An Adenovirus Vector Incorporating Carbohydrate Binding Domains Utilizes Glycans for Gene Transfer

Background

Vectors based on human adenovirus serotype 5 (HAdV-5) continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting.

Methodology/Principal Findings

As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4). This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells.

Conclusions/Significance

These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers.     

Primordial germ cell-mediated chimera technology produces viable pure-line Houbara bustard offspring: potential for repopulating an endangered species

Background

The Houbara bustard (Chlamydotis undulata) is a wild seasonal breeding bird populating arid sandy semi-desert habitats in North Africa and the Middle East. Its population has declined drastically during the last two decades and it is classified as vulnerable. Captive breeding programmes have, hitherto, been unsuccessful in reviving population numbers and thus radical technological solutions are essential for the long term survival of this species. The purpose of this study was to investigate the use of primordial germ cell-mediated chimera technology to produce viable Houbara bustard offspring.

Methodology/Principal Findings

Embryonic gonadal tissue was dissected from Houbara bustard embryos at eight days post-incubation. Subsequently, Houbara tissue containing gonadal primordial germ cells (gPGCs) was injected into White Leghorn chicken (Gallus gallus domesticus) embryos, producing 83/138 surviving male chimeric embryos, of which 35 chimeric roosters reached sexual maturity after 5 months. The incorporation and differentiation of Houbara gPGCs in chimeric chicken testis were assessed by PCR with Houbara-specific primers and 31.3% (5/16) gonads collected from the injected chicken embryos showed the presence of donor Houbara cells. A total of 302 semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germline chimeras. Semen samples from these eight roosters were used to artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara eggs were obtained and incubated, two of which were fertile. One egg hatched as a male live born Houbara; the other was female but died before hatching. Genotyping confirmed that the male chick was a pure-line Houbara derived from a chimeric rooster.

Conclusion

This study demonstrates for the first time that Houbara gPGCs can migrate, differentiate and eventually give rise to functional sperm in the chimeric chicken testis. This approach may provide a promising tool for propagation and conservation of endangered avian species that cannot breed in captivity.     

Expression of multiple artificial microRNAs from a chicken miRNA126-based lentiviral vector

Background

The use of RNAi in both basic and translational research often requires expression of multiple siRNAs from the same vector.

Methods/Principal Findings

We have developed a novel chicken miR126-based artificial miRNA expression system that can express one, two or three miRNAs from a single cassette in a lentiviral vector. We show that each of the miRNAs expressed from the same lentiviral vector is capable of potent inhibition of reporter gene expression in transient transfection and stable integration assays in chicken fibroblast DF-1 cells. Transduction of Vero cells with lentivirus expressing two or three different anti-influenza miRNAs leads to inhibition of influenza virus production. In addition, the chicken miR126-based expression system effectively inhibits reporter gene expression in human, monkey, dog and mouse cells. These results demonstrate that the flanking regions of a single primary miRNA can support processing of three different stem-loops in a single vector.

Conclusions/Significance

This novel design expands the means to express multiple miRNAs from the same vector for potent and effective silencing of target genes and influenza virus.     

The Cytoplasmic Location of Chicken Mx Is Not the Determining Factor for Its Lack of Antiviral Activity

Background

Chicken Mx belongs to the Mx family of interferon-induced dynamin-like GTPases, which in some species possess potent antiviral properties. Conflicting data exist for the antiviral capability of chicken Mx. Reports of anti-influenza activity of alleles encoding an Asn631 polymorphism have not been supported by subsequent studies. The normal cytoplasmic localisation of chicken Mx may influence its antiviral capacity. Here we report further studies to determine the antiviral potential of chicken Mx against Newcastle disease virus (NDV), an economically important cytoplasmic RNA virus of chickens, and Thogoto virus, an orthomyxovirus known to be exquisitely sensitive to the cytoplasmic MxA protein from humans. We also report the consequences of re-locating chicken Mx to the nucleus.

Methodology/Principal Findings

Chicken Mx was tested in virus infection assays using NDV. Neither the Asn631 nor Ser631 Mx alleles (when transfected into 293T cells) showed inhibition of virus-directed gene expression when the cells were subsequently infected with NDV. Human MxA however did show significant inhibition of NDV-directed gene expression. Chicken Mx failed to inhibit a Thogoto virus (THOV) minireplicon system in which the cytoplasmic human MxA protein showed potent and specific inhibition. Relocalisation of chicken Mx to the nucleus was achieved by inserting the Simian Virus 40 large T antigen nuclear localisation sequence (SV40 NLS) at the N-terminus of chicken Mx. Nuclear re-localised chicken Mx did not inhibit influenza (A/PR/8/34) gene expression during virus infection in cell culture or influenza polymerase activity in A/PR/8/34 or A/Turkey/50-92/91 minireplicon systems.

Conclusions/Significance

The chicken Mx protein (Asn631) lacks inhibitory effects against THOV and NDV, and is unable to suppress influenza replication when artificially re-localised to the cell nucleus. Thus, the natural cytoplasmic localisation of the chicken Mx protein does not account for its lack of antiviral activity.     

The C-Terminal Sequence of RhoB Directs Protein Degradation through an Endo-Lysosomal Pathway

Background

Protein degradation is essential for cell homeostasis. Targeting of proteins for degradation is often achieved by specific protein sequences or posttranslational modifications such as ubiquitination.

Methodology/Principal Findings

By using biochemical and genetic tools we have monitored the localization and degradation of endogenous and chimeric proteins in live primary cells by confocal microscopy and ultra-structural analysis. Here we identify an eight amino acid sequence from the C-terminus of the short-lived GTPase RhoB that directs the rapid degradation of both RhoB and chimeric proteins bearing this sequence through a lysosomal pathway. Elucidation of the RhoB degradation pathway unveils a mechanism dependent on protein isoprenylation and palmitoylation that involves sorting of the protein into multivesicular bodies, mediated by the ESCRT machinery. Moreover, RhoB sorting is regulated by late endosome specific lipid dynamics and is altered in human genetic lipid traffic disease.

Conclusions/Significance

Our findings characterize a short-lived cytosolic protein that is degraded through a lysosomal pathway. In addition, we define a novel motif for protein sorting and rapid degradation, which allows controlling protein levels by means of clinically used drugs.     

Dual-affinity avidin molecules

A recently reported dual-chain avidin was modified further to contain two distinct, independent types of ligand-binding sites within a single polypeptide chain. Chicken avidin is normally a tetrameric glycoprotein that binds water-soluble d-biotin with extreme affinity (K(d) approximately 10(-15) M). Avidin is utilized in various applications and techniques in the life sciences and in the nanosciences. In a recent study, we described a novel avidin monomer-fusion chimera that joins two circularly permuted monomers into a single polypeptide chain. Two of these dual-chain avidins were observed to associate spontaneously to form a dimer equivalent to the wt tetramer. In the present study, we successfully used this scaffold to generate avidins in which the neighboring biotin-binding sites of dual-chain avidin exhibit two different affinities for biotin. In these novel avidins, one of the two binding sites in each polypeptide chain, the pseudodimer, is genetically modified to have lower binding affinity for biotin, whereas the remaining binding site still exhibits the high-affinity characteristic of the wt protein. The pseudotetramer (i.e., a dimer of dual-chain avidins) has two high and two lower affinity biotin-binding sites. The usefulness of these novel proteins was demonstrated by immobilizing dual-affinity avidin with its high-affinity sites. The sites with lower affinity were then used for affinity purification of a biotinylated enzyme. These "dual-affinity" avidin molecules open up wholly new possibilities in avidin-biotin technology, where they may have uses as novel bioseparation tools, carrier proteins, or nanoscale adapters.     

Immunization of Chickens with Newcastle Disease Virus Expressing H5 Hemagglutinin Protects against Highly Pathogenic H5N1 Avian Influenza Viruses

Background

Highly-pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are the two most important poultry viruses in the world. Natural low-virulence NDV strains have been used as vaccines over the past 70 years with proven track records. We have previously developed a reverse genetics system to produce low-virulent NDV vaccine strain LaSota from cloned cDNA. This system allows us to use NDV as a vaccine vector for other avian pathogens.

