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- Timmusk, Sirje, et al.
(author)
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Regulator of G protein signalling 16 is target for a porcine circorvirus type 2 protein
- 2009
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In: Journal of General Virology. - : Microbiology Society. - 0022-1317 .- 1465-2099. ; 90, s. 2425-2436
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Journal article (peer-reviewed)abstract
- Interaction studies have suggested that the non-structural protein encoded by open reading frame 3 (ORF3) of porcine circovirus type 2 (PCV2) binds specifically to a regulator of G protein signalling (RGS) related to human RGS16 (huRGS16). The full-length clone of RGS16 was generated from porcine cells and sequence analysis revealed a close relationship to huRGS16 and murine RGS16. In vitro pull-down experiments verified an interaction between porcine RGS16 (poRGS16) and ORF3 from PCV2. Using GST-linked ORF3 proteins from three different genogroups of PCV2 and from porcine circovirus type 1 (PCV1) in the pull-down experiments indicated that there were differences in their ability to bind poRGS16. Quantitative RT-PCR demonstrated that the expression of poRGS16 mRNA could be induced by a number of cell activators including mitogens (LPS and PHA), interferon inducers (ODN 2216 and poly I : C) and the neurotransmitter norepinephrine. Immunofluorescence labelling confirmed the induced expression of poRGS16 at the protein level and suggested that the PCV2 ORF3 protein co-localized with poRGS16 in LPS-activated porcine PBMC. Furthermore, poRGS16 appeared to participate in the translocation of the ORF3 protein into the cell nucleus, suggesting that the observed interaction may play an important role in the infection biology of porcine circovirus.
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| 3. |
- Timmusk, Sirje
(author)
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Studies of immunoglobulin light chains in rainbow trout (Oncorhynchus mykiss): new insight into the evolution of immunoglobulin genes
- 2002
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Doctoral thesis (other academic/artistic)abstract
- In this thesis, the structure and the expression of immunoglobulin (Ig) light chain genes were studied in rainbow trout and including an evolutionary perspective. A novel Ig light chain locus (IgL2) was identified and characterized. As an expansion of this study, the genomic organization of two Ig light chains was settled in genomic clones, some putative transcription regulatory elements were identified and the expression of L1 and L2 was studied in several tissues. In order to study expression of L1 and L2 at the protein level, monoclonal antibodies were generated, using DNA immunization. The contribution of different IgL isotypes in the antibody response against a fish rhabdovirus was investigated. This study demonstrated that the variability of L1 and L2 was modified in different manners. Genomic clones for a third trout light chain (IgL3) were isolated, and the sequence of the putative promoter regions of the three isotypes were compared. The L1 and L3 promoters have a classical Ig promoter structure (i.e. with TATA-box, E-box, and octamer) whereas the L2 promoter lacks the typical features of an Ig promoter. The IgL2 promoter region contained a κ-Y motif and an E-box. Cell transfection studies using different deletion constructs demonstrated that the κ-Y element drives the transcriptional activity of L2 promoter. Both the L1 and L2 promoters can cooperate with a B cell specific enhancer from cod, suggesting that common transcription factors are used.L1 is most similar to the κ isotype found in mammals whereas L2 shows many features that deviate from this branch and the λ branch and forms a separate branch. The similarity between the L1C and L2C of trout Ig is about 30%. Thus, a sequence comparison of L1C regions with L3C regions shows a continuous transition in identity percentages, whereas the L2C appears not closely related to any C segment from another isotype. These results pave the way for a comprehensive understanding of the evolution, regulation and functions of light chains in a primitive vertebrate the rainbow trout, and other fish species.
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| 4. |
- Wikström, Frida Hasslung, et al.
(author)
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Structure-dependent modulation of alpha interferon production by porcine circovirus 2 oligodeoxyribonucleotide and CpG DNAs in porcine peripheral blood mononuclear cells.
- 2007
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In: Journal of virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 81:10, s. 4919-27
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Journal article (peer-reviewed)abstract
- DNA sequences containing CpG motifs are recognized as immunomodulators in several species. Phosphodiester oligodeoxyribonucleotides (ODNs) representing sequences from the genome of porcine circovirus type 2 (PCV2) have been identified as potent inducers (ODN PCV2/5) or inhibitors (ODN PCV2/1) of alpha interferon (IFN-alpha) production by porcine peripheral blood mononuclear cells (poPBMCs) in vitro. In this study, the IFN-alpha-inducing or -inhibitory activities of specific phosphodiester ODNs were demonstrated to be dependent on their ability to form secondary structures. When a poly(G) sequence was added to a stimulatory self-complementary ODN, high levels of IFN-alpha were elicited, and the induction was not dependent on pretreatment with the transfecting agent Lipofectin. In addition, the IFN-alpha-inducing ODN required the presence of an intact CpG dinucleotide, whereas the inhibitory activity of ODN PCV2/1 was not affected by methylation or removal of the central CpG dinucleotide. Of particular significance, the IFN-alpha inhibition elicited by ODN PCV2/1 was only effective against induction stimulated by DNA control inducers and not RNA control inducers, indicating activity directed to TLR9 signaling. The PCV2 genome as a whole was demonstrated to induce IFN-alpha in cultures of poPBMCs, and the presence of immune modulatory sequences within the genome of PCV2 may, therefore, have implications with regard to the immune evasion mechanisms utilized by PCV2.
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