Escherichia coli strain in which RecBCD has been genetically replaced by the bacteriophage λ Red system engages in efficient recombination between its chromosome and linear double-stranded DNA species sharing sequences with the chromosome. Previous studies of this experimental system have focused on a gene replacement-type event, in which a 3.5 kbp dsDNA consisting of the cat gene and flanking lac operon sequences recombines with the E. coli chromosome to generate a chloramphenicol-resistant Lac- recombinant. The dsDNA was delivered into the cell as part of the chromosome of a non-replicating λ vector, from which it was released by the action of a restriction endonuclease in the infected cell. This study characterizes the genetic requirements and outcomes of a variety of additional Red-promoted homologous recombination events producing Lac+ recombinants.An cat was not frequently co-inherited with cat. (5) Recombination between phage sequences in the linear DNA and cryptic prophages in the chromosome was responsible for most of the observed Lac+ recombinants. In addition, observations were made concerning recombination events between the chromosome and circular DNAs: (6) Formation of recombinants depended upon both RecA and, to a lesser extent, Red. (7) The linked tetracycline-resistance marker was frequently co-inherited in this case.A number of observations concerning recombination events between the chromosome and linear DNAs were made: (1) Formation of Lac+ and Lac- recombinants depended upon the same recombination functions. (2) High multiplicity and high chromosome copy number favored Lac+ recombinant formation. (3) The Lac+ recombinants were unstable, segregating Lac- progeny. (4) A tetracycline-resistance marker in a site of the phage chromosome distant from lac and cryptic prophage sequences in the chromosome generates a partial duplication of the bacterial chromosome. When the incoming DNA species is circular rather than linear, cointegrates are the most frequent type of recombinant.The Lac+ recombinants arise from events in which homologous recombination between the incoming linear DNA and both  Escherichia coli strain in which RecBCD has been genetically replaced by Red exhibits greatly elevated levels of recombination between its chromosome and short linear double-stranded DNA species sharing sequences with the chromosome [The Red recombination system of bacteriophage λ promotes efficient double strand break repair/recombination. An romosome .cat gene and flanking lac operon sequences recombines with the E. coli chromosome to generate a chloramphenicol-resistant Lac- recombinant [red and the bacterial recombination genes recA, recF, recO, recR, recQ, ruvAB, and ruvC. Large numbers of chloramphenicol-resistant Lac+ recombinants were generated in these crosses as well. In this study, we characterize the Lac+ recombinants and the processes which generate them. Most appear to arise from events in which homologous recombination between the incoming DNA and the chromosome generates a partial duplication of the bacterial chromosome.In previous studies, we have characterized a recombination event, pictured in Figure ombinant -4. The drecA, recF, recO, recR, recQ, ruvAB, and ruvC. Deletion of recG increased the production of both Lac+ and Lac- recombinants. Lac+ recombinants accounted for 10–60% of the total chloramphenicol-resistant offspring of the crosses. These results strongly suggest that formation of the Lac+ recombinants takes place via homologous recombination, though they do not rule out the possibility that non-homologous end joining, or other varieties of "illegitimate" recombination, might be involved as well.Data presented in Table lac::cat sequences would be attached to phage sequences. Heitman et al. [in vivo are rapidly repaired by DNA ligase. The results of previous physical studies with other substituted λ phages cut in vivo by PaeR7 lead to the expectation that the λ lac::cat819 chromosome would be uncut, or only singly cut, much of the time in the infected cell [The reason for considering mechanisms other than homologous recombination in the formation of the Lac+ recombinants is because of the expectation that some of the phage-borne n et al.  showed tted cell ,6.lacZ (not shown). When the lac::cat dsDNA was delivered into the cell by a λ vector bearing a tetracycline resistance determinant (Δ nin::tet859 – described below) neither the Lac+ nor Lac- recombinants acquired tetracycline resistance. These findings suggest that the process which formed the recombinants did not involve the entire phage chromosome.Preliminary characterization of the Lac+ recombinants formed in cells which were wild-type for all the recombination functions revealed that most, possibly all, were unstable. When streaked on plates containing chloramphenicol and X-Gal, they segregated Lac- (colorless) progeny at variable frequencies. Southern gel analysis of chromosomal DNA from unstable Lac+ recombinants revealed that some of them were multiploid for lac locus, will produce a Lac+, chloramphenicol-resistant recombinant. Data presented below, in which simpler lac::cat recombining substrates generate Lac+ recombinants at a frequency approximately 1000-fold lower than Lac-, suggest the frequency of spontaneous lac duplications in Red+ but otherwise wild type E. coli is approximately 10-3, consistent with estimates of spontaneous duplication frequency in Salmonella made by Roth et al. [A baseline level of Lac+ recombinants was to be expected in these crosses. The recombination event pictured in Figure h et al. . The Laclac::cat segment were to recombine with more than one chromosome at a time. Further complicating interpretation of the experiment, the cells were infected at a multiplicity of 10 phages per cell.The crosses described above were done by infecting log phase host cells grown in rich medium. Such cells contain multiple copies of their chromosome. Complications might arise if the lac::cat segment and single copies of the bacterial chromosome. As shown in Table To reduce the complexity of the system, we switched to a cross procedure involving low multiplicity infection (0.1 phage per cell) of stationary phase cells. Presumably, under these conditions, most of the events producing chloramphenicol-resistant recombinants take place between single copies of the lac::cat, which are diagrammed in Figure lac::cat819, the progenitor of this series, has two PaeR7 sites. Upon infecting a host cell bearing the Δ recBCD::Ptac-gam-bet-exo-pae-cI substitution, it injects its chromosome, which circularizes. Expression of its lytic genes, including those necessary for phage DNA replication, is blocked by the action of cI repressor. Cutting of its chromosome by the PaeR7 restriction endonuclease in the infected cell releases a 3.5 kbp linear dsDNA, consisting of the cat gene and 1.3 kbp flanks of lac sequences on the right and left. . The ends of the flanking lac sequences match precisely their counterparts in the chromosome.To examine the dependence of Lac- and Lac+ recombinant formation on the structure of the DNA substrate, we constructed variants of λ lac::cat930 and λ lac::cat931 have only single PaeR7 sites, on the right and left, respectively. Cutting by PaeR7 in the infected cell should produce large linear dsDNAs related to the lac::cat819 fragment, but with long tails of non-homologous DNA extending from the left and right sides, respectively. Interestingly, both of these single-cut phages produce recombinants more efficiently than the double-cut λ lac::cat819, in both recG+ and Δ recG backgrounds  Some of the λ lac::cat929 chromosomes might be cut by the EcoK restriction endonuclease. The infected cells contained a functional EcoK restriction-modification system. λ lac::cat929 was grown in a similarly EcoK+ host, and therefore was presumably EcoK-modified. However, if the modification were not complete, there might be residual EcoK restriction activity, resulting in 10% of the phage chromosomes being cut. . (2) The production of the recombinants might not be Red-mediated. (3) The linear partner might be a broken bacterial chromosome.Approximately half of the strongly chloramphenicol-resistant recombinants generated by the uncuttable phage λ  partner . How, thhsdR, with a tetracycline resistance determinant. This replacement had little effect on the formation of recombinants by λ lac::cat929 . A fourth event, also not shown, involves recombination between λ genes and homologues in cryptic prophages in the E. coli chromosome (discussed below). As suggested in the figure, cointegrates formed by recombination in the right-side lac flank are phenotypically Lac-, while recombination in the left-side lac flank (or other loci) forms Lac+ recombinants. To test this idea, we constructed variants of λ lac::cat missing either or both lac flanks; they are diagrammed in Figure Cointegrates could in principle be formed by four different single reciprocal recombination events between the circular λ cat cassette with 1000 bp homologous flanks was only 10-fold more efficient than one with 40 bp flanks. Their experiments were done with cells which had been subjected to heat shock and electroporation. To test the efficiency of short-flank recombination under less extreme conditions, we constructed a short-flank λ lac::cat variant.The ability of the λ Red system to promote recombination events involving short sequence homologies makes it particularly useful for genetic engineering. Yu et al.  reportedlac::cat921 consists of the cat gene flanked by 40 bp sequences corresponding exactly to the terminal 40 bp at each end of the 1.3 kbp flanks of λ lac::cat819. As shown in Table lac::cat921 exhibits a several hundred-fold lower efficiency of recombinant formation than its long-flank counterpart, in both recG+ and Δ recG backgrounds. λ lac::cat921 was similarly inefficient in log phase cells (data not shown). These observations suggest that short-flank