide Spectral Matches (PSM) were filtered using percolator based on q-values at a 0.01 FDR (high confidence). Proteins were considered identified with 2 unique high confident peptides . MS (peptides  with Scapeptides . Protein4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% TritonX-100 at 37°C for 60 min (unless specified in figure legends). Finally, the reaction mixtures were stopped by addition of standard denaturing gel loading buffer , heated at 95°C for 5 min and loaded on 7 M urea 12% polyacrylamide (PAA) gel, run at 40 W for 2 h in TBE buffer. Substrates and products were quantified by laser scanning densitometry .The nuclease activity of both human RNaseH2 and DDX3X enzymes was monitored on a double stranded (ds) DNA bearing a single ribonucleotide in the strand labelled at the 5′-end with a 6-FAM fluorescent group. A final concentration of 50 nM DNA substrate was used in the experiments. The amounts of RNaseH2 and DDX3X proteins are indicated in the respective figure legends. Reactions were performed in 20 mM Tris–HCl pH 8.8, 10 mM (NH2 at 37°C for 30 min (unless otherwise stated in figure legends) and stopped by adding EDTA 50 mM pH 8. Products were separated through non-denaturing 7% PAGE at 5 W for 3 h in TBE buffer at 4°C.The helicase activity of DDX3X was monitored by measuring the conversion of a double stranded (ds) RNA (labeled at the 5′-end of one strand with a 6-FAM fluorescent group) into single stranded (ss) nucleic acid. A final concentration of 50 nM RNA substrate was used in the experiments, unless otherwise stated. The amounts of DDX3X proteins are indicated in the respective figure legends. Reactions were performed in 20 mM Tris–HCl pH 8, 2 mM DTT, 70 mM KCl and 1 mM ATP, 2 mM MgClSubstrates and products were quantified by laser scanning densitometry .The FRET helicase assay was performed as described . Briefly-UUUUUUUUUUUUUUAGUACCGCCACCCUCAGAACC-3′Fluo-FAM 5′-GGUUCUGAGGGUGGCGGUACUA-3′Qu-BHQ1 5′-TAGTACCGCCACCCTCAGAACC-3′DNA capture 5′The sequence in Fluo-FAM complementary to Qu-BHQ1 is underlined. Flu-FAM carries a 6- carboxyfluorescein fluorophore at its 3′ end, while Qu-BHQ1 carries a Black Hole quencher group at its 5′ end. The DNA capture oligonucleotide is complementary to the Qu-BHQ1 oligonucleotide but bears no modifications.2, 2 mM dithiothreititol, 12 units RNasin , 2 mM ATP, 100 nM capture strand and different and increasing concentration of dsRNA in 20 μl of reaction volume. The unwinding reaction was started by adding DDX3X recombinant protein and R351A mutant and carried out at 37°C for 40 min using a LightCycler 480 (Roche). The fluorescence intensity was recorded every 30 s. Data of fluorescence signals were analyzed by linear interpolation and the corresponding slope values were used to determine the apparent unwinding rate.Helicase assay using the dsRNA substrate was performed in 20 mM Tris-HCl pH 8, 70 mM KCl, 2 mM MgClin vitro by reproducing all steps of physiological RER: incision of a single rMNP embedded in a dsDNA by RNaseH2 or DDX3X, was followed by DNA synthesis with Pols δ, β or λ, 5′ rMNP-terminated flap removal by Fen-1 and ligation by DNA ligase 1 to generate as final product a dsDNA without embedded rMNP. A final concentration of 50 nM DNA substrate was used in the experiments. The amounts of RNaseH2, DDX3X, Pols δ and β, PCNA and dNTPs are indicated in the respective figure legends. Reactions were performed in 20 mM Tris–HCl pH 8.8, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100 at 37°C for 60 min (unless specified in the figures). To prove that no more ribonucleotides were present into the fully repaired DNA substrate, at the end of the RER reaction we inactivated all the enzymes at 75°C for 10 min. Then, we left the reaction at room temperature for 10 min and we proceeded adding 100 nM of human RNaseH2 for 45 min. The dsDNA product without embedded rMNP, will be resistant to the digestion of RNaseH2 