Construction And Identification Of A KSHV Encoded K13 Or K9 Deletion Mutant

Background: Kaposi's sarcoma-associated herpesvirus(KSHV)is a ?2 typeherpesvirus and encodes about 90 open reading frames(ORFs).KSHV K13 coded viral Fas-associated death domain-like interleukin 1? converting enzyme inhibitory protein(v FLIP)and K9 coded viral interferon regulatory factor 1(v IRF1)have been demonstrated to participate in KSHV induced tumorigenesis through virous pathways.Previously,most studies are based on overexpression systems,while the influence when K13 or K9 deleted from KSHV genome on KSHV tumorigenesis is not clear.Objective: To construct K13 or K9 deleted KSHV mutants and apply them for the functional research of K13 or K9 based on the KSHV whole genome.Methods: In this study,we generated recombinant viruses lacking the CDS of K13 or K9 gene from KSHV RGB-BAC16 genome through two-step red-mediated recombination which is manipulated in E.coli GS1783.PCR,sequencing and digestion by restriction endonuclease were used to verify whether the mutant was constructed successfully.The RGB-BAC16 were transfected into i SLK-puro cells and screened by antibiotics until the rate of fluorescence was up to 90%.The wide type and mutant KSHV viruses was induced in i SLK cell lines and the viruses were used in de novo infection of HUVECs.RT-q PCR,western blot and dual luciferase reporter assay system have been used to verify the function of KSHV infected HUVECs.Results: In this project,we generated recombinant viruses lacking the CDS of K13 or K9 gene from KSHV RGB-BAC16 genome.The mutant viruses producer,i SLK cells were stably transfected with K13 or K9-deleted mutant bacmids.Importantly,the v FLIP or v IRF1 knockout viruses possessed the ability of de novo infection of HUVECs.Conclusions: This project generated recombinant KSHV viruses lacking the CDS of K13 or K9,and it can help study the function and molecular mechanism of both oncoprotein v FLIP and v IRF1.