Screening Of Efficient SiRNA Target Sites Directed Against UL54 Gene Of Human Cytomegalovirus With SiRNA Expression Vectors

Human cytomegalovirus(HCMV) is a main pathogen responsible for a variety of severe diseases in immunocompromised patients, as well as a major cause of congenital defects in newborn children. Current antiviral agents can't surppress the virus thoroughly, let alone the side effects and drug resistance. Therefore, it is urgent to develop a more effective and safe anti-viral treatment. RNA interference, a new technology for silencing genes, is a promising therapeutic approach to deal with virus infection. The gene coding DNA polymerase of HCMV, UL54, is essential to the replication of the virus, which makes it possible to be the target for gene therapy. Short interfering RNA(siRNA), a key intermediate in the RNAi pathway, can silence gene expression by directing the sequence-specific cleavage of target messenger RNA . One of the challenges in applying small interfering RNAs is the identification of a potent siRNA/target sequence combination. That is, the complicated secondary structure of target mRNA is likely to hamper the recognition of target m&NA by siRNAs. Hence, it is necessary to design and pick out the functional siRNAs. In this study, we developed 6 short hairpin RNAs (shRNAs) expression vectors named psiUL54-1～6, each of which contains a human U6 small nuclear RNA promoter. Then -the shRNA expression vectors were co-transfected into human AD293 cells with the fusion protein expression vectors pUL54S1-EGFP and pUL54S2-EGFP. The fluorescent signals produced by fusion protein UL54S1/2-EGFP were detected byfluorescence microscopy at 24h, 48h after transfection respectively. We also applied the method of reverse transcription-polymerase chain reaction(RT-PCR) to evaluate the mRNA level of UL54S1 and UL54S2 at 24h, 48h after transfection. Furthermore, EGFP-positive rate and the mean fluorescence intensity of cell samples were analyzed by flow cytometry so as to determine whether siRNAs can inhibit the expression of fusion proteins. We finally established an effective and economic screening system of efficient siRNAs targeting UL54 gene in human cytomegalovirus with siRNA expression vectors. The reseach was composed of two parts, Part one:Construction of UL54S1-EGFP/UL54S2-EGFP fusion protein expression vectors and their expression in Human AD293 cells; Part two: Establishment of a system for screening of efficient siRNAs targeting UL54 gene with siRNA expression vectors.Part OneConstruction of UL54S1-EGFP/UL54S2-EGFP fusion protein expression vectors and their expression in Human AD293 cellsMethods:We designed primers targeting 784-bp (967-1750) and 763-bp (2308-3070) region of the HCMV UL54 gene , named UL54S1, UL54S2 respectively. Fragments produced by the restriction endonucleases Hind III, EcoR I from UL54S1 and UL54S2 gene were each cloned into the pEGFP-N1 vector. Then, the resulting plasmids were transformed into competent cells of E. coli DH5 α . The positive clones were screened initially by polymerase chain reaction(PCR) and restriction endonucleases which cut asymmetrically in the purified plasmids from individual colonies. Moreover, DNA sequencing confirmed the exact location of the fragments. Subsequently, we applied the fluorescence microscopy and flow cytometry to detect the expression of fusion proteins at 24h, 48h posttransfection. The method of RT-PCR was also utilized to evaluate the mRNA levels of UL54S1 and UL54S2 gene at 24h, 48h after transfection. Results:1.DNA sequencing confirmed that the sequence of the cloned fragments was consistent with UL54 gene(gene identification number: 1487749)at site 967-1750,2308-3070 from GeneBank databases.2.Fluorescent signals could be detected in pEGFP-N1, pUL54S1-EGFP group as well as pUL54S2-EGFP group at 24h, 48h after transfection by fluorescence microscopy; However, there were no enhanced green fluorescent proteins detected in the control group (vector negative cells).3.The transfection efficiency and mean fluorescence intensity of each group at 48h posttransfection were observed with the flow cytometry:38%, 590 (pEGFP-N1); 47%, 547 (pUL54S1-EGFP); 56%,578 (pUL54S2-EGFP).Conclusions: We succeeded to construct the fusion protein expression vectorspUL54S1-EGFP and pUL54S2-EGFP. It's convenient to detect the expression of enhanced green fluorescent proteins in AD293 cells by fluorescence microscopy. EGFP-positive rate and the mean fluorescence intensity of cell samples can be analyzed by flow cytometry, which makes it possible to detect UL54S1, UL54S2 gene quantitively. On the other hand, it will contribute to screen of efficient siRNA target sites directed against UL54 gene of human cytomegalovirus.Part twoEstablishment of a system for screening of effective siRNAs targeting UL54 gene with siRNA expression vectorsMethods: We took advantage of the online tool to design six pairs of 52-nucleotideoligonucleotides each encoding a 19-nucleotide shRNA, which contained extra Sall and Xball restriction sites to facilitate cloning. The single strains of 52-nucleotide oligonucleotides were annealed and cloned into the Sail and Xbal sites of the pAV_U6+27 vector, containing one U6 small nuclear RNA promoter and the first 27 nucleotides of human U6 RNA. The fusion protein expression vectors (pUL54S1-EGFP, pUL54S2-EGFP) were cotransfected with the plasmids coding shRNAs (named psiUL54) into AD293 cells using lipofectamine 2000 reagent. The levels of mRNA and enhanced green fluorescent protein were evaluated at 24,48h after transfection by means of RT-PCR, fluorescence microscopy and flowcytometry so as to assess the efficiency of inhibition by siRNA.Results:1.shRNA expression vectors psiUL54-1～6 were identified using PCR, and the results of DNA sequencing showed the inserted fragments of psiUL54-2～6 had expected known sequences, while fragments inserted into psiUL54-l could not be detected completely.2.EGFP expression analysis by fluorescent microscope revealed that the intensity of EGFP fluorescence in AD293 cells cotransfected pUL54S1-EGFP with psiUL54-3 decreased at 48h after transfection compared to the control group (pUL54Sl-EGFP , pAV_U6+27); While, psiUL54-4～6 /pUL54S2-EGFP group and psiUL54-2/pUL54S1-EGFP group had no noticeable inhibitory effects at 48h posttransfection.3.It was demonstrated that siRNA generated from psiUL54-3, not from psiUL54-2, could down regulate the level of UL54S1 mRNA assessed by RT-PCR 48h after transfection;other shRNA expression vectors psiUL54-4～6 displayed no significant reduction in UL54S2 mRNA level at the same time. None vectors affected UL54S1/UL54S2 mRNA levels at 24h posttransfection.4.The total fluorescence intensity of cells cotransfected pUL54S1-EGFP with psiUL54-3 was significantly lower than that of the control group at 48h after, transfection(P<0.01);The psiUL54-4～6 /pUL54S2-EGFP group almost made no difference to the total fluorescence intensity at 48h posttransfection compared to the control(P>0.05), likewise the psiULS4-21pUL54S1-EGFP group.Conclusion: siRNAs generated from shRNA expression vector psiUL54-3 could down regulate the mRNA level of UL54S1 gene, inhibit the expression of fusion protein UL54S1-EGFP effectively; The selected siRNA site corresponding to nt 1532-1550 of the UL54 coding sequence is an efficient site; We successfully established a system for screening of efficient siRNAs targeting UL54 gene with siRNA expression vectors.