enzyme. Finally, the reaction mixtures was stopped by addition of standard denaturing gel loading buffer , heated at 95°C for 5 min and loaded on 7 M urea 12% polyacrylamide (PAA) gel at 40 W for 2 h in TBE buffer. Substrates and products were quantified by laser scanning densitometry .The ribonucleotide excision repair (RER) assay was reconstituted 7 cells were ruptured with a Dounce homogenizer in Lysis Buffer . The lysate was incubated 30 min on ice, sonicated for 5 min  and centrifuged at 15 000 × g for 10 min at 4°C. The protein concentration in the supernatant (crude extract) was quantified with Bradford. For DDX3X quantification, increasing concentrations of crude extract  were loaded on a SDS-PAGE alongside known concentrations  of recombinant purified DDX3X. Separated proteins were subjected to Western Blot analysis with anti-DDX3X A300-475A (BETHYL) at 1:4000 dilution in 5% milk. The blot was next incubated with an HRP-conjugated secondary antibody diluted to 1:5000 and the bands corresponding to DDX3X were visualized with Enhanced Chemiluminescent Substrate  using a ChemiDoc™ XRS (Bio-Rad) apparatus. The intensity of the bands was measured by densitometry and the values obtained for the purified DDX3X were plotted as a function of the protein concentration and analysed by linear interpolation to derive a reference curve, whose slope corresponded to the estimated intensity (I) × ng−1 (DDX3X) value. This parameter was used to calculate the I × ng−1 values for the DDX3X in the cell extract, from the intensities of the DDX3X bands in the corresponding cell extract (CE) samples. The mean I × ng−1 (CE) value was used to calculate the ng of DDX3X × μl−1 of extract, and then the total ng of DDX3X present in the extract. This value was divided for the total number of cells used (107), to derive the ng DDX3X/cell. Based on the known molecular weight of DDX3X, the Avogadro number and assuming a mean cellular volume of 6.55 × 10−11 l, the final molar concentration of DDX3X per cell was calculated. Each experiment was repeated three times, with each blot carrying a reference curve alongside the extract samples, to account for variations in loading/transfer efficiency. The reference I × ng−1 (DDX3X) value obtained was anyway comparable across the different experiments (±20% variation).As already described  107 cellA is the burst constant, appk is the apparent reaction rate and t is time.Time-dependent product accumulation was fitted to the simple exponential equation:Eobs is the observed enzymatic activity in the presence of each enzyme dose E0; Emax is the maximal enzymatic activity; nH is an exponential term to take into account sigmoidal dose–response curves.Dependence of the reaction products from DDX3X concentrations was fitted to the equation:Eobs is the observed enzymatic activity in the presence of each ATP dose ([ATP]); Emax is the maximal enzymatic activity in the absence of ATP; ID50 is the ATP concentration inhibiting the enzymatic reaction by 50%; nH is an exponential term to take into account sigmoidal dose–response curves.Dose-dependent inhibition of the DDX3X nuclease reaction by ATP was fitted to the equation:Vmax and Km constants, data (in triplicate) were plotted and analyzed by least-squares nonlinear regression and fitted to the following equation:v is the apparent reaction velocity at each substrate concentration, Vmax is the maximum rate of the reaction, Km is the Michaelis–Menten constant, S is the variable substrate concentration, n is an exponential term to take into account sigmoidal dose–response curves, due to the positive cooperativity of DDX3X binding to RNA  and the RNA substrate binding efficiency of our enzymes was calculated as the kcat/Km ratio.The turnover constant Data were fitted to Equations , 2), ,  with the6 HEK293T cells were seeded in 10 cm tissue culture dishes in 10 ml Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal bovine serum (Sigma) and 20 μg/ml gentamycin (Sigma) and grown for 24 h in a humidified 5% CO2 atmosphere. After 2 h before transfection, cell medium was replaced. Then, cells were transfected with 13.2 μg Inducible Dharmacon™ TRIPZ™ Lentiviral shRNA , 10 μg pSPAX2 vector  and 4 μg pMD2 vector  diluted in 50 mM Hepes pH 7.12, 140 mM NaCl, 750 nM Na2HPO4 and 125 mM CaCl2. To enhance transfection efficiency, 100 μM Chloroquine (Sigma) was added to the cells. Cell medium was changed 14–16 h after transfection and viruses were collected for the two following days.For lentivirus preparation, 3–3.5 × 10All this work was done in Biosafety Level 2 (BSL2) laboratory.2 atmosphere. Then, cell medium was replaced with 2 ml of viral particles and 4 μg/ml protamine sulfate (Sigma) to enhance the transduction efficiency. After 3–4 h, new fresh medium containing viral particles was added for each plate and left O/N. Finally, cells were washed with Phosphate Saline Buffer (Sigma) and new fresh medium Dulbecco's Modified Eagle's medium (Sigma) containing 10% fetal bovine serum (Sigma) and 20 μg/ml gentamycin (Sigma) was added. Cells grown for 24 h in a humidified 5% CO2 atmosphere without any additional selection. Then, since cells that have integrated the Inducible Dharmacon™ TRIPZ™ Lentiviral shRNA  possess Puromycin resistance, a dose-escalation curve of Puromycin (Sigma) was used to select resistant cells. All this work was done in Biosafety Level 2 (BSL2) laboratory. Knockdown (KD) of DDX3X in U2OS cells (shDDX3X cells) or in the non-silencing vector shRNA (NSC) control cells were both induced adding 1 μg/ml Doxycycline (Sigma) into the cell medium and simultaneously maintaining cells in Puromycin selection (2 μg/ml). Doxycycline was maintained for 48–72 h and then cells were harvested and KD efficiency was checked by western blot.One day prior to transduction, 500 000–600 000 U2OS cells were seeded in 2 ml Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal bovine serum (Sigma) and 20 μg/ml gentamycin (Sigma) in 6 well plates and grown in a humidified 5% COAll siRNAs were purchased from Dharmacon™-Horizon Discovery and were transfected using RNAiMAX  according to manufacturer instructions. siRNAs were used at final concentration of 5 nM and 25 nM for DDX3X and RNaseH2 silencing, respectively. U20S cells grow in Dulbecco's modified Eagle's medium  containing 10% fetal bovine serum  and 20 μg/ml gentamycin (Euroclone). Medium was changed 24 h after transfection and samples collected after 72 h. The following mRNA target sequences were used:RNASEH2A siRNAs pool: Target sequences 5→3′CGGGAAAGGCUGUUUGCGA; AAAUGGAGGACACGGACUU;AUGCAUUGGACCAGGGCGU; AGACCCUAUUGGAGAGCGADDX3X siRNAs pool: Target sequences 5′→3′GCAAAUACUUGGUGUUAGA; ACAUUGAGCUUACUCGUUA;CUAUAUUCCUCCUCAUUUA; GGUAUUAGCACCAACGAGANon-Targeting siRNAs pool: Target sequences 5′→3′UAGCGACUAAACACAUCAA; UAAGGCUAUGAAGAGAUAC;AUGUAUUGGCCUGUAUUAG; AUGAACGUGAAUUGCUCAA® Turbo TM device (Bio-Rad). Finally, the following primary antibodies were used: rabbit polyclonal anti-DDX3X A300-475A antibody (1:2000 dilution) (BETHYL), mouse 201055 anti-actin (1:1000 dilution) , mouse MAB3574 vinculin (1:10 000 dilution)  and rabbit ab83943 anti-RNaseH2A (1:500 dilution) .U20S cell pellets were lysed for 30 min on ice in 50 mM pH 8 Tris–HCl, 0.1% SDS, 350 mM NaCl, 0.25% Triton X-100 in the presence of Protease Inhibitor Cocktail 1X (Sigma). Then, samples were sonicated  and centrifuged at 4°C for 10 min at 15 000 × g. The soluble portion of the extract was then quantified by Bradford assay (Bio-Rad) and loaded on SDS-Page gels. After gels running, proteins were transferred to a nitrocellulose membrane with a pore size of 0.45 μm  using Trans-Blot® Tissue kit . Then, 1 μg of genomic DNA was digested with 1 U of E. coli RNaseH2  or with 50 nM human recombinant RNaseH2 and the reaction was led in a dry heating block (Eppendorf® Thermomixer®) at 37°C with constant shaking at 550 rpm