recombination may not be a significant activity of the Red system in nature. However, they do not speak to the question of whether long flanks work better because they provide a larger homology target for synapsis, or because they provide non sequence-specific protection, perhaps delaying exonucleolytic degradation of the recombining sequences long enough to permit recombination to take place.The fragment released by PaeR7 from the chromosome of λ lac::cat930, but not by the left side-cut λ lac::cat931  cloning the cat gene from the deletion mutant, along with flanking chromosomal sequences; (3) using the cloned, plasmid-borne flanking sequences, and Red-mediated gap repair, to clone the lac operon in a plasmid; (4) inverting the lac operon, relative to its flanking sequences, in the plasmid; (5) replacing the Δ lac::cat chromosomal allele with the plasmid-borne lac-inv allele.The strategy for inverting the chromosomal lac::cat930 and lac::cat931 alleles were constructed by replacing the left-side and right-side PaeR7 sites, respectively, of pTP819, with XbaI sites. To produce inverted derivatives of these two alleles, the plasmids pTP930 and pTP931 were digested with XbaI and XhoI (a PaeR7 isoschizomer); lac::cat inserts and bla-ori backbone fragments from the two plasmids were exchanged. The inverted alleles were then crossed into phage λ, as described in the Methods section.The single-cut E. coli K-12 [lac::cat930 and λ lac::cat931, and the efficiency of Lac+ recombinant formation by λ lac::cat930, were slightly elevated relative to those in the corresponding AB1157-derived strain.In constructing the chromosomal inversion, we switched genetic backgrounds, from the AB1157-derived strains with which most recombination studies have been done, to MG1655, the sequenced wild type oli K-12 . In the lac-inverted E. coli) were tested for Lac+ recombinant formation. The results are shown in Figure lac::cat930  in lac-wild type, and lac::cat1033  in lac-inv.The eight combinations of normal and inverted phages and bacteria  lacking its sequences derived from phage P22 was equally proficient at generating Lac+ recombinants. A derivative missing the left lac flank made Lac+ recombinants almost exclusively; the small number of Lac- recombinants in this case probably represent cointegrates made by uncut plasmid DNA in the fragment preparation.We constructed several other linear DNA substrates to test the generality of the model in Figure ion  and λ lac::cat930 (cut right) behave like their counterparts generated by electroporation of linear dsDNAs into Red+ cells: they segregate Lac- chloramphenicol-resistant clones at low frequency, and Lac+ chloramphenicol-sensitives at high frequency (data not shown). In addition, their formation also depends upon the presence of cryptic prophages in the chromosome: λ lac::cat819 was found to produce Lac+ chloramphenicol-resistant recombinants as only 0.13% (average of six measurements) of the total chloramphenicol-resistant progeny in crosses with TP750. These properties suggested both kinds of recombinant might have the same structures as well, but PCR tests of 50 of the phage-generated recombinants with the primers used to demonstrate type I, II, and III recombinants  when electroporated directly into cells .One aspect of Lac+ recombinant formation by λ ls Table . How doelac::cat-bearing λ phages and the bacterial chromosome, as well as the mechanism of their formation. Our studies uncovered a diversity of recombinant structures, including complex duplications and cointegrates. Recombination between λ and cryptic prophage sequences in the bacterial chromosome was found to be the most significant mechanism generating Lac+ recombinants. All of the Lac+ recombinants could be generated by homologous recombination events of types which have been previously described. In particular, there was no evidence for end-joining or other non-homologous recombination events. Perhaps the most surprising finding was the high frequency with which large duplications in the bacterial chromosome – up to 1 Mbp – could be generated by the Red system.The bacteriophage λ Red recombination system is of general interest for two main reasons. First, it is an intensively characterized and relatively simple system, which serves as a model for studies of homologous recombination -17. SecoE. coli strain DH5α (λ pir) [hsdR::tet allele in TP842 was constructed by using a Tn 10-containing E. coli strain as template in PCR with primers hsdRut (5'-TTGGACAGGCCCGCACAGCAATGGATTAATAACAATGATGCTCGACATCTTGGTTACCGT-3') and hsdRdt (5'-GCTGAATTTGCCCAGCAGGGTATCGAGATTATCGTCAAAGCGCGGAATAACATCATTTGG-3'). The Δ lac::cat allele in TP872 was constructed by using a Tn 9-containing E. coli strain as template in PCR with primers cat15 (5'-TCTGGTGGCCGGAAGGCGAAGCGGCATGCATTTACGTTGAATGAGACGTTGATCGGCACG-3') and cat16 (5'-AGAGTACATCTCGCCGTTTTTTCTCAATTCATGGTGTACAATTCAGGCGTAGCACCAGGC-3'). (λ pir)  was usedcI857 Sam7 containing the nin region genes was cloned into the EcoR1 site of pBR322. In the resulting plasmid, pnin, the nin genes are read clockwise in the conventional map of pBR322. pTP772 was constructed by deleting sequences between the two HindIII sites of pnin. pTP859 was constructed by cutting pTP772 with SacII and ClaI, blunting the ends with T4 DNA polymerase, ligating with NotI linkers (5'-AGCGGCCGCT-3'), cutting with NotI, and ligating with a NotI fragment of pTP857 [tetR and tetA genes of transposon Tn10.An EcoR1 fragment of λ f pTP857  containicat gene is flanked on both sides by lac sequences, PaeR7 sites, and λ sequences (for crossing into the phage), has been described [lac flank and PaeR7 site. pTP922 was constructed by deleting the lac and cat sequences between the two PaeR7 sites in pTP819. . pTP926, pTP927, and pTP928 were constructed by ligating the oligonucleotide 5'-TCGACAGTCTAGACTG-3' into the PaeR7 sites of pTP828, pTP829, and pTP922, respectively, eliminating the PaeR7 sites and replacing them with XbaI sites. pTP929 was constructed by ligating the XbaI-NcoI fragment of pTP926 containing the N-terminal coding sequences of cat and the NcoI-XbaI fragment of pTP927 containing the C-terminal coding sequence of cat into the XbaI site of pTP928. The orientation of the reconstructed cat gene is the same as in pTP819. pTP930 was constructed by ligating together the large ApaI-SacII fragment of pTP929 and the small ApaI-SacII fragment of pTP819. pTP931 was constructed by ligating together the small ApaI-SacII fragment of pTP929 and the large ApaI-SacII fragment of pTP819.pTP819, in which the escribed . pTP828 cat gene from a Tn9-containing E. coli strain with primers 5'-GACGCACTCGAGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGCCGGCCACTGGAGCACCTCAAAAACACCA-3' and 5'-GACGCACTCGAGGCACACAGCGCCCAGCCAACACAGCCAAACATCCGCGCGGGCCCGACCGGGTCGAATTTGCTTTCGAA-3'. The presence of the expected sequences from the synthetic oligonucleotides in pTP921 DNA was verified by automated sequencing (data not shown).pTP921 was constructed by ligating together two XhoI-digested DNAs: pTP922 and a PCR product made by amplifying the lac flank only) was constructed by replacing the XbaI-ApaI lacZY segment of pTP930 with a mixture of two oligonucleotides, 5'-CTAGTTGCAAGCTTGGGCC-3' and 5'-CAAGCTTGCAA-3'. pTP989 (right lac flank only) was constructed by replacing the NgoMI-XhoI lacZ segment of pTP930 with a mixture of two oligonucleotides, 5'-CCGGCAAGCTTGCTGGTGGGCAA-3' and 5'-TCGATTGCCACCAGCAAGCTTG-3'. pTP995 (no lac flank) was constructed by ligating together the small NcoI fragment of pTP988 and the large, ori-containing NcoI fragment of pTP989.pTP988 (left cat-containing fragment of pTP930 and the XbaI- and PaeR7-ended ori-containing fragment of pTP931. pTP1033 was constructed by ligating together the XbaI- and PaeR7-ended cat-containing fragment of pTP931 and the XbaI- and PaeR7-ended ori-containing fragment of pTP930.pTP1032 was constructed by ligating together the XbaI- and PaeR7-ended cat gene and flanking sequences from strain TP872 (Δ lac::cat) with primers 5'-CATCATCACGCGGCCGCGACGTTTGCCGCTTCTGAA-3' and 5'-ATCATCCACGCGGCCGCTGCGTTTTGCACCAGTACG-3'. In pTP1016, the cat gene is flanked closely by unique SphI and BsrGI sites. pTP1027 was constructed by electroporating SphI- and BsrGI-digested pTP1016 into strain TP829 (lac+); a plasmid formed by gap repair was isolated. pTP1028 was constructed by ligating SphI-digested pTP1027 with the oligonucleotide 5'-GTTGTACAACCATG-3', converting the SphI site into a BsrGI site. pTP1034 was constructed by cutting pTP1028 with BsrGI, ligating the two fragments back together, and screening for plasmids in which the lac genes were inverted relative to their flanking sequences.pTP1016 was constructed by ligating into the NotI site of pTP809  the NotIpir function, was constructed by ligating together two AatII- and Bam-digested DNA species: a PCR product made by amplifying a Tn10-containing E. coli strain with primers 5'-TCAACGTAAATGCATGGACGTCCTCGACATCTTGGTTACCGT-3' and 5'-TGTACACCATGAATTGGATCCCGCGGAATAACATCATTTGG-3'; and the ori-containing fragment of plasmid pLD54 [lac::cat-containing fragments of pTP819, 930, 931, 1032, and 1033, respectively, into the BamHI site of pTP1029.pTP1029, a tetracycline resistance bearing vector capable of replicating only in cells expressing R6K id pLD54 . pTP1018cat-containing fragment of pTP1051 into the BamHI site of pTP1029.pTP1047 was constructed by ligating the complementary oligonucleotides 5'-TCGTCTAGAGT-3' and 5'-CCGGACTCTAGACGAAGCT-3' between the SacI and NgoMIV sites of pTP1019. pTP1051 was constructed by ligating the complementary oligonucleotides 5'-CAGCATGCAT-3' and 5'-CTAGATGCATGCTGGTAC-3' between the KpnI and XbaI sites of pTP930. pTP1052 was constructed by ligating the complementary oligonucleotides 5'-CAGCATGCAGGGCC-3' and 5'-CTGCATGCTGGTAC-3' between the KpnI and ApaI sites of pTP1019. pTP1053 was constructed by ligating the complementary oligonucleotides 5'-GGCATGCAGGTTCTTTGAGTCCTTTGGGCGGCCGCGGGCC-3' and 