Bacterial artificial chromosome-based reverse genetics system for cloning and manipulation of the full-length genome of infectious bronchitis virus.

Avian infectious bronchitis virus (IBV) causes highly contagious respiratory reproductive and renal system diseases in chickens, and emergence of serotypic variants resulting from mutations in the viral S gene hampers vaccine management for IBV infection. In this study, to facilitate the molecular analysis of IBV pathogenesis and the development of a new-generation IBV vaccine, we established a reverse genetics system (RGS) for cloning the full-length cDNA of the IBV C-78E128 attenuated strain in a bacterial artificial chromosome (BAC). The BAC-cloned C-78E128 cDNA generated infectious viruses with biological properties of the parental C-78E128 strain with regard to an avirulent phenotype, tissue tropism and induction of virus neutralizing (VN) antibody in vivo. To assess the feasibility of genetic manipulation of the IBV genome using the BAC-based RGS, the S gene of the BAC-cloned C-78E128 cDNA was replaced with that of the IBV S95E4 virulent strain, which differs from the C-78E128 strain in serotype and tissue tropism, by bacteriophage lambda Red-mediated homologous recombination in Escherichia coli (E. coli). The resultant S gene recombinant virus was found to be avirulent and fully competent to induce a serotype-specific VN antibody against the S95 strain; however, the S gene recombinant virus did not fully recapitulate the tissue tropism of the S95E4 strain. These data imply that serotype-specific VN immunogenicity, but not tissue-tropism and pathogenicity, of IBV is determined by the viral S gene. The IBV BAC-based RGS that enables cloning and manipulation of the IBV virus genome entirely in E. coli provides a useful platform for the molecular analyses of IBV pathogenesis and the development of rationally designed IBV recombinant vaccines.

Repeated avian infectious bronchitis virus infections within a single chicken farm.

Genotyping of avian infectious bronchitis virus (IBV) was performed on trachea and kidney samples of six chickens obtained from a single farm in Japan. Using two primer sets targeting the spike (S) protein gene, the S1 and S2 regions of DNA fragments were amplified. Sequences of amplified S1 fragments extracted from both organs were identical among the six chickens, showing a JP-I genotype. Sequences of amplified S2 fragments differed between trachea and kidney samples. The kidney profile showed a group IV genotype, whereas the trachea profile showed an unclassified group. This result showed that two different IBVs infected the six chickens. The first IBV infection induced poor protective immunity in this farm, permitting a second IBV infection to occur.

Galactosylation of human erythropoietin produced by chimeric chickens expressing galactosyltransferase.

Human erythropoietin produced in the egg white of chimeric chicken contains N-glycan with lower amounts of terminal galactose and sialic acid; therefore, the chicken galactosyltransferase gene was introduced together with the human erythropoietin gene by a retroviral vector. We found that erythropoietin accumulated in the egg white was partially galactosylated.

Moloney murine leukemia virus integrase and reverse transcriptase interact with PML proteins.

Pull-down assay and co-immunoprecipitation of cell extracts in which the integrase or reverse transcriptase of Moloney murine leukemia virus was transiently expressed showed that both enzymes interacted with PML proteins. In infected cells, interaction between the integrase and PML was also observed. Transient expression of PIASy and SUMO proteins facilitated SUMOylation of the integrase but had no apparent effects on the interaction with PML. A FLAG-tagged integrase co-localized with PML protein possibly in the PML body. Knockdown of PML by small interfering RNA resulted in reduced viral cDNA levels and integration efficiency. This suggested that PML proteins activated reverse transcription.

Genetic modification of a chicken expression system for the galactosylation of therapeutic proteins produced in egg white.

As a tool for large scale production of recombinant proteins, chickens have advantages such as high productivity and low breeding costs compared to other animals. We previously reported the production of erythropoietin, the tumor necrosis factor receptor fused to an Fc fragment, and an Fc-fused single-chain Fv antibody in eggs laid by genetically manipulated chickens. In egg white, however, the incomplete addition of terminal sugars such as sialic acid and galactose was found on N-linked glycans of exogenously expressed proteins. This could be a draw back to the use of transgenic chickens since the loss of these terminal sugars may affect the functions and stability of recombinant proteins purified from chicken egg white for pharmaceutical usage. To overcome this problem, we studied galactosyltransferase (GalT) activity in the magnum where the majority of egg-white proteins are secreted. In the magnum, lower ß1,4-GalT1 expression and poor galactose-transfer activity were observed. Thus, we supposed that the lack of GalT1 activity may partly cause the incomplete glycosylation of egg-white proteins, and generated genetically manipulated chickens expressing GalT1 by retrovirus-mediated gene transfer. In a Golgi fraction prepared from magnum cells of the genetically manipulated chickens, significant GalT activity was detected. The series of analyses revealed a considerable improvement in the galactosylation of native egg-white proteins as well as an exogenously expressed single-chain Fv antibody fused to an Fc fragment. We conclude that chickens with genetically modified GalT activity in the magnum could be an attractive platform for producing galactosylated therapeutics.