Methodology/Principal Finding

Here, we constructed two recombinant NDVs (rNDVs) each of which expresses the hemagglutinin (HA) gene of HPAIV H5N1strain A/Vietnam/1203/2004 from an added gene. In one, rNDV (rNDV-HA), the open reading frame (ORF) of HA gene was expressed without modification. In the second, rNDV (rNDV-HAF), the ORF was modified so that the transmembrane and cytoplasmic domains of the encoded HA gene were replaced with those of the NDV F protein. The insertion of either version of the HA ORF did not increase the virulence of the rNDV vector. The HA protein was found to be incorporated into the envelopes of both rNDV-HA and rNDV-HAF. However, there was an enhanced incorporation of the HA protein in rNDV-HAF. Chickens immunized with a single dose of either rNDV-HA or rNDV-HAF induced a high titer of HPAIV H5-specific antibodies and were completely protected against challenge with NDV as well as lethal challenges of both homologous and heterologous HPAIV H5N1.

Conclusion and Significance

Our results suggest that these chimeric viruses have potential as safe and effective bivalent vaccines against NDV and. HPAIV. These vaccines will be convenient and affordable, which will be highly beneficial to the poultry industry. Furthermore, immunization with these vaccines will permit serological differentiation of vaccinated and avian influenza field virus infected animals.     

Targeted expression of Cre recombinase provokes placental-specific DNA recombination in transgenic mice

Background

Inadequate placental development is associated with a high incidence of early embryonic lethality and serious pregnancy disorders in both humans and mice. However, the lack of well-defined trophoblast-specific gene regulatory elements has hampered investigations regarding the role of specific genes in placental development and fetal growth.

Principal Findings

By random assembly of placental enhancers from two previously characterized genes, trophoblast specific protein α (Tpbpa) and adenosine deaminase (Ada), we identified a chimeric Tpbpa/Ada enhancer that when combined with the basal Ada promoter provided the highest luciferase activity in cultured human trophoblast cells, in comparison with non-trophoblast cell lines. We used this chimeric enhancer arrangement to drive the expression of a Cre recombinase transgene in the placentas of transgenic mice. Cre transgene expression occurred throughout the placenta but not in maternal organs examined or in the fetus.

Significance

In conclusion, we have provided both in vitro and in vivo evidence for a novel genetic system to achieve placental transgene expression by the use of a chimeric Tpbpa/Ada enhancer driven transgene. The availability of this expression vector provides transgenic opportunities to direct the production of desired proteins to the placenta.     

Structural studies of the tandem Tudor domains of fragile X mental retardation related proteins FXR1 and FXR2

Background

Expansion of the CGG trinucleotide repeat in the 5′-untranslated region of the FMR1, fragile X mental retardation 1, gene results in suppression of protein expression for this gene and is the underlying cause of Fragile X syndrome. In unaffected individuals, the FMRP protein, together with two additional paralogues (Fragile X Mental Retardation Syndrome-related Protein 1 and 2), associates with mRNA to form a ribonucleoprotein complex in the nucleus that is transported to dendrites and spines of neuronal cells. It is thought that the fragile X family of proteins contributes to the regulation of protein synthesis at sites where mRNAs are locally translated in response to stimuli.

Methodology/Principal Findings

Here, we report the X-ray crystal structures of the non-canonical nuclear localization signals of the FXR1 and FXR2 autosomal paralogues of FMRP, which were determined at 2.50 and 1.92 Å, respectively. The nuclear localization signals of the FXR1 and FXR2 comprise tandem Tudor domain architectures, closely resembling that of UHRF1, which is proposed to bind methylated histone H3K9.

Conclusions

The FMRP, FXR1 and FXR2 proteins comprise a small family of highly conserved proteins that appear to be important in translational regulation, particularly in neuronal cells. The crystal structures of the N-terminal tandem Tudor domains of FXR1 and FXR2 revealed a conserved architecture with that of FMRP. Biochemical analysis of the tandem Tudor doamins reveals their ability to preferentially recognize trimethylated peptides in a sequence-specific manner.

Enhanced version

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Impact of GC content on gene expression pattern in chicken

Background

GC content varies greatly between different genomic regions in many eukaryotes. In order to determine whether this organization named isochore organization influences gene expression patterns, the relationship between GC content and gene expression has been investigated in man and mouse. However, to date, this question is still a matter for debate. Among the avian species, chicken (Gallus gallus) is the best studied representative with a complete genome sequence. The distinctive features and organization of its sequence make it a good model to explore important issues in genome structure and evolution.