5'-CGCGGCCGCCCAAAGGACTCAAAGAACCTGCATGCCGC-3' between the SacII and ApaI sites of pTP1019. pTP1054 was constructed by ligating the complementary oligonucleotides 5'-CCGGCGCGGCCGCAGGTTCTTTGAGTCCTTTGGTGTACAGAGCT-3' and 5'-CTGTACACCAAAGGACTCAAAGAACCTGCGGCCGCG-3' between the NgoMIV and SacI sites of pTP1020. pTP1055 was constructed by ligating the small SphI-NotI fragment of pTP1016 between the SphI and NotI sites of pTP1053. pTP1056 was constructed by ligating the small NotI-BsrGI fragment of pTP1016 between the NotI and BsrGI sites of pTP1054. pTP1057 was constructed by ligating the BamHI Ptac-gam-bet-exo-pae-cI operon flanked by sequences upstream from recC on one side and sequences internal to recD on the other, has been described [cI gene from E. coli strain TP507 with the same primers used in the construction of pTPP822. This construction has the effect of simply deleting the pae genes from pTP822. pTP996 was constructed by deleting the C-terminal coding sequences of bet and the N-terminal coding sequences of exo between the two HpaI sites of pTP978. pTP979 was constructed by ligating together two NcoI- and XbaI-digested DNAs: pTP822 and a PCR product made by amplifying the bla gene from pBR322 with primers 5'-CCACCAATCATCCATGGCGCGGAACCCCTATTTGTTT-3' and 5'-TTGTTGGACGATCTAGAGGTCTGACAGTTACCAATGC-3'. This construction has the effect of replacing pae and cI with bla.pTP822, which bears a synthetic lac::cat819 and λ lac::cat819 nin5 have been described [nin::tet859 was made by crossing λ wild type with plasmid pTP859, infecting a tetracycline-sensitive strain with the resulting lysate, and selecting a tetracycline-resistant lysogen. The Δ nin::tet859 substitution replaces λ bp 40388–43825 with the tetR and tetA genes of transposon Tn 10.λ escribed . λΔ nin:lac::cat921, 929, 930, 931, 988, 989, 995, 1032, and 1033 substitutions were crossed into λ wild type and/or λ Δ nin::tet859. The parent phages were crossed with the cat substitution-bearing plasmids. Phages which had acquired the substitution were either selected by plating on a strain lysogenic for phage P2 (Spi- phenotype), or identified by their clear-plaque morphologies . The structures of the substituted phages were all verified by their ability to generate specific products when used as templates in PCR (not shown).The 4), and shaken with chloroform. Two methods for crosses monitoring recombination between phage-injected DNA and the bacterial chromosome, both previously described, were employed: high-multiplicity infection of log phase cultures [Phages were crossed with plasmids by spotting enough phage to make a confluent zone of lysis on a lawn of a sensitive bacterial strain bearing the parent plasmid. After overnight incubation at 37°C, material from the zone was collected in TM  dimethylsulfoxide, 20 mM Tris-HCl pH 8.4, 50 mM KCl, and primers at 0.7 μM. Samples were heated to 95C for 5 min, then put through 30 cycles of 94C for 1 min, 55C for 1 min, 72C for 2–4 min, depending upon the length of the expected products. Primers for demonstrating the type I, II, and III tfa junctions were combined in a single mixture of 4 oligonucleotides: Jsp1 (GTTGAATGGGCGGATGCTAA), Jsp2 (TCTTCCACCAGAAAGCTACC), JncA (TGCCGTGTGAACGGTTTAC), and Jnc2 (TTCTAGCCCCATCATCTGTG). Primers for spanning the ACF-generated duplication  and Csj2 (CGAATAGTCGGCTCAACGTGGGTT).The structures of various Red-generated duplications in the PCR: polymerase chain reaction. X-Gal: 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. IPTG: isopropyl β-D-thiogalactopyranoside. bp: base pairs.ACF carried out the experiments summarized in Table


Feline leukemia virus (FeLV) induces degenerative, proliferative and malignant hematologic disorders in its natural host, the domestic cat. FeLV-945 is a viral variant identified as predominant in a cohort of naturally infected animals. FeLV-945 contains a unique sequence motif in the long terminal repeat (LTR) comprised of a single copy of transcriptional enhancer followed by a 21-bp sequence triplicated in tandem. The LTR is precisely conserved among independent cases of multicentric lymphoma, myeloproliferative disease and anemia in animals from the cohort. The 21-bp triplication was previously shown to act as a transcriptional enhancer preferentially in hematopoietic cells and to confer a replicative advantage. The objective of the present study was to examine the molecular mechanism by which the 21-bp triplication exerts its influence and the selective advantage responsible for its precise conservation.Potential binding sites for the transcription factor, c-Myb, were identified across the repeat junctions of the 21-bp triplication. Such sites would not occur in the absence of the repeat; thus, a requirement for c-Myb binding to the repeat junctions of the triplication would exert a selective pressure to conserve its sequence precisely. Electrophoretic mobility shift assays demonstrated specific binding of c-Myb to the 21-bp triplication. Reporter gene assays showed that the triplication-containing LTR is responsive to c-Myb, and that responsiveness requires the presence of both c-Myb binding sites. Results further indicated that c-Myb in complex with the 21-bp triplication recruits the transcriptional co-activator, CBP, a regulator of normal hematopoiesis. FeLV-945 replication was shown to be positively regulated by CBP in a manner dependent on the presence of the 21-bp triplication.Binding sites for c-Myb across the repeat junctions of the 21-bp triplication may account for its precise conservation in the FeLV-945 LTR. c-Myb binding and CBP recruitment to the LTR positively regulated virus production, and thus may be responsible for the replicative advantage conferred by the 21-bp triplication. Considering that CBP is present in hematopoietic cells in limiting amounts, we hypothesize that FeLV-945 replication in bone marrow may influence CBP availability and thereby alter the regulation of CBP-responsive genes, thus contributing to altered hematopoiesis and consequent hematologic disease. The variable clinical outcome of FeLV infection is thought to reflect this genetic diversity .Feline leukemia virus (FeLV) is a simple gammaretrovirus that induces degenerative, proliferative and malignant hematologic disorders in its natural host, the domestic cat. Like other natural retroviruses, FeLV is not a single genomic species but is a genetically complex family of closely related viruses subject to selective pressures in the host. Variant genomes are generated during virus replication as a result of both error-prone reverse transcription and recombination. The consequence of this variation is a diverse population that is continuously shaped iversity ,2. FeLV-iversity ,4, ChandThe 21-bp triplication was previously shown to provide transcriptional enhancer function to the LTR that contains it, and to function preferentially in primitive hematopoietic cells . In K-56Regarding the molecular mechanism by which the 21-bp triplication functions in the context of the LTR, at least two possibilities have been considered. One possibility is that the 21-bp triplication functions to maintain the appropriate spacing in the LTR between the enhancer and the promoter. A spacer function might be particularly relevant in an LTR like FeLV-945 in which the enhancer is not tandemly repeated. Substitution of the 21-bp repeat element with unrelated sequence of the same length, however, was observed to ablate the replicative advantage, thus indicating that the 21-bp triplication does not perform solely a spacer function . An alteAs described above, previous studies suggested that preserving the protein binding sites may confer a selective advantage that accounts for the precise sequence conservation of the 21-bp triplication in the FeLV-945 LTR . In the To evaluate whether c-Myb binding to the 21-bp triplication regulates LTR function, reporter plasmids were constructed in which expression of the firefly luciferase gene was driven by the U3 region of an FeLV LTR containing one, two or three copies of the 21-bp element. Reporter gene constructs were introduced by lipid-mediated transfection into feline embryonic fibroblasts (FEA) along with increasing amounts of a c-Myb expression vector. Fibroblasts were selected because the level of endogenous c-Myb expression in those cells is low or absent ,15. The Previous studies had shown that the 21-bp triplication contributes enhancer function to the LTR in a cell type-specific manner, and that it is significantly more active in K-562 cells as compared to a fibroblast line ,15. WhenSupershift EMSA was used to evaluate whether c-Myb and CBP might be present in the specific complex that forms on the 21-bp triplication. Specific complex formation was observed using a radiolabeled probe representing the 21-bp triplication in the presence of nuclear extracts from either K-562 cells or feline 3201 T-cells. In both cases, mobility of the complex was reduced (supershifted) when reacted with antibody to c-Myb. When the same reaction was performed in the presence of an antibody to CBP, an identical supershift was observed Figure . No compet al., manuscript submitted]. The 21-bp triplication was shown to enhance transcription from the FeLV LTR and to confer a replicative advantage to the virus, at least in part through the specific binding of unidentified nuclear proteins to the repeat motif , it is likely that the tropism of FeLV-945 in vivo included the hematopoietic progenitors in which c-Myb is expressed. Considering this possibility, we hypothesized that c-Myb may act as a transcriptional regulator of FeLV-945. In support of this hypothesis, reporter gene assays showed that an LTR containing the triplication was responsive to c-Myb in a dose-dependent manner  and was maintained in RPMI 1640 medium with 10% FBS. The FEA cell line, a continuous line of feline embryonic fibroblasts, was obtained from Dr. Jennifer Rojko and was grown in Eagle minimal essential culture medium supplemented with 10% fetal bovine serum (FBS), 0.1 mM non essential amino acids and 50 μg/ml gentamicin reagent solution . 