GA context and were relatively infrequent, thereby allowing for spreading infection. The mutations were absent in cells lacking A3C but were induced after transient expression of A3C in the infected target cell. Inhibiting endogenous A3C by RNA interference in Magi cells prevented the viral mutations. Thus, A3C is necessary and sufficient for G-to-A mutations in some HIV-1 strains. A3C-induced mutations occur at levels that allow replication to persist and may therefore contribute to viral diversity. Developing drugs that inhibit A3C may be a novel strategy for delaying viral escape from immune or antiretroviral inhibition.The evolutionary success of primate lentiviruses reflects their high capacity to mutate and adapt to new host species, immune responses within individual hosts, and, in recent years, antiviral drugs. APOBEC3G (A3G) and APOBEC3F (A3F) are host cell DNA-editing enzymes that induce extensive HIV-1 mutation that severely attenuates viral replication. The HIV-1 virion infectivity factor (Vif), expressed in vivo, counteracts the antiviral activity of A3G and A3F by inducing their degradation. Other APOBECs may contribute more to viral diversity by inducing less extensive mutations allowing viral replication to persist. Here we show that in APOBEC3C (A3C)-expressing cells infected with the patient-derived HIV-1 molecular clones 210WW, 210WM, 210MW, and 210MM, and the lab-adapted molecular clone LAI, viral G-to-A mutations were detected in the presence of Vif expression. Mutations occurred primarily in the  HIV has shown a chameleon-like nature, always changing to adapt to its environment. Defining the factors that drive and regulate genetic changes in HIV over time is key to understanding how HIV causes disease and escapes from the body's immune responses and drug treatment. The diversity of HIV has implications for the development of effective drugs and for fostering better immune responses. In this study, we showed that a human protein, called APOBEC3C (A3C), could induce certain mutations in some HIV-1 strains, including those derived from a patient who had developed rapid drug resistance. Eliminating the expression of this protein prevented the mutations in two different types of cells. Furthermore, short-term A3C expression was sufficient to cause mutation. We conclude that A3C is necessary and sufficient to induce signature mutations in HIV-1. A3C-induced mutations may provide potential benefit for the virus if the mutation rate is low enough such that the majority of viruses are able to replicate, while accumulating a limited number of novel mutations that may allow the virus to survive in the face of antiviral drugs or immune responses. The evolutionary success of primate lentiviruses is evident from their prevalence in Old-World primates and their capacity to spread to new host species, frequently leading to the emergence of zoonotic disease ,2. EstabGG-to-AG mutations [GA-to-AA mutations [GA and GG dinucleotides, with a preference for GA over GG [An important mechanism of HIV genetic variation is G-to-A mutation during reverse transcription ,6. Such utations –15 whereutations ,11,12. I over GG ,12,16. G over GG .A3G- and A3F-induced mutations are suppressed by HIV-1 virion infectivity factor (Vif), which is typically expressed in vivo, thus limiting their contribution to the adaptation of viral populations ,18–22. Ivif-expressing viruses is caused by an APOBEC that does not have a strong antiviral activity, is relatively resistant to Vif action, and is expressed in HIV-1 target cells. To contribute to the adaptation of viral populations, the APOBEC activity should be weak/moderate, such that mutations are sufficiently infrequent to allow some mutant progeny to survive. APOBEC3C (A3C) is a candidate because it is expressed in cells targeted by HIV-1, including PBMC, macrophages, and thymocytes [GA and GG contexts [We hypothesized that G-to-A mutation in ymocytes . A3C cancontexts ,12. A3C contexts ,9,24,25.contexts . Low-levvif-expressing NL4–3 molecular clones  contain combinations of pre- (W) and post-therapy (M) gag and pro genes from an HIV-1-infected patient who rapidly developed resistance to a protease inhibitor–containing regimen [In this study, we assessed the influence of A3C on G-to-A mutation and HIV-1 replication using multiple infectious viruses differing in Gag and/or Pro. The first group of isogenic  regimen . A seconenv and VSV-G-pseudotyped). After an additional 24 h in culture, cellular DNA and RNA were extracted. A3C mRNA was undetectable by RT-PCR (unpublished data).To determine whether A3C is responsible for inducing G-to-A mutation in HIV-1, we inhibited A3C expression in HIV-1-infected Magi cells and examined the viral sequences. Magi cells, which normally express A3C , were trGA versus GG contexts , which targets residues 107–125, and causes a partial knockdown of A3C mRNA expression . When trThus, specific inhibition of A3C expression by siRNA eliminated 210WW G-to-A mutations after infection of Magi B and 293env VSV-G pseudotyped NL4–3 and 210WW viruses produced by 293T cells. After an additional 24 h, DNA was extracted, amplified using the sensitive mutation assay for detecting G-to-A mutation in the viral population [GA context with 210WW showing significantly higher levels of mutation in comparison to NL4–3 , with only one to two G-to-A mutations each in the region analyzed. Of 47 NL4–3 clones, 43 were wild type and four were mutated (8%). Of 41 clones of Δvif virus, four were wild type and 37 were mutated (90%). We then determined the frequency of mutations leading to lethal premature stop codons: four of 45 (9%) contained premature stop codons consistent with the result of the blue and white β-galactosidase complementation assay and the non-attenuated replication kinetics of 210WW  210WW clones in the absence of drug selection but in none of the NL4–3 or Δvif clones. These results suggest that limited G-to-A mutation could be deleterious for some viral progeny, but surviving progeny may have greater genetic diversity that could allow adaptation to antiviral drugs.To investigate whether G-to-A mutation could enhance the capacity of patient-derived viruses to adapt to antiviral drugs, we screened mutated clones for drug-resistance mutations. We looked for reatment  and is cgag and pro genes from a patient who had developed resistance to ritonavir within 4 wk of treatment and that were cloned into a NL4–3 genetic background [GA as opposed to the GG context was seen in 210WW, 210WM, 210MW, and 210MM  that consists of reconstructed HIV-1 molecular clones derived from combinations of pre- and post-therapy ckground . These mckground ,29,30. Tckground . There and 210MM C. SuscepSS cells C. No mutTo determine whether A3C from the producer cell, target cell, or both induces G-to-A mutations, we measured mutations in A3C-positive and A3C-negative target cells from viruses derived from A3C-positive 293T cells directly, or after propagation for 7 d in A3C-negative cells (SupT1). Viral infection titers were normalized by supernatant Gag p24. Infections were carried out and G-to-A mutations were determined as described in GA and GG contexts: Both mutational patterns are widely observed in vivo with G-to-A mutation in the GA context more predominant than in the GG context [GA context reported by previous studies [GA context  supplemented with L-glutamine (2 mM), 20% FBS (Gemini Bioproducts), and IL-2  before and after infection.The cell lines H9, CEMSS, SupT1, Magi, and 293T were directly obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. Cell line identity was confirmed using DNA fingerprinting by PCR amplification with allele-specific primers (Research Genetics). PBMCs from leukocyte-enriched fractions of whole blood from HIV-1-seronegative donors were isolated by density-gradient centrifugation . Cells were stimulated with PHA-P  for 24 h, washed, and maintained at 2 × 10pNL4–3, obtained from the NIH AIDS Research and Reference Reagent Program , is a laboratory wild-type HIV-1 molecular clone. pM46I/L63P/V82T/I84V , and pL10R/M46I/L63P/V82T/I84V  are NL4–3-based infectious molecular clones with protease mutations introduced in vitro. pJF4A  is a full-length infectious molecular clone that is identical to pNL4–3 except for two nucleotide substitutions introduced by site-directed mutagenesis at nt 2754 (A-G) and 2755 (C-A) that render it resistant to ddC. pLAI.2  is a laboratory wild-type molecular clone.gag and pro gene segments from a patient who had developed resistance mutations to ritonavir 4 wk after treatment [gag, gag, and pro . The DNA polymerase I Klenow fragment (New England Biolabs) was used to fill in the ends and the plasmids were religated using the T4 DNA ligase (Promega). Pseudotyped VSV-G ΔEnv NL4–3 and 210WW viruses were produced by cotransfecting 293T with the pMD.G plasmid expressing the VSV-G envelope protein [NL4–3  protein  and the Δvif is a NL4–3 variant with three stop codons introduced into vif by directed mutagenesis .NL43 5 cells) with VSV-G Δenv pseudotyped viruses for 24 h.Virus stocks were prepared by Lipofectamine-mediated transfection of 293T cells. The clarified viral supernatants were quantified and normalized by HIV-1 Gag p24 ELISA (Perkin Elmer). DNase treatment of viral supernatant was carried out at 37 °C for 30 min with the equivalent of 10 units DNase (Sigma)/1 ml of the clarified crude virus. Spreading infections were monitored over time by accumulation of p24 Gag in the culture supernatants. Single-cycle infectivity was performed by challenging the cell lines (5 × 10PolyA+ RNA was isolated from cells with the QuickPrep Micro mRNA Purification Kit (Pharmacia). The open reading frames of A3B, A3C, A3F, and A3G were amplified by RT-PCR with the following primers: 3B forward 5′-ATGAATCCACAGATCAGAAATCC-3′ and reverse 5′-TCAGTTTCCCTGATTCTGG-3′; 3C: forward 5′-ATGAATCCACAGATCAGAAACC-3′ and reverse 5′-TCACTGGAGACTCTCCCGTA-3′; 3F: forward 5′-ATGAAGCCTCACTTCAGAAAC-3′ and reverse 5′-TCACTCGAGAATCTCCTGC-3′; 3G: forward 5′-ATGAAGCCTCACTTCAGAAACACAG-3′ and reverse 5′-TCAGTTTTCCTGATTCTGGAGAATGG-3′. The amplicons were cloned into pGEM-T easy vectors (Promega) and confirmed by sequencing. The A3C gene with HA tag sequence at its 3′ terminus was amplified from mRNA of H9 cells by RT-PCR, and the identity of the product was confirmed by sequencing. It was then cloned into pTT-IRES-GFP  for protein expression.The 293T or Magi cells were transfected with Lipofectamine 2000 transfection reagent (Invitrogen), according to the manufacturer's recommendations. The SupT1 cells were transfected by Amaxa electroporation technology with the program T29, according to the manufacturer's recommendations.DNA was extracted from infected cells with the DNA Easy kit (Invitrogen) and quantified by spectrometry. PolyA + RNA was isolated with QuickPrep Micro mRNA Purification kit (Pharmacia). Reverse transcription reaction was performed with random hexamers using SuperScript III reverse transcriptase (Invitrogen). An RT minus control was always performed in parallel to check for DNA contamination. PCR was performed with Easy-A High Fidelity PCR Cloning Enzyme (Stratagene). APOBEC reading frames  were amplified with primers specific for a single gene and listed above in the APOBEC plasmid construction section. The specificity of the primers was verified by sequencing the PCR products. The PCR products were sequenced by dye terminator cycle sequencing with Big Dye v3.1 (Applied Biosystems) or ET Terminators (Amersham Biosciences) on an ABI 3100 microcapillary sequencer. Raw sequence data was analyzed and aligned using Sequencher (Gene Codes).GA and GG context using the HIV-1 pro primers Hypa 10 and Hypa 11 in the first PCR and DP 16 and DP 17 in the second round PCR as described [This assay is designed to detect G-to-A viral mutated sequences present in a high wild-type background. A nested PCR procedure specific for a 230-nt region in the protease gene amplifies both G (wt) and A (mut) species in the The first round PCR uses primers specific for the mutated sequences (Hypa 10 and Hypa 11). The