P300 Promotes B Cell Lymphoma Proliferation Through Suppressing Kaposi's Sarcoma-Associated Herpesvirus(KSHV) Reactivation

Primary Effusion Lymphoma(PEL)is a B-cell lymphoma associated with Kaposi's sarcoma herpesvirus(KSHV)infection.However,the mechanism of oncogenesis of PEL is still unclear.Studies have shown that the cellular transcriptional coactivator p300 regulates the interaction between host and virus,which plays a vital role in viral infection.In this study,we investigated the role of p300 in BCBL1 cells during the KSHV life cycle.Result show that:(1)knockout p300 result in a significant increase in KSHV early lytic genes expression and promote gene expression with BCBL1 cells proliferation;(2)knockout p300 significantly inhibited the lytic activation of KSHV,in other words,the expression of early gene RTA and late lytic gene K8;(3)the number of copies of the KSHV genