Unconserved C Terminal Of Human Cytomegalovirus Tegument Protein PUL76 Induces Chromosome Damage In Transfected Cells

Objective To define the inducing DNA breaks is determinated by which part of the p UL76,one tegument protein of HCMV. Full-length, conserved N terminal and unconserved C terminal of p UL76 were constructed to eukaryotic expression plasmid p EGFP-N1 and then transfected to cells.Method DNA fragments that encoding full-length, conserved N terminal and unconserved C terminal of p UL76 were acquired by PCR method. Primers using here to amplify these fragments were designed to contain restriction endonuclease site HindⅢ and Bam HIat the 5’ end and 3’ end respectively. These fragments were constructed to p EGFP-N1 in frame with sequence encoding enhanced green fluorescent protein.The recombinant plasmids were designate p EGFP-UL76, p EGFP-UL76 N, p EGFP-UL76 C respectively. Double digestion and sequencing were performed to verify the accuracy of recombinant plasmids construction. These plasmids and p EGFP-N1 were transfected to HEK293 cells and HF cells. Indirect immunofluorescent assay was performed to detect colocalization of chromosome damage signal γ-H2 AX and EGFP-fusion proteins. Also the relative expression level of γ-H2 AX in different transfection groups were measured by Western blot. Comet assay was used to evaluate DNA damage in each group.Results:1. Double digestion and sequencing firmed the accuracy of different recombinant plasmids construction. Anti-EGFP antibody deteced three bands of EGFP-fusion protein with expected molecular weight in WB assay. 2. Colocalization of γ-H2 AX and EGFP-fusion protein was found in cells transfected with either p EGFP-UL76 or p EGFP-UL76 C. But these was merely to be found in p EGFP-N1 or p EGFP-UL76 N transfection groups. The relative expression level ofγ-H2 AX were elvated in p EGFP-UL76 or p EGFP-UL76 C transfection groups compared to the control. The p EGFP-UL76 N transfection group has the equal level of γ-H2 AX compared to the control. 3. Precentage of cells with comet tails were higher in cells transfected with either pEGFP-UL76 or pEGFP-UL76 C compared to the p EGFP-N1. Precentage of cells with comet tails were comparative to the control in cells transfected with p EGFP-UL76 N. 4. Flourescent signal were concentrated to form aggresomes in nucleus in either p EGFP-UL76 or p EGFP-UL76 C transfection group, while it diffused along the cells in cells transfected with p EGFP-N1 or p EGFP-UL76 N.Conclusion: In this study we further demonstrated that the unconserved C terminal(not the conserved N terminal) of p UL76 was sufficient to induce DNA damage in transfected cells.