Chikungunya fever is a pandemic disease caused by the mosquito-borne Chikungunya virus (CHIKV). E1 glycoprotein mediation of viral membrane fusion during CHIKV infection is a crucial step in the release of viral genome into the host cytoplasm for replication. How the E1 structure determines membrane fusion and whether other CHIKV structural proteins participate in E1 fusion activity remain largely unexplored.A bicistronic baculovirus expression system to produce recombinant baculoviruses for cell-based assay was used. Sf21 insect cells infected by recombinant baculoviruses bearing wild type or single-amino-acid substitution of CHIKV E1 and EGFP (enhanced green fluorescence protein) were employed to investigate the roles of four E1 amino acid residues  in membrane fusion activity.Western blot analysis revealed that the E1 expression level and surface features in wild type and mutant substituted cells were similar. However, cell fusion assay found that those cells infected by CHIKV E1-H230A mutant baculovirus showed little fusion activity, and those bearing CHIKV E1-G91D mutant completely lost the ability to induce cell-cell fusion. Cells infected by recombinant baculoviruses of CHIKV E1-A226V and E1-V178A mutants exhibited the same membrane fusion capability as wild type. Although the E1 expression level of cells bearing monomeric-E1-based constructs (expressing E1 only) was greater than that of cells bearing 26S-based constructs , the sizes of syncytial cells induced by infection of baculoviruses containing 26S-based constructs were larger than those from infections having monomeric-E1 constructs, suggesting that other viral structure proteins participate or regulate E1 fusion activity. Furthermore, membrane fusion in cells infected by baculovirus bearing the A226V mutation constructs exhibited increased cholesterol-dependences and lower pH thresholds. Cells bearing the V178A mutation exhibited a slight decrease in cholesterol-dependence and a higher-pH threshold for fusion.Cells expressing amino acid substitutions of conserved protein E1 residues of E1-G91 and E1-H230 lost most of the CHIKV E1-mediated membrane fusion activity. Cells expressing mutations of less-conserved amino acids, E1-V178A and E1-A226V, retained membrane fusion activity to levels similar to those expressing wild type E1, but their fusion properties of pH threshold and cholesterol dependence were slightly altered. Chikungunya virus (CHIKV) is an enveloped plus-stranded RNA virus classified into the genus Alphavirus of family Togaviridae. The CHIKV genomic organization is arranged as with other alphaviruses: "5'-nsP1-nsP2-nsP3-nsP4-junction region-C-E3-E2-6K-E1-poly (A)-'3 " . The E1 Aedes mosquito, and is associated with clinical symptoms including fever, headache, myalgia, and joint pains [A. albopictus[A. albopictus. E2-I211T and E1-T98A mutants have an epistatic interaction with the E1-A226V mutation that influences CHIKV fitness in A. albopictus [A. albopictus.Chikungunya fever manifests after a bite by a CHIKV-infected nt pains . An expant pains . A recenlbopictus-19. The bopictus ,21. Addibopictus . Thus, tsrf-3 (sterol requirement in function) mutant (P226S) and srf-5 mutant (V178A), two substituted amino acids located outside of fusion sequences, were identified by cholesterol-depletion experiments in mosquito cell lines; both mutants show decreased fusogenicity related cholesterol-dependence [A. albobilicus[Viral fusion proteins contain a "fusion" amino acid sequence. For example, a recent study demonstrated that the HIV fusion peptide at the gp41 N-terminus, a glycine-rich hydrophobic sequence, is a critical determinant of HIV-infected cell membrane fusion . Substit mutant P6S and srpendence . The rec mutant V8A, two sbobilicus,30. Althmutant P2S and srfTo explore the structure and fusogenicity of the E1 protein, four E1 residues  were selected for change by mutation. Mutants were tested for their ability to induce cell membrane fusion. Constructs expressing the wild type protein E1, or a single-amino-acid substitution, either in monomeric-E1 form or with two other structural proteins (capsid protein C and envelope protein E2), were introduced into a bicistronic baculovirus expression system  to produDNA preparations and manipulations were performed as described by Sambrook et al.  or folloThe production and titer determination of recombinant viruses were as described previously . Baculov6 cells/well in a 6-well plate and infected with recombinant baculoviruses at a multiplicity of infection (M.O.I.) of one in Sf-900 II SFM, containing 8% fetal calf serum (FCS) at pH 6.4. At two days post infection (dpi), cells were washed twice with PBS , then incubated with 200 μl PBS (pH 7.4) containing EZ-Link Sulfo-NHS-LC-Biotin (0.5 mg/ml)  at 4°C for 30 minutes. The incubated cells were washed twice with glycine in PBS  to stop biotinylation. Cells were then lysed in 250 μl RIPA buffer  containing an EDTA-free Protease Inhibitor Cocktail . The lysates were sonicated on ice using a Microson XL 2005 ultrasonic cell disruptor equipped with a P1 microprobe  at 14 W for 10 seconds. Samples were then centrifuged at 15000 rpm for 15 minutes. The resulting pellets were dissolved into urea  and re-centrifuged at 15000 rpm for 15 minutes to remove un-dissolved materials. Fifty-microliter aliquots of the supernatant were mixed with PBS . Biotinylated cell surface proteins in supernatant were separated from non-biotinylated proteins by precipitation with 50 μl M-280 streptavidin magnetic beads  at 4°C for 60 minutes. Beads were washed three times with 0.5 ml PBS. Biotinylated E1 proteins were then eluted by boiling the beads in 50 μl 2 × Laemmli sample buffer containing 4 M urea, resolved by 10% SDS-PAGE, and detected by Western blot using rabbit anti-CHIKV E1. The same blot was re-probed by mAb anti-gp64  as a control for total protein loading.Cell surface proteins were biotinylated using literature methods . Sf-21 c6 cells/well in a 24-well plate and infected with recombinant viruses at an M.O.I. of one. Total proteins harvested at 2 dpi were dissolved in Laemmli sample buffer containing 4 M urea and separated onto 10% SDS-PAGE. The blotted membranes were incubated with rabbit anti-CHIKV E1 serum (1/500), anti-CHIKV E2 serum (1/500), or anti-gp64 monoclonal antibody (1/2000) . The membranes were then incubated at a 1:1000 dilution with Peroxidase-conjugated Goat anti-Rabbit IgG  or peroxidase-conjugated anti-mouse IgG  for one hour at room temperature. Peroxidase was detected on the membrane using a LumiFast Plus Chemiluminescence Detection Kit  following the manufacturer's protocol. The UVP AutoChemi Image System was used for capturing and processing the various images.Western blot analysis was performed as previously reported  with modFor CHIKV E1 protein staining the cell surface, Sf21 cells were seeded in a 96-well plate and infected with recombinant baculoviruses at an M.O.I. of one in Sf-900 II (pH 6.4) containing 2% FCS. At two dpi, cells were fixed with 3% formaldehyde and stained with rabbit anti-whole CHIKV serum at a dilution of 1:100 for 30 minutes at room temperature. After washing twice with cold PBS, cells were incubated with the secondary antibody, Alexa Fluor 594-conjugated goat anti-rabbit IgG  at a dilution of 1:500 for 30 minutes at room temperature and then washed twice with cold PBS. The stained cells were recorded using an inverted fluorescence microscope , red-channel for CHIKV E1 staining and green-channel for EGFP.CHIKV E1 fusion activity, expressed on the baculovirus-infected Sf21 cell surfaces was examined using a cell-cell fusion assay as previously described . BrieflyThe roles in membrane fusion activity and cholesterol requirement of four amino acid residues at positions 91, 178, 226, and 230 in SFV and SINV E1 proteins have been reported ,37. HoweTo evaluate the influence of residues at positions 91, 178, 226, and 230 of E1 protein on cell fusion, we used site-directed mutagenesis to generate pE1-A226V, pE1-V178A, pE1-G91D, and pE1-H230A mutants in the CHIKV E1 gene. Mutants were cloned into the bicistronic baculovirus expression vector: pBac-Rhir-E  and the gp64 control protein were labeled with biotin Figure , indicatTo examine whether single-amino-acid substitution mutants retain the ability to mediate membrane fusion, Sf21 cells, infected by various recombinant baculoviruses were incubated under optimal conditions for CHIKV E1-mediated membrane fusion, in a medium containing 100 μg/ml cholesterol at pH 5.8 . Green fTo analyze whether E1-A226 and E1-V178 protein's pH and cholesterol dependency differ from those of wild-type E1 protein, cells were incubated with medium containing 200 μg/ml cholesterol at various pH values (ranging from pH 5.8 to pH 6.9), or at a constant pH of 5.9 with varying concentrations of cholesterol (ranging from 0 to 100 μg/ml). Cell fusion capacity was analyzed by fluorescence microscopy and fusion indexes were determined. To attain 100% cell fusion, cells expressing E1-A226V required a slightly lower pH than those expressing wild type E1 or E1-V178A (pH = 6.2 vs. pH = 6.4) Figure . Cells eE2 plays a role in E1 fusogenicity with other alphaviruses; we infected cells with baculovirus constructs expressing the C protein E2, and wild type or mutant E1, and examined the cell fusion capability. Cells infected by recombinant baculoviruses were first analyzed for expression of E2 and E1. Western blot results showed that E2 was expressed in cells infected by baculoviruses bearing S-WT, S-A226S, and S-V178A, but not in cells bearing vector or monomeric-E1 base (E1-WT) Figure .Immunofluorescence microscopy showed that all E1 proteins expressed from 26S-based constructs were present on the cell surface Figure  similar To determine whether structural proteins change the fusogenic capacity of mutated E1 protein with respect to pH and cholesterol dependence, we performed experiments similar to those described in Figure In this study, we studied the roles of four amino acids of CHIKV E1 protein  in cell fusion using an insect cell-based system . This cesrf-5 mutants (V178A) are associated with decreased cholesterol-dependence on viral membrane fusion [A. albopictus mosquitoes does not correlate with acquisition of cholesterol dependence or low-pH thresholds for membrane fusion [Second, the less-conserved amino acid residues (V178 and A226) can be replaced without losing E1 fusion capacity Figure . Howevere fusion . Limitate fusion . The obsBlissard and Wenz classified fusion induced by viral membrane fusion as "fusion from within" (FFWI), requiring viral fusogen synthesis. The authors defined "Fusion from without" (FFWO) as exogenous fusogen . FFWI caIn summary, we demonstrated that mutation of highly conserved amino acids of CHIKV E1 across 15 other alphaviruses, i. e., G91 and H230, lost the membrane fusion activity. In contrast, the less conserved amino acid residues (V178 and A226) were replaceable by other amino acid without losing fusion activity but changing in cholesterol and pH dependency slightly. The presence of other structural protein, E2, could enhance E1 fusion activity.CHIKV: Chikungunya virus; SFV: Semiliki forest virus; SINV: Sindbis virus; VEEV: Venezuelan equine encephalitis virus; srf: Sterol requirement in function; MOI: Multiplicity of infection; EGFP: Enhanced green fluorescence protein; FCS: Fetal calf serum; dpi: Days post infection.The authors declare that they have no competing interests.SCK designed and performed the experiments and drafted the manuscript. YJC, YMW, PYT and MDK performed the experiments. TYW and SJL participated in the design of the study, data analysis, and writing the manuscript. All authors read and approved the final manuscript.


Chlamydia trachomatis is an obligate intracellular bacterial pathogen of humans that uses a type III secretion (T3S) system to manipulate host cells through the delivery of effector proteins into their cytosol and membranes. The function of T3S systems depends on small bacterial cytosolic chaperone-like proteins, which bind T3S substrates and ensure their appropriate secretion. To find novel T3S chaperone-substrate complexes of C. trachomatis we first searched its genome for genes encoding proteins with features of T3S chaperones. We then systematically tested for interactions between candidate chaperones and chlamydial T3S substrates by bacterial two-hybrid. This revealed interactions between Slc1 (a known T3S chaperone) or CT584 and several T3S substrates. Co-immunoprecipation after protein expression in Yersinia enterocolitica and protein overlay binding assays indicated that Slc1 interacted with the N-terminal region of the known T3S substrates Tarp (a previously described substrate of Slc1), CT694, and CT695, and that CT584 interacted with a central region of CT082, which we identified as a C. trachomatis T3S substrate using Y. enterocolitica as a heterologous system. Further T3S assays in Yersinia indicated that Slc1 or CT584 increased the amount of secreted Tarp, CT694, and CT695, or CT082, respectively. Expression of CT584 increased the intra-bacterial stability of CT082, while Slc1 did not affect the stability of its substrates. Overall, this indicated that in C. trachomatis Slc1 is a chaperone of multiple T3S substrates and that CT584 is a chaperone of the newly identified T3S substrate CT082. Type III secretion (T3S) systems are used by many Gram-negative bacterial pathogens to manipulate eukaryotic host cells through the delivery of effector proteins into their cytosol and membranes Secretion of some, but not all, T3S substrates requires the assistance in the bacterial cytosol of characteristic chaperone-like proteins Chlamydia trachomatis causes ocular and genital infections in humans that are a significant clinical and public health concern C. trachomatis is part of a family of obligate intracellular bacteria sharing a characteristic developmental cycle that involves the inter-conversion between an infectious form, the elementary body (EB), and a non-infectious form, the reticulate body (RB) Chlamydiae reside and multiply within a membrane-bound compartment, the inclusion, and use a T3S system to translocate effector proteins into host cells C. trachomatis infections are still hampered by the lack of straightforward methods for its genetic manipulation.C. trachomatis T3S substrates have been found by using SalmonellaShigellaYersiniaC. trachomatis genomes may encode ∼100 T3S substrates C. trachomatis T3S effectors have been identified by immunofluorescence microscopy using specific antibodies against chlamydial proteins, which enabled to visualize their translocation during host cell infection. These include Tarp C. trachomatis class I chaperones have been identified: CT260 (known as Mcsc), which binds to and stabilizes at least Cap1 and the Inc protein CT618 Chlamydia pneumoniae orthologs of C. trachomatis CT088 and CT663, respectively) function as a heterodimeric chaperone of CopN C. trachomatis class II and class III chaperones have also been identified Numerous C. trachomatis type III substrate.In this work, we found that Slc1 chaperones not only Tarp 2. HeLa HtTA1 cells were transfected with plasmid DNA by using the jetPEI reagent (Polyplus Transfection), as detailed by the manufacturer.HeLa 229 (ATCC) and HeLa HtTA1  cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% (v/v) foetal bovine serum (Invitrogen) at 37°C in a humidified atmosphere of 5% (v/v) COC. trachomatis L2/434 (from ATCC) was propagated in HeLa 229 cells using standard techniques Escherichia coli TOP10 (Invitrogen) was used for construction and purification of the plasmids, E. coli BTH101 (Euromedex) was used for bacterial two-hybrid, and E. coli BL21 DE3 (Novagen) was used for protein expression. Yersinia enterocolitica ΔHOPEMT , deficient for the Yersinia Yop T3S effectors H, O, P, E, M, and T, but T3S-proficient Y. enterocolitica ΔHOPEMT ΔYscU  yscU gene encodes an essential component of the Y. enterocolitica T3S system, and the Δ1–354yscU mutation is non-polar E. coli or Y. enterocolitica were routinely grown in liquid or solid Luria-Bertani (LB) medium with the appropriate antibiotics and supplements. Plasmids were introduced into E. coli or Y. enterocolitica by electroporation.C. trachomatis genes, or parts of genes, were amplified by PCR from genomic DNA of strain L2/434 using custom oligonucleotide primers , goat polyclonal anti-green fluorescent protein (GFP) , and mouse monoclonal anti-α-tubulin . The generation and purification of the rabbit polyclonal anti-CT082 and anti-CT584 used is described below. Immunoblot detection was done with horseradish peroxidase-conjugated secondary antibodies , Western Lightning Plus-ECL (Perkin Elmer), and a ChemiDoc XRS+ system (BioRad).The following antibodies were used for immunoblotting: rabbit polyclonal anti-Myc , rat monoclonal anti-HA , mouse monoclonal anti-TEM-1 , rabbit polyclonal anti-SycO (1∶1000) Bordetella pertussis. For bacterial two-hybrid, adenylate cyclase (Cya)-deficient E. coli BTH101 was transformed with plasmids expressing the different T18 and T25 hybrid proteins. Co-transformants were grown on solid LB supplemented with isopropyl β-D-1-thiogalactopyranoside , the chromogenic substrate 5-bromo-4-chloro-indolyl-β-D-galactoside  and antibiotics. Plates were incubated for 24 h at 28°C. The appearance of blue colonies, resulting from β-galactosidase -mediated cleavage of X-gal, was taken as a first indication of a protein-protein interaction. In these cases, we further quantified β-gal activity by monitoring the β-gal-dependent cleavage of ortho-nitrophenyl-β-galactoside (ONPG) into the colorimetric molecule ortho-nitrophenol, as described by the manufacturer (Euromedex). In all experiments, we used as positive control strain BTH101 transformed with plasmids pUT18C-zip and pKT25-zip, encoding two leucine zipper domains, and as negative control strain BTH101 transformed with plasmids pUT18C and pKT25. The final results were based on three independent experiments, using three different single bacterial colonies.We used the bacterial adenylate cyclase two-hybrid (BACTH) system Y. enterocolitica ΔHOPEMT strains carrying pBAD/Myc-His A (Invitrogen) derivatives encoding C-terminally Myc-tagged C. trachomatis T3S substrates  under the control of the E. coli arabinose operon promoter (BADP), and pBBR1MCS-2 Y. enterocolitica T3S chaperone SycE, were diluted from overnight cultures to an optical density at 600 nm (OD600) of 0.1 in brain heart infusion broth supplemented with 5 mM of CaCl2 and the appropriate antibiotics (non-secreting conditions). The bacterial cultures were incubated for 2 h at 26°C and at 150 rpm, and then shifted to 37°C (to induce expression from the sycE promoter) and incubated for more 4 h at 150 rpm. At the time of the temperature shift, to induce expression from BADP, L-arabinose was added to the cultures to a final concentration of 0.2% (w/v). After the 4 h incubation at 37°C, bacterial cultures were transferred to ice and the equivalent of 5 OD600 units were centrifuged at 4,500 g for 10 min at 4°C. The bacterial cells were washed once with ice-cold phosphate buffered saline (PBS) and lysed in PBS using the BugBuster reagent (Novagen). After centrifugation at 17,000 g for 10 min at 4°C, the lysate supernatant was pre-cleared with 20 µl of Pierce Protein G agarose (Thermo Scientific), which had been previously washed with ice-cold co-immunoprecipitation (co-IP) buffer , by incubation for 1 h at 4°C with end-over-end rotation. A sample of the pre-cleared lysate was collected for further analysis (input), and the remaining was incubated with 10 µl of Pierce Protein G agarose (Thermo Scientific) that had been previously incubated with 5 µg of mouse monoclonal anti-Myc  or with 5 µg of mouse monoclonal anti-HA  for 1 h 30 min at 4°C with end-over-end rotation. The IPs were carried out at 4°C with end-over-end rotation for 2 h. The samples were then centrifuged at 500 g for 2 min at 4°C and washed three-times with ice-cold co-IP buffer. Finally, 25 µl of Laemmli SDS-PAGE loading buffer were added to the agarose beads and the samples were incubated for 5 min at 95°C (output). Input and output samples were analysed by immunoblotting.Y. enterocolitica ΔHOPEMT or ΔHOPEMT ΔYscU strains carrying the plasmids described above (in the co-immunoprecipitation section). In addition, we also used plasmids expressing C-terminally Myc-tagged Yersinia YopE and/or C-terminally HA-tagged C. trachomatis CT790. The amounts of protein in bacterial pellets and culture supernatants were analysed by immunoblotting and Coomassie staining. The amounts of protein in bacterial pellets and/or culture supernatants were estimated from images of immunoblots and of Coomassie-stained SDS-PAGE with Image Lab (Bio-Rad). Briefly, using “Volume tools” of Image Lab, the band to be quantified was manually selected and the background was subtracted using the local subtraction method. The quantification value was given by a mean value, which corresponds to all the pixels inside the volume boundary. These data are represented as the ratio between when T3S substrates  were expressed together with Slc1, CT584, or CT790, relative to when they were expressed alone. Because YopB, YopD, LcrV and YopN could not be easily distinguished as independent bands in Coomassie-stained SDS-PAGE, the amounts of these proteins in the supernatant fraction were quantified all together (as if they corresponded to a single secreted protein). The results from the quantifications are the average ± standard error of the mean (SEM) from three or four independent experiments, as indicated.T3S assays were done as previously described Y. enterocolitica ΔHOPEMT strains carrying plasmids encoding Myc-tagged C. trachomatis T3S substrates  and HA-tagged candidate T3S chaperones (Slc1 and CT584), were diluted from overnight cultures and incubated as described above for co-IP experiments. After 2 h (strains expressing CT694-Myc or CT695-Myc) or 4 h (strains expressing CT082-HA or Tarp-HA) of incubation at 37°C, chloramphenicol was added to a final concentration of 50 µg/ml. Samples were then taken at different time points and analysed by immunobloting.E. coli BL21 strains harbouring plasmids encoding (6x)His-CT584 or a fusion of maltose binding protein to the N-terminus of CT082 (MBP-CT082) were grown at 37°C [(6x)His-CT584] or at 26°C [MBP-CT082] in auto-induction conditions g for 1 h at 4°C. His-tagged fusion proteins [(6x)His-CT584] or MBP-CT082 were purified from the soluble fraction using Ni-nitrilotriacetic acid (NTA) resin (Qiagen) or amylose resin (New England Biolabs), respectively, according to the instructions of the manufacturers.Purified (6x)His-CT584 and MBP-CT082 were used to immunize New Zealand White rabbits for the production of polyclonal antisera . The anti-CT584 serum was affinity purified on (6x)His-CT584 immobilized on a nitrocellulose membrane. The anti-CT082 serum was first depleted of anti-MBP antibodies by repeated passages on an amylose resin with bound purified MBP, and then affinity purified on MBP-CT082 immobilized on a nitrocellulose membrane. The affinity purified antibodies were concentrated using Amicon Ultra-4 (Millipore) and quantified using NanoDrop 1000 (Thermo Scientific).E. coli BL21 DE3 strains harbouring plasmids encoding glutathione S-transferase (GST) or fusions of GST to the N-terminus of CT082 were grown at 37°C under auto-induction