goal is to enrich the mutated sequences that are usually present in low concentration. The second round PCR uses primers that can amplify both mutated (if present from the first round PCR) and the input wild-type DNA (carried over from the first PCR) . Under tThe clonal analysis was performed using the neutral primers DP16–2 and DP17–2. The sequence of DP16–2 is 5′-TATCCTTTAGCTTCCCTCA-3′. The sequence of DP17–2 is 5′-TAATGGGAAAATTTAAAGTGCAG-3′. PCR products were cloned in pCR 4-TOPO (Invitrogen). The plasmids were then sequenced and analyzed for their G-to-A content.pol fragment that includes six tryptophan codons susceptible to become stop codons if G-to-A mutations occur. Viral DNA was amplified using the primers Nsi-Trp6 Fw: 5′-CCA ATGCATACTCCTAAAT TTAAATTACC CATACAAAA-3′ and Trp6 Rev Spe: 5′-GACTAGTTAAGGGAGGG GTATTGA-3′. The amplified DNA was phenol extracted, precipitated with ethanol, subjected to treatment with Nsi1 and SpeI, and then purified from a 1% agarose gel. This fragment encoding six Trp codons was cloned in frame into the lacZ fragment of the pGEM-T vector. For this purpose pGEM-T was digested first with Nsi1 and then with SpeI to obtain the linearized vector, which was purified from a 1% agarose gel and treated with alkaline phosphatase before ligation to the PCR products. Escherichia coli XL-1 Blue was transformed and plated onto minimal agar plates containing 8% X-Gal  and 20% IPTG . The plates were incubated at 37 °C for approximately 15 h. We validated the assay by first mixing 0%–5%–25%–50%–75%–100% of G-to-A mutated clones with wild-type clones. We then amplified each mix, cloned the PCR product, and counted the number of blue and white colonies. We confirmed that the number of blue and whites clones obtained after every specific mixture with the level of mutated sequences included in the mix. We also determined that the background of this assay was around 0.3%, which corresponded to the percentage of white colonies detected using 100% wild-type plasmids. The sequences of the white clones obtained in this specific mix revealed insertions or deletions interrupting the reading frame, and not stop codons. We also sequenced 20 white and 20 blue clones obtained from the other mix to verify their mutation content and the presence of stop codons. 20/20 blue clones were wild-type sequences and 20/20 white clones were mutated sequences. Blue and white clones were counted using the EagleSight (Stratagene) instrument and confirmed by manual counting.The genetic assay was carried out with a 111-nucleotide viral DNA containing a HIV-1 A3C protein expression in 293T transfected cells was detected by western blot with an HA-probe (Y-11) and HRP polyclonal antibody specific for the HA tag (Santa Cruz Biotechnology).siRNAs targeting A3C from positions 167–185 (siRNA 1) and 107–125 (siRNA 2) bases relative to the start codon were transcribed in vitro  from an oligo DNA template containing the T7 promoter synthesized by Invitrogen. The sequence of the scrambled RNA for both siRNAs was generated with the Promega siRNA target designer.4 cells/ml. After about 2 h, purified A3C siRNAs along with RNA oligo conjugated to FITC  were transfected into the cells with Lipofectamine 2000, according to the manufacturer's protocols. A scrambled siRNA was transfected separately as a control.For the siRNA transfection, 293T and Magi cells were seeded into six-well plates at a density of 10The efficiency of transfection was monitored by fluorescent microscopy at 24, 48, and 72 h after siRNA transfection. For flow cytometry analysis, the cells were analyzed with a Becton Dickinson FACS cytometer. Samples were counted and analyzed with FlowJo software (Tree Star). Non-transfected cells were used as a control. FITC-positive cells were sorted  for further analysis and infection.Figure S1gag, gag, and pro (nt 702-2563 relative to NL4–3). The dots indicate the nucleotides that match.The alignment includes the segment of the 5′ untranslated region upstream of (704 KB AI).Click here for additional data file.Figure S2(A) Gag amino acid alignment of 210WW, 210MM, NL4–3, and LAI viruses. Amino acid position relative to polyprotein start in HXB2: 1–513. Residue numbers for each line of sequence are provided. The dots indicate the residues that match.(B) Protease alignment of 210WW, 210MM, NL4–3, and LAI viruses. Amino acid position relative to polyprotein start in HXB2: 557–655.(500 KB AI).Click here for additional data file.Figure S3(A) 293T cells were transfected with siRNA 2 (50 nM) against A3C or with scrambled RNA. A FITC-conjugated oligo was cotransfected and FITC-positive cells were sorted 48 h following transfection. PolyA+ RNA was isolated, and RT-PCR was performed with primers specific for A3B (lanes 1 and 2), A3C (lanes 3 and 4), A3F (lanes 5 and 6), and A3G (lanes 7 and 8). β-Actin (lanes 9 and 10) was used as an internal control. A sample lacking template DNA (lane 11) was used as a negative control. Lane 12 is a molecular weight standard.(B) The transfected cells were infected with the VSV-G-pseudotyped 210WW and collected 24 h later. Viral DNA was amplified using the sensitive mutation assay and population sequencing was performed to analyze G-to-A mutation. The G-to-A mutation typically appeared as a mixture of G and A peaks at a given position, with G, the wild-type sequence, as the predominant peak. A change from G-to-A was considered a true mutation only if A represented at least 20% of the peak.(C) Base composition of 210WW plasmid, 210WW DNA infection of 293T + scrambled RNA, and 210WW DNA infection of 293T + siRNA 2 in the region amplified by the sensitive mutation assay.(786 KB AI).Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank/index.html) accession numbers for the genes and proteins mentioned in the text are: A3B (NM_004900), A3C (NM_014508), A3F (NM_001006666), A3G (NM_021822), LAI gag-pro (K02013), NL4–3 gag-pro (AF324493), 210MM gag-pro (EU100418), and 210WW gag-pro (EU100417).The GenBank (


N-linked sugars to an Asn-X-Cys motif in cystatin F and provide evidence that the mannose 6-phosphate sorting machinery is used to divert cystatin F from the secretory pathway and to mediate its uptake from extracellular pools. These studies identify a function for the oligosaccharides on cystatin F and raise the possibility that cystatin F might regulate proteases in transby secretion in an inactive form by one cell and subsequent internalization and activation by another cell.Cystatin F is a cysteine protease inhibitor that is selectively expressed in immune cells and unlike other cystatin family members is targeted to a significant extent to intracellular compartments. Initially made as an inactive glycosylated disulfide-linked dimer, cystatin F is converted to an active monomer by proteolytic cleavage following transport to the endosomal/lysosomal system. This active form of cystatin F targets cathepsin C/DPPI and probably other cathepsins in immune cells. We show that efficient targeting of cystatin F to the endocytic pathway is dependent not on its unique dimeric conformation but rather on its oligosaccharide chains. We demonstrate the unusual addition of  The cystatins are a family of naturally occurring cysteine protease inhibitors comprising at least 11 members . Some arThe cystatins are structurally well conserved and comprise a single α-helix associated with a 5-stranded β-sheet whose loops combine with residues from the N-terminus to form an ‘edge’ that engages the protease active site N-linked oligosaccharides. Finally, the amino acids present in the protease-binding loops and N-terminal region of cystatin F are not homologous with those of other family members, indicating that cystatin F might bind distinct protease targets  because of carbohydrate addition on a cryptic noncanonical (Asn-X-Cys) N-glycosylation site. Either this site, or a neighboring canonical site, sufficed to specify sorting of cystatin F to lysosomal compartments. We further show that cystatin F made and secreted by one cell can be internalized and activated within a different cell through mannose 6-phosphate (M6P)-mediated targeting. Thus, immune cells expressing cystatin F may regulate in transthe protease activity of bystander cells.Here, we investigate how cystatin F is retained within cells by analysis of cystatin F mutants that either cannot dimerize or lack specific oligosaccharide chains. We show that cystatin F molecules that cannot dimerize are still efficiently targeted to the endosomal system. Moreover, a mutant of cystatin F lacking both canonical N-glycosylation accounted for the resolution of distinct forms of cystatin F on SDS–PAGE To investigate the trafficking of cystatin F, we generated various mutant forms and analyzed these initially in the human kidney epithelial cell line 293T, which does not express cystatin F. The cells were transfected, and after 48 h, the relative amount of cystatin F in cell lysates (L) and in the culture medium (M) was assessed. Expression of wild-type cystatin F leads to significant secretion of the protein into the culture medium as we and others have previously reported ,13,16. NCystatin F dimerizes through two disulfide bridges involving Cys26 on one subunit and Cys63 on the other To confirm targeting to the endocytic pathway, transfected cells were fixed and stained with antibodies against cystatin F and a marker of the late endosomal/lysosomal compartment (CD63). As shown in Although covalent dimer formation was circumvented by substitution of the two cysteines involved in disulfide bond formation with serine, noncovalent dimer formation was still plausible. In other words, dimerization of C26,63S through noncovalent interaction may account for its lysosomal localization and secretion. To address this, wild-type and C26,63S cystatin F were subject to size exclusion chromatography on a column calibrated with standard proteins of known molecular weight and stokes’ radii. Elution of cystatin F was initially followed by absorption (280nm) of the eluted fractions and then confirmed by western blot . As expeN-linked sugar at a site homologous to Asn62 in humans. We generated mutant forms of human cystatin F where one or both Asn residues were altered to Ser  and transfected these into 293T cells. Culture medium and cell lysates were then analyzed for the presence of cystatin F and digested with PNGase to assess its glycosylation status. Similar to wild-type cystatin F, all mutant forms of cystatin F migrated as a single band following treatment with PNGase. Puzzling, however, the gel mobility of the N62S mutant in the absence of PNGase digestion was similar to that of wild type , the N61,62S mutant and the triple mutant were largely absent and found mostly in the medium . Consistecretion . These rN-linked glycans that become modified with M6P and use the cation-dependent or -independent M6P receptors  for targeting the endocytic pathway 32P] orthophosphate in 293T cells, and this label was removed by treatment with endo-β-N-acetylglucosaminindase H (EndoH), suggesting phosphorylation occurred on high mannose glycans. In contrast, [35S]-labeled cystatin F migrated faster following treatment with EndoH, but the label was not removed. Next, we infected mouse fibroblasts, including those lacking the CI-MPR, with a retrovirus expressing wild-type cystatin F. Whereas L929 cells, which express wild-type levels of both MPR, retained a proportion of cystatin F and converted it to the monomeric form, no intracellular cystatin F could be detected in L cells lacking the CI-MPRs . As expected, the cell lysates also contained cystatin F, much of which was converted to the active monomeric form  analysis to distinguish wild type (CD45.1+) from cystatin F null (CD45.2+) cells  are secreted from cells, suggesting that their primary function is to inactivate potentially dangerous proteases that have been secreted or lost from cells into the extracellular space thereby protecting from inflammatory reactions associated with aberrant release of these proteases –25. WhilThe mechanism by which cystatin F is retained within cells