3201 is an FeLV-negative thymic lymphoma cell line of feline origin [me sites . The FeLme sites .TAACGGTTCCGTAGTGA) or a CREB consensus binding site (5'-AGAGATTGCCTGACGTCAGAGAGCTAG). Protein-DNA complexes were resolved by 6% polyacrylamide gel electrophoresis in 0.25X TBE buffer . Gels were then dried at 80°C and exposed to radiographic film for varying periods of time. In some reactions, monoclonal antibody (4 μg) to either c-Myb or CBP, or isotype control antibody, was added after the 30-minute incubation period and incubated overnight at 4°C. Complexes were then resolved by 6% polyacrylamide gel electrophoresis as described above. The mouse monoclonal IgG1 antibody to c-Myb was raised against a recombinant protein corresponding to amino acids 500–640 of the human protein . The mouse monoclonal IgG1 antibody to CBP was raised against a peptide corresponding to amino acids 2422–2441 of CBP of human origin .A double-stranded oligonucleotide probe containing the 21-bp triplication and 40 bp of flanking sequence from the FeLV-945 LTR was radiolabeled using the synthetic oligonucleotide GS945 as template (5'- GCTGAAACAGCAGAAGTTTCAAGGCCACTGCCAGCAGTTTCAAGGCCACTGCCAGCAGTTTCAAGGCCACTGCCAGCAGTCTCCAGGCTCCCCAGTTGAC -3'), the filling primer (5'-CTGGTCAACTGGGGAGCCT-3') and the Klenow fragment of DNA polymerase  to complete the duplex. A homologous probe containing only a single copy of the 21-bp element was similarly synthesized using the oligonucleotide GS61E as template (5'-GCTGAAACAGCAGAAGTTTCAAGGCCACTGCCAGCAGTCTCCAGG CTCCCCAGTTGAC-3'). Nuclear extract from K-562 cells was obtained from Active Motif . Nuclear extracts from FEA and 3201 cells were prepared using the Nuclear Extract Kit from Active Motif  according to manufacturer specifications. Nuclear extracts were also prepared from FEA cells following the lipid-mediated transfection  of FL-Myb, a c-Myb expression vector in which full length murine c-Myb cDNA was inserted into the multiple cloning site of pcDNA3.1 . DNA-protein binding reactions included 5 μg of nuclear extract and 2.4 ng of radiolabeled probe in a 20 μl reaction containing 1 mM Tris pH 7.5, 7.5 mM NaCl, 1 mM EDTA, 0.7% glycerol, 0.1 mM DTT and 2 μg poly(dI-dC). Reactions containing nuclear extracts from K-562 cells were incubated at 4°C for 30 minutes. Reactions containing nuclear extracts from 3201 or FEA cells were incubated at 30°C for 30 minutes. In some reactions, unlabeled probe was added to the reaction as a specific competitor, or HindIII/HaeIII-digested bacteriophage lambda DNA was included as non-specific competitor. Some reactions included as competitor a double-stranded oligonucleotide containing a known high-affinity c-Myb consensus binding site  and +/- (5'-GCTGAAACAGCAGAAGTTTCAAGGCCACTGCCAGCAGTTTCAAGGCCACTGCCAGCAGATTCAAGGCCACTGCCAGCAGTCTCCAGGCTCCCCAGTTGAC-3') that contained a point mutation in the first or second binding site, respectively (mutated base indicated by boldface and underline). The indicated point mutation had previously been shown to ablate c-Myb binding [AluI/HincII and substituted into the LTR of FeLV-A/61E. The KpnI/PstI fragment of the resulting recombinant LTR was then excised and cloned into the pGL2-Basic luciferase reporter plasmid. Finally, a luciferase reporter plasmid was developed that contained the isolated 21-bp triplication cloned upstream of the SV40 promoter in pGL2-Promoter . A double-stranded DNA fragment containing the 21-bp triplication was generated by PCR amplification using the oligonucleotide GS945 (described above) as template and primers fw945-kpn1 (5'- GCTCGGTACCAGCTGAAACAGCAGAAGTTTC) and rv945-sac1 (5'- ATGCTGAGCTCAACTGGGGAGCCTGGAGACT). The resulting amplification product was digested with KpnI/SstI and inserted into the multiple cloning site upstream of the SV40 promoter in the reporter plasmid.Luciferase reporter plasmids were constructed to contain the U3 region of an FeLV LTR containing one, two or three copies of the 21-bp element. The U3 region of the FeLV-A/61E LTR, containing one copy of the 21-bp element, was cloned into the firefly luciferase reporter plasmid pGL2-Basic . The LTR was then substituted between  binding . A doubl5 cells were seeded in triplicate into 6-well tissue culture plates. The next day, reporter plasmids (500 ng) were introduced into cultured cells by lipid-mediated transfection  in the presence of pRL-TK (5 ng) in a 100:1 ratio. pRL-TK encodes Renilla luciferase and was used as an internal control for transfection efficiency. Firefly and Renilla luciferase activities were quantified 24 hours later using the Dual-Luciferase Reporter Assay System . Data from triplicate wells were analyzed statistically using one-way ANOVA and Bonferroni post test. Statistical significance was considered as p < 0.05. In some assays, the c-Myb expression vector FL-Myb (described above) was added to the transfection in increasing amounts (50 ng – 500 ng). The 5XMRE plasmid was used in reporter gene assays as a positive control. This plasmid contains five tandem c-Myb binding sites cloned upstream of a luciferase gene .For reporter gene assays, 2 × 105 K-562 cells, uninfected or chronically infected with recombinant FeLVs GA-945L and GA-61EL (described above) were seeded in triplicate into 24-well tissue culture plates. A full-length CBP expression vector  was introduced into the cells by lipid mediated transfection using Lipofectamine 2000 reagent . Culture supernatants were collected three days later and reverse transcriptase activity was quantified as previously described [5 × 10escribed . Data frNone declared.SLF developed reporter gene constructs and performed binding assays, gene expression and virus replication assays. SP identified and initially demonstrated c-Myb binding sites. KRR developed the reporter gene assays. LSL directed the experimental design, implementation and interpretation of data. All authors read and approved the final manuscript.


Drugs and other xenobiotics alter gene expression of cytochromes P450 (CYP) by activating the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in mammals. In non-mammalian species, only one xenosensor gene has been found. Using chicken as a model organism, the aim of our study was to elucidate whether non-mammalian species only have one or two xenosensors like mammals.To explore the evolutionary aspect of this divergence, we tried to identify additional xenobiotic sensing nuclear receptors in chicken using various experimental approaches. However, none of those revealed novel candidates. Ablation of chicken xenobiotic receptor (CXR) function by RNAi or dominant-negative alleles drastically reduced drug-induction in a chicken hepatoma cell line. Subsequently, we functionally and structurally characterized CXR and compared our results to PXR and CAR. Despite the high similarity in their amino acid sequence, PXR and CAR have very distinct modes of activation. Some aspects of CXR function, e.g. direct ligand activation and high promiscuity are very reminiscent of PXR. On the other hand, cellular localization studies revealed common characteristics of CXR and CAR in terms of cytoplasmic-nuclear distribution. Finally, CXR has unique properties regarding its regulation in comparison to PXR and CAR.Our finding thus strongly suggest that CXR constitutes an ancestral gene which has evolved into PXR and CAR in mammals. Future studies should elucidate the reason for this divergence in mammalian versus non-mammalian species. A gene superfamily of heme-proteins, the cytochromes P450 (CYP), encodes the main enzymatic system for metabolism of structurally diverse lipophilic substrates . A subseFugu rubripes -radiolabeled CYP2H1 264-bp PBRU.Electrophoretic mobility shift assays have been described in detail . ProteinGGATGGGGCTCTGGCCGGCTTCAAGAGAGCCGGCCAGAGCCCCATCCTTTTTGGAAA) and the anti-sense strand (AGCTTTTCCAAAAAGGATGGGGCTCTGGCCGGCTCTCTTGAAGCCGGCCAGAGCCCCATCCGGG) and subsequent ligation into pSUPER cut with BglII and HindIII (underlined letters refer to CXR-specific targeting sequence). After verification of the ligation product the pSUPER-CXR-RNAi expression cassette was cut out using BamHI and XhoI and subcloned into BglII/XhoI-digested pcDNA3 . The ScaI-linearised construct was transfected into LMH cells using FUGENE 6 . Stable transfectants were selected by addition of 175 μg/ml G418  to the cell culture medium. A control cell line was selected in parallel which was stably transfected with pcDNA3 carrying the empty pSUPER expression cassette. Reporter gene assays in LMH cells using the CXR-RNAi clones were performed using reporter constructs for CYP2H1, CYP3A37, CYP2C45 and ALAS-1 described previously [Expression of CXR in LMH cells was repressed by RNAi as described . In brieeviously -26.LMH cells were cultivated on glass cover slips and subsequently transfected with pEGFP-C1 or pEGFP-N1 expression plasmids  before cells were either treated with vehicle, 400 μM PB or 0.1 μM okadaic acid for 16 hours. Cells were washed with PBS, fixed in 3% formaldehyde for 30 minutes, washed again with PBS, stained with 300 nM DAPI and subsequently mounted on glass slides. Digital images were captured using a Leica DC 300F camera  mounted on a Leitz DMRB microscope with the Leica IM50 Image Manager program version 1.20. Figures were assembled with Adobe Photoshop version 5.0.CXR ligand-binding domain was expressed in bacteria, purified and injected into rabbits for antibody production according to standard procedures. Anti-CXR-ligand-binding domain antibody from rabbit serum was subsequently used in Western blots. LMH cells were grown under standard conditions and treated with vehicle or 400 μM PB overnight. Cells were subsequently washed with PBS and protein extracts prepared using RIPA buffer. As control, CV-1 cells were transfected with empty pSG5 