A Preliminary Study Of The Poly-cistronic Translational Control In Avian Polyomavirus-1 Late Genes

The purpose of this study is to explore the mechanism of the poly-cistronic translational control in avian polyomavirus late genes,especially the role of APV-1 Agno-1a protein regulating downstream VPs protein synthesis.A series of the new recombinant cDNA viruses were constructed by inserting one and two in-frame 27 bp ATG DNA segment(s)with the better Kozak translational initiation signal in the ATG region of APV-1 agno-1a mRNA encoding sequence.The start codon ATG of agno-1a mRNA was mutated to ACG or CTG by PCR.The ATG mutated recombinant clones might shut down the Agno-1a protein translation and change the poly-cistronic mRNA key structure more or less.The artificial fragment inserted clones: the artificial synthesized and phosphorylated 27 bp target fragment was linked with the AccI partially digested DNA fragment of cDNA pHL1003 by T4 ligase.The successful fragment inserted cDNA clones,pHL1003+1 and pHL1003+2,were screened and verified by DNA sequencing.Point mutated clones: The DNA fragment with the agno-1a start codon ATG mutated to ACG or CTG by PCR were inserted into the pHL1003 vector DNA fragment cutted by NcoI and AccI(partially digestion).Screening positive clones and verifying by DNA sequencing.A eukaryotic bi-cistronic expression vector with downstream EGFP reporter gene was designed and constructed by replacing the late structural genes VPs of wild-type APV-1 cDNA pHL1003 with EGFP gene.pHL1003-1,an Agno-1a expression clone,was built by removed the APV-1 VPs genes region,which contains APV-1 Ori,early and late Agno-1a region.The EGFP coding sequence of pEGFP-N1(+)was ampified by PCR was inserted into the downstream of agno-1a gene of pHL1003-1.The recombinant pHL1003-GFP,a eukaryotic bi-cistronic APV-1 late genes expression vector with downstream EGFP reporter gene,was got by screening and sequencing.pHL1003+1,pHL1003+2 and pHL1003-GFP were cut by Nru I and Ned I.The DNA fragment with the extra one or two ATG(s)was linked with the vector pHL1003-GFP DNA fragment respectively so as to get pHL1003+1-GFP and pHL1003+2-GFP clones.Through the transfection method of calcium phosphate transfection and liposome,the plasmid DNA of the constructed cDNA mutant was transfected into embryonic chicken and quail fibroblast cells.The APV-1 late VPs and EGFP genes expression were observed by Western blot(the antibody against full APV-1 virion)and fluorescence microscopy respectively.Western blot results showed that there were some viral specific bands,including VP1,VP2,VP3 and Agno-1a.After 72 h transfection,the strong fluorescence in the DNA transfeced chicken and quail embryo fibroblast cells(CEF and QEF)was observed by fluorescence microscopy.These results reveal that the EGFP gene located at downstream of APV-1 eukaryotic bi-cistronic expression vector could be expressed in chicken and quail embryonic fibroblast cells.The culture time after the transfection,the transfection amount of plasmid DNA and the culture medium pH in the transfection process were optimized for improving the transfection efficiency.Based on the GFP expression difference in the condition of different cDNAs and its different combinations to transfect CEF and QEF and statistical analysis of the results,the data imply that the insertion of additional initiation codon ATG in the upstream of APV-1 late polycistronic genes would influence the downstream cistron translation;The viral Agno-1a protein could positively control the downstream genes expression,especially in its translational initiation.