conditions 2, 1 mM dithiothreitol (DTT), 0.1% (v/v) Nonidet P-40] for 16 h at 4°C with gentle rocking. The two membranes were then processed by immunoblotting with anti-(6x)His and anti-GST antibodies, as appropriate.C. trachomatis L2/434 is predicted to encode 889 proteins Chlamydiae and Proteobacteria, we reasoned that primary and secondary structures of C. trachomatis T3S chaperones could have unique features. Based on this, we identified 21 C. trachomatis proteins as possible T3S chaperones: four previously identified chaperones  C. pneumoniae (Cpn0803) interacts with components of the injectisome C. trachomatis chaperones. To assess whether these small and acidic proteins could form dimers, we analyzed if they self-interacted by using a bacterial two-hybrid system (BACTH) E. coli, by using the two fragments (T18 and T25) that constitute the catalytic domain of CyaA from B. pertussis fused to two potentially interacting proteins. Interaction between the two proteins fused to T18 or T25 reconstitutes activity of CyaA, which leads to an increase in cytosolic cyclic AMP levels and activation of the lacZ reporter gene. We therefore analyzed the activity of lacZ-encoded β-gal in bacteria expressing each of the selected 21 C. trachomatis proteins as T18 or T25 hybrid proteins  C. trachomatis putative T3S substrates  . These experiments confirmed that Slc1 interacts with Tarp and also revealed that it can bind CT694 (C. trachomatis T3S substrate CT082 (see below), but not with CT695 or with any other protein tested .To analyze if CT274, CT584, CT790, and CT845 could function as T3S chaperones, and whether Slc1, Mcsc, and Scc4 have additional interacting partners among nd CT694 . By the In the BACTH experiments described above we did not monitor expression of the proteins and therefore a negative result does not imply that two proteins do not interact. For example, by bacterial two-hybrid we did not detect the previously described binding of the Mcsc chaperone to Inc protein CT225 Y. enterocolitica as a heterologous host. We describe first the studies done with Slc1.We next aimed to confirm the possible interactions between Slc1 and CT584 and its substrates by co-IP experiments and analyze their possible roles as T3S chaperones, by using ct694Y. enterocolitica ΔHOPEMT strains harboring plasmids expressing the relevant proteins and grown in non-secreting conditions (in the presence 5 mM Ca2+). A mouse monoclonal anti-Myc antibody was used to pull-down proteins from extracts of ΔHOPEMT co-expressing Tarp, CT694, or CT695 with a C-terminal Myc tag  and Slc1 with a C-terminal HA tag (Slc1-HA). This showed that the IP of Tarp-, CT694-, or CT695-Myc pulled down Slc1-HA  normally bind to the N-terminal region of their substrates  of Slc1 . For unkY. enterocolitica carrying plasmids encoding Tarp-Myc, CT694-Myc, or CT695-Myc (described above in the co-IP experiments). T3S of these proteins was analyzed when they were expressed alone or co-expressed with Slc1-HA. Bacterial strains were incubated in T3S-inducing conditions (without Ca2+) Myc-His A (Invitrogen), encoding TEM-1 β-lactamase. Immunodetection of TEM-1 only in the bacterial pellet fraction controlled that detection of proteins in the supernatant fraction did not result from bacterial lysis or contamination with the bacterial pellet  or T3S-deficient (ΔHOPEMT ΔYscU) l pellet . Immunobl pellet . Howeverl pellet . Specifil pellet . In paral pellet . As expecolitica . The smabstrates . However Slc1-HA . In contcolitica . OverallY. enterocolitica ΔHOPEMT co-expressing Slc1-HA and Tarp-Myc, CT694-Myc, or CT695-Myc, or expressing Tarp-Myc, CT694-Myc, or CT695-Myc alone, were grown in non-secreting conditions allowing for protein expression. Chloramphenicol was then added to stop bacterial protein synthesis and samples were periodically taken in the subsequent 120 min. The protein levels were then analyzed by immunoblotting with anti-Myc and anti-HA antibodies, as well as with anti-TEM-1 antibodies (as a loading control). This revealed that the intra-bacterial stability of Tarp-, CT694-, and CT695-Myc was not significantly affected by the presence of Slc1-HA  provided a first confirmation of the interaction between CT584 and CT082  and C-terminally HA-tagged CT584 (CT584-HA), showed co-IP of CT584-HA  using co-IP, GST pull-downs, co-purifications, or overlay assays (data not shown). Because appropriate internal controls were used in these experiments (not shown), this suggests that the interaction between CT584 and DUF proteins observed by BACTH was probably an artifact. On the other hand, a protein overlay binding assay using purified (6x)His-CT584 to probe extracts of nd CT082 . In addiCT584-HA . CT584-HCT584-HA  -His- or a GST-tagged protein, as suggested by the appearance on SDS-PAGE of multiple putative proteolytic fragments His-CT584 revealed interactions only with GST-CT082101–560 and GST-CT082201–560, but not with GST-CT0821–100 or GST-CT0821–200. This indicated that the interacting region of CT584 within CT082 should lay between amino acid residues 201 and 560  to the N-terminus of CT082 (EGFP-CT082)   and a C-P-CT082) , respectP-CT082)  and CT08P-CT082) , respectnfection  and of Cnfection . In furtextracts  and 6, Cextracts . Putativextracts , but theC. trachomatis. CT695 remains to be immunodetected during the chlamydial developmental cycle; however, as mentioned above, quantitative proteomics clearly detected CT695 on EBs C. trachomatis.In summary, in agreement with previous quantitative proteomics data Y. enterocolitica. We analyzed this possibility, although, if anything, expression of Slc1-HA or CT584-HA slightly reduced secretion by Yersinia of chlamydial T3S substrates  that are not their binding partners , in the presence or absence of Slc1-HA or CT584-HA. YopE-Myc was expressed under the control of arabinose-inducible BADP, as all chlamydial T3S substrates examined in this work. As control, we also analyzed the consequences of expressing the Chlamydia protein CT790 with a C-terminal HA tag (CT790-HA) on Yersinia T3S. In BACTH assays, CT790 showed to self-interact (as Slc1 and CT584) but did not bind to any of the C. trachomatis T3S substrates tested  or Coomassie staining (to monitor secretion of endogenous Y. enterocolitica T3S substrates encoded by strain ΔHOPEMT). This showed that expression of Slc1-HA, CT584-HA or CT790-HA did not affect the amount of secreted YopE-Myc  and of a Yersinia T3S regulator (YopN) . Considering that Slc1 and CT584 did not evolve in Yersinia, their possible interaction with components of the Y. enterocolitica injectisome may also hamper the normal functioning of this T3SS. Therefore, these analyses supported the idea that Slc1 and CT584 are T3S chaperones.A possible alternative explanation for the results described above would be that expression of Slc1-HA or CT584-HA caused a general up-regulation of T3S by partners . We perfYopE-Myc . ExpressYopE-Myc , a Yersir (YopN) . The amoC. trachomatis T3S system likely mediates bacterial uptake, intracellular survival and proliferation during infection The Chlamydia effectors, i.e., it has been shown that they are translocated across the inclusion membrane in infected cells and interfere with host cell functions It is well-established that, in addition to effectors, T3SSs also secrete components of the injectisome 1XXX(IV)5XX(IV)8X(N)10] has been identified as a “conserved chaperone-binding domain” (CCBD) recognized by class IB chaperones Proteus mirabilis, Burkholderia dolosa, Y. enterocolitica, Salmonella, and Shigella) as well as from endosymbionts  Chlamydia and of those of Proteobacteria.Effector T3S chaperones (class I chaperones) normally bind to the N-terminal region of their substrates and are distinguished according to whether they have one (class IA) or several substrates (class IB) Regarding the mode of action of Slc1, Slc1-substrate complexes could act as a 3D T3S signal ct695 gene is possibly co-transcribed with ct694C. trachomatis infection.Tarp and CT694 are immediate early effectors, stored in EBs and likely involved in the invasion-associated re-organization of the host cell cytoskeleton Chlamydiaceae, but not in those of other members of the Chlamydiae phylum. Their amino acid sequences do not show significant similarity to any other proteins in the databases. Furthermore, the 3D structure of Cpn0803, the ortholog of CT584 in C. pneumoniae (Cpn0803 and CT584 display 83% of identity and 92% of similarity), does not significantly resemble other proteins in structural databases Orthologs of CT584 and CT082 can be found in sequenced genomes of Y. enterocolitica probably by stabilizing CT082 in the cytosol prior to secretion (C. pneumoniae (Cpn0803) and chlamydial injectisome proteins CdsN and CdsQ CT584 binds CT082  and promecretion . These rC. pneumoniae) is unique E. coli EspA filament protein, binds to two discrete regions within the N- and C-terminus of EspA and its 3D structure does not resemble that of other classic T3S chaperones Chlamydiae than in Proteobacteria. We are currently analyzing these possibilities by examining the subcellular localization of CT082 in C. trachomatis infected cells and testing if it can interact with components of the Chlamydia T3SS.The characteristics of CT584 and of its binding to CT082 suggest that it is not a class I chaperone. This implies that CT082, albeit being a T3S substrate, may not be an effector. First, the 3D structure of Cpn0803 (the ortholog of CT584 in www.predictprotein.org) no regular secondary structure (NORS) regions are widespread in nature and this apparent lack of structure is crucial for protein function A characteristic of CT082 is a long region (between amino acids 319 and 456) predicted to be intrinsically unstructured, as analyzed with PredictProtein  were immunoprecipitated with mouse monoclonal anti-Myc antibodies bound to Protein G agarose beads (output). The input (Inp.) and output (Outp.) fractions from the immunoprecipitations (IPs) were analyzed by immunoblotting with rabbit polyclonal anti-Myc and rat monoclonal anti-HA antibodies.(PDF)Click here for additional data file.Figure S2Slc1 interacts with Tarp, CT694 and CT695 (A), and CT584 interacts with CT082 (B).Y. enterocolitica ΔHOPEMT strains expressing the indicated proteins were grown in T3S-inducing conditions (without Ca2+). The bacterial cells were lysed and proteins in the lysate supernatants (input) were immunoprecipitated with mouse monoclonal anti-HA antibodies bound to Protein G agarose beads (output). The input (Inp.) and output (Outp.) fractions from the immunoprecipitations (IPs) were analyzed by immunoblotting with rabbit polyclonal anti-Myc and rat monoclonal anti-HA antibodies.(PDF)Click here for additional data file.Figure S3Expression of CT082 and CT584 during host cell infection by C. trachomatis. (A and B) To analyze the specificity of rabbit polyclonal anti-CT082 (α-CT082) (A) and anti-CT584 (α-CT584) (B) antibodies, HeLa HtTA1 cells were transfected with plasmids encoding EGFP, EGFP-CT082, CT082-HA, and CT584-HA (as indicated). Extracts from the transfected cells were analyzed by immunoblotting, comparing α-CT082 and α-CT584 antibodies relative to commercial α-GFP and α-HA antibodies, respectively. (C and D) HeLa 229 cells were left uninfected (UI) or infected with C. trachomatis L2/434 for the indicated times. Protein extracts were analyzed by immunoblotting with α-CT082, α-CT584, α-C. trachomatis major outer membrane protein (MOMP), and α-tubulin (loading control) antibodies.(PDF)Click here for additional data file.Table S1Plasmids used and constructed in this study.(PDF)Click here for additional data file.Table S2Primers used in this work.(PDF)Click here for additional data file.