Chicken oviduct-specific expression of transgene by a hybrid ovalbumin enhancer and the Tet expression system.

We generated genetically manipulated chickens and quail by infecting them with a retroviral vector expressing the human growth hormone under the control of chicken ovalbumin promoter/enhancer up to -3861 bp from the transcriptional start site. The growth hormone was expressed in an oviduct-specific manner and was found in egg white, although its level was low. The DNA sequence of the integrated form of the viral vector in the packaging cells was shown to be truncated and contained only the sequence spanning -3861 to -1569 bp. This represented only the DNase I hypersensitive site (DHS) III of the 4 DHSs and lacked the proximal promoter of the ovalbumin control region. We found several TATA-like and other promoter motifs of approximately -1800 bp and considered that these promoter motifs and DHS III may cause weak but oviduct-specific expression of the growth hormone. To prove this hypothesis and apply this system to oviduct-specific expression of the transgene, the truncated regulatory sequence was fused to an artificial transactivator-promoter system. In this system, initial weak but oviduct-specific expression of the Tet activator from the promoter element in the ovalbumin control sequence triggered a self-amplifying cycle of expression. DsRed was specifically expressed in oviduct cells of genetically manipulated chickens using this system. Furthermore, deletion of a short region possibly containing the promoter elements (-2112 to -1569 bp) completely abrogated oviduct-specific expression. Taken together, these results suggest that weak expression of this putative promoter causes oviduct-specific expression of the transgene.

Transcription factor YY1 interacts with retroviral integrases and facilitates integration of moloney murine leukemia virus cDNA into the host chromosomes.

Retroviral integrases associate during the early viral life cycle with preintegration complexes that catalyze the integration of reverse-transcribed viral cDNA into the host chromosomes. Several cellular and viral proteins have been reported to be incorporated in the preintegration complex. This study demonstrates that transcription factor Yin Yang 1 binds to Moloney murine leukemia virus, human immunodeficiency virus type 1, and avian sarcoma virus integrases. The results of coimmunoprecipitation and in vitro pulldown assays revealed that Yin Yang 1 interacted with the catalytic core and C-terminal domains of Moloney murine leukemia virus and human immunodeficiency virus type 1 integrases, while the transcriptional repression and DNA-binding domains of the Yin Yang 1 molecule interacted with Moloney murine leukemia virus integrase. Immunoprecipitation of the cytoplasmic fraction of virus-infected cells followed by Southern blotting and chromatin immunoprecipitation demonstrated that Yin Yang 1 associated with Moloney murine leukemia virus cDNA in virus-infected cells. Yin Yang 1 enhanced the in vitro integrase activity of Moloney murine leukemia virus, human immunodeficiency virus type 1, and avian sarcoma virus integrases. Furthermore, knockdown of Yin Yang 1 in host cells by small interfering RNA reduced Moloney murine leukemia virus cDNA integration in vivo, although viral cDNA synthesis was increased, suggesting that Yin Yang 1 facilitates integration events in vivo. Taking these results together, Yin Yang 1 appears to be involved in integration events during the early viral life cycle, possibly as an enhancer of integration.

Y65C missense mutation in the WW domain of the Golabi-Ito-Hall syndrome protein PQBP1 affects its binding activity and deregulates pre-mRNA splicing.