expression vector or vector expressing CXR and subsequently lysed with RIPA buffer. Nuclear extracts were prepared as published .LMH cells were treated with the indicated compounds for 16 hours before total RNA was isolated using the TRIZOL Reagent . Twenty μg of total RNA were subjected to electrophoresis and analysed in Northern hybridisations as described ,33.CYP, cytochrome P450; PB, phenobarbital; PXR, pregnane X receptor; CAR, constitutive androstane receptor; CXR, chicken xenobiotic receptor; PBRU, phenobarbital-responsive enhancer unit; ALAS-1, δ-aminolevulinate synthase; AF-1/2, activation function-1/2; LXR, liver X receptor; PB, phenobarbital; XRS, xenochemical response signal; GFP, green fluorescent protein; PPAR, peroxisome-proliferator activated receptor; FXR, farnesoid X receptor.The authors declare that they have no competing interests.CH carried out the cellular localization assays, cloned the various CXR mutants, performed the reporter gene and the electrophoretic-mobility shift assays as well as the transcriptional regulation studies. SB did the various screens for further chicken xenobiotic-sensing nuclear receptors. AR performed the RNAi experiments. RL and MO isolated the CXR antibody and carried out the protein stabilization and localization assays. MRK helped with the RNAi experiments. MP and CG helped with the CV-1 cell transactivation assays. UAM conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Dendritic cells (DC) are known to present exogenous protein Ag effectively to T cells. In this study we sought to identify the proteases that DC employ during antigen processing. The murine epidermal-derived DC line Xs52, when pulsed with PPD, optimally activated the PPD-reactive Th1 clone LNC.2F1 as well as the Th2 clone LNC.4k1, and this activation was completely blocked by chloroquine pretreatment. These results validate the capacity of XS52 DC to digest PPD into immunogenic peptides inducing antigen specific T cell immune responses. XS52 DC, as well as splenic DC and DCs derived from bone marrow degraded standard substrates for cathepsins B, C, D/E, H, J, and L, tryptase, and chymases, indicating that DC express a variety of protease activities. Treatment of XS52 DC with pepstatin A, an inhibitor of aspartic acid proteases, completely abrogated their capacity to present native PPD, but not trypsin-digested PPD fragments to Th1 and Th2 cell clones. Pepstatin A also inhibited cathepsin D/E activity selectively among the XS52 DC-associated protease activities. On the other hand, inhibitors of serine proteases  or of cystein proteases (E-64) did not impair XS52 DC presentation of PPD, nor did they inhibit cathepsin D/E activity. Finally, all tested DC populations  constitutively expressed cathepsin D mRNA. These results suggest that DC primarily employ cathepsin D (and perhaps E) to digest PPD into antigenic peptides. Dendritic cells (DC) are professional antigen presenting cells that induce primary antigen specific T cell responses  and exhiIn the present study, we sought to characterize the Ag processing capacity of DC, as well as the enzymes previously involved in this process. In this regard, several groups have previously reported that epidermal LC and splenic DC, both of which contain small numbers of non-DC contaminants, exhibit significant Ag processing capacities -12. LC fStaphylococcus aureus nuclease [With respect to the mechanisms by which DC process complex protein Ags, chloroquine has been shown to inhibit this process; this suggests that Ag processing primarily occurs within acidic compartments -8, 10-1-110-12. nuclease -35. Thesnuclease . Little The XS52 DC cell line , a long-term DC line established from the epidermis of newborn BALB/c mice , were ex+ cells , and T cells (with GK1.5 and 3.155 mAbs). The remaining I-A- cells were then cultured in the presence of GM-CSF (10 ng/ml). The purity of bone marrow derived DC was more than 95% as determined by flow cytometry using anti-CD11c and anti-I-A antibody (not shown).Splenic DC were purified from BALB/c mice  by a series of magnetic bead separations as before ,25. BrieCells were lysed in 0.1% Triton X-100 in 0.9% NaCl; extracts were then examined for protease activities using the following substrates: a) Z-Arg-Arg-βNA , b) denatured hemoglobin , c) Arg-βNA , d) Z-Phe-Arg-MCA , e) Z-Phe-Arg-MCA , f) Gly-Phe-βNA , g) BLT ester , and h) Suc-Ala-Ala-Pro-Phe-SBz and Suc-Phe-Leu-Phe-SBz . Samples were incubated at the indicated pH and enzymatic activities were assessed by colorimetric or fluorogenic changes . Enzymat4CL (15 mM). Subsequently, the XS52 cells were washed 3 times with PBS to remove unbound PPD and then cultured in 96 round-bottom well-plates (104 cells/well) with either the PPD-reactive Th1 or Th2 clone (105 cells/well) in the presence of the same inhibitor at the above concentration. In some experiments, XS52 DC were pulsed overnight with PPD in the presence of an inhibitor and then fixed with 0.05% glutaraldehyde in PBS for 30 seconds at 4°C; the fixation reaction was stopped by adding 0.1 M L-lysine. These XS52 cells were then washed with PBS and examined for their ability to activate Th1 or Th2 clones in the absence of protease inhibitors. In order to determine the mechanism of action for pepstatin A, XS52 cells were pulsed in its presence with PPD either in a native form or following digestion with trypsin-conjugated sepharose beads  for 15 minutes at 37°C. We also examined the effect of added pepstatin A on the capacity of XS52 cells to activated allogeneic T cells isolated from CBA mice (Jackson Laboratories). Samples were pulsed for 18 hr with 1 μCi of 3H-thymidine and then harvested using an automated cell harvestor.XS52 DC were γ-irradiated (2000 rad) and then pulsed for 8 hr with 100 μg/ml of PPD  in the presence of each of the following inhibitors (or vehicle controls): a) pepstatin A , b) DCI 100 μM, Sigma), c) E-64 , d) DMSO (1%), and e) NHmRNA expression for cathepsin D was examined by RT-PCR. RNA isolation, reverse-transcription, and cDNA amplification were carried out as previously described . The fol+ splenic DC, and GM-CSF-propagated bone marrow DC. As noted in Table In the first set of experiments we sought to identify which protease activities were expressed by DC. A panel of synthetic peptide and protein substrates was incubated with extracts prepared from three DC populations: the XS52 DC line, 4F7We next asked whether DC are capable of digesting a complex protein Ag into antigenic products. In this regard, it has been reported previously that the original XS52 DC, as well as clones derived from this line, are capable of presenting KLH to the KLH-specific Th1 clone HDK-1 . These rTo identify the proteases responsible for processing PPD, we employed three inhibitors: pepstatin A (aspartic acid protease inhibitor), DCI (serine protease inhibitor), and E-64 (cysteine protease inhibitor). XS52 DC were pulsed for 8 hr with native PPD in the presence of each inhibitor and then examined for the capacity to activate PPD-reactive Th1 and Th2 clones. To ensure a maximal effect, inhibitors were also added to cocultures of XS52 DC and T cells. As noted in Figure To identify the protease(s) inhibited by pepstatin A, XS52 DC were pretreated for 1 hr with pepstatin A (100 μg/ml), and extracts prepared from these cells were then examined for enzymatic activities. As noted in Figure + splenic DC and a bone marrow derived DC line, indicating that DC do possess the capacity to produce cathepsin D  during Ag processing by DC. Taken together, these results reinforce the concept that DC are fully capable of processing complex protein Ag into antigenic peptides.The experiments reported in this study provide new information with respect to complex Ag processing by DC. First, the long-term DC line, XS52 DC, was capable of processing PPD into immunogenic peptides, in the complete absence of other cell types. Although previous studies using several different DC preparations have documented similar results (3–12), this is the first report validating the Ag processing capacity of DC, in the absence of contaminating cells. Second, we have characterized the protease profiles expressed by DC. XS52 DC, 4F7Staphylococcus aureus nuclease (17–22). Here we report that DC also employ cathepsin D and/or E to digest PPD into an immunogenic Ag-product. This conclusion is supported by several lines of evidence: a) pepstatin A, but not other protease inhibitors, completely blocked the presentation of intact PPD by XS52 DC to PPD-reactive Th1 and Th2 clones, whereas it did not affect the presentation of PPD fragments; b) pepstatin A pretreatment inhibited cathepsin D/E activity selectively among the DC-associated protease activities; and c) all tested DC preparations expressed cathepsin D mRNA constitutively. In this regard, DC isolated from the mouse thoracic duct have been reported to produce neglible, if any, cathespin D immunoreactivity (assessed by immunofluorescence staining), whereas peritoneal macrophages produced relatively large amounts [As described before, macrophages and B cells have been reported to employ cathepsins B, D, and E primarily to digest complex protein Ag, such as ovalbumin (OVA), hen egg white lysozyme (HEL), myoglobin, exogenous IgG, and  amounts . Also co amounts  may be uIt is important to emphasize that different protein Ag may be degraded by different proteases in DC. Moreover, DC isolated from different tissues or in different maturational states may employ different proteases. For example, murine DC isolated from the thoracic are unable to digest human serum albumin effectively , and murNone declared.Dr. Mohamadzadeh is the major contributor (15%) of the experimental data and a rough draft of the paper. The next three intermediate authors' contributed remaining data and advice. Dr. Luftig was the overall individual who directed the several drafts and contributed to providing a new set of references to the manuscript.