and matured in the endocytic compartment is addressed here. We demonstrate that dimerization is not necessary for intracellular cystatin F to be directed to the lysosomal pathway but rather a carbohydrate-dependent sorting mechanism operates. Our findings provide an explanation for why cystatin F is glycosylated when almost all other family members are not. Cystatin M/E is also glycosylated and is found in the lamellar granules of the cells of the stratum granulosum of the skin as well as in extracellular locations To our knowledge, cystatin F is the first lysosomal protease inhibitor shown to be M6P modified. This appears to be integral for proper targeting to the lysosomal compartment as judged by the failure of cells lacking MPRs to retain cystatin F. An earlier report identified cystatin F as one of several secreted proteins recovered from an MPR column, although it was not clear if the association was direct or indirect, for example through a binding partner modified with M6P N-linked oligosaccharides at Asn62 and 115, whereas murine cystatin F lacks the second sugar. Consistent with conservation of glycosylation at position 62 in mammalian cystatins  in the Asn-X-Cys motif is involved in the dimerizing disulfide bond, presumably recognition of the motif and addition of core oligosaccharide to Asn61 must occur before dimerization and loss of the free thiol. However, this has not been formally demonstrated.Human cystatin F carries two ystatins , we showthus far . Other pthus far ,21,33.Whin vivo.High concentrations of reducing agents are required to reduce the cystatin F dimer making a proteolytic mechanism for monomerization more likely and linking cystatin F activation to endosomal proteolytic activity rather than reducing potential ,16. BaseSequestration of cystatin F from the secretory pathway by an M6P/MPR mechanism raised the possibility that secreted dimeric cystatin F might be taken up into cells using the same system, as first described by Neufeld and colleagues for lysosomal enzymes + T cells often show heterogeneous levels of expression. Therefore, secretion and internalization of cystatin F in the closely confined in vivomilieu may even out these differences in expression. Cystatin F may be transferred efficiently from one cell to another not only because of its M6P tag but also because, unlike other cystatins found in the external milieu, it is unable to bind protease targets and therefore will not be sequestered by extracellular cysteine proteases. Several precedents exist for in vivoacquisition of lysosomal enzymes by one cell type following secretion by another. For example, cathepsin L-deficient thymocytes acquired this enzyme from surrounding epithelial cells rescuing a defect in antigen presentation and development of natural killer T cells in vivofrom the glomerular filtrate into kidney proximal tubule cells and reconstituted a cathepsin B deficiency in vivo, our results clearly indicate that this may take place. Conceivably, given that cathepsin C is one of the targets of cystatin F, this may be a way of attenuating granule protease-driven inflammatory reactions.These results raise the possibility that immune cells secreting cystatin F may be able to regulate protease activity in neighboring cells that either do not express cystatin F or express insufficient levels. Cystatin F expressing primary cell populations such as CD8N-linked M6P-modified sugar can be added to Asn61. Although the dimeric form is not crucial for intracellular targeting, it appears to be necessary for endocytic uptake. Moreover, dimerization ensures the latency of the inhibitor, which only becomes active following activation by proteases. The possibility that cystatin F is activated by the same protease systems that it inhibits is currently under investigation.In summary, our study sheds light on the unusual features of the cystatin F molecule. Glycosylation and more specifically M6P modification serve to target it to the endocytic pathway during biosynthesis, and for a proportion of molecules, by endocytosis from the external medium . In the Cell culture media were purchased from Gibco unless otherwise indicated. H-Gly-Phe-AMC and Z-Phe-Arg-AMC were purchased from Bachem. PNGase and EndoH were purchased from Roche. Rabbit polyclonal antibodies directed against full-length human cystatin F (FL Ab) or against an N-terminal peptide  have been described previously ml- glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. L cells devoid of the CI-MPR (CI-MPR KO), and those reconstituted with the CI-MPR (KO + CI-MPR) were kindly provided by T. Braulke  and were cultured in the same medium as L929 cells. Wild-type MEF, MPR knockout fibroblasts and those reconstituted with the human CI-MPR were generous gifts from R. Pohlmann  and B. Hoflack  and were cultured as described + T lymphocytes were cultured from the bone marrow and spleen, respectively, of C57BL/6 mice as described previously (+ (CD45.1+) mice were a kind gift from P. Crocker . Cystatin F knockout mice were generated under contract by Artemis Pharmaceuticals GmbH and will be fully described elsewhere.The cell lines 293T and L929 were cultured in DMEM supplemented with 10% fetal calf serum (FCS), 2 meviously ,47. C57BTCCTGCACGAACGACATGTTCTTGTTC and GAACAAGAACATGTCGTTCGTGCAGGAGTTGAACTTTTCAACACTG; N61,62S, CAGTGTTGAAAAGTTCAGCTCCTGCACGAACGACATGTTCTTGTTC and GAACAAGAACATGTCGTTCGTGCAGGAGCTGAACTTTTCAACACTG and N115S, GACTTCCAAACCAGCCACACCTTGAAGCAGACTCTGAGCTGCTAC and GTAGCAGCTCAGAGTCTGCTTCAAGGTGTGGCTGGTTTGGAAGTC. Underline denotes substituted residues. All mutagenesis was performed using the Quik Change Site-Directed Mutagenesis kit. Glycosylation of cystatin F was assessed by treating cell lysates, conditioned medium or immunoprecipitated cystatin F with PNGase to remove the entire glycan or EndoH to selectively remove high mannose forms of the glycans according to manufacturer’s guidelines.Mutagenesis was performed using plasmids encoding full-length human cystatin F cDNA in a pcDNA-DHFR expression vector as described previously mcitrate, pH 5.4, 150 mmNaCl, 2 mmMgCl2 and 1% Triton-X-100. Culture media and cell lysates were analyzed for cystatin F expression by western blot using antibodies directed against the whole protein (FL Ab) unless otherwise noted. Protein concentrations in cell lysates were quantified by Bio-Rad Protein Assay (Bio-Rad). Equivalent concentrations of cystatin F in culture medium were determined by western blot and normalized before internalization assays, as described below.293T cells were transfected with plasmids containing either full-length (wild type) or mutant cystatin F following manufacturer’s instructions . Human cystatin F was transduced into murine L929 and L cells using the retroviral vector pBMN-I-GFP  as described previously mH-Gly-Phe-AMC (cathepsin C) or 40 mmZ-Phe-Arg-AMC (cathepsin L) in assay buffer containing 150 mmNaCl, 2 mmethylenediaminetetraacetic acid, 5 mmDTT and 100 mmcitrate, pH 5.5. All fluorometric assays were performed on a FLUOstar Optima Fluorimeter (BMG) reading excitation at 360 nm and emission at 460 nm. Cathepsin activity in 293T cells (cathepsin C) and L929 cells (cathepsin L) are expressed as the rate of AMC release over time.Cathepsin activities were determined from 293T or L929 cell lysates following transfection or incubation with cystatin F-enriched media, as described below. Ten micrograms of postnuclear cell lysates was assayed using either 50 mmphosphate buffer and 0.15 M NaCl, pH 7.2. Protein was loaded in 0.8 mL of equilibration buffer in detergent-free conditions at a flow rate of 0.5 mL/min at room temperature on the ÄKTA fast protein liquid chromatography system (Amersham Pharmacia) collecting 1 mL fractions. The elution volumes of cystatin F and standard proteins (Ve) were converted into Kav using the following equation; Kav = (Ve − Vo)/(Vc − Vo), where Vo is the void volume as determined by blue dextran and Vc is the calculated geometric column volume (Vc = r2∏L). The Stokes’ radius of cystatin F was determined using a linear calibration plot of stokes’ radii versus (−log Kav)1/2Stokes’ radius determination of wild-type dimeric cystatin F, C26,63S cystatin F and standard proteins were performed by size exclusion chromatography using an Amersham HiLoad 26/60 Superdex 75 column (Amersham Pharmacia). The column was pre-equilibrated in 50 m35S] labeling, transfected 293T cells were preincubated in Met/Cys-free media (Sigma), labeled for 45 min with 0.4 mCi/mL [35S] Met/Cys  and chased for 3 h in complete RPMI-medium containing 100 μg/mL methionine and 500 μg/mL cysteine. For [32P] orthophosphate labeling, transfected 293T cells were incubated for 30 min at 37°C in phosphate-free MEM containing 10% dialyzed FCS and for another 4 h in the same medium supplemented with 0.5 mCi/mL [32P] orthophosphate (Perkin Elmer). Culture medium was removed and cells were washed extensively and then lysed in buffer containing 1% Triton-X-100. Radiolabeled cystatin F from culture medium and lysates were immunoprecipitated using cystatin F antibodies (FL Ab) as described 125I-labeling experiments, 20 μg of purified recombinant cystatin F was labeled with 0.5 mCi [125I]-Na (Amersham) using the IODO-GEN reagent (Pierce) following manufacturer’s instructions.For -labeled cystatin F, L929 cells were incubated with 500 μg/mL [125I] cystatin F in the presence or absence of 10 mmM6P at 4°C. The cells were washed three times with PBS to remove unbound protein and incubated at 37°C for 30 min to allow for internalization. Before lysis, cystatin F remaining on the plasma membrane was removed by acid stripping . Control cells were maintained at 0°C throughout. Percent internalization was determined by dividing the acid stable fraction following acid washing at 30 min from the acid labile fraction at 0 min. Primary murine CD8+ T lymphocytes (CTL) and dendritic cells were isolated from the spleen and bone marrow of C57Bl/6 mice as described  and CD45.2 (APC) were detected by flow cytometry using directly conjugated antibodies. Internalization of cystatin F was determined by surface staining of either CD45.1 (APC) or CD45.2 (APC) followed by intracellular staining for cystatin F (FL Ab) using the Fix & Perm cell permeabilization kit .To follow the internalization of cystatin F, L929 cells were cultured at 5 × 10escribed ,47. Priml-lysine-coated coverslips for 48 h before staining. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton-X-100 and costained with rabbit anti-cystatin F (FL Ab), and either mouse anti-CD63 or sheep anti-TGN46 in the presence of 1% (w/v) BSA as described l-lysine-coated coverslips for 18 h and treated with cystatin F-containing conditioned media or control media as described above. Primary antibodies were detected using Alexa-488 or Alexa-594-conjugated anti-rabbit (Invitrogen), TR-conjugated anti-sheep (pseudo-colored green) (Jackson Laboratory) and Alexa-488 anti-mouse (Invitrogen) secondary antibodies. Stained cells were mounted on glass slides and imaged on a LSM 510 META confocal microscope using a 100× PlanFluar/NA 1.45 objective and lsm510 software (Carl Zeiss Inc.) followed by processing with Photoshop (Adobe).Transfected 293T cells were grown on poly-