Escherichia coli Sci1 TssJ lipoprotein, a two four-stranded β-sheets protein that exhibits a transthyretin fold with an additional α-helical domain and a protruding loop. We showed that TssJ contacts TssM through this loop since a loop depleted mutant failed to interact with TssM in vitro or in vivo. Biophysical analysis of TssM and TssJ-TssM interaction suggest a structural model of the membrane-anchored outer shell of T6SS. Collectively, our results provide an improved understanding of T6SS assembly and encourage structure-aided drug design of novel antimicrobials targeting T6SS.Type VI secretion systems (T6SS) are trans-envelope machines dedicated to the secretion of virulence factors into eukaryotic or prokaryotic cells, therefore required for pathogenesis and/or for competition towards neighboring bacteria. The T6SS apparatus resembles the injection device of bacteriophage T4, and is anchored to the cell envelope through a membrane complex. This membrane complex is composed of the TssL, TssM and TagL inner membrane anchored proteins and of the TssJ outer membrane lipoprotein. Here, we report the crystal structure of the enteroaggregative  Type VI secretion systems (T6SS) are specialized secretion machines responsible for the transport of virulence factors. T6SS are versatile as they are able to target both eukaryotic and prokaryotic cells. They therefore play an important role in pathogenesis by acting directly on the host, as well as eliminating competing bacteria from the niche. At a molecular level, T6SS are composed of a minimum of 13 proteins called core-components, all required for the activity of the secretion system. These core-components can be divided in two groups: soluble proteins having a common evolution history with bacteriophage T4 subunits, and membrane or membrane-associated proteins required for anchoring the bacteriophage-like structure to the envelope. Here, we report the crystal structure of one of the membrane-associated core component, the TssJ lipoprotein. The structure exhibits a transthyretin fold supplemented with additional structural elements. One of these, a loop connecting two beta-strands, is responsible for the interaction of the TssJ lipoprotein with the C-terminal domain of the inner membrane protein TssM. We propose that these two proteins link the two membranes and form a channel accommodating the bacteriophage-like structure. These results provide important new insights to understand the biogenesis of these secretion apparati. Vibrio cholerae, the T6SS is required to escape amoeba predation, or for killing host cells by modification of the host cell cytoskeleton and subsequent impairing phagocytic activity Pathogenic bacteria have evolved numerous and original strategies to invade host tissues, colonize new niches or to kill predators. Bacteria are able to adhere to various surfaces and to actively release protein toxins. The delivery of effectors in the milieu, into host cells or bacteria involves dedicated machineries called secretion systems. Among the six secretion systems identified in Gram negative bacteria, the recently identified Type VI secretion system (T6SS) is composed of 13 core components which form a trans-envelope apparatus Vibrio cholerae TssB/TssC (VipA/VipB) tubular structures are disassembled by TssH, an AAA+ traffic ATPase of the Clp family A hallmark of T6SS is that two proteins are found in culture supernatants of bacteria producing T6SS: Hcp and VgrG tssJ and tssH encode an outer membrane (OM) lipoprotein and an AAA+ ATPase, two components regularly found in bacterial secretion systems or in trans-envelope structures allowing the assembly of cell surface appendages Escherichia coli (EAEC) Agrobacterium tumefaciens and Edwardsiella tarda have been shown to interact with homologues of the TssL inner membrane protein and of the outer membrane lipoprotein TssJ in vivo data have been accumulated on the topology of the membrane complex subunits and their interactions, little is known on the structural organization of these proteins. To gain structural information on the assembly of this complex, we initiated the purification of the different subunits. We report here the crystal structure of the TssJ protein of the enteroaggregative Escherichia coli Sci1 T6SS which likely constitute a prototype for all TssJ-like proteins. We also present biochemical data on the TssM protein and on the TssJ-TssM interaction. We provide in vitro and in vivo evidence for the function of a specific loop of TssJ in mediating contact with the TssM subunit, which therefore provide fundamental insight into T6SS biogenesis and topology.Beside bacteriophage-derived components, a number of membrane-associated proteins were shown to be critical for T6SS. Among these components, TssL and TssM have close homologues in Type IVb secretion systems tssJ gene of the sci1 cluster from enteroaggregative Escherichia coli consisting of amino-acid residues 2-155 of the processed TssJ lipoprotein (residues 25-178 of the full-length protein) was cloned in the Gateway vector pETG20A, with an N-terminal fusion hexahistidine tagged thioredoxin for purification A fragment of the dn/dc value of 0.185 mL/g indicated a mass of 17260±800 Da, close to the theoretical mass of 16,899 Da  experiments 6,899 Da  & S2.via Cys1 and its phospholipid thioester as well as the last four residues were not ordered in the crystal.TssJ crystallized readily with 2.2 M ammonium sulfate as a precipitant at pH 6.0 in sitting nano-drops A unique TssJ molecule is contained in the asymmetric unit, and the PISA server TssJ has the topology of a β-sandwich formed by two four-stranded β-sheets . Sheet oP. aeruginosa Type III secretion system (T3SS) Searching the protein database for structurally related proteins with Dali E. coli Sci1 have been performed with the 49 closest sequences in the non redundant (NR) database  bearing three trans-membrane helices and a periplasmic domain of 744 residues . JPRED Enteroaggregative in vivo co-immunoprecipitation assay. TssM-ekto and the two deletion variants, TssM-ekto-Nt and TssM-ekto-Ct were cloned downstream a signal peptide allowing their targeting to the periplasm. All TssM constructs were fused to a FLAG epitope. The full-length, acylated TssJ protein was produced with a C-terminal hemagglutinine (HA) tag E. coli K12  to test for direct interaction. Cells expressing both TssM-ekto and TssJ proteins were treated with formaldehyde and subjected to membrane solubilization. Solubilized extracts were then used in an immunoprecipitation assay with the anti-FLAG antibody. TssJ co-precipitated with TssM, whereas no TssJ was found associated with the resin in absence of TssM-ekto  with a Biacore X100. TssJ was covalently coupled to a CM5 chip and TssM-ekto was injected in the microfluidic channel. The sensorgrams indicated that TssM-ekto was released in a short time without the necessity of chip regeneration . The Kd ±0.25 µM . We thenmeasured . In contin vivo and in vitro. The TssJ-ΔL1-2 mutant was found to be soluble, monomeric, and with a CD spectrum closely similar to that of the native protein  might have been exposed to solvent, probably inducing aggregation.Interactions between proteins require surface complementarities. The three dimensional structure of TssJ shows that it lacks any crevice able to host any putative TssM-ekto protruding domain. In contrast, TssJ exhibits extensions, compared to transthyretin and ExsB, which could interact with crevices on TssM. The helical domain is located on the face of the β-sandwich opposite to the N- and C-termini, while the extended loop is located at the apical side of the β-sandwich, opposite to the N-terminus, and hence to the outer membrane. These sites are however close enough to participate to a large interaction area with TssM. We therefore constructed TssJ mutants deleted of residues 39-42 from the L1-2 loop (TssJ-ΔL1-2) and of the helical domain (TssJ-Δαdom). The TssJ-ΔL1-2 mutant was expressed at similar level that the native protein, both  protein , indicatin vivo and in vitro interaction with TssM-ekto. The presence of Hcp in the culture supernatant has been previously shown to reflect the correct assembly of the machine. Whole cells and supernatants of ΔtssJ cells expressing full-length wild-type or mutant TssJ were separated by centrifugation and the presence of Hcp in both fractions was assessed by western-blot. As shown in tssJ mutation for Hcp release. We then tested whether the deletion mutant of TssJ interacts with the periplasmic domain of TssM in an in vivo co-immunoprecipitation assay. The TssJ-ΔL1-2 mutant was tested (i) for function in a complementation assay and (ii) in vitro analyses. First, analysis of a mixture of TssM-ekto and TssJ-ΔL1-2 by gel filtration revealed a unique peak eluting at the same volume as TssM-ekto alone. SDS-PAGE analysis of the different fractions demonstrated the presence of TssM only  is anchored to the membrane through a thioether linkage between Cys1 and a diacylglycerol. Residues 2-22 have been found to be non-ordered in the crystal structure, providing to TssJ a wide radius of periplasm exploration for catching TssM. Interestingly, the TssM-ekto-Ct domain is predicted to be essentially formed by β-strands. One interesting hypothesis that remains to be tested is that the TssJ lipoprotein may contribute to the folding or the stability of the TssM-ekto-Ct domain. A chaperone-like role for lipoproteins has already been demonstrated in most bacterial secretion systems: in Type II secretion systems (T2SS), the GspS lipoprotein acts as a pilotine to allow the passage of the GspD secretin through the periplasm and help to its proper insertion in the outer membrane d value in the µM range, suggesting that the interaction does not involve a large surface area of interaction, such as the 600-900 Å2 typical of Fab/protein interfaces that would assemble with Kd values down to the nM range Our data showed that the 4 amino-acids protruding loop L1-2 of TssJ is required for a functional TssJ-TssM interaction. This should be analyzed in light of the SPR experiments, reporting a KEscherichia coli K12 DH5α was used for cloning procedures. The enteroaggregative E. coli strain 17-2  and its ΔtssJ derivative  were used for this study. EAEC strains were routinely grown in LB medium at 37°C with shaking. Plasmids were maintained by the addition of ampicillin (200 µg/ml) or kanamycin (100 µg/ml).HA  and pHcpFLAG  have been previously reported HA by site-directed mutagenesis using mutagenic primers annealing upstream and downstream of the sequence to be deleted (5′-CCAGGGAGGCCATTAACACCGGTGGCGCCTCGGTTGTGGTGCGGATTTATC and 5′-GATAAATCCGCACCACAACCGAGGCGCCACCGGTGTTAATGGCCTCCCTGG). pSciSp, encoding the EAEC TssM-ekto domain (amino-acids 386 to 1129) fused to a signal peptide for periplasm targeting, has been constructed by a double PCR technique using pASK-IBA4 as template and oligonucleotides IBA-Sp5 (GATTATAAAGACGACGATGACAAAAGTCTGGTTGCTGAAGTACAGGAACAGATTCGTCCG5′-CGCTACCGTAGCGCAGGCCGCTAGC [sequence encoding the FLAG