In this study, we analyzed TL-3 efficacy using a highly pathogenic FIV-C isolate, which causes a severe acute phase immunodeficiency syndrome, with high early mortality rates.The protease inhibitor, TL-3, demonstrated broad efficacy p ≤ 0.05) higher viral RNA loads as compared to asymptomatic animals. A marked and progressive increase in CD8+ T lymphocytes in animals surviving acute phase infection was noted, which was not evident in symptomatic animals (p ≤ 0.05). Average viral loads were lower in TL-3 treated animals and of the 6 animals requiring euthanasia, four were from the untreated cohort. At eight weeks post infection, half of the TL-3 treated animals and only one of six untreated animals had viral loads below detection limits. Analysis of protease genes in TL-3 treated animals with higher than average viral loads revealed sequence variations relative to wild type protease. In particular, one mutant, D105G, imparted 5-fold resistance against TL-3 relative to wild type protease.Twenty cats were infected with uncloned FIV-C and half were treated with TL-3 while the other half were left untreated. Two uninfected cats were used as controls. The general health and the immunological and virological status of the animals was monitored for eight weeks following infection. All infected animals became viremic independent of TL-3 treatment and seven of 20 FIV-C infected animals developed severe immunodepletive disease in conjunction with significantly (The findings indicate that the protease inhibitor, TL-3, when administered orally as a monotherapy, did not prevent viremia in cats infected with high dose FIV-C. However, the modest lowering of viral loads with TL-3 treatment, the greater survival rate in symptomatic animals of the treated cohort, and the lower average viral load in TL-3 treated animals at eight weeks post infection is indicative of a therapeutic effect of the compound on virus infection. Infected cats exhibit similar disease patterns as human immunodeficiency virus (HIV) infected patients by developing multiple immuno-depletive symptoms collectively referred to as acquired immunodeficiency syndrome (AIDS). As with HIV, differences in virulence among the different FIV subgroups are evident -4. Thus,in vitro with similar effectiveness -L-alanyl-L-valyl]amino])  treatmenUninfected and FIV-infected animals were intermittently scheduled for analyses of evoked potentials in conjunction with the blood sampling, including testing for both auditory and visual evoked potentials as previously described . Once reAnimals were examined daily and in case of health concerns further therapy/diagnostics were initiated. Animals with abnormal weight, or on antibiotics were placed on supplemental feeding with moist food and Nutrical. Dehydrated animals received subcutaneous fluid therapy. More affected animals received supportive care and medications, consisting of BID administration of antibiotics (Baytril), BID subcutaneous fluid therapy, BID temperature evaluation, BID application of antibiotic ophthalmic ointment supportive care. Any animals that required extensive supportive care  were euthanized. Euthanasia of research animals was conducted with strict adherence to NIH Office of Laboratory Animal Welfare protocols.Blood samples  were collected weekly during the first month of the study and then every two weeks thereafter, as described . SamplesPlasma was separated from blood by centrifugation at 3000 rpm for 5 minutes at room temperature. Blood cells were resuspended in 3 ml PBS and PBMC were separated from buffy coats by density gradient centrifugation using Ficoll-Hypaque Plus . PBMC were washed once in PBS and twice in PBS/2% FBS for flow cytometry analyses.Statistical p values for the FIV-C viral load were determined by the Student's two-tailed t-test . Statistical p values for the weekly total CD4 and CD8 cell counts were also determined by the Student's two-tailed t-test .1κ FITC and PE  were used as isotype controls. Cells were fixed with 2% PFA prior to analysis performed on a FACScan flow cytometer (Beckton Dickenson Immunocytometry Systems) using the Cell Quest Software program.Two-color flow cytometry analysis was performed on cells stained with mouse α-feline CD4 FITC and mouse α-feline CD8 PE . Anti-mouse IgG9 copies RNA /280 μl plasma), corresponding to the 1.2 kb KAN gene , was used to normalize plasma volumes between samples and to correct for sample loss from viral RNA extraction and cDNA synthesis. An on-column DNase/ RNase free (Qiagen) incubation step for 10 minutes at room temperature was added to remove residual cellular DNA. Complimentary DNA (cDNA) was generated in a 20 μl total reaction using 13 μl of sample RNA, 0.5 μl (2 μM stock) each of KAN and FIV specific reverse primers (sequences below) and StrataScript reverse transcriptase, following the manufacturer's protocol . After incubation, each cDNA sample was diluted in water to 30 μl and stored at -80°C for use in real-time PCR.Plasma for weeks 0, 2, 4, 6 , 6.5 , and 8, were isolated from whole blood by centrifugation and stored at -20°C. Viral RNA was extracted using the QiaAmp Viral RNA Isolation Kit  according to manufacturer's instructions with slight modifications: Plasma samples (280 μl) were lysed in buffer AVL  (Qiagen) for 10 minutes at room temperature in the presence of carrier RNA (10 μg/ml) and an external Kanamycin (KAN) RNA spike. Equal amounts of the external RNA spike  , forward, reverse primers and probe mix (7.5 μl), and cDNA target (5 μl). The mixture was incubated at 50°C for 2 minutes, 95°C for 10 minutes, then cycled at 95°C for 15 seconds and 60°C for 60 seconds 55 times, using the ABI Prism 7700 Sequence Detection System . Data were analyzed using the ABI 7700 Sequence Detection Software. Forward and reverse primers  used in real-time PCR were made at IDT, , while the fluorescein-dabsyl Amplifluor UniPrimer  was purchased from Serologicals . The primer sequences used for real-time PCR are as follows:FIV reverse-transcriptase forward: 5'-ACTGAACCTGACCGTACAGATAAATTACAGGAA GAACCCCCATA-3'FIV reverse-transcriptase reverse: 5'-TGTTAATGGATGTAATTCA TAACCCATC-3'KAN forward: 5'-ACTGAACCTGACCGTACACGCTCAGGCGCAATCAC-3'KAN reverse: 5'-CCAGCCATTACGCTCGTCAT-3'8 to 5 × 102 copies per well) of dsDNA plasmids of known copy number , against the cycle threshold (Ct) determined for that value. The pET28 vector (Novagen) was used as the target plasmid for the KAN gene, while a plasmid containing the molecular clone of FIV-C was used for the FIV reverse-transcriptase gene. Calculated values for each plasma sample represent relative copy numbers for the purposes of evaluation between individual samples.To determine the relative copy numbers of KAN and FIV from plasma samples, a linear standard curve was generated by plotting 10-fold dilutions (5 × 10Complementary DNA (cDNA) was synthesized from isolated plasma viral RNA of infected cats. The cDNA pool was used as a template for PCR reactions using 5' primer MFIVCPL5' (5'-GATTTATAAATCATATG GCATATAATAAAGTGGGTACCACTACAACATTAG-3'), which adds an NdeI restriction site, methionine, and alkaline to the N-terminal of the protease and 3' primer MFIVCPL33' (5'-CTGAGATCTGAGCAAGCTTTTACATTACTAATCT AATATTAAATTTAACCATG TTATC-3'), which adds a stop codon and a Hind III restriction site to the C-terminus of the protease. The amplified PCR product was gel purified and cloned into pCR-TOPOII vector  for sequencing. Selected DNA of mutants, N55D, H72R, D105G, and M107R were digested with Nde I/Hind III and ligated into pET21a expression vector (Novagen). The mutant FIV-C proteases were expressed in Rosetta pLysS cells (Novagen) and purified as previously described .2)-Val-Gln-Ser-Lys-Gly-Arg-NH2. The concentration was determined by active-site titration with inhibitor TL-3. Inhibitor constant (Ki) of TL-3 against mutant FIV-C protease was analyzed as described [Enzyme kinetics of FIV-C protease were assayed on the flourogenic substrate Arg-Ala-Leu-Thr-Lys(Abz)-Val-Gln/Phe(NOescribed .None of the authors have commercial interests or direct association with a company that is marketing TL-3. J.H.E. is a co-author of a patent application regarding use of protease inhibitors like TL-3 reported here, containing small P3 residues, as inhibitors of HIV and FIV.S.R. and C.H.S contributed equally to this work and carried out all tissue culture analyses, purification and characterization of cell populations isolated from PBMC, statistical analyses, and preparation of results for publication. Quantitative PCR analyses was carried out by D.A.S. K.J.C. was responsible for all in vivo animal work, including TL-3 administration and oversight of veterinary care for the animals. S.H-R. carried out measurements of brainstem auditory evoked potential changes. S.H., B.E.T, and J.H.E. acted as mentors for the various facets of the project, oversaw the writing of the manuscript, and provided space and funding to carry out the work.