Methods

Only nuclear genes with complete information on protein-coding sequence with no evidence of multiple-splicing forms were included in this study. Chicken protein coding sequences, complete mRNA sequences (or full length cDNA sequences), and 5^′ untranslated region sequences (5^′ UTR) were downloaded from Ensembl and chicken expression data originated from a previous work. Three indices i.e. expression level, expression breadth and maximum expression level were used to measure the expression pattern of a given gene. CpG islands were identified using hgTables of the UCSC Genome Browser. Correlation analysis between variables was performed by SAS Proprietary Software Release 8.1.

Results

In chicken, the GC content of 5^′ UTR is significantly and positively correlated with expression level, expression breadth, and maximum expression level, whereas that of coding sequences and introns and at the third coding position are negatively correlated with expression level and expression breadth, and not correlated with maximum expression level. These significant trends are independent of recombination rate, chromosome size and gene density. Furthermore, multiple linear regression analysis indicated that GC content in genes could explain approximately 10% of the variation in gene expression.

Conclusions

GC content is significantly associated with gene expression pattern and could be one of the important regulation factors in the chicken genome.     

Mapping and genotypic analysis of the NK-lysin gene in chicken

Background

Antimicrobial peptides (AMP) are important elements of the first line of defence against pathogens in animals. NK-lysin is a cationic AMP that plays a critical role in innate immunity. The chicken NK-lysin gene has been cloned and its antimicrobial and anticancer activity has been described but its location in the chicken genome remains unknown. Here, we mapped the NK-lysin gene and examined the distribution of a functionally significant single nucleotide polymorphism (SNP) among different chicken inbred lines and heritage breeds.

Results

A 6000 rad radiation hybrid panel (ChickRH6) was used to map the NK-lysin gene to the distal end of chromosome 22. Two additional genes, the adipocyte enhancer-binding protein 1-like gene (AEBP1) and the DNA polymerase delta subunit 2-like (POLD2) gene, are located in the same {"type":"entrez-nucleotide","attrs":{"text":"NW_003779909","term_id":"358468969","term_text":"NW_003779909"}}NW_003779909 contig as NK-lysin, and were thus indirectly mapped to chromosome 22 as well. Previously, we reported a functionally significant SNP at position 271 of the NK-lysin coding sequence in two different chicken breeds. Here, we examined this SNP and found that the A allele appears to be more common than the G allele in these heritage breeds and inbred lines.

Conclusions

The chicken NK-lysin gene mapped to the distal end of chromosome 22. Two additional genes, AEBP1 and POLD2, were indirectly mapped to chromosome 22 also. SNP analyses revealed that the A allele, which encodes a peptide with a higher antimicrobial activity, is more common than the G allele in our tested inbred lines and heritage breeds.     

The phytocyanin gene family in rice (Oryza sativa L.): genome-wide identification, classification and transcriptional analysis

Background

Phytocyanins (PCs) are plant-specific blue copper proteins involved in electron transport, and a large number of known PCs are considered to be chimeric arabinogalactan proteins (AGPs). To date there has not been a genome-wide overview of the OsPC gene family. Therefore, as the first step and a useful strategy to elucidate the functions of OsPCs, there is an urgent need for a thorough genome-wide analysis of this gene family.

Methodology/Principal Findings

In this study, a total of 62 OsPC genes were identified through a comprehensive bioinformatics analysis of the rice (Oryza sativa L.) genome. Based on phylogeny and motif constitution, the family of OsPCs was classified into three subclasses: uclacyanin-like proteins (OsUCLs), stellacyanin-like proteins (OsSCLs) and early nodulin-like proteins (OsENODLs). Structure and glycosylation prediction indicated that 46 OsPCs were glycosylphosphatigylinositol-anchored proteins and 38 OsPCs were chimeric AGPs. Gene duplication analysis revealed that chromosomal segment and tandem duplications contributed almost equally to the expansion of this gene family, and duplication events were mostly happened in the OsUCL subfamily. The expression profiles of OsPC genes were analyzed at different stages of vegetative and reproductive development and under abiotic stresses. It revealed that a large number of OsPC genes were abundantly expressed in the various stages of development. Moreover, 17 genes were regulated under the treatments of abiotic stresses.

Conclusions/Significance

The genome-wide identification and expression analysis of OsPC genes should facilitate research in this gene family and give new insights toward elucidating their functions in higher plants.