E. coli have been unsuccessful.Caspase-3, a principal apoptotic effector that cleaves the majority of cellular substrates, is an important medicinal target for the treatment of cancers and neurodegenerative diseases. Large amounts of the protein are required for drug discovery research. However, previous efforts to express the full-length caspase-3 gene in E. coli and easily purified by affinity chromatography, to levels of 10–15 mg from 1 L of E. coli culture, and readily activated by thrombin digestion. Kinetic evaluation disclosed that thrombin digestion enhanced catalytic activity (kcat/KM) of the precursor proteins by two orders of magnitude.Overproducers of thrombin-activatable full-length caspase-3 precursors were prepared by engineering the auto-activation sites of caspase-3 precursor into a sequence susceptible to thrombin hydrolysis. The engineered precursors were highly expressed as soluble proteins in E. coli. The precursor protein was easily purified and activated through specific cleavage at the engineered sites by thrombin, generating active caspase-3 in high yields.A novel method for a large-scale preparation of active caspase-3 was developed by a strategic engineering to lack auto-activation during expression with amino acid sequences susceptible to thrombin, facilitating high-level expression in  Multicellular organisms maintain homeostasis through a balance between cell proliferation and death. Apoptosis is a controlled cell death process crucial in a wide range of biological activities, such as normal cell turnover, immune system, embryonic development, metamorphosis, and chemical-dependent cell death . Neuronac released from mitochondria (intrinsic apoptosis pathway). Activated caspases subsequently convert procaspase-3 and -7 to fully active enzymes by specific proteolytic cleavage. Caspase-6 is activated after caspase-3. The three former caspases are known as apoptotic initiators, whereas the latter three are known as apoptotic effectors or executioners. Caspase-3  is the first reported apoptotic effector, and cleaves the majority of cellular substrates in apoptotic cells [Caspases, a family of cysteine proteases, play crucial roles in apoptosis, pro-inflammatory cytokine activation, and presumably, keratinocyte differentiation ,7. Folloic cells -14. Caspic cells ,15-18.E. coli, full-length caspase-3 (procaspase-3) undergoes presumable autoprocessing to yield the appropriate subunits characteristic of the active enzyme with only a marginal expression level, probably due to its cytotoxicity [Pichia pastoris [E. coli. The most frequently employed large-scale caspase-3 preparation method includes separate expression of the two insoluble domains in E. coli and subsequent refolding of the two combined domains for the active enzyme [E. coli. The precursor protein was activated through specific cleavage at the engineered sites by thrombin, generating active caspase-3. Furthermore, this protein efficiently digested endogenous caspase-3 substrate.Drug discovery research, including screening chemical libraries as well as structural and kinetic analyses, requires large-scale caspase-3 preparation. When expressed in toxicity . While fpastoris , the proe enzyme . This meE. coli hampers large-scale preparation with conventional methods [II) was depleted of 28 amino acids at the N-terminus of procaspase-3 (I), while Δ28/175TS-caspase-3 (III) contained LVPRGS instead of wild-type residues at positions 172–177 of construct II , LVPRGS mutations were introduced at two sites (positions 25–30 and 172–177) of wild-type caspase-3 (I). In 28TS/180TI-caspase (V), the LVPRGS motif replaced residues 25–30, and was inserted between Asp180 and Asp181, along with a D175A mutation to prevent auto-cleavage. Since caspase-3 activation occurs mainly via cleavage at Asp 28 and 175, constructs were designed to ensure cleavage by thrombin at the corresponding sites (between R and G of LVPRGS). The LVPRGS motif was inserted between Asp180 and 181 in 28TS/180TI-caspase (V) to maintain the structural integrity of the sequence encompassing residues 165–174 after cleavage, in view of the crystal structure of active caspase-3, which discloses tight interactions between this region and the other dimer within a heterotetrameric arrangement [Caspases are translated in cells as inactive precursors, which are sequentially activated following internal or external cell death signals. Apoptotic initiators, such as caspases-8, -9 and -10, activated procaspases-3 and -7 by specific hydrolysis, thereby triggering cell death by specific apoptotic executioners ,7. Durin methods . We plan II Fig. . In the angement .E. coli BL21 Rosetta (Novagen), and purified by cobalt affinity chromatography. Compared with wild-type, engineered proteins III, IV and V were highly expressed , respectively. However, the C163S caspase-3 precursor did not display significant cleavage under similar conditions (lanes 7 and 8), indicating that fragmentation of III and IV is due to specific thrombin activity at the mutated sites. Notably, the cleavage sites of protein V during expression were distinct to those of thrombin. These specific sites remain to be identified. Since engineered caspase-3 precursors have His6-tag at their C-terminus, the thrombin can be easily removed by cobalt affinity chromatography after activation of caspase-3 (data not shown). Next, the size and activity of activated engineered caspases were compared with endogenous active caspase-3 using PAGE analysis. As shown in Fig. VI and VIII cleaved PARP, a well-known substrate of caspase-3, at the similar level to wild-type caspase-3 . The engineered protein precursors III and IV containing LVPRGS exhibited negligible catalytic activity (no higher than 0.1% that of the wild-type enzyme). Thrombin digestion of proteins III and IV resulted in a drastic increase in catalytic activity , in agreement with a previous report [kcat/KM values representing the substrate specificities of IV and VII were determined as 1.7 × 102 M-1s-1 and 2.9 × 104 M-1s-1, respectively. The kcat/KM value of protein V containing an insertion of the LVPRGS motif between D180 and D181 was calculated as 6.7 × 104 M-1s-1, which was not significantly increased upon thrombin digestion. The data indicate that protein V is already activated during expression. This finding is supported by SDS-PAGE analysis results showing four smaller bands instead of the 30 kDa protein . Amino acid sequences of the engineered proteins were numbered based on wild-type procaspase-3.Primer synthesis and DNA sequencing were performed by Bioneer  and Genotech , respectively. The primers used for plasmid construction are listed in Table CATATGGAGAACACTGAAAACTCAG-3' (P1) containing an NdeI site (marked in bold type letters), and reverse primer, 5'-CCGCTCGAGGTGATAAAAATAGAGTTCTTTTGT-3' (P2), with a XhoI site (underlined), and inserted into the corresponding sites of the pET21a plasmid (Novagen). The resulting construct was designated plasmid I. A Δ28 caspase-3 deletion mutant devoid of the N-terminal 28 amino acids of wild-type caspase-3 (I) was prepared using the forward primer, 5'-GGGAATTCCATATGTCTGGAATATCCCTGGACAACAGT-3' (P3) with an NdeI site (marked in bold type letters), and reverse primer, P2. The resulting DNA was inserted into the pET21a expression vector, and denoted plasmid II. For preparation of Δ28/175TS caspase-3, the 175TS mutant of plasmid I was initially prepared using the megaprimer PCR method with slight modifications [P1, and reverse primer, 5'-ATCAACGCTGCCGCGCGGCACCAGGCCACAGTCCAGTTCTGTACCACG-3' (P4). C-terminal megaprimer DNA substituted at positions 172–177 (IETDSG) with LVPRGS was prepared using the forward primer, 5'-TGTGGCCTGGTGCCGCGCGGCAGCGTTGATGATGACATGGCGTGTCAT-3', (P5) and reverse primer, P2. The thrombin recognition sites in P4 and P5 are italicized. Each megaprimer was extended by PCR, using the other as a template. The resulting DNA was inserted into pET21a using the NdeI and XhoI restriction sites, and designated plasmid IX. The Δ28/175TS caspase-3 mutant of plasmid IX was prepared using forward P3 and reverse P2 primers. The amplified product was inserted into the NdeI and XhoI sites of pET21a, and the construct denoted plasmid III. For generating the 28TS/175TS mutant caspase-3 construct in plasmid IX, the megaprimer with substitutions at positions 24–30 (LVPRGS in place of ESMDSG) was prepared using the forward primer, P1, and reverse primer, 5'-ACTGTTGTCCAGGGATATGCTGCCGCGCGGCACCAGGCTTCCATGTATGATCTT-3' (P6). The thrombin recognition site in P6 is italicized. The resulting megaprimer, in combination with P2, was applied for the next PCR reaction to prepare 28TS/175TS caspase-3 DNA, which was cloned into pET21a, and designated plasmid IV. The 28TS/D175A/180TI caspase-3 construct was generated in plasmid I using a combination of megaprimer PCR and the QuikChange site-directed mutagenesis kit (Stratagene). To prepare the 28TS caspase-3 construct in plasmid I, a corresponding megaprimer produced using the P1 forward and P6 reverse primers was employed together with P2. The resulting construct was designated plasmid X. A 28TS/D175A caspase-3 construct in plasmid X was prepared with the QuikChange kit using the forward primer, 5'-GACTGTGGCATTGAGACAGCGAGTGGTGTTGATGAT-3' (P7), and reverse primer, 5'-CAGATCATCAACACCACTCGCTGTCTCAATGCCACA-3' (P8), and designated plasmid XI. The Ala mutation sites in P7 and P8 are italicized. The thrombin recognition site was successfully inserted into the plasmid XI background with the megaprimer method. N- and C-terminal megaprimers were prepared using the forward primer, P1, and reverse primer, 5'-TATTTTATGACACGCCATGTCGCTGCCGCGCGGCACCAGATCATCAACACCACT-3' (P9), as well as the forward primer, 5'-ACAGACAGTGGTGTTGATGATCTGGTGCCGCGCGGCAGCGACATGGCGTGTCATAAA-3' (P10) and reverse primer, P2, respectively. The thrombin recognition sites are italicized. Each prepared megaprimer was extended by PCR, using the other as a template, and the resulting product inserted into the NdeI and XhoI sites of pET21a (designated plasmid V).Wild-type caspase-3 gene was amplified using the forward primer, 5'-GGGAATTCications . N-termiE. coli BL21 Rosetta cells (Novagen) containing the specified expression plasmids were grown at 37°C in LB medium until an A600 of 0.6–0.8. Engineered caspase-3 precursors were expressed by adding 1 mM IPTG at 18°C for 18 h. Cells were harvested, washed with buffer A , and lysed by ultrasonication. After centrifugation , the supernatant was incubated with a cobalt affinity resin  on a rocker at 4°C for 1 h, and washed with buffer A containing 10 mM imidazole. Proteins were eluted from the metal affinity resin with buffer A supplemented with 100 mM imidazole. Following dialysis against 20 mM Tris, pH 7.5, 250 mM NaCl, 5% glycerol, and 1 mM dithiothreitol, caspase-3 precursors were concentrated to 2 mg/ml and stored at -80°C.2 at 4°C. Digestion was performed for 18 h, and monitored by 15% SDS-PAGE.Caspase-3 precursors were activated by digestion with bovine thrombin. Briefly, 100 μg of each precursor protein was incubated with 1 NIH unit of thrombin in 50 mM Tris, pH 7.5, 250 mM NaCl, 5% glycerol, 0.05% β-mercaptoethanol, 3 mM CaClCaspase-3 precursors were treated with wild-type caspase-3 to test autocleavage during activation. Briefly, 100 μg of each precursor protein was incubated with 1 μg of wild-type caspase-3 in 50 mM Tris, pH 7.5, 250 mM NaCl, 5% glycerol, 0.05% β-mercaptoethanol, and 5 mM DTT at RT. Cleavage was performed for 18 h, and monitored by 15% SDS-PAGE.pNA. Briefly, caspase-3 was activated in reaction buffer  for 18 h before use. The appropriate amount of activated caspase-3  was added to the substrate solution at a series of final concentrations . The p-nitroanilide released by the caspase reaction was monitored at 405 nm using a DU 800 UV-VIS Spectrophotometer (Beckman Coulter). Km and Vmax values were obtained using Hyper32 version 1.0.0 , and kcat values calculated from Vmax and the used enzyme concentration.The activity of each caspase-3 protein was measured using the colorimetric substrate, Ac-DEVD-fmk were used [Human promyelocytic leukemia HL60 cells were cultured in RPMI. If necessary, apoptosis was induced by treatment of the cells with 100 μM etoposide for 12 h. Cell lysates (60 μg) were incubated with engineered caspases at 37°C for 2 h. Then, the cleavage of endogenous PARP was determined by immunoblot analysis with an anti-PARP antibody. For negative control, cell lysates treated together with engineered caspases and z-DEVD-ere used ,27.HJK, SGP and KHB participated in the experimental design, carried out the molecular genetic and biochemical experiments, participated in data interpretation and helped draft the manuscript. YML, YJJ, KP, MJ and MK conceived the study. SJC directly supervised the project, participated in its experimental design and data interpretation and was responsible for writing the manuscript. All authors have read and approved the manuscript.