epitope tag underlined]) and IBA-Sp3 5′-GCCTTTTTCGAACTGCGGGTGGCTCCATCAGTCAGTCTCCTCCACGGTATCCCCGG). Plasmid encoding TssM-ekto-Nt (amino-acids 386 to 973) has been obtained by site-directed mutagenesis by introduction of a stop codon at codon Asn974 using pSciSp as template and oligonucleotides 5′- GGATGTGGCGTTCACCACAGGTTAAGCGGGGCTGCATTTTGAGCTGC and 5′- GCAGCTCAAAATGCAGCCCCGCTTAACCTGTGGTGAACGCCACATCC. Plasmid encoding TssM-ekto-Ct (amino-acids 972 to 1129) has been constructed by a double PCR technique using pASK-IBA4 as template and oligonucleotides IBA-Spβ5 (GATTATAAAGACGACGATGACAAAGGTAACGCGGGGCTGCATTTTGAGCTGCG5′-CGCTACCGTAGCGCAGGCCGCTAGC) and IBA-Sp3. Primers were obtained from custom oligonucleotides synthesized by Eurogentec. Polymerase Chain Reactions (PCR) were performed with a Biometra thermocycler, using the Pfu Turbo DNA polymerase . All constructs have been verified by DNA sequencing (GATC).Constructions of plasmid pSciN9 cells producing FLAG epitope-tagged Hcp were harvested and collected by centrifugation at 2,000 × g for 5 min. The supernatant fraction was then subjected to a second low-speed centrifugation and then at 16,000 × g for 15 min. The supernatant was then filtered on sterile polyester membranes with a pore size of 0.2 µm  before precipitation with trichloroacetic acid (TCA) 15%. Cells and precipitated supernatant were then resuspended in loading buffer and analyzed by SDS-PAGE and immunoblotting with the anti-FLAG M2 monoclonal antibody (Sigma-Aldrich). As control for cell lysis, Western blots were probed with antibodies raised against the periplasmic TolB protein.Supernatant and cell fractions have been separated as previously described 9 exponentially growing cells were harvested, washed with 20 ml of 10 mM sodium phosphate buffer , and resuspended in NaPi buffer supplemented with para-formaldehyde 1%. After incubation at room temperature for 20 minutes, the cross-linking reaction was quenched by the addition of 0.3 M Tris-HCl pH 6.8, and the cells were washed twice in Tris-HCl 20 mM pH 6.8. The cell pellet was then subjected to solubilization for 30 min at 37°C in TES  in presence of protease inhibitors , and diluted 15-fold in TNE  supplemented with 1% Triton X-100. After incubation 2 hours at room temperature with vigorous shaking, the extract was centrifuged 15 min at 18000 x g to remove unsolubilized material. Supernatants were then incubated overnight at 4°C with anti-HA antibody coupled to Agarose-Protein G beads (Roche). Beads were then washed twice with TNE supplemented with 1% Triton X-100, once in TNE supplemented with 0.1% Triton X-100 and Tween 0.1% and once in TNE supplemented with 0.1% Triton X-100. The immunoprecipitated material was heated in loading buffer prior to analyses by SDS-PAGE and immunoblotting.Co-immunoprecipitation experiments were performed essentially as previously described tssJ and tssM-ekto of enteroaggregative Escherichia coli strain 17-2 were cloned into pETG-20A  expression vector according to standard Gateway™ protocols. The final constructs encoded the target genes and an N-terminal fusion with hexahistidine tagged thioredoxine followed by a TEV protease cleavage site. Both plasmids were transformed in Escherichia coli T7 Iq pLysS (New England Biolabs) expression strain. Cells were grown at 37°C in Terrific Broth until the OD600 reached 0.9 and the tssJ or tssM expression was inducted with 0.5 mM isopropyl-β-thio-galactoside (IPTG) overnight at 25°C and 17°C, respectively. After cells harvesting, the lysis was done by adding 0.25 mg/ml lysozyme, followed by sonication. Soluble protein was separated from inclusion bodies and cell debris by 30 min of centrifugation at 20,000 g. We used an AKTA FPLC system to four steps of purification: a Ni2+ affinity chromatography  with a step gradient of 250 mM Imidazole, an overnight TEV His protease digestion at 4°C with a 1∶10 (w/w) protease: protein ratio, a second Ni2+ affinity chromatography; a preparative Superdex 75  gel filtration run in 20 mM Tris pH 8, 100 mM NaCl. Gel filtration of TssM-ekto was performed in a preparative Superose 6 in 20 mM Tris pH 8.0, 200 mM NaCl, 5% glycerol.2+ affinity chromatography in a step gradient of 250 mM Imidazole of the mixture Trx-(His6)-TssM-ekto and TssJ, an overnight TEV His protease digestion at 4°C with a 1∶10 (w/w) protease: protein ratio and a preparative Superose S6  gel filtration run in 20 mM Tris pH 8.0, 200 mM NaCl, 5% glycerol.Co-purification of TssM-ekto and TssJ was performed in three steps on a AKTA FPLC system: a Nidn/dc value of 0.185 mL/g.Size exclusion chromatography (SEC) was performed on an Alliance 2695 HPLC system (Waters) using a KW803 and KW804 columns (Shodex) run in 10 mM HEPES pH 7.5, 150 mM NaCl at 0.5 ml/min. MALS, UV spectrophotometry, QUELS and RI were achieved with MiniDawn Treos (Wyatt Technology), a Photo Diode Array 2996 (Waters), a DynaPro (Wyatt Technology) and an Optilab rEX (Wyatt Technology), respectively, as described 2PO4 pH 7,2, 150 mM NaCl using a Jasco J-810 spectropolarimeter.Circular dichroism spectra of TssJ, TssJ-ΔL1-2 mutant and Trx-TssM-ekto were recorded in 20 mM NaHD values were obtained using the fitting tool of the BIAevaluation software (BIAcore). A 1∶1 binding model was assumed in all cases.The Surface Plasmon Resonance measurements were performed in a 10 mM Tris pH 8, 150 mM NaCl, 0.005% detergent TWEEN using a BIACORE X100 (BIAcore). The chip CM5 (BIAcore) was coated with TssJ N-terminal thioredoxine fusion with 600 response units (RU). We also used the inverse set-up, coating the chip CM5 with TssM-ekto N-terminal thioredoxine fusion with 3000 response units. Binding assays with TssJ N-terminal thioredoxine fusion covalently linked to the chip CM5 were performed with TssM N-terminal thioredoxine fusion at 10, 5, 2.5, 1.25, 0.625, 0.312 µM. We also performed the binding assays with the chip CM5 coated with TssM N-terminal thioredoxine fusion at 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.312 µM. The signal from the uncoated reference cell and the buffer response was subtracted from all measurements. The K4, 0.2 M Na+-thiocyanate pH 6.0. Crystals were cryo-protected with mother-liquor supplemented with 20% ethylene glycol and flash frozen in liquid nitrogen. Some crystals were soaked in the cryo-protecting solution that also contained 0.5 M NaI and 0.5 M CsI. Two data sets were collected: a native data set (λ = 0.98011) and I-SAD (λ = 1.37760) at Proxima I beamline  using an ADSC Q315r detector. Data processing and scaling were done using XDS, XSCALE 121 with unit-cell parameters a = b = 78.07 Å, c = 46.95 Å. TssJ structure was solved by SIRAS with Sharp TssJ crystallization trials were carried out in sitting-drop vapor diffusion method at 20°C in 96-well Greiner crystallization plates using a nanodrop-dispensing robot (Cartesian Inc.) The atomic coordinates and structure factors have been deposited at the Protein Data Bank with accession code 3RX9.Figure S1EAEC TssJ analyzed by MALS/QELS/UV/RI experiments. The protein mass was measured at 17260 ± 800 Da, a value close to that of the theoretical mass of 16,899 Da Click here for additional data file.Figure S2Stereo view of the Fo-Fc electron density map of TssJ depicted at 1 sigma level around Tyr 65.(TIF)Click here for additional data file.Figure S3Superimposition of the EAEC T6SS TssJ lipoprotein with the P. aeruginosa T3SS ExsB lipoprotein (PDB 2yjl ). TssJ and ExsB exhibit a common transthyretin fold, but TssJ possesses an extra α-domain and an extended loop between strands 1 and 2 (indicated by the arrows). Panels A and B are rotated 90° from each other.(TIF)Click here for additional data file.Figure S4Sequence alignment of enteroaggregative E. coli TssJ (gi218696620) with the 49 first hits obtained by blasting the NR database. The secondary structures are depicted according to the TssJ crystal structure. The red arrow indicates the position at which the electron density starts in the X-ray structure. Note that the 33 first sequences do not present any insertion/deletions compared to the EAEC TssJ protein. The conserved residues are boxed in red. Other semi-conserved residues are depicted in red and boxed in blue. Sequence alignment has been performed with Multalin  Salmonella enterica subsp. arizonae; gi288550304, Enterobacter cancerogenus; gi262041848, Klebsiella pneumoniae subsp. rhinoscleromatis; gi238895309, K. pneumoniae; gi 152970795 K. pneumoniae subsp. pneumoniae MGH 78578; gi206578921, K. pneumoniae; gi290508950, Klebsiella sp.; gi238792667, Yersinia intermedia; Y. bercovieri; Y. mollaretii; Y. frederiksenii; Y. mollaretii; Y. rohdei; Proteus penneri; gi212712391, Providencia alcalifaciens; gi261347002, Providencia rustigianii; gi156932370, Cronobacter sakazakii; gi253991289, Photorhabdus asymbiotica; gi37528038, Photorhabdus luminescens; gi161504491, Salmonella enterica subsp. arizonae; gi292898109, Erwinia amylovora ATCC 49946; gi292489675, Erwinia amylovora CFBP1430; gi260597748, Cronobacter turicensis; gi156934209, Cronobacter sakazakii; gi206579068, K. pneumoniae; gi262039776, K. pneumoniae subsp. rhinoscleromatis; gi194434338, Shigella dysenteriae; gi26249236, E. coli CFT073; gi194426146, E. coli B171; gi307554802, E. coli ABU 83972; gi227888372, E. coli 83972; gi317491536, Enterobacteriaceae bacterium; gi149366543, Y. pestis CA88-4125; gi22126561, Y. pestis KIM10; Y. pseudotuberculosis IP31758; gi270486848, Y. pestis KIMD27; gi170024810, Y. pseudotuberculosis YPIII; gi51595841, Y. pseudotuberculosis IP32953; gi108806780, Y. pestis Antiqua; gi153950286, Y. pseudotuberculosis IP31758; gi300715009, Erwinia billingiae; gi157370044, Serratia proteamaculans; gi296102178, Enterobacter cloacae; gi304398550, Pantoea sp.; gi290509839, Klebsiella sp.; gi238894433, K. pneumoniae; gi288935813, K. variicola; gi317049653, Pantoea sp.).(TIF)Click here for additional data file.Figure S5Representation of TssJ conserved residues. TssJ is shown in ribbon representation and rainbow coloring from blue (N-terminus) to red (C-terminus); the sequence is represented above. Figure made with Pymol (TIF)Click here for additional data file.Figure S6Surface representation of TssJ.(A) TssJ B-factors. The surface is colored according to B-factors values, from blue (low B-factors) to red (high-B-factors). (B) TssJ electrostatic potential. The surface is colored according to the contact electrostatic potential calculated with Pymol (JPG)Click here for additional data file.Figure S7Circular dichroism spectra of TssJ and TssM. (A) CD spectra of TssM-ekto (black line) and TssM-ekto-Nt (blue line). (B) Comparison of the Wild-type TssJ  compared to the loop depleted mutant .(TIF)Click here for additional data file.Table S1Biochemical characteristics of the proteins and domains reported in this work.(DOC)Click here for additional data file.Table S2Biophysical characteristics of the proteins and domains reported in this work.(DOC)Click here for additional data file.Table S3Data collection (PROXIMA 1 at SOLEIL) and refinement statistics.(DOC)Click here for additional data file.


Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamic acid to gamma-aminobutyric acid (GABA), was fused to the cellulose-binding domain (CBD) and a linker of Trichoderma harzianum endoglucanase II. To prevent proteolysis of the fusion protein, the native linker was replaced with a S3N10 peptide known to be completely resistant to E. coli endopeptidase. The CBD-GAD expressed in E. coli was successfully immobilized on Avicel, a crystalline cellulose, with binding capacity of 33 ± 2 nmolCBD-GAD/gAvicel and the immobilized enzymes retained 60% of their initial activities after 10 uses. The results of this report provide a feasible alternative to produce GABA using immobilized GAD through fusion to CBD. GADs are ubiquitously found throughout the biological world from microorganisms to mammals  was used as the host strain for cloning and plasmid maintenance. E. coli BL21   harboring a lambda derivative, DE3, was used for the expression of GAD.BglII and NdeI in pET-28b(+) was replaced by the region between same enzyme sites in pET-22b(+), which created a modified pET-28b(+). For the CBD-GAD fusion, the gene encoding CBD and linker of THEG were amplified from pGAL-CBD-Lipase [GAAGGAGATATACATATGCAGCAAACTGTTTGGGGGCAA-3′) and a reverse primer (5′-ACGGAGCTCGAATTCGGATCCAGAGCTAGTTGGCGGAGTAGC-3′) in which the underlined characters were introduced for in-fusion ligation. The polymerase chain reaction (PCR) product was ligated into the modified pET-28b(+) plasmid  digested with NdeI and BamHI using the In-fusion™ Advantage PCR cloning kit . The resulting plasmid was named pEKPM-CBD. The GAD gene was amplified from E. coli genomic DNA by PCR using a forward primer (5′-ACTCCGCCAACTAGCTCTGGATCCGATAAGAAGCAAGTAACG-3′) and a reverse primer (5′-GTGGTGGTGGTGGTGGTGCTCGAGGGTATGTTTAAAGCT-3′) in which the underlined base pairs were introduced for in-fusion ligation. The PCR product was ligated into the pEKPM-CBD plasmid previously digested with BamHI and XhoI using the In-fusion™ Advantage PCR cloning kit, which created pEKPM-CBD-Lk-EcGAD-H6. To replace the native linker peptide with S3N10 linker sequence as suggested by Kavoosi et al. [3N10 linker are amplified from pEKPM-CBD-Lk-EcGAD-H6 using the specific primers . The PCR products containing CBD and the S3N10 linker were ligated into pEKPM-CBD-Lk-EcGAD-H6 previously digested with NdeI and BamHI, which created pEKPM-CBD-mLk-EcGAD-H6.For the efficient cloning work and use of kanamycin as a selective marker, DNA region between D-Lipase  using a i et al. , CBD witE. coli genomic DNA by PCR using a forward primer (5′-GAAGGAGATATACATATGGATAAGAAGCAAGTAACG-3′) and a reverse primer (5′-GTGGTGGTGGTGGTGGTGCTCGAGGGTATGTTTAAAGCT-3′) in which the underlined base pairs were introduced for in-fusion ligation. The PCR product was ligated into the modified pET-28b(+) plasmid previously digested with BamHI and XhoI using the In-fusion™ Advantage PCR cloning kit, which created pEKPM-GAD-H6.For GAD protein without CBD, the GAD gene was amplified from Transformants were cultured overnight in a test tube containing 2 mL of Luria-Bertani (LB) media with kanamycin (50 μg/mL) at 37 °C. The culture was then transferred to 1 L baffled flasks containing 200 mL of LB media supplemented with kanamycin (50 μg/mL). When cell density reached an optical density (OD) of 0.5 at 600 nm as measured by a spectrophotometer , induction was initiated with 0.4 mM IPTG for 3 h. After IPTG induction, the cells were harvested and lysed by sonication for further analysis. Proteins in the lysates were separated by SDS-PAGE according to the method by Laemmli  on 10% pN-terminal amino acid sequencing was performed by the Korea Basic Science Institute (Republic of Korea).CBD-GAD-H6 was isolated using SDS-PAGE, and the corresponding bands were transferred to a PVDF (polyvinylidene fluoride) membrane using a semi-dry electroblotter . The membrane was stained with Ponceau S  to visualize the GAD bands and ®II reagent  to extract soluble proteins. The supernatant was collected for purification; nickel affinity chromatography using Ni-NTA resin  was performed to purify CBD-GAD and GAD proteins. The bound proteins were eluted with a three-step gradient of imidazole  in 20 mM sodium phosphate buffer with 0.5 M NaCl, pH 7.4. Fractions collected at each step were analyzed by 10% SDS-PAGE. Purified proteins were dialyzed overnight against 20 mM sodium phosphate buffer (pH 7.4) at 4 °C.The transformants were harvested by centrifugation. The cells were then resuspended in B-PER+ per minute under assay conditions.Enzymatic activities were measured by a 718 Stat Titrino  with an aqueous solution of 0.1 M HCl. The solution containing MSG of 0.1M and PLP of 0.02 mM was adjusted to final pH of 4.0 and an enzyme was added. The enzyme reaction was performed at 37 °C while recording the titration. Activities were calculated by the slope of the titration curve over 5 min. One unit was defined as the amount of enzyme required for being added 1 μmol HTo estimate the binding capacity of CBD-GAD attached to the Avicel, adsorption isotherm measurements were taken at room temperature in 1.5 mL Eppendorf tubes. Samples containing 0–60 nM of CBD-GAD and 0.2 mg of Avicel were mixed with 20 mM sodium acetate buffer (pH 6.0) in a final aqueous volume of 1.2 mL. Each solution was incubated for 1 h to allow the adsorption to saturate. The supernatants were collected by centrifugation and the protein concentration was measured using a BCA protein assay kit . This value was then subtracted from the initial amount of protein added to the reaction tube and the difference was considered to be amount of proteins adsorbed onto Avicel. All values were measured in triplicate.n-butanol/acetic acid/water . The plates were developed by applying ninhydrin reagent  then dried at 80 °C for 5 min. The image analysis of the spot on the plate was processed by TotalLap TL100 Image Analysis Software .Relative quantification of retained activities after use of immobilized GAD was performed by TLC combined with image analyzer. Samples were prepared as follows; Enzyme was added into the 100 mM MSG and 0.02 mM PLP in 20 mM sodium acetate buffer (pH 4.0). After enzyme reaction at 37 °C for 30 min, immobilized enzyme was recovered by centrifugation and re-used after washing with sodium acetate buffer (pH 4.0). The loss of enzyme molecules due to the recovery process was calculated as 0.3% at each step. The supernatants after enzyme reaction and GABA standard solution were spotted on 10 cm × 10 cm glass TLC plates precoated with 60F254 silica gel . The mobile phase was prepared by mixing E. coli which can express relatively high-level of GAD fused to CBD with a histag to facilitate purification as soluble forms through the modification of linker peptide and low temperature culture condition. This bifunctional chimeric protein simultaneously showed both GAD activity and cellulose-binding ability, which allowed it to be immobilized on a cellulose matrix. To our knowledge, this is the first report on the immobilization of GAD using affinity-tag to obtain GABA by bio-conversion. Applying of low-priced cellulose as matrix and reutilizing of enzyme due to immobilization can be accompanied by decrease in cost. The presented process, therefore, can be potentially applicable to produce GABA in large-scale.In conclusion, we succeeded in producing recombinant


Pseudomonas aeruginosa. The proteins encoded by these genes are both members of the EFG I subfamily. The gene encoding one of the forms of EF-G is located in the str operon and the resulting protein is referred to as EF-G1A while the gene encoding the other form of EF-G is located in another part of the genome and the resulting protein is referred to as EF-G1B. These proteins were expressed and purified to 98% homogeneity. Sequence analysis indicated the two proteins are 90/84% similar/identical. In other organisms containing multiple forms of EF-G a lower degree of similarity is seen. When assayed in a poly(U)-directed poly-phenylalanine translation system, EF-G1B was 75-fold more active than EF-G1A. EF-G1A pre-incubate with ribosomes in the presence of the ribosome recycling factor (RRF) decreased polymerization of poly-phenylalanine upon addition of EF-G1B in poly(U)-directed translation suggesting a role for EF-G1A in uncoupling of the ribosome into its constituent subunits. Both forms of P. aeruginosa EF-G were active in ribosome dependent GTPase activity. The kinetic parameters (KM) for the interaction of EF-G1A and EF-G1B with GTP were 85 and 70 μM, respectively. However, EF-G1B exhibited a 5-fold greater turnover number (observed kcat) for the hydrolysis of GTP than EF-G1A; 0.2 s-1 vs. 0.04 s-1. These values resulted in specificity constants (kcatobs/KM) for EF-G1A and EF-G1B of 0.5 x 103 s-1 M-1 and 3.0 x 103 s-1 M-1, respectively. The antibiotic fusidic acid (FA) completely inhibited poly(U)-dependent protein synthesis containing P. aeruginosa EF-G1B, but the same protein synthesis system containing EF-G1A was not affected. Likewise, the activity of EF-G1B in ribosome dependent GTPase assays was completely inhibited by FA, while the activity of EF-G1A was not affected.Genes encoding two proteins corresponding to elongation factor G (EF-G) were cloned from  EF-G is a member of the GTPase superfamily and functions in ribosome-dependent hydrolysis of GTP ,2,3. EarhEFG1 and hEFG2) encoding two different forms of EF-G were discovered in mammalian mitochondria. Both of these were chromosomal genes encoding mitochondrial EF-G proteins: EF-G1mt and EF-G2mt phenylalanine (75 cpm/pmol), 0.3 mg/ml poly(U) RNA, 0.03 mM spermine, 1 mM DTT, 0.05 μM P. aeruginosa Elongation Factor-Ts (EF-Ts), 1.0 μM P. aeruginosa Elongation Factor-Tu (EF-Tu), 0.1 μM P. aeruginosa phenylalanyl-tRNA synthetase (PheRS), 0.2 μM P. aeruginosa ribosomes and the indicated amounts of EF-G1A or EF-G1B. Reactions were started by the addition of E. coli tRNA to a final concentration of 0.5 μM tRNAPhe and continued for 1 h at 37 °C. Reactions were stopped by the addition of 2 ml 10% trichloroacetic acid (TCA) and filtered through glass fiber filters (Whatman) as previously described [3H]Phe represents the amount of poly-phenylalanine, poly(Phe), synthesized. The effects of FA on protein synthesis were determined using the same assays but contained from 4 to 250 μM FA. In these assays, the concentration of EF-G1A and EF-G1B were 1 μM and 0.2 μM, respectively. Assays to determine the effect of RRF on the activity of EF-G1B were the same as described above, with RRF titrated into the assay between 0.2 and 6.4 μM. Assays to determine the ability of EF-G1A/RRF to affect the activity of EF-G1B contained 0.2 μM EF-G1B, 1 μM EF-G1A and 2 μM RRF. In these reactions EF-G1A and/or RRF were pre-incubated in the reaction mix at 37 °C for 5 min, EF-G1B was then added and incubation was continued for 1 h.Protein synthesis assays were carried out in 50 μl reactions containing: 50 mM Tris-HCl, (pH 7.5), 10 mM MgClT. thermophilus has been solved bound to GDP and in the nucleotide free form; it appears to be an elongated protein composed of five structural domains [P. aeruginosa EF-G1 was determined at 2.9 Å [T. thermophilus EF-G1. The structure of EF-G2 from T. thermophilus has also been determined and is similar to the structure to EF-G1 even though the sequence homology is only 30% [P. aeruginosa (EF-G1A and EF-G1B) have a much higher level of amino acid sequence conservation; with the amino acid sequences being 84% identical and 90% similar (Bordetella bronchiseptica and Burkholderia rhizoxinica) contained two EF-G molecules that contained a high level of amino acid sequence conservation. When P. aeruginosa EF-G1A and EF-G1B were compared to the four homologs from