for 2.5 h. Reaction was stopped on ice for 30 min and nick translation reaction started trough the addition of 1 U of E. coli DNA pol I  and 2 μl of Atto647N-dUTP pH 7.5 NT Labelling Kit  at 16°C for 1 h.The protocol is based on the one previously published  with furP values were calculated using the Student's t-test.Finally, the reaction was stopped by addition of standard gel loading buffer and loaded on a 1% agarose gel at 100 V for 2 h in TBE buffer. Ethidium bromide and Atto647N-dUTP signals were detected by laser scanning densitometry  and quantified with ImageQuant TL  normalizing Atto647N-dUTP signals with the Ethidium bromide ones. Quantitation of undigested genomic DNAs were subtracted from quantitation of digested genomic DNAs and 39R1D15: D55D Figure −1 μM−1 for DDX3X and 3.3 min−1 μM−1 for RNaseH2, respectively. To the best of our knowledge, no other human enzyme besides RNaseH2 has been described so far to catalyze single rNMP excision from dsDNA.It is already known that DDX3X is able to resolve DNA:RNA hybrids and RNA secondary structures and it has been implicated in RNA degradation processes . We have19R1D4: D24D and *18R1D5: D24D), also generated the expected products , RNaseH2 preferentially cut 5′ of the last rNMP , DDX3X cuts at the rNMP position with the same efficiency as the control substrate  failed to detect RNaseH2-like activity, suggesting that it was peculiar to DDX3X  even at high DDX3X concentrations in the presence of ATP   , suggesting that binding of ATP triggered DDX3X oligomer formation, as suggested for other DEAD-box helicases  Figure . In the ) Figure . The nuc) Figure , with a elicases . Thus, Delicases ,32.Attempting to rationalize the observed nuclease activity, previously unnoticed in human DDX3X or its orthologs in other organisms, a structural modeling comparison was made between catalytically important regions of RNaseH2 and DDX3X. Because of the lack of complete crystal structures of human RNaseH2, we selected the structure of the murine enzyme (amino acid identity 86.62%). The DDX3X crystal structure in its open conformation was used  since oukcat/Km reduction of 5.5-fold in helicase activity if compared to wild type  lacking ATPase/helicase activity  also disin vitro, we incubated human RNaseH2, human Fen-1 and human DNA ligase 1 not only with the canonical human Pol δ, but also in combination with the X-family DNA repair enzymes human Pols β and λ on a dsDNA substrate bearing a single rCMP at position +39 (Substrate *39R1D15: D55D) and a dsDNA oligonucleotide with an embedded stretch of four rNMPs (Substrate *19R4D18: D41D) both long enough to allow productive binding by Fen-1. Starting from Substrate *39R1D15: D55D, RNaseH2 generated the expected 39 nt product  and RNaseH2, reactions with Pol β and δ in the conditions previously described, generated a fully ligated 41 nt repair product only in presence of Fen1 and DNA ligase 1 , RNaseH2 generated the expected products . These products were resistant to degradation by RNaseH2 added back after the ligation steps, indicating that they did not contain rNMPs .We observed that also the DNA repair Pol λ could operate in RER. Performing assays with both Substrates *39R1D15: D55D and using Pol β and Pol δ under the same conditions employed for RNaseH2, also with DDX3X a 55 nt repair product was generated in the presence of Fen-1 and DNA ligase 1 , in cells silenced for DDX3X. If our hypothesis is true, we expected to see an accumulation of rNMPs in silenced cells compared to the control. Stable DDX3X knockdown (KD) U2OS cells were generated by lentiviral transduction of a specific shRNA and silencing efficiency was assessed by Western Blot Figure .E. coli RNaseH2 and human RNaseH2 treatments with a P value significance < 0.05 (Student's t-test) . This result demonstrated that DDX3X KD U2OS cells are more prone to accumulate rNMPs in their genome compared to not silenced cells, even in