The Gag capsid (CA) is one of the most conserved proteins in highly-diversified human and simian immunodeficiency viruses (HIV and SIV). Understanding the limitations imposed on amino acid sequences in CA could provide valuable information for vaccine immunogen design or anti-HIV drug development. Here, by comparing two pathogenic SIV strains, SIVmac239 and SIVsmE543-3, we found critical amino acid residues for functional interaction between the N-terminal and the C-terminal domains in CA.206-216 (IINEEAADWDL) epitope-specific cytotoxic T lymphocytes (CTLs) were previously shown to respond to SIVmac239 but not SIVsmE543-3 infection. A mutant SIVmac239, SIVmac239Gag205E, whose Gag205D is replaced with Gag205E showed lower replicative ability. Interestingly, however, SIVmac239Gag205E passaged in macaque T cell culture often resulted in selection of an additional mutation at Gag residue 340, a change from SIVmac239 valine (Gag340V) to SIVsmE543-3 methionine (Gag340M), with recovery of viral fitness. Structural modeling analysis suggested possible intermolecular interaction between the Gag205 residue in the N-terminal domain and Gag340 in the C-terminal in CA hexamers. The Gag205D-to-Gag205E substitution in SIVmac239 resulted in loss of in vitro core stability, which was recovered by additional Gag340V-to-Gag340M substitution. Finally, selection of Gag205E plus Gag340M mutations, but not Gag205E alone was observed in a chronically SIVmac239-infected rhesus macaque eliciting Gag206-216-specific CTL responses.We first examined the impact of Gag residue 205, aspartate (Gag205D) in SIVmac239 and glutamate (Gag205E) in SIVsmE543-3, on viral replication; due to this difference, GagThese results present in vitro and in vivo evidence implicating the interaction between Gag residues 205 in CA NTD and 340 in CA CTD in SIV replication. Thus, this study indicates a structural constraint for functional interaction between SIV CA NTD and CTD, providing insight into immunogen design to limit viral escape options. One of the characteristics of human immunodeficiency virus (HIV) is to induce persistent viral replication resulting in AIDS progression. HIV has enormous capacity to mutate and escape from host immune recognition, driving genetic diversification of the circulating viruses -3. The GVirus-specific cytotoxic T-lymphocyte (CTL) responses play a central role in the control of immunodeficiency virus infection -12. CTLs90-120-Ia ) containing the entire gag region was amplified from the RNA by RT-PCR and sequenced to determine dominant sequences as described previously [HSC-F cells were coinfected with two SIVs at a ratio of 1:1 or 1:4, and the culture supernatants harvested every other day were used for RT assays. On day 6, the supernatant was added to fresh HSC-F cells to start the second culture. Similarly, on day 12 after the initial coinfection, the second culture supernatant was added to fresh HSC-F cells to start the third culture. RNAs were extracted using the High Pure viral RNA kit  from the initial culture supernatant on day 6 and from the third culture supernatant on day 18 post-coinfection. The fragment , HIV-1 1M9C[1A8O), and he1A8O[3H47) were ta1A8O[3H47. Three-d1A8O[3H47,51. We o1A8O[3H47. The fin1A8O[3H47 combined1A8O[3H47. Physica1A8O[3H47.Detergent treatment of wild-type and mutant SIV particles was performed essentially as described previously . BrieflyThe authors declare that they have no competing interests.NI and TM designed the study. NI, HT, and AR performed virological analyses in vitro. MY and HS performed structure modeling analyses. HY and MK examined viral genome sequences. NI and TM analyzed the data and wrote the paper. All authors read and approved the final manuscript.


To tackle this issue, we generated mice with global 2CR5-HT deficiency (2C null) and mice with 5-HT2CRs re-expression only in pro-opiomelanocortin (POMC) neurons (2C/POMC mice). We show that 2C null mice predictably developed hyperphagia, hyperactivity, and obesity and showed attenuated responses to anorexigenic 5-HT drugs. Remarkably, all these deficiencies were normalized in 2C/POMC mice. These results demonstrate that 5-HT2CR expression solely in POMC neurons is sufficient to mediate effects of serotoninergic compounds on food intake. The findings also highlight the physiological relevance of the 5-HT2CR-melanocortin circuitry in the long-term regulation of energy balance.Drugs activating 5-hydroxytryptamine 2C receptors (5-HT This is best demonstrated by the hyperphagia and obesity in mice with global 2CR5-HT deficiency   system, including the 5-HTficiency . 5-HT2CR (d-Fen) , a drug  (d-Fen) . Nonethe2CR gene . Furthersyndrome . CollectPro-opiomelanocortin (POMC) neurons in the arcuate nucleus of hypothalamus (ARC) produce α-melanocyte-stimulating hormone (α-MSH), an endogenous agonist of melanocortin 4 receptors (MC4Rs) . The cen2CRs and mediate the effects of 5-HT2CRs on feeding behavior. Particularly, POMC neurons in the ARC coexpress 5-HT2CRs  and mice with 5-HT2CRs re-expressed specifically and only in POMC neurons (2C/POMC). Taking advantage of these unique genetic models, we directly tested the hypotheses that 5-HT2CRs in POMC neurons mediate the anorexigenic effects of 5-HT agents and that the re-establishment of the 5-HT2CR-melanocortin circuitry is sufficient to rescue the obese phenotypes caused by global 2CR5-HT deficiency.To directly address these questions, we generated mice with global deficiency of 2C null (2CRloxTB 5-HT) mice were generated by inserting a loxP-flanked transcription blocker (loxTB)   into theCre mice  producedombinase A.2CR5-HT mRNA, we found that expression of 2CR5-HT mRNA was disrupted in the cerebral cortex, whole hypothalamus, ARC, and brainstem of 2C null mice. In 2C/POMC mice, 2CR5-HT mRNA was re-expressed only in samples of the whole hypothalamus or brainstem. Further, we found specific re-expression in samples of microdissected arcuate nucleus, where POMC neurons are located  mice. It should be noted that POMC is also expressed by a small population of neurons in the nucleus of solitary tract (NTS) in the brainstem  (2C null mice (2CR5-HT knockout mice that showed attenuated anorexigenic effects in response to both d-Fen and mCPP (To further confirm that our ull mice A. Similae (mCPP) , decreasull mice B. These and mCPP .2CRs in POMC neurons is sufficient to restore the anorexigenic responses to 5-HT drugs, we assessed 2C/POMC mice, whose 5-HT2CRs were re-expressed selectively in POMC neurons. Notably, the anorexigenic effects induced by both d-Fen and mCPP were restored to levels that were indistinguishable from those in WT mice -fed 2C null mice compared to their WT littermates at 11 weeks .In addition to hyperphagia and obesity, another characteristic phenotype of mice with ung mice . Althougout mice , our 2C chow see . This ischow see . Notablychow see . Therefo2CRs, has been one of the priorities in the obesity field because of the potent effects of serotoninergic agents on feeding. Indeed, drugs generally influencing 5-HT bioavailability, such as sibutramine, are among the few approved current pharmacotherapies for obesity. However, due to the widespread distribution of 5-HT2CRs in the brain  mice were created by inserting a loxP-flanked transcriptional blocking cassette (loxTB) (X-linked 2CR5-HT gene (2CR5-HT gene (Mouse RPCI.22 BAC clone 32 B 10) . The 2CRloxTB 5-HT gene was placed in a pGEM-T plasmid using the following PCR template (left homology arm [TAATTATAAACACTATTATACACAGAGATTTCCAATTTATTAACTAAAATTACTTTCAAAGTCATGCCTTACCGG TCCAACGCGTTGGATGCATAG] and right homology arm [GAGCTTAAAACATTAGCAATCAGCAGCAAAGATGCA AATATTCCTCAACCGTGTCAGTACTATAGACTAAACCGGTGTATTTTCTCCTTACGCATC]) and then linearized. The final targeting construct, which consisted of the 2CR-loxTB5-HT flanked by 4 kb 2CR5-HT homology arms, was electroporated into ES cells, and correct targeting was confirmed by Southern blot analyses. For Southern blot analysis, ES cells were digested with NCO1 for 4 hr at 37°C. Primers were labeled with Megaprime DNA labeling kit. The right probe was 202 bp and amplified using TCACAATTGAAGACATTTCCTG and TGTTGGGTTTCTTTGTGGTTCTC. Detection of a nontargeted ES cell would produce an 11,095 bp band, and a target ES cell would produce a 6718 bp band. The left probe was 265 bp and amplified using TGTGAGAAATGCTGCAGGAATAAA and TTGGGAAGTTTTGTTTTTGTGGA. Detection of a nontargeted ES cell would produce an 11,095 bp band, while a targeted ES cell would produce a 6570 bp band. Targeted ES cells were injected into blastocysts, and, after germline transmission was established, the chimera carrying the recombinant allele was crossed onto a C57BL/6J background. (loxTB)  between 2CR gene A. The ta2CRloxTB 5-HT heterozygous mice (backcrossed to the C57BL/6J background for eight generations) were crossed with male POMC-Cre hemizygous mice (backcrossed to the C57BL/6J background for five generations), and only male offspring were collected for the experiments described below. These male mice carry one of the following genotypes: wild-type (WT), hemizygous 2CRloxTB 5-HT (2C null), hemizygous 2CRloxTB 5-HT and hemizygous POMC-Cre (2C/POMC), as well as hemizygous POMC-Cre (POMC-Cre). All mice were housed in their home cages with food and water available ad libitum in a temperature-controlled room with 12 hr light, 12 hr dark cycle in the animal facility of University of Texas Southwestern Medical Center at Dallas.Female For the HFD-feeding study, mice were group housed (two to five mice per cage) and provided with a 42% fat diet  from 5 weeks of age, and body weight was measured weekly. Another HFD cohort was individually housed, and a similar growth pattern was observed as in the group-housed cohort. Food intake was measured weekly from mice that were individually housed.2C null, 2C/POMC, and POMC-Cre mice. Total RNA was extracted with the QIAzol Lysis Reagent  and reverse-transcribed to cDNA using SuperScript II First-Strand cDNA Synthesis kit  according to the manufacturer's instructions. Expression of 2CR5-HT was detected in all the tissues with primers that span exon 3 and 4 of 2CR5-HT gene . As positive controls, expression of NPY was detected in the cerebral cortex, whole hypothalamus, and ARC with NPY primers ; POMC was detected in the brainstem and pituitary using POMC primers . Due to the low level of 2CR5-HT mRNA in the ARC of 2C/POMC mice, the first PCR products of ARC tissues from all mice were purified and subjected to the second PCR amplification using the same primers. 