these two bacteria a high level of homology was observed in which the similarity only varied from 76 to 81% . These three residues are located in a loop region in domain IV and would therefore probably not affect function. The only region of lower sequence similarity is the region of domain IV at the C-terminus of the protein. This region contains the highest degree of divergence between EF-G1A and EF-G1B; however, the variations seen in these amino acids are moderate. In the structure of EF-G1 (EF-G1A) from P. aeruginosa this region forms an α-helix and is detached from the body of the protein [In a comparison of EF-G1A and EF-G1B, the functional regions  of domain I are strictly conserved with only one residue variation from a methionine to a tyrosine . The fiv protein .T. thermophilus [S. typhimurium [S. aureus [P. aeruginosa are shown in Fusidic acid inhibits protein synthesis by trapping EF-G in the post-translocation step during elongation ,30. Mutamophilus ,32, S. thimurium  and S. a. aureus . The posP. aeruginosa were cloned and expressed. The purification yielded both forms of EF-G in preparations that were greater than 98% homogeneous . The kinetic parameters KM and Vmax, for the GTPase activity of P. aeruginosa EF-G1B, were determined from Lineweaver-Burk analysis to be 71 μM and 3.7 μM/min, respectively (kcatobs) was calculated to be 0.2 s-1 and the specificity constant (kcat/KM) was calculated to be 3.0 x 103 s-1 M-1  of EF-G1B is 5-fold greater than that of EF-G1A.To determine the kinetic parameters governing the ribosome dependent hydrolysis of GTP by the two forms of ectively . From th s-1 M-1 . The sam s-1 M-1 . The KMTo determine the inhibitory effect of the antibiotic fusidic acid on the GTPase activity of both forms of EF-G, assays were performed containing FA at concentrations between 5 and 250 μM . The GTPP. aeruginosa protein synthesis components has been developed in our laboratory [An aminoacylation/translation (A/T) system composed of boratory  . Regions of the sequence that have been shown to be involved in hydrolysis of GTP are almost strictly conserved between the two forms. This is complemented by our data that show that the two EF-G molecules can both efficiently hydrolyze GTP in ribosome dependent reactions. If these findings are taken together, one could surmise that the two molecules are indeed different copies of EF-G performing the same role. This is supported by the fact that the many residues that have been shown to mutate in conferring resistance to FA are also almost strictly conserved between the two forms of EF-G from P. aeruginosa . In many organisms this form of EF-G is the major translocase involved in protein biosynthesis. Next, the spdEFG subfamilies are restricted to three taxonomic divisions: Spriochaetes, Planctomycetes and δ-proteobacteria and to mitochondria [B. burgdorferi [P. aeruginosa EF-G1A and EF-G1B are both members of the EFG I subfamily. The Pseudomonassp., along with the Burkholderia sp.and Bordetellasp. are shown to have a second EF-G (located outside the str operon) that is termed a recent duplication from EFG I and the genes encoding these proteins are thought to have been acquired by lateral gene transfer [P. aeruginosa, of the two highly conserved proteins has not been retained. P. aeruginosa EF-G1A is encoded by the fus gene located in the str operon and appears to function in ribosome recycling, while EF-G1B, which functions in translocation, is encoded in a secondary fus like gene  and is located in the opposite side of the genome from the str operon. Perhaps as suggested for spdEFG1 and 2 [Evolutionary studies have identified four subfamilies of EFG: EFG I, spdEFG1, spdEFG2 and EFG II . First, chondria . The twogdorferi ,15. Finatransfer . The auttransfer  indicateG1 and 2 , the dupP. aeruginosa has recently been solved by groups from Novartis [fusA gene that is located within the str operon. The natural compound argyrin B was shown to inhibit growth of P. aeruginosa and when the genome of one mutant was sequenced a single point mutation in the fusA gene (labeled as fusA1) was detected. The fusA1 gene was then sequenced from five additional fusA mutants and at least seven mutations  were detected. There were no details from this work that indicated that the gene (fusA2) encoding the second EF-G (EFG 2) was sequenced from any other argyrin B resistant mutants after the initial genome that was screened. From the work described here, we know that the protein that was labeled EF-G1 (encoded by fusA1) is the form of EF-G (EF-G1A) that is not functional in elongation but appears to be involved in ribosome recycling. Comparison of the protein primary sequence indicates that the mutated residues identified in conferring resistance to argyrin B in EF-G1 (EF-G1A) are conserved in EF-G1B. Also, the residues in EF-G1 (EF-G1A) shown in the Novartis structure that directly interact with argyrin B are also conserved in EF-G1B. Another group [fusA1 gene, at positions Ile457 and Met685. The amino acids at these two positions are also conserved in EF-G1B. This group also sequenced the fusA2 gene (encoding EF-G1B) and found no mutations in argyrin B resistant mutants. The possibility exists that this compound, like FA, may inhibit the activity of one form of EF-G yet have no effect on the other form. If this is the case, argyrin B only inhibits the form of EF-G that may be involved in ribosome recycling. This study [P. aeruginosa that was taken from clinical isolates. RNA transcripts encoding EF-G1 (EF-G1A) was observed to be more highly transcribed in clinical isolates than were transcripts encoding EF-G2 (EF-G1B). The sources of the clinical isolates were not given. This is contrary to what would be expected in actively growing cultures. In growing cultures, if EF-G1B functions exclusively in translocation and EF-G1A functions exclusively in ribosome recycling, since there are many more rounds of elongation than termination events one would expect to find more mRNA encoding EF-G1B than EF-G1A. However, if the samples were taken from respiratory passages in cystic fibrosis patients in which the bacteria form biofilms, a different set of mRNA might be expected. In biofilm formation the bacteria under goes a shift in behavior in which large suites of genes are differentially regulated [The structure of what is referred to as EF-G1 from Novartis . This prer group  carried is study  also anaegulated . In the P. aeruginosa, which contain a high level of homology, perform distinctly different functions during protein biosynthesis. From an in-depth inspection of the primary amino acid sequences no viable reason for this was determined. It would be of interest in future studies to exchange the domains of the two forms of EF-G and determine which domain is responsible for the variation of activity as well as which region is responsible for the resistance to the antibiotic FA.In this work, we have shown that the two forms of EF-G from


The p12 protein of the murine leukemia virus (MLV) is a constituent of the pre-integration complex (PIC) but its function in this complex remains unknown. We developed an imaging system to monitor MLV PIC trafficking in live cells. This allowed the visualization of PIC docking to mitotic chromosomes and its release upon exit from mitosis. Docking occurred concomitantly with nuclear envelope breakdown and was impaired for PICs of viruses with lethal p12 mutations. Insertion of a heterologous chromatin binding module into p12 of one of these mutants restored PICs attachment to the chromosomes and partially rescued virus replication. Capsid dissociated from wild type PICs in mitotic cells but remained associated with PICs harboring tethering-negative p12 mutants. Altogether, these results explain, in part, MLV restriction to dividing cells and reveal a role for p12 as a factor that tethers MLV PIC to mitotic chromosomes. Retroviruses, including the murine leukemia virus (MLV), reverse transcribe their RNA genome to a DNA copy, which travels from the cytoplasm to the nucleus as part of a ‘pre-integration complex’ (PIC), to integrate into cellular chromosomes. The viral p12 protein is a constituent of the MLV PIC, but its function in this complex has remained unknown. We developed a real-time imaging system to detect p12 and MLV PICs in live cells. This revealed that p12 tethers the MLV PIC to mitotic chromosomes. Accordingly, PICs derived from viruses with specific lethal mutations in p12 failed to attach to the chromosomes, and insertion of a heterologous chromatin binding module into p12 restored PICs attachment to the chromosomes and rescued virus replication. In addition, docking of wild type PICs to chromosomes coincided with nuclear envelope breakdown during mitosis, and detachment occurred upon exit from mitosis. Capsid - another viral component of the PIC - dissociated from wild type PICs in mitotic cells but remained associated with PICs harboring tethering-negative p12 mutants, suggesting interplay between these two proteins in regulating targeting of mitotic chromosomes by the PIC. These results highlight steps contributing to the high tropism of MLV to dividing cells. To integrate, reverse transcribed retroviral genomes are imported from the cytoplasm to the chromosomes as part of a pre-integration complex (PIC). Differences in retrovirus PIC trafficking influence their ability to infect resting and/or dividing cells. Lentiviruses, including the human immunodeficiency virus (HIV), infect both dividing and resting cells. In contrast, simple oncoretroviruses, such as murine leukemia viruses (MLV), are restricted to dividing cells This dissimilarity between HIV and MLV PIC trafficking likely stems from their different composition in vitro integration activity shown by the PIC of one of these mutants p12, a cleavage product of MLV Gag precursor, is thought to influence MLV integration. p12 acts in the budding of assembled Gags, and mutations in this domain hamper particle morphogenesis and release p12 is a component of the MLV PIC, crucial for the progression of the PIC towards integration: p12, observed as discrete puncta, associates with CA and the viral genomic DNA and this p12- genome association occurs in the cytoplasm and adjoining chromosomes, suggesting that p12 escorts the viral genomic DNA throughout early stages of infection To investigate the role of p12 in PIC trafficking we labeled the PIC with p12 fused to enhanced green fluorescent protein (GFP). An in-frame insertion of GFP to the central region of p12 was lethal to the virus (data not shown). Thus, we generated chimeric virions, composed of both wt and modified Gag molecules; the latter containing GFP fused to p12 N-terminus. In the modified Gag (MA-GFP/p12-CA-NC), the GFP sequence was inserted in-frame, downstream of the protease-cleavable matrix (MA)-p12 junction, and upstream of a short, non-cleavable linker fused to p12 . MA-GFP/+ cells were counted by fluorescence-activate cell sorting (FACS). A 1∶1 molar ratio resulted in only a minor reduction (∼15%) in infectivity compared to the wt virus (with no MA-GFP/p12-CA-NC) control , encoding for the puromycin-resistance gene and GFP was expressed together with different ratios of MA-GFP/p12-CA-NC to wt virus. Virions, normalized by reverse transcriptase (RT) activity, were used to infect NIH3T3 cells and GFP control . CountinAt 12 hr post-infection (hpi) of U/R cells  To monitor the PICs in mitotic cells, U/R/RFP-laminA cells were arrested at metaphase with 2-methoxyestradiol (2ME2) and infected with wt GFP virions. 