Background: Histone-like proteins are small molecular weight DNA-binding proteins that are widely distributed in prokaryotes. These proteins have multiple functions in cellular structures and processes, including the morphological stability of the nucleoid, DNA compactness, DNA replication, and DNA repair. Deinococcus radiodurans, an extremophilic microorganism, has extraordinary DNA repair capability and encodes an essential histone-like protein, DrHU.Objective: We aim to investigate the phosphorylation regulation role of a histone-like HU protein from Deinococcus radiodurans.Methods: LC-MS/MS analysis was used to determine the phosphorylation site of endogenous DrHU. The predicted structure of DrHU-DNA was obtained from homology modeling (Swiss-model) using Staphylococcus aureus HU-DNA structure (PDB ID: 4QJU) as the starting model. Two types of mutant proteins T37E and T37A were generated to explore their DNA binding affinity. Complemented-knockout strategy was used to generate the ΔDrHU/pk-T37A and ΔDrHU/pk-T37E strains for growth curves and phenotypical analyses.Results and Discussion: The phosphorylation site Thr37, which is present in most bacterial HU proteins, is located at the putative protein-DNA interaction interface of DrHU. Compared to the wild-type protein, one in which this threonine is replaced by glutamate to mimic a permanent state of phosphorylation (T37E) showed enhanced double-stranded DNA binding but a weakened protective effect against hydroxyl radical cleavage. Complementation of T37E in a DrHU-knockout strain caused growth defects and sensitized the cells to UV radiation and oxidative stress.Conclusions: Phosphorylation modulates the DNA-binding capabilities of the histone-like HU protein from D. radiodurans, which contributes to the environmental adaptation of this organism. As the name suggests, these proteins are nucleoid-association proteins because of their functional similarity to eukaryotic histone proteins. Based on their abundance in living cells and capacity to bind DNA, histone-like proteins are associated with nucleoid stability, DNA compaction, DNA replication, and DNA repair -3.Streptococcus pneumoniae, HU is required for cell viability, and conditional inactivation of this protein impairs DNA supercoiling in vivo [Escherichia coli cells only show modest phenotypes in the absence of HU, which might be due to the expression of redundant histone-like proteins [E. coli, HU is involved in the regulation of SOS genes and is able to displace the LexA repressor from the SOS box [HU is a histone-like protein that is essential for some gram-positive bacteria. In  in vivo . In contproteins . In addiproteins -10. In t SOS box .Mycobacterium smegmatis promotes the DNA end-joining reaction, an important DNA double-strand break repair pathway, which is consistent with the proposed DNA repair function of HU proteins [1A). Most of these extensions are rich in lysine residues and participate in the modulation of HU DNA binding [Bacillus stearothermophilus [As a DNA-binding protein, HU can bind to different types of DNA in a non-specific manner. However, for canonical HU proteins, binding affinity for abnormal DNA, such as Holliday junctions, DNA forks, and gapped or nicked duplex DNA, appears to be higher than that for regular duplex DNA . These Dproteins . Sequenc binding , 16. Thu binding -20. Cons binding , 22. Formophilus .Deinococcus radiodurans, a non-spore-forming gram-positive bacterium, is well known for its extreme resistance to environmental stresses, including ionizing radiation, oxidative stress, and DNA damage. It has been shown that the robustness of this bacterium is the result of multiple mechanisms, e.g., enhanced DNA repair capabilities, novel DNA damage responses, effective antioxidation systems, and cellular protection of damaged DNA and proteins [D. radiodurans encodes one HU protein, DrHU (DR_A0065), which is highly expressed from a leaderless mRNA [E. coli HU-knockout strain, DrHU is required for cell viability under normal growth conditions. Progressive depletion of DrHU using a thermosensitive vector causes diffuse and enlarged nucleoid morphology, further leading to cell death [proteins -26. Wholess mRNA . In contll death . Sequencll death . Moreovell death . Furtherll death .D. radiodurans, lysine succinylation regulates a variety of biological processes and enzymatic activities of important DNA repair proteins, such as PprI and DdrB [D. radiodurans [Mycobacterium tuberculosis HU protein modulate its ability to compact DNA, but mutations of PTM sites reduce the drug-tolerant population, linking PTM of bacterial HU to antibiotic treatment [D. radiodurans.Posttranslational modifications (PTMs) play fundamental roles in various cellular processes and are critical for appropriate protein functions and biochemical activities. The role of methylation and phosphorylation in histone regulation is well documented . In D. rand DdrB . Phosphoiodurans . Despiteiodurans . Lysine reatment , 35. To 22.1S1. All primers used are given in Table S2. The D. radiodurans wild-type R1 strain (ATCC13939) and mutants were grown in TGY broth  or on TGY agar plates (1.5% agar) at 30°C. Full-length DR_A0065 (encoding 122 amino acids) was cloned into the shuttle vector pRADK containing the D. radiodurans groEL promoter for complementation. Site-directed mutagenesis was carried out using PrimeSTAR HS DNA polymerase (Takara) to generate complementary plasmids containing T37A and T37E DrHU. The complementation-knockout ΔDrHU/pk-T37A and ΔDrHU/ pk-T37E strains were constructed by firstly transforming the complementary plasmid into the wild-type R1 strain, followed by the tripartite ligation method, and the genomic DR_A0065 gene was substituted with the streptomycin gene, as described previously [DR_A0065 were firstly amplified by PCR. Following digestion with BamHI and HindIII, the fragments were ligated to a streptomycin gene, which was predigested with BamHI and HindIII. The triplet ligation product was amplified and transformed into competent cells. The mutants were selected with streptomycin and chloramphenicol, and confirmed by sequencing.All strains and plasmids used in this study are listed in Table eviously . Briefly2.2600 = 1.0) were harvested by centrifugation at 8000 × g and resuspended in sterile phosphate-buffered saline (PBS) three times. Four volumes of lysis buffer (100 mM NH4HCO3 (pH 8), 6 M urea, and 0.2% SDS) were added, followed by sonication on ice. After concentration at 20000 × g at 4°C, the protein extracts were subjected to disulfide reduction with 2 mM DTT  and subsequently alkylated with 20 mM iodoacetamide . Four volumes of ice-cold acetone were incubated with samples at -20°C for 2 h. After centrifugation, the precipitate was collected and washed twice with cold acetone, and the pellet was dried and redissolved in a dissolution buffer containing 0.1 M triethylammonium bicarbonate  and 6 M urea. The supernatant was digested with Trypsin Gold (Promega) at a 1:50 enzyme-to-substrate ratio at 37°C for 16 h, and then the peptides were desalted with a C18 cartridge to remove high levels of urea and dried by vacuum centrifugation. The dried fractions were redissolved in 250 mM acetic acid with 30% acetonitrile, and 1 M HCl was used to adjust the pH to 2.5-3.0. Enrichment was carried out using Phos-select iron affinity gel  following the manufacturer’s instructions. The bound peptides were eluted, dried, and desalted using peptide desalting spin columns . The enriched phosphopeptide mixture was analyzed using an EASY-nLCTM 1200 UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Extractive HF-X mass spectrometer (Thermo Fisher) operating in data-dependent acquisition (DDA) mode. The full-scan MS spectra were acquired at a resolution of 60000 (at 200 m/z) and ranged from 350 to 1500 m/z with automatic gain control (AGC) target value of 3 × 106 with a maximum ion injection fill time of 20 ms. The 40 most abundant precursor ions were sequentially isolated and fragmented by high-energy collision dissociation (HCD) at a resolution of 15000 (at 200 m/z) with an AGC target value of 5E4, normalized collision energy of 27%, a maximum ion injection time of 80 ms, a dynamic exclusion parameter of 30 s, and an intensity threshold of 1.3E4. The data obtained were combined as the raw data. The search engine Proteome Discoverer 2.2  was used to analyze the raw MS spectra acquired from the protein database of D. radiodurans (strain R1) in UniProt (https://www.UniProt.org/).Identification of the DrHU phosphorylation site was performed using a similar method as described but with modification . Briefly2.3S. aureus HU-DNA structure (PDB ID: 4QJU) as the starting model.For homology modeling, the Swiss model was used to predict the structure of DrHU-DNA using the 2.4Wild-type T37A and T37E DrHU were cloned into the modified pET28a expression vector pET28a-HMT, which contains an N-terminal cleavable 6×His tag, as described previously [2.5S3. Annealing reactions of duplex DNA were carried out by heating at 95°C for 5 min, followed by slow cooling to room temperature in oligo annealing buffer . The final reaction mixture consisted of 50 mM NaCl, 20 mM Tris-HCl 8.0, 0.1 mg/ml albumin from bovine serum (BSA), 3 mM DTT, 0.1 mM EDTA, 10% glycerol, 10 mM MgCl2, 50 nM duplex DNA and DrHU . The mixtures were incubated at room temperature for 20 min and separated using 22% native polyacrylamide gels in 1 × TB buffer. The gels were imaged in fluorescence mode (FAM) using a Typhoon FLA 9500 . Quantifications and curve fitting were performed as described [All oligonucleotides labeled with 5’-FAM were purchased from Sangon , as listed in Table escribed . All rea2.6600 = 1.0) was resuspended in 50 mL new fresh TGY medium and incubated at 30°C. Cell density was monitored at various incubation times by measuring the OD600. For UV treatment, cells at an OD600 of 1.0 were diluted and plated on TGY agar plates and illuminated with 100, 200, 300, and 400 J/m2 UV radiation. For H2O2 treatment, cells at an OD600 of 1.0 were incubated with 20, 40, 60, and 80 mM H2O2, diluted and plated on TGY agar plates. Colonies were counted after incubation for 2-3 days. Three independent