2CR5-HT PCR amplicons from the hypothalami of WT, 2C/POMC, and POMC-Cre mice were gel purified and sequenced in the local core facility, and we confirmed that 2CR5-HT mRNA re-expressed in 2C/POMC hypothalamus was identical to those of WT and POMC-Cre hypothalami.The cerebral cortex, whole hypothalamus, ARC, brainstem, and pituitary were taken from WT, Body composition was measured with the Bruker minispec mq10 MRS system. For fat distribution, mice were anesthetized with 1% isoflurane inhalation and then the trunk  of each mouse was scanned at an isotropic voxel size of 93 μm  using the eXplore Locus micro-CT scanner . Three-dimensional images were reconstructed from two-dimensional grayscale image slices and visualized using Microview Software . Density values for soft tissue and bone were calibrated from a phantom  containing air bubble, water, and hydroxyl apatite rod. The separation of fat regions was obtained from the appropriate grayscale value . The abdominal muscular wall was used as the differentiation line to separate visceral adipose tissue from subcutaneous adipose tissue. The contour lines were drawn around the viscera and three-dimensional ROI was generated. The visceral fat was determined from the histogram of these segmented viscera using the same thresholds. Subcutaneous fat was obtained by subtracting visceral fat from the total body fat.2CRs in POMC neurons are sufficient to mediate anorexigenic effects of 5-HT compounds, 4-month-old HFD-fed mice were individually housed and weight matched. After overnight fasting, the mice received intraperitoneal injections of saline, d-Fen (3 mg/kg), or mCPP (5 mg/kg). HFD was represented to their cages 30 min after the injections. Food intake in the next hour was measured and normalized by their body weight. Each mouse was tested with all three treatments , administered in a counterbalanced order, with a minimum of 5 days between the treatments. Data were presented as mean ± SEM. Differences among genotypes were determined by two-way ANOVA analysis, followed by the post hoc Student-Newman-Keuls test.To assess whether 5-HTFed and/or fasted serum leptin levels were measured in HFD-fed mice at 4 or 8 months of age. At month 4, food was removed from the home cages for 2 hr (fed condition) or for overnight (fasted condition), and blood was collected from the saphenous vein. At month 8, trunk blood was collected 2 hr after mice were deprived of food. Leptin levels were measured using the leptin ELISA kit  according to the manufacturer's instruction.2 consumption and CO2 production were measured to determine the energy expenditure. In addition, meal patterns were determined continuously by integration of weighing sensors fixed at the top of the cage from which the food containers have been suspended into the sealed cage environment. Meals were defined as food intake events with a minimum duration of 60 s and a break of 300 s between food intake events.Physical activity, energy expenditure, and meal patterns were monitored using a combined indirect calorimetry system  . After aData were presented as mean ± SEM. Statistical analyses were carried out with SigmaStat software. Unless otherwise mentioned, all data were analyzed by one-way ANOVA analysis, followed by the post hoc Student-Newman-Keuls test when the ANOVA analysis indicated significant differences. p < 0.05 indicated statistical significance.


RIG-I is a pivotal receptor that detects numerous RNA and DNA viruses. Thus, its defectiveness may strongly impair the host antiviral immunity. Remarkably, very little information is available on RIG-I single-nucleotide polymorphisms (SNPs) presenting a functional impact on the host response.i) a frameshift mutation (P229fs) that generates a truncated, constitutively active receptor and (ii) a serine to isoleucine mutation (S183I), which drastically inhibits antiviral signaling and exerts a down-regulatory effect, due to unintended stable complexes of RIG-I with itself and with MAVS, a key downstream adapter protein.Here, we studied all non-synonymous SNPs of RIG-I using biochemical and structural modeling approaches. We identified two important variants: (229fs and S183I SNPs as major functional RIG-I variants and potential genetic determinants of viral susceptibility. This work also demonstrated that serine 183 is a residue that critically regulates RIG-I-induced antiviral signaling.Hence, this study characterized P Among all viral components that trigger the antiviral screen of the host, nucleic acids have been viewed as the most important Via its amino-terminal tandem Caspase Recruitment Domains (CARDs), RIG-I homocomplexes relay a signal by binding MAVS , an adapter protein that mediates CARD-dependent interactions with RIG-I. This signaling complex further activates the transcription factors NF-κB and interferon regulatory factor (IRF)-3 to ultimately upregulate the expression of pro-inflammatory and antiviral mediators and the subsequent induction of adaptive immune responses Current knowledge posits RIG-I as a particularly critical surveillance molecule that detects numerous viruses such as the human pathogens influenza and hepatitis C (HCV) viruses Importantly, the receptor function of RIG-I is non-redundant, as confirmed by knock-out studies i.e. R7C, S144F, S183I, T260P, I406T, D580E, F789L (229fs as it includes the first 229 residues (instead of 925 residues in the WT RIG-I protein) followed by 4 unintended residues , in agreement with the fact that RIG-I is especially prominent in signaling pathways leading to type I IFNs 260P, I406T or F789L mutants was similar to that induced by WT RIG-I and lower in cells expressing the R7C, S144F, or D580E RIG-I . With regard to P229fs RIG-I, we observed a salient constitutive IFN-β and NF-κB reporter activities, at a level well above that induced by the full-length form of WT RIG-I , IFN-β and NF-κB reporter activities elicited by the viral mimetic poly(I∶C), in agreement with previous studies that have shown that poly(I∶C) is a potent RIG-I stimulus (p<0.0001). To confirm the pathophysiological relevance of the above findings, we sought to address the responsiveness of the mutant proteins to viral infection. We clearly demonstrated that S183I mutation had a deleterious effect on RIG-I antiviral activity as it drastically reduced IFN-β and NF-κB-mediated responses triggered by intact, replicative Sendai or influenza A viruses . Nevertheless, as S183I SNP uniquely resulted in the strongest inhibition of RIG-I-dependent signaling induced by all stimuli, we decided to focus the rest of our study on this specific mutation. Thus, the clear loss-of-function effect of S183I RIG-I SNP was confirmed by measuring the secretion of IL-8  was not down-modulated by S183I RIG-I, in comparison with WT RIG-I  and BEAS-2B cells (not shown). This loss-of-function effect was confirmed by measuring the secretion of endogenous mediators in supernatants of HEK 293T cells  domain, we next postulated that this paradoxical enhancing effect of S183I SNP might be due to CARD structure alteration. Using sequence alignment information . Additionally, comparison of the movement of secondary structure elements over the course of the simulation suggests the S183I structure is less flexible than the WT structure  or infected by Sendai virus . We also observed that this S183I 2CARD mutant reduced IFN-β activity of WT 2CARD by ∼50% when transfected at a 1∶1 ratio and up to 70% at a fourfold excess of transfected plasmid DNA  or tandem 2CARD or Sendai virus, consistently with the primary role of RIG-I in type I-IFN-inducing antiviral signaling pathways Phenotypes of several heritable disorders are linked to missense mutations in single alleles. In some cases, the mutant protein exhibits a regulatory effect whereby heterozygous co-expression of mutant and WT gene has a deleterious consequence, relatively to the case in which two WT alleles are expressed 55I) The efforts conducted by international consortiums – such as the HapMap Project and Perlegen – to identify and characterize the levels of polymorphic variation in humans has yielded an ever-growing list of SNPs 229fs as a SNP which results in a truncated constitutively active RIG-I. This finding is particularly important as it suggests that individuals carrying such mutation may constitutively produce exaggerated amounts of antiviral and pro-inflammatory mediators. Conversely, in agreement with a very recent study from T. Fujita's laboratory 183I SNP. Interestingly, this natural mutation allowed us to further demonstrate the importance of S183 in the transient complex formation that is required for proper RIG-I signaling. Thus, our findings strongly support the hypothesis that regulation of RIG-I/RIG-I and RIG-I/MAVS association/dissociation constitutes a major checkpoint of this antiviral signaling pathway.Among the eight RIG-I SNPs reported in NCBI SNP database, we characterized two distinct functional SNPs which strongly alter RIG-I-mediated signaling. First, we identified Pe.g. MAVS, Apaf-1, RAIDD, Nod1 et al.  183I SNP. In our model, serine 183 maps to helix H6  subfamily and the DED (death effector domain) subfamily. All these domains have a six-helical bundle (H1–H6) structural fold and mediate homotypic interactions within each domain subfamily 183I SNP inhibits RIG-I-induced signaling pathways despite its enhancing effect on RIG-I complexes formation. Based on our biochemical assays and structural modeling showing that this mutation does affect hydrophobicity and flexibility of the CARD#2 domain of RIG-I but does not influence its ligand binding activity, we hypothesize that S183I rather induces an abortive conformation of RIG-I, rendering it incapable of downstream signaling. Concerning the inhibitory effect of S183I on RIG-I/MAVS-dependent signal transduction, a recent study clearly supports the concept that MAVS association with RIG-I is not per se sufficient for inducing immune gene expression 183I, expression of MAVS 1a interferes with the formation of productive RIG-I/MAVS signaling complexes, which likely contributes to its inhibitory outcome.Collectively, on the basis of the data presented here, we consider that serine 183 residue plays a central role in the molecular ordering that leads to RIG-I-mediated NF-κB and IRF-3 activation pathways. Nevertheless, one can wonder how S183I SNP frequency in control healthy individuals and patients infected by viruses will certainly clarify the contribution of RIG-I variation to the pathogenesis of viral diseases. Likewise, investigating the clinical relevance of the potent immunostimulatory P229fs SNP may be particularly interesting in patients with autoimmune diseases where cytokines play a pivotal pathogenic role. Among them, evidence linking IFN-α/β with the pathogenesis of lupus and insulin-dependent diabetes mellitus in humans are the most convincing Elucidating the functional role of RIG-I SNPs may enhance our understanding of the pathogenesis of viral infections, to ultimately decrease morbidity and mortality through improved risk assessment and early administration of prophylactic therapies Influenza/A/Scotland/20/74 (H3N2) virus was prepared as previously described (PMA) were from Sigma. Human recombinant TNFα was purchased from Peprotech.cf.http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=23586). RIG-I sequences from human to platypus were aligned using EMBL ClustalW software and manually arranged.RIG-I SNPs were as described in NCBI's SNP database  was used to generate biotin-tagged WT or S183I RIG-I, as previously described 183I) expression vectors were generated by PCR using pEFBOS(+)-Flag RIG-I as a template and the following forward (5′-ggggacaactttgtacaaaaaagttggcatgACCACCGAGCAGCGACGCA-3′) and reverse primers (5′-ggggacaactttgtacaagaaagttggttaTTTGGACATTTCTGCTGGATCAAATGG-3′), as well as the Gateway technology for a final cloning in pcDNA6/BioEase-DEST plasmid (Invitrogen), according to manufacturer's instructions.The pEFBOS(+)-Flag-RIG-I (amino acids 2–925) or 2CARD (amino acids 2–229) vectors were previously described All constructs were entirely sequenced to confirm that no unintended mutations were generated during PCR reaction.