the presence of a functional RNaseH2, suggesting that in cells, DDX3X and RNaseH2 operate in alternative, non-redundant RER pathways. To complement this approach we then silenced DDX3X and RNaseH2, alone or in combination, also using siRNA technology. Silencing efficiency was assessed by Western Blot  cell line after both t Figure  and agait Figure . RNaseH2t Figure  as previt Figure  while tht Figure . This ret Figure ,8.According to the current models, error-free removal of rNMPs by RER absolutely relies on one key enzyme: the ribonuclease RNaseH2, the only enzyme known so far to be able to recognize and incise the DNA-RNA junction at the 5′ side of a single rNMP embedded into a double stranded DNA. This elegant model, however, raises additional questions. For example, it has been shown so far that in the absence of RNaseH2, DNA topoisomerases can initiate and aberrant, highly mutagenic rNMP removal reaction, leading to DNA deletions. Thus, it could be relevant for the cell to have other less mutagenic, backup alternative mechanisms for rNMPs removal, which leads to the question as to whether other enzymes besides RNaseH2 could initiate RER. In addition, experimental evidence, also provided by our group, suggest that repair Pols can incorporate rNMPs ,29. ThisIn the present manuscript, we offer a possible answer to both these questions, by disclosing two absolutely novel, to the best of our knowledge, findings: the existence of a second RNaseH2-like enzyme in human cells, the RNA helicase DDX3X, and the ability of repair Pols β and λ to allow full RER reactions.in vitro systems, DDX3X operated with an apparent lower cutting efficiency than RNaseH2  failed to show RNaseH2-like activity, also suggesting that it was peculiar to DDX3X. In addition, our kinetic data show that the RNaseH2-like activity of DDX3X did not require its RNA helicase activity nor ATP. Moreover, they suggest that the helicase vs. nuclease activities of DDX3X are regulated by ATP, through the induction of different oligomeric states, thus providing a possible mechanism for the modulation of these two activities into the cell. Such a role of ATP as a regulator of the stoichiometry of the interaction of DDX3X and RNA is in agreement with the findings of Kim and Myong . The curin vitro by various combinations of RNaseH2 and DDX3X with at least three different Pols , along with the observation that silencing DDX3X causes rNMPs accumulation in the genome, even in a RNaseH2 wild type background, suggests that RNaseH2 and DDX3X operate in a non-redundant fashion. Probably, in cells the local availability of a particular Pol along with either RNaseH2 or DDX3X, can be exploited by the different alternative RER pathways. We are nonetheless well aware of the fact that our in vitro system represents an oversimplification of the in vivo situation, which need to be validated in more physiological systems.The possibility shown here, to fully reconstitute RER reactions in vitro systems (However, the potential relevance of our findings stretches from showing the possibility of an efficient mechanism of RER in non-replicating cells, to the suggestion that RNaseH2-deficient cells might still, at least in part, counteract rNMPs accumulation, but less efficiently. Indeed, accumulation of rNMPs in the genome above a certain threshold, induces cellular stress also in the presence of a completely functional RNaseH2 . DDX3X s systems . Thus, aAs already mentioned, loss-of-function mutations of RNaseH2 cause the neurodegenerative disease Aicardi-Goutieres Syndrome  through Overall, the results presented here challenge the current RER model, significantly broadening the landscape of possible alternative mechanisms that can be used by human cells to cope with the burden of misincorporated rNMPs.All the original raw data are available from the authors upon reasonable request.gkaa948_Supplemental_FileClick here for additional data file.