2ME2 impairs microtubule dynamics without gross microtubule depolymerization and arrests cells at the spindle assembly checkpoint To test for the docking of immobile PICs to mitotic chromosomes, we generated U/R/RFP-H2A cells [U/R cells stably expressing RFP fused to histone H2A (RFP-H2A)], infected them with wt GFP virions and imaged unsynchronized, interphase and mitotic cells. Indeed, motionless PICs were attached to the mitotic chromosomes, while cytoplasmic PICs in interphase cells were motile , using MLV with the D184A mutation in the catalytic site of IN, which disrupts its activity To monitor the transition between the cytoplasmic movements of the PICs to their docking to mitotic chromosomes, we viewed infected cells as they entered mitosis. Confluent U/R/RFP-H2A and U/R/RFP-laminA cells were infected with wt GFP particles and 2 hr later, the cells were trypsinized and replated at a lower density. After additional 5 hr we detected infected cells that entered mitosis as judged by the growing condensation of their chromosomes (U/R/RFP-H2A), or by the progressive dissolution of the NE (U/R/RFP-laminA). Docking of the GFP-labeled PICs to the RFP-labeled chromosomes could readily be detected; the time frame between the first observed docking event and the docking of the rest of the PICs was ∼2 min . This result provides further support for the affinity of the MLV PIC towards mitotic, and not interphase, chromosomes. Of note, a minor fraction of PICs (integration-competent wt GFP or integration-incompetent D184A GFP) remained associated with the chromatin following exit from mitosis, A mutations] are lethal to the virus and dramatically reduce the levels of circular forms of the viral genomic DNA, suggesting a defect in nuclear entry of this genome Of note, chimeric PICs resulting from the expression of wt MLV genome and modified Gag containing the PM14 mutation docked to mitotic chromosomes (not shown), demonstrating that the docking activity of wt p12 is dominant over the lack of such activity of PM14 GFP-p12, and providing genetic evidence for the ability of GFP-p12 to mark the incoming MLV PIC.Viruses carrying the SA mutations are phenotypically undistinguishable from the PM14 virus. However, a replication-competent revertant with an additional compensatory mutation (M63I) in p12 exists for this virus To evaluate if addition of a foreign chromatin-binding element rescues the docking of mutant p12 PICs, we inserted such a module of the herpes LANA protein (LANA31), into p12 of the PM14 clone, generating the PM14/LANA31 virus. LANA31 consists of 31 residues, 23 of which bind the groove between histones 2A and 2B Further support for the rescue of PM14 infectivity by LANA31 came from single-cycle infection assays, in which NIH3T3 cells were infected with wt, wt/LANA31, PM14 or PM14/LANA31 virus-like particles (VLPs), harboring puromycin or neomycin -resistance markers, and normalized by RT activity . QuantifWe also tested how the exit from mitosis affects LANA31-mediated docking of the PICs to the chromosomes. In unsynchronized U/R cells co-infected with wt/LANA31 GFP and wt mCherry, and stained with Hoechst; wt mCherry PICs regained their movement upon exit from mitosis, while wt/LANA31 GFP PICs remained motionless A p12 mutants, which sharply contrasted to the stable docking of PICs from wt and the SA/M63I revertant viruses. This was best visualized upon the co-infection of PM14 and wt viruses, which showed lack of docking, or stable chromosomal association, respectively, in the same cell. Moreover, insertion of a heterologous chromatin-binding module (LANA31) into p12 of the PM14 virus rescued both its infectivity and the anchorage of its PIC to the chromosomes, strongly implying a chromosome-tethering function for p12. The discrepancy between the full restoration of docking to the chromosomes of PM14/LANA31 PICs and the partial restoration of infectivity of this virus likely results from the adverse effect caused by the addition of 31 residues into p12 on other stages of the replication cycle; for example, we observed a reduction in assembly of the wt/LANA31virus (data not shown). A similar-sized insertion (a triple Myc epitope) in the same location also attenuated replication LEDGF/p75 was identified as the factor that tethers the HIV IN to chromatin: it interacts with unknown chromatin ligands and with IN, and is essential for the chromosomal targeting of HIV IN What kind of interactions may be involved in the p12-mediated tethering activity? The specific recognition of mitotic chromosomes by p12-labled PICs, and their detachment upon exit from mitosis suggest that the MLV PIC recognizes chromatin features, such as post-translational modifications that are associated with cell cycle progression. For example, phosphorylation of histone H3 at Thr3 (H3T3) and methylation of the adjacent Lys4 (H3K4), serve as a motif for the binding of cellular factors, such as TFIID, to mitotic chromosomes MLV integration peaks in cells after exit from metaphase and decondensation of chromosomes Using immunofluorescence analysis, we demonstrated here that CA, a known component of the PIC In summary, we identified p12 - a PIC component essential for integration - as a factor that tethers the MLV PICs to mitotic chromosomes. MLV-based vectors have been used successfully in gene therapy trials in humans, yet with the risk of leukemogenesis Moloney MLV clones wt (pNCS), PM14, 1xMycR, SA, SA/M63I, and pQCXIP-gfp-C1 vector were described before Culture conditions and serum starvation/aphidicolin treatment were described before + cells. For live-cell imaging, chimeric particles were generated by co-transfecting 293T cells with a 1∶1 ratio of the indicated virus and the modified Gag as described above. Infections were carried out as described above with the following modifications: ∼8 hr before infection, the cells were plated (∼10% cofluency) in a 4-compartments-cell-view-glass-bottom-dish  and were infected with MOI of approximately 10 (based on the comparison of the RT activity of the samples to a standard MLV stock). For imaging, HEPES pH 7.0 (20 mM) was added to growth medium. Imaged samples were maintained at 37°C and supplied with CO2 when imaging exceeded 2 hr. In some experiments, chromosomes were stained with Hoechst 33342 . Imaging was with spinning disk confocal  microscope , 100× lens  and Evolve or HQ2 (Photometrics) cameras.Generation of, and infection with, the 1xMycR virus were described before Single-cycle infection assays were carried out with the pQCXIP-GFP-C1 or pQCXIN (Clontech) vectors. For VLPs carrying the wt p12 sequence the following plasmids were co-transfected into 293T cells: vector plasmid (10 µg), VSV-G expression plasmid (2.5 µg), pGag-PolGpt helper plasmid For Western blotting, virions were purified through sucrose cushions using ultracentrifugation For the calculation of the spatial retention of PICs over time, time-lapse sequences were processed . Objects were identified through intensity-based segmentation, with no further selection for specific PICs. For each movie, the second, third or fourth frame were superimposed on the first frame; the areas of overlapping PICs identified, and the signal intensity in overlapping areas was presented as the percentage of total intensity of objects in the first frame.Figure S1Co-localization of CA and GFP-p12 in interphase and mitotic cells. wt GFP-infected U/R cells were immunostained with anti-CA antibodies (red), 12 hpi. Chromosomes were stained with DAPI (blue). Interphase (A) and mitotic (B) cells are shown. Bars represent 10 µm.(TIF)Click here for additional data file.Figure S2Close association between mitotic chromosomes and wt GFP, but not PM14 GFP, derived PICs. U/R/RFP-H2A cells were infected with either wt GFP (A) or PM14 GFP (B), fixed and imaged. Serial optical sections of mitotic chromosomes were reconstituted into 3D images, with a 10 µm grid. Chromosomes are in red; free and chromosome-imbedded PICs are in dark and light green, respectively.(TIF)Click here for additional data file.Movie S1PICs cytoplasmic movement in an interphase cell. Time-lapse microscopy analysis of PICs in aphidicolin-treated, wt GFP-infected, U/R/RFP-laminA cell. Arrowheads mark PICs with directed cytoplasmic movement. Rectangle surrounds a group of extracellular virions attached to the glass. Cells were visualized 18 hpi. The decrease in the red and green signals, shown in this movie and in the following movies, is due to fluorescence bleaching over time (indicated in seconds). Frequency: 1 frame per second (fps).(MOV)Click here for additional data file.Movie S2Cytoplasmic movement versus docking to mitotic chromosomes of MLV PICs. 2ME2-treated, wt GFP-infected, mitotic (A) and interphase (B) U/R/RFP-laminA cells were visualized by time-lapse microscopy 12 hpi. The same procedure was applied to mitotic and interphase  unsynchronized U/R/RFP-H2A cells; unsynchronized NIH3T3/RFP-H2A cells (E); unsynchronized, D184A GFP-infected U/R/RFP-H2A cells (G); and nocodazole-treated, wt GFP-infected, mitotic U/R/RFP-H2A cells (F). Frequency: 1, 1.2, 0.5, 0.4, 1.3, 0.9 and 1.1 fps for A to G, respectively.(MOV)Click here for additional data file.Movie S3Transition from cytoplasmic movement to docking to chromosomes of MLV PICs in cells entering mitosis. wt GFP-infected, U/R/RFP-H2A (A) and U/R/RFP-laminA (B) cells, were visualized by time-lapse microscopy 5 hpi as they entered mitosis. Arrowheads mark PICs docked to the chromosomes. Empty triangles mark gaps in the disassembled NE. Frequency: 0.2 fps for A and B.(MOV)Click here for additional data file.Movie S4Transition from docking to chromosomes to intra-nuclear movement of MLV PICs in cells exiting mitosis. Mitotic, 2ME2-arrested, wt GFP-infected U/R/RFP-H2A cells were visualized by time-lapse microscopy before (A) and after (B) the addition of Reversine. The same procedure was applied for infected, unsynchronized U/R/RFP-H2A cells before (C) and after (D) natural exit from mitosis; and for unsynchronized U/R/RFP-laminA cells, before and after exit from mitosis . The U/R/RFP-H2A cells that exit mitosis and that were infected with D184A GFP (G) are the same cells shown during mitosis in part F of Movie S2. Frequency: 1.8, 1.8, 0.9, 0.9, 1.1, 1.1 and 1.1 fps for A to G, respectively.(MOV)Click here for additional data file.Movie S5PICs with mutant p12 proteins fail to dock to mitotic chromosomes. Interphase (A) and mitotic (B) cells of unsynchronized, PM14 GFP-infected U/R/RFP-H2A culture were visualized by time-lapse microscopy 12 hpi. The same procedure was applied to visualize mitotic cells of unsynchronized, PM14-infected NIH3T3/RFP-H2A culture (C); 2ME2-arrested U/R cells, co-infected with wt mCherry and PM14 GFP (D); and unsynchronized U/R/RFP-H2A cells infected with SA GFP (E) or with SA/M63I GFP (F). Broken lined circle marks chromosomal region with moving PM14 PICs with no apparent docking (B). Arrowheads point to representative wt mCherry PICs with stable chromosomal docking (D), or to an SA GFP PIC showing only transient, unstable association with the chromosomes (E). Frequency: 0.4, 0.4, 1.2, 0.8, 1.2 and 1.2 fps for A to F, respectively. Scale bar, 10 µm.(MOV)Click here for additional data file.Movie S6Docking to the chromosomes of PICs with p12 proteins containing the LANA31 peptide. Unsynchronized U/R/RFP-H2A cells were infected with PM14/LANA31 GFP (A) or wt/LANA31 GFP (B) and mitotic cells were visualized by time-lapse microscopy 12 hpi. Unsynchronized U/R cells were co-infected with wt mCherry together with wt/LANA31 GFP and cells that exit mitosis (C) were visualized as above. Frequency: 1.2, 1.2 and 0.8 fps for A to C, respectively. Scale bar, 10 µm.(MOV)Click here for additional data file.Text S1Supplementary methods for plasmids construction and generation of cell lines with labeled NE and chromosomes.(DOC)Click here for additional data file.