−3) of relative luciferase units (RLU) normalized with β-galactosidase activity minus basal activity measured in empty vector-transfected cells.Detailed protocols were described before EK 293T cells were transiently co-transfected with 1 µg of Flag-tagged MAVS or V5-tagged 2CARD vectors  or 3 µg of Flag-tagged 2CARD and biotin-conjugated RIG-I vectors (for tandem 2CARD/RIG-I interaction). After cell disruption and a pre-clearing step, pull-down of biotin-tagged RIG-I was performed using streptavidin sepharose beads . For co-immunoprecipitation assay, cell lysates were incubated with a monoclonal anti-Flag M2 antibody, followed by the addition of protein G sepharose beads. More detailed protocols can be provided upon request. After centrifugation and protein denaturation, samples were analyzed by immunoblot as described in reference 183I) was previously described 183I). 48 h post-transfection, cells were disrupted in 1.5 ml of RIPA lysis buffer and 400 µg of cell lysates were incubated with poly(I∶C)- or control poly(C)-coated agarose beads (Sigma) in RIPA lysis buffer supplemented with proteases inhibitors cocktail and 50 U/ml of RNAse inhibitor (Promega) for 1 h at 4°C. Agarose beads were then collected by centrifugation and washed three times with lysis buffer before resuspension in 30 µl sample denaturating buffer.Assay of dsRNA binding activity of RIG-I .To evaluate RIG-I (WT or SNP) protein expression levels and subcellular localization, BEAS-2B and HEK 293T cells were transfected and processed as previously described et al.www.pymol.org) for visualization; HotPatch Homology modeling and molecular dynamics of the human RIG-I CARD#2 domain were carried out as previously described by Kubarenko p<0.05.Statistical differences were tested using a one-way ANOVA followed by a Fisher test, with a threshold of Table S1Plasmids containing SNPs were made by site-directed mutagenesis using the QuickChange II XL Site-Directed Mutagenesis kit (Stratagene), 125 ng of specific forward and reverse primers and 25 ng of RIG-I WT vector as a template in 50 µl reaction volume. After an initial denaturation step at 95°C for 1 min, mutagenesis was performed by 18 cycles of amplification , followed by a final elongation step at 68°C for 7 min. After PCR, template digestion by DpnI restriction enzyme and transformation of bacteria were performed according to manufacturer's instructions.(0.03 MB DOC)Click here for additional data file.Figure S1Alignment of amino acid sequence of CARD#2 domain of RIG-I with other CARD structures. Alignment of CARD domain sequences from different CARD proteins which were used for RIG-I CARD#2 modeling. Helix 1 colored in red, helix 2 in orange, helix 3 in yellow, helix 4 in green, helix 5 in blue and helix 6 in brown. For Apaf1, MAVS, RAIDD, ICEBERG and NOD1 CARDs, helix boundaries were determined directly from the respective PDB files 1cww, 2vgq, 3crd, 1dgn and 2b1w, based on a 3D alignment of these structures.(0.91 MB DOC)Click here for additional data file.Figure S2Intermediate frames of WT and S183I CARD#2 structures during molecular dynamic simulation. Eleven frames from the molecular dynamics simulation of RIG-I CARD#2 WT (A) or S183I mutant (B), showing one frame per picosecond. First frame (0 ps) corresponds to the initial raw model conformation. Helixes from 1 to 6 are rainbow-colored, helix 6 which harbors S183 is colored red.(4.28 MB DOC)Click here for additional data file.Figure S3Comparison of the stability of WT and S183I CARD#2 structures over the course of molecular dynamic simulation. Root mean square deviation (RMSD) calculated from 10 ps molecular dynamics simulation mapped onto the initial raw models of WT RIG-I CARD#2 (A) or S183I mutant (B) structures. More stable elements are colored green, more flexible regions in red.(1.37 MB DOC)Click here for additional data file.


None of these were significantly different from the parental Newcastle disease virus (NDV) LaSota strain (p > 0.05). Vaccination of white leghorn chickens at one day of age with 106 EID50 rNDV-IBV-T/B provided 90% protection against virulent IBV M41 challenge at three weeks of age, which was significantly higher than the protection of the control group vaccinated with phosphate-buffered saline (PBS) (p < 0.05). The ciliostasis scores of rNDV-IBV-T/B-vaccinated and LaSota-vaccinated groups were 4.2 and 37.6, respectively, which indicated that rNDV-IBV-T/B vaccination reduced the pathogenicity of IBV toward the trachea. Furthermore, real-time RT-PCR assay showed that the rNDV-IBV-T/B vaccination resulted in low levels of viral load (647.80 ± 49.65 RNA copies) in the trachea four days post-challenge, which is significantly lower than groups vaccinated with PBS (8591.25 ± 311.10 RNA copies) or LaSota (7742.60 ± 298.50 RNA copies) (p < 0.05). Meanwhile, the same dose of rNDV-IBV-T/B vaccination provided complete protection against velogenic NDV F48E9 challenge. These results demonstrate that the rNDV-IBV-T/B strain is a promising vaccine candidate to control both IB and ND simultaneously. Furthermore, epitope-based live vector vaccines provide an alternative strategy for the development of cost-effective and, broadly, cross-protective vaccines.Infectious bronchitis (IB) and Newcastle disease (ND) are two major infectious diseases that are a threat to the domestic poultry industry. In this study, we successfully generated a recombinant LaSota candidate vaccine strain, rNDV-IBV-T/B, which expresses a short, synthetic, previously identified IBV S1 multi-epitope cassette using the reverse genetic system. The recombinant virus was propagated in nine-day-old embryonated chicken eggs for 20 passages and genetic stability was confirmed by whole genome DNA sequencing. The recombinant virus had a hemagglutination (HA) titer of 2 Nidovirales, family Coronaviridae, genus Gammacoronavirus. IBV causes upper respiratory illness, nephritic syndrome, and a drop in egg production in layer chickens [Avian infectious bronchitis (IB) is an acute and highly contagious viral respiratory disease of chickens. IB occurs worldwide and causes substantial economic losses in the poultry industry ,2,3,4,5.chickens . The morchickens ,7,8,9,10Orthoavulavirus of the family Paramyxoviridae, often referred to as Newcastle disease virus (NDV) [Currently, novel serotype and variant IBV have emerged due to rapid mutation rates, viral recombination, and host selection pressure, which have resulted in IB prevention becoming a global challenge ,12. Vaccus (NDV) , which hus (NDV) ,17. Thesus (NDV) ,19,20,21In recent years, epitope-based vaccines have been widely studied for their advantages, including safety, genetic stability, ease of production, and the ability to elicit immune responses against multi serotype pathogens. ,23. ThusThe technology of the reverse genetic system (RGS) for NDV was established in Europe two decades ago, which has been successfully applied to research on pathogenicity, replication regulatory mechanisms, and vaccine vectors . MeanwhiThe aim of this study was to develop a live vaccine using recombinant NDV expressing previously identified IBV multi-epitope cassette. The protective efficacy of the recombinant NDV vaccine was evaluated by heterologous IBV and velogenic NDV challenge. The findings could provide an alternative and promising strategy for the development of a cost-effective and extensively immune-protective vaccine for the control of ND and IB. 50) of a virus in harvested allantoic fluid was calculated by the Reed and Muench mathematical technique [The chicken embryonic fibroblast cell line (DF-1) and baby hamster kidney fibroblasts clone 13 cell line (BHK-21) were obtained from the American Type Culture Collection . They were grown in Dulbecco’s minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS). Fertile white leghorn SPF embryonated eggs were purchased from the Beijing Merial Vital Laboratory Animal Technology Co., Ltd. The recombinant avirulent NDV LaSota strain was developed previously in our laboratory by a reverse genetics system ,36. The echnique . All expWe employed NDV as virus vector and rescued the recombinant NDV expressing an IBV S1 protein multi-epitope cassette via a reverse genetics system. Briefly, five overlapping cDNA fragments covering the complete genome of the NDV LaSota strain were amplified by RT-PCR using genomic RNA as a template. The five cDNA fragments were subsequently assembled into a modified pBR322 vector under the control of the T7 promotor and followed by a partial Hepatitis D virus (HDV) ribozyme and T7 terminator, resulting in a full-length cDNA clone, named pBR-LS.The IBV S1 multi-epitope cassette S-T/B, constructed previously, was inserted between P and M genes in the LaSota genome as an additional transcriptional unit . The fulThe recombinant LaSota virus, named rNDV-IBV-T/B, P1-P20 passages were detected by reverse transcription polymerase chain reaction (RT-PCR) and all the passages rNDV-IBV-T/B whole genome were analyzed by DNA sequencing . Briefly, RNA was extracted from allantoic fluid of rNDV-IBV-T/B-infected chick embryos  and reverse transcribed to cDNA with AMV Reverse Transcriptase  and random primers  at 42 °C for 30 min. To amplify the target gene IBV multi-epitope cassette, RT-PCR was performed using 2 µL of rNDV-IBV-T/B cDNA, 10 µmol each of the primers , 10 µL PCR Master Mix (2X)  in a total volume of 20 µL. PCR cycling procedure began with an initial hold at 95 °C for 10 min, followed by 30 cycles containing 94 °C for 20 s, 53 °C for 30 s and 72 °C for 20 s and a final step of 72 °C for 10 min. RT-PCR amplification product was examined by agarose-gel electrophoresis.50) assay on DF-1 cells in 96-well plates, and the 50% egg infective dose (EID50) assay in nine-day-old SPF chicken embryos, according to A Basic Laboratory Manual for the Small-Scale Production and Testing of I-2 Newcastle Disease Vaccine [http://www.oie.int/en/standard-setting/terrestrial-manual).The infectivity of the recombinant virus rNDV-IBV-T/B and parental LaSota strain were characterized using the standard hemagglutination test (HA) and the 50% tissue infectious dose  of 1 with P1 and P20 passages of rNDV-IBV-T/B and the parental virus LaSota strain. DF1 cells were harvested 36 h post-infection, lysed, and proteins were separated on a 10% SDS polyacrylamide gel (SDS-PAGE) under denaturing conditions. After electrophoresis, proteins were transferred onto polyvinylidene fluoride (PVDF) filter membrane. Then, the membrane was incubated in 5% skim milk with PBST buffer  for 1 h at room temperature, followed by incubation with a 1:5000 dilution of rabbit anti-IBV strain polyclonal antibody or mouse monoclonal NDV NP protein antibody in PBST buffer for 1 h at 37 °C. After triplicate washes with PBST buffer, the PVDF membrane was incubated with an HRP-conjugated goat anti-rabbit or rabbit anti-mouse secondary antibody. The protein was visualized using the Pierce ECL substrate kit  and imaged using a Tanon 5200 automatic chemiluminescence image analysis system .g for 20 min to remove the debris and collect the supernatant. Four milliliters of rNDV-IBV-T/B were layered on top of a step gradient of 60%, 50%, 40%, and 25% w/v sucrose prepared in Milli-Q water. Ultracentrifugation was carried out at 120,000 ×g in a SW41 Ti rotor  for 4 h, at 4 °C. The virus-containing band between the 40% and 50% sucrose layers was collected and 5 μl of rNDV-IBV-T/B virus was placed onto the shiny side of an EM grid and then adsorbed for ~3 min. The grid was stained immediately after virus adsorption 3 times with 2% uranyl acetate for 45 s. After negative staining, observation was by an 80 kV FEI Tecnai Spirit TEM T12 (Thermo Fisher) at 98,000× magnification.The rNDV-IBV-T/B was propagated in nine-day-old SPF chicken embryos and purified by sucrose gradient centrifugation according to a previous report . Briefly6 EID50 per chicken, and groups C and D were inoculated with LaSota at the same dose via the oculonasal route. The control groups E and F were inoculated with PBS by the same route.To analyze the protective efficacy of the rNDV-IBV-T/B vaccine against NDV and IBV infection, a total of 90 one-day-old SPF white leghorn chicks were randomly divided into six groups and housed in negative pressure isolators with sterile water and enough feed. Groups A and B were administered with rNDV-IBV-T/B at dose of 106 EID50 of the IBV M41 strain via the oculonasal route, and groups B and D, and control group F were challenged with 106 EID50 of the velogenic NDV strain F48E9 by the same route. The lethal dose of the challenge virus had been determined by an experimental chicken infection study. Five challenged chickens from groups A and C, and control group E were subjected to ciliostasis tests at 4 days post-challenge (dpc). The other chickens of each group were monitored daily for symptoms and mortality for another two weeks. The immune-protection and challenge experiments were conducted in triplicate.Three weeks post-vaccination, groups A and C, and control group E were challenged with 102 EID50 of M41 strain, followed by incubation for 1 h at 37 °C. Neutralizing antibody titer was estimated by inoculating nine-day-old SPF embryonated chicken eggs with the incubated virus-serum mixture via the allantoic cavity route. PBS was used as the negative control. The embryonated eggs were examined daily for 5 days. VN titers were defined as the reciprocal of the highest dilution without any mortality and IBV specific lesions on chicken embryos. All the assays were performed in triplicate and VN titers were expressed as the mean ± SEM.In order to evaluate the antibody responses elicited by rNDV-IBV-T/B, the vaccinated chickens were bled through the wing veins, and sera were isolated for serological tests 21 days post-immunization. HI assays were conducted in V-bottomed microwell plates according to the standard protocol of the OIE Manual to determine the level of antibody titers mounted against NDV in chickens vaccinated by rNDV-IBV-T/B. . Ten serTo evaluate the efficacy of rNDV-IBV-T/B in preventing shedding of IBV in immunized chickens, tracheal swab samples were randomly taken from five chickens from each group 4 dpc. RNA was extracted using an using a High Pure RNA Isolation Kit  according to the manufacturer’s instructions. The primers, probe, and PCR conditions for the assay used to detect IBV RNA using RT-qPCR were previously described in the literature ,42. TaqmThe ciliostasis activity of the tracheal explants was examined on 10 tracheal ring explants per chicken (5 chickens of each group) 5 dpc. The tracheas were carefully removed and examined for ciliary activity within 2 h after collection as described in the referenced papers ,44. Briep values < 0.05 were considered statistically significant. The data are expressed as the means ± SEM.GraphPad Prism v5.01 was used for statistical analysis. One-way ANOVA with repeated measures was used to evaluate the effect of the vaccines through HI and VN tests. The survival rates of immune protection experiments were determined by log-rank tests, 2. The cell lysates were harvested, filtered, and inoculated into SPF embryonated chicken eggs. The rNDV-IBV-T/B was serially passaged 20 times (P20), and the sequence of the full-length multi-epitope cassette S-T/B was examined by RT-PCR (The multi-epitope cassette A containy RT-PCR C and seqOne MOI of two different passages (P1 and P20) of rNDV-IBV-T/B and the parental strain LaSota were used to infect DF1 cells for 24 h, and the cell lysates were harvested and analyzed by Western blot using rabbit polyclonal anti-IBV serum and a mouse monoclonal anti-NDV/NP antibody. The protein expressed from the IBV S1 protein multi-epitope cassette and the NDV NP protein were found at ~35 and 53 kDa bands in P1 and P20 of rNDV-IBV-T/B lysates, however, the parental virus strain LaSota only had the ~53 kDa NP protein band , confirm50 titers of the parental virus LaSota and rNDV-IBV-T/B reached 6.5 × 108/ml and 6.7 × 108/mL, respectively. The TCID50 titer of rNDV-IBV-T/B (3.7 × 107/mL) was slightly higher than that of LaSota (3.6 × 107/mL) at 48 h post-infection. The HA titers of LaSota and rNDV-IBV-T/B were the same (210). The statistical analysis of EID50, TCID50, and HA results indicated that there were no significant differences between rNDV-IBV-T/B and the parental LaSota virus (p > 0.05). The recombinant NDV remained stable, with no apparent changes in MDT and virus titers after 20t passages in SPF chicken embryonated eggs. Furthermore, rNDV-IBV-T/B has a lower ICPI (0.05) than LaSota (0.10) (p > 0.05).To determine whether inserting the IBV S1 protein multi-epitope cassette into the NDV LaSota genome affects the biological properties of the recombinant virus, the growth characteristics and pathogenicity after 20 passages of rNDV-IBV-T/B were evaluated through virus titration, MDT, and ICPI tests in vitro and in vivo. The titers of the recombinant virus, determined either in embryonated eggs or in DF-1 cells, were comparable to those of the parental LaSota strain. The EIDa (0.10) . These rTo observe the morphology of the recombinant virus rNDV-IBV-T/B, the 20th passage (P20) of rNDV-IBV-T/B and parental LaSota virions were purified by sucrose density gradient centrifugation. Following negative staining, virions were observed by TEM at 98,000× magnification. The rNDV-IBV-T/B virus appeared as circles or ovals about 100 to 150 nm in diameter. The envelope located on the surface layer of the virus can be clearly observed, which is consistent with the morphology of LaSota . TEM indp < 0.05)  . This rep < 0.05). The VN antibodies induced by rNDV-IBV-T/B administration could effectively neutralize heterologous IBV strain, indicating that the recombinant vaccine could protect against heterologous IBV challenge.Virus neutralization (VN) assays were performed according to the standard protocol of OIE. The VN titer of rNDV-IBV-T/B group against IBV M41 strain showed approximately 6.82 ± 0.26, which was significantly higher than the LaSota and PBS groups’ (no VN activity detected) (p < 0.05). The rNDV-IBV-T/B immunized group exhibited protective efficacy equivalent to the parental LaSota group (p < 0.05). The same protection rate was observed in two additional immune-protection tests conducted in parallel. These results confirm that chickens vaccinated with rNDV-IBV-T/B vaccine are substantially protected against NDV and IBV challenge.To evaluate the efficacy of the multi-epitope vaccine in protecting against IBV and NDV, one-day-old SPF chicks were immunized with rNDV-IBV-T/B virus or PBS via the oculonasal route. At three weeks post-immunization, the chickens were challenged with lethal doses of the NDV F48E9 strain or IBV M41 strain. Chickens inoculated with PBS manifested clinical symptoms on day two post NDV challenge, and all these chickens died within four days. Compared to chickens immunized with parental LaSota and chickens inoculated with PBS, chickens immunized with rNDV-IBV-T/B showed no apparent symptoms post NDV challenge (ta group A. The clta group B. In conp < 0.05) (p < 0.05). It is probable that rNDV-IBV-T/B effectively activated the immune response and suppressed viral replication to protect chickens against lethal dose of IBV challenge.Tracheal swab samples taken from five chickens of IBV challenge groups on 4 dpc were analyzed for IBV virions shedding via Taqman probe Real-time qRT-PCR (expressed as means ± SEM). Chickens vaccinated with rNDV-IBV-T/B showed low levels of viral load (647.80 ± 49.65) in the trachea, which was significantly lower than those inoculated with PBS (8591.25 ± 311.10) or LaSota (7742.60 ± 298.50) ( < 0.05) . The resThe ciliostasis scores between chickens immunized with rNDV-IBV-T/B, LaSota, and PBS control after a lethal dose of IBV challenge were calculated . The maxInfectious bronchitis (IB) and Newcastle disease (ND) are two major infectious diseases of the poultry production industry. Especially in layer and breeder chickens, a decline in egg production alone can cause significant economic losses. However, IBV and NDV live vaccines probably mutually interfere with concurrent immunization ,46,47. FpV-S1B+S1T could provide effective protection against a lethal dose of homologous IBV SH1208 challenge [p > 0.05). In previous study, the immune-protection experiment demonstrated that multi epitope-based DNA vaccine hallenge ,32. ThisThe immune-protection experiment showed that a single immunization of one-day-old chicks with rNDV-IBV-T/B could protect 100% and 90% chicks against lethal doses of F48E9 and M41 strain, respectively. The IBV protection rate was analogous to that of a recombinant NDV expressing the full-length S protein of the IBV M41 strain, reported by Shirvani, et al. ,55. WhetAbozeid et al.  reportedIn summary, the findings of this study support that NDV is an outstanding vaccine vector for expressing an IBV multi-epitope protein as a bivalent vaccine against NDV and IBV. Furthermore, the rNDV-IBV-T/B provides an alternative strategy for the development of a cost-effective and extensively immune-protective vaccine for the control of variant IBV infection.