The PQBP1 (polyglutamine tract-binding protein 1) gene encodes a nuclear protein that regulates pre-mRNA splicing and transcription. Mutations in the PQBP1 gene were reported in several X chromosome-linked mental retardation disorders including Golabi-Ito-Hall syndrome. The missense mutation that causes this syndrome is unique among other PQBP1 mutations reported to date because it maps within a functional domain of PQBP1, known as the WW domain. The mutation substitutes tyrosine 65 with cysteine and is located within the conserved core of aromatic amino acids of the domain. We show here that the binding property of the Y65C-mutated WW domain and the full-length mutant protein toward its cognate proline-rich ligands was diminished. Furthermore, in Golabi-Ito-Hall-derived lymphoblasts we showed that the complex between PQBP1-Y65C and WBP11 (WW domain-binding protein 11) splicing factor was compromised. In these cells a substantial decrease in pre-mRNA splicing efficiency was detected. Our study points to the critical role of the WW domain in the function of the PQBP1 protein and provides an insight into the molecular mechanism that underlies the X chromosome-linked mental retardation entities classified globally as Renpenning syndrome.

Mammalian chromatin remodeling complex SWI/SNF is essential for enhanced expression of the albumin gene during liver development.

The chromatin remodeling complex SWI/SNF is known to regulate the transcription of several genes by controlling chromatin structure in an ATP-dependent manner. SWI/SNF contains the Swi2p/Snf2p like ATPases BRG1 or BRM exclusively. We found that the expression of BRM gradually increases and that of BRG1 decreases as liver cells differentiate. Chromatin immunoprecipitation assays revealed that the ATPase subunits of SWI/SNF and tumor suppressor retinoblastoma (RB) family proteins bind to the promoter region of the albumin gene in hepatocytes, and that the replacement of BRG1 with BRM and pRB with p130 at this site occurs over the course of differentiation. Small interfering RNA experiments showed that blocking the expression of BRG1 and BRM in fetal and adult hepatocytes, respectively, causes a reduction in albumin expression. In luciferase reporter assays with a pREP4-based reporter plasmid that forms a chromatin structure, BRG1 showed activity stimulating the expression of the albumin promoter mediated by CCAAT/enhancer-binding protein alpha (C/EBPalpha). This enhancement was facilitated by the RB family members pRB and p130. ATPase assays showed that both pRB and C/EBPalpha proteins directly stimulate the ATPase activity of BRG1. Our findings suggest that the mechanism by which the activity of transcription factors is enhanced by RB family members and SWI/SNF includes an increase in the ATPase activity of the chromatin remodeling complex.

Repression of GR-mediated expression of the tryptophan oxygenase gene by the SWI/SNF complex during liver development.

The chromatin remodeling complex, SWI/SNF, is known to regulate the transcription of several genes by altering the chromatin structure in an ATP-dependent manner. SWI/SNF exclusively contains BRG1 or BRM as an ATPase subunit. In the present study, we studied the role of SWI/SNF containing BRM or BRG1 in the expression of the liver-specific tryptophan oxygenase (TO) and tyrosine aminotransferase genes. Chromatin remodeling factors significantly repressed the expression of these genes induced by glucocorticoid receptor and dexamethasone. Since the repression was not reversed by trichostatin A treatment, it seemed to be independent of the well-known histone deacetylase pathway. Knock-down of BRG1 by small interfering RNA reversed the repression in primary fetal hepatocytes. These results support a model in which SWI/SNF containing BRG1 represses late stage-specific TO gene expression at an early stage of liver development.

Transcriptional coactivators CBP and p300 cooperatively enhance HNF-1alpha-mediated expression of the albumin gene in hepatocytes.

Transcriptional coactivators, CREB-binding protein (CBP) and p300, exhibit high homology in structure and similar functions. In the present study, we analyzed the function of CBP and p300 proteins as transcriptional coactivators in the expression of albumin in hepatocytes. The expression levels of CBP and p300 were high in fetal hepatocytes, but low in adult ones. Immunoprecipitation assays showed that both CBP and p300 interacted with hepatocyte nuclear factor-1alpha (HNF-1alpha) in primary hepatocytes. Furthermore, CBP and p300 were co-precipitated without HNF-1alpha. Chromatin immunoprecipitation (ChIP) assays revealed that both CBP and p300 are located in the albumin promoter region in hepatocytes. These results suggested that HNF-1alpha, CBP and p300 were incorporated into a preinitiation complex of RNA polymerase II at the albumin promoter. Luciferase reporter assays showed that CBP and p300 cooperatively triggered HNF-1alpha-mediated transcription of the albumin promoter. In addition, inhibition of CBP or p300 using small interfering RNAs (siRNAs) resulted in a reduction in albumin expression. These results suggest that both CBP and p300 are required for enhanced expression of albumin.