experiments were performed. Growth and survival fraction curves were plotted using GraphPad Prism 8.Phenotypic assays were performed as previously described . For gro2.7Wild-type and mutant DrHU proteins  were incubated with 1 kb double-stranded linear DNA fragments PCR-amplified from genomic DNA (100 ng) in 20 µl reaction containing 30 mM Tris-HCI pH 8.0 and 100 mM KCl for 30 minutes at room temperature. Next, 150 µM ferrous sulfate and 10 mM hydrogen peroxide were added for 5 minutes at room temperature. The reaction was stopped with buffer containing 0.8% (w/v) SDS and 2.5% glycerol, and the samples were loaded onto a 1% agarose gel in glue (0.5 × TBE). The gel was stained with ethidium bromide and viewed under ultraviolet light.2.8Results of growth curves, phenotypical analyses, and DNA binding assays were determined using GraphPad Prism 8. The results represented the means and standard deviations (SD) of three independent experiments. One-way ANOVA method followed by Tukey’s post-hoc test was performed to compare the significant differences between wild-type strain and complementary strains.33.11)). In addition to the HU core domain comprising the DNA-binding residues, the N-terminal lysine-rich extension (residues 1-31) appears to be highly conserved among Deinococcus species. Phosphoproteome profiling of D. radiodurans under normal conditions was performed to identify possible phosphorylation sites of DrHU. After enrichment of phosphopeptide, LC–MS/MS (liquid chromatography-tandem mass spectrometry) analysis showed that DrHU contained a phosphorylation modification at the Thr37 site, which has a mass shift of +79.9663 Da based on a search against the UniProt database . This phosphorylation site is located at the N-terminal region of the core domain, which is present in not only all Deinococcus species but also HU proteins from other bacteria, e.g., threonine in HU from Staphylococcus aureus and threonine/serine in E. coli HUα/β . Notably, a lysine residue (Lys36 in DrHU and Lys3 in SauHU) upstream of Thr37 is conserved in almost all HU proteins, which undergoes acetylation modification in M. tuberculosis HU protein .As noted by a previous study, DrHU, containing 122 amino acids, is translated from a natural leaderless mRNA  . Despite being biologically irrelevant, the phosphorylation site Thr37 of DrHU is exposed on the surface and interacts with the DNA of a symmetric-related HU-DNA complex. Lys36, the conserved residue adjacent to the phosphorylation site, interacts with Ser59 . It has been proposed that this Lys-Asp salt bridge interaction of S. aureus HU is maintained upon DNA binding [1 and Figure S3). In addition to the wild-type protein, we generated two types of mutant proteins: one in which this threonine is replaced by glutamate to mimic a permanent state of phosphorylation (T37E), and one in which this threonine is mutated to alanine to prevent phosphorylation (T37A).A homology model of the DrHU-DNA complex was predicted using the  binding . To explKd) in the millimolar range . When longer duplex DNA was tested, DrHU exhibited relatively higher binding affinity (449.3 ± 29.6 nM for 48-bp duplex DNA). However, no complex was detectable with the phosphorylation-defective T37A mutant protein incubated with 24-, 36- or 48-bp duplex DNA, indicating that Thr37, per se, is required for the DNA-binding activity of DrHU. Compared to the wild-type protein, the phosphorylation mimic DrHU (T37E) showed increased DNA-binding abilities with all duplex DNA tested, especially for longer duplex DNA, which was approximately 2.9-fold higher than that for 36-bp duplex DNA (457.4 ± 62.9 nM for T37E vs. 1352.0 ± 200.5 nM for wild-type).Although the binding affinity was rather low, wild-type DrHU was able to form a stable single complex with duplex DNA in a length-dependent manner. Incubation of DrHU with a short DNA duplex (24 bp and 36 bp) showed a dissociation constant (3.3DR_A0065 (encoding wild-type (WT), T37A, and T37E DrHU) was transformed into the D. radiodurans wild-type R1 strain, followed by knockout mutagenesis as described above. Compared to the wild-type R1 strain, the WT complemented-knockout strain (ΔDrHU/pk-WT) did not show growth defects  and displayed no sensitivity to DNA-damaging agents, including ultraviolet (UV) irradiation  and hydrogen peroxide (H2O2) , indicating that genomic-expressed DrHU can be fully complemented by plasmid-based copies. In addition to reduced growth , the T37A (ΔDrHU/pk-T37A) and T37E (ΔDrHU/pk-T37E) complemented-knockout strains were both sensitive to DNA-damaging agents to various degrees . Interestingly, although the survival rates of ΔDrHU/pk-T37A were 1-2 orders of magnitude lower than those of the wild-type strain in the presence of high DNA-damaging agent concentrations, the phosphorylation mimic ΔDrHU/pk-T37E displayed survival rates that were more than three orders of magnitude lower . Given the enhanced duplex DNA-binding capability of T37E DrHU, these results suggest the toxic effects of over phosphorylation of DrHU in vivo.As noted by previous studies, complete inactivation of DrHU is lethal . We were3.44)2(SO4)2 and H2O2  in the presence or absence of DrHU ). Linear DNA was completely degraded in the absence of DrHU , but it was substantially protected by subsequent addition of wild-type DrHU . A similar protective effect was also observed with the T37A mutant protein at low concentration . However, the phosphorylation mimic T37E exhibited much weaker protection against hydroxyl radical cleavage, and the linear DNA was entirely degraded at a low concentration of T37E mutant protein .As HU proteins have been reported to protect DNA against hydroxyl radical attack, we next examined whether phosphorylation of DrHU affects its DNA protection features. A 1-kb linear DNA duplex was incubated with Fe orchestrating nucleoid dynamics. HU proteins, the most abundant NAP in some bacteria, contribute to both nucleoid organization and gene expression under certain conditions, such as environmental stress and virulence , 39. D. acterium . Such co1B). Interestingly, this lysine is not only required for effective short-length DNA wrapping of HU but is also involved in the regulation of DNA compaction through acetylation in M. tuberculosis HU [in vivo and in vitro. For example, phosphorylation of Ser10 at histone H3 enhances acetylation at Lys14, contributing to gene regulation [In addition to gene expression, PTM plays key roles in the regulation of protein structure, biochemical activity, and protein-protein interactions. Phosphorylation, one of the most important and well-studied PTMs, is involved in nucleoid/mitochondrial nucleoid organization in both bacterial and eukaryotic organisms. In this study, the sole phosphorylation site Thr37 of DrHU was identified; it is adjacent to a lysine residue (Lys3 in other bacteria) highly conserved among HU proteins  to assess their DNA-binding capabilities (Table 1). Wild-type DrHU was able to form a stable complex with a binding site of 24 bp of short duplex DNA, which is generally comparable with HU homologous from other bacteria, e.g., 37 bp for E. coli TF1 and 19 bp for Helicobacter pylori HU [2B, modeled using S. aureus HU-DNA structure 4QJU), which is consistent with the proposed DNA-binding role of the α-helical body of HU proteins [S. aureus HU, an internal salt bridge between Lys3 (Lys36 in DrHU) and Asp26 was observed [Deinococcus HU proteins (Ser59 in DrHU). Thus, the enhanced DNA-binding capability of T37E may be due to a possible salt bridge formation between Lys36 and phosphorylated Thr37, modulating the DNA-binding process of DrHU.According to the original annotation, DrHU contains 137 amino acids. Given the suggested leaderless translation ylori HU , 43. Howproteins . Moreoveproteins , 44. In observed . HoweverE. coli and Bacillus subtilis favor DNA recombination and repair intermediates. D. radiodurans, the model organism for DNA repair studies, has the extraordinary capability to repair hundreds of DNA double-strand breaks (DSBs) after heavy irradiation. Compared to other types of DNA damage, DSBs are considered the most lethal form, lacking an intact undamaged template strand for repair. Thus, preventing diffusion of these unrepaired DSBs appears to be a promising strategy for D. radiodurans to survive under extreme conditions. Given that depletion of DrHU causes diffuse nucleoid structure, we performed phenotypical assays to evaluate the possible effect of DrHU phosphorylation in the absence of a gene encoding DrHU. Consistent with the almost abolished short DNA-binding capability, ΔDrHU/pk-T37A exhibited growth defects and was sensitive to environmental stresses ). Unexpectedly, the phenotypes of ΔDrHU/pk-T37E were much more severe than those of ΔDrHU/pk-T37A. These toxic effects might be due to the following: (1) in the absence of wild-type DrHU, although the enhanced DNA binding of T37E DrHU may facilitate restraining DSBs after DNA damage, this phosphorylation-mimic protein is defective in normal dissociation from DNA, leading to suppression of genes involved in DNA repair and chromosome segregation; (2) compared to wild-type DrHU, T37E shows much reduced protective effects against oxidative attack, which can easily cause DNA damage; and (3) the HU protein has been shown to regulate the formation of higher-order protein complexes involved in DNA replication and gene expression by interactions with multiple protein partners [In addition to DNA compaction, HU proteins can form DNA-protein-DNA bridges and protect DNA from digestion, regulating DNA transcriptions and facilitating DNA repair. For example, HU proteins from partners . PhosphoD. radiodurans, which may contribute to the robustness of this organism. However, physiological phosphorylation is dynamic and transient in response to various stimuli or cellular events. Given the toxicity of the phosphorylation-mimic protein in the absence of genomic-expressed wild-type DrHU, phosphorylation of DrHU must be very tightly controlled in response to environmental stresses.Collectively, our findings provide the first evidence that phosphorylation modulates the DNA-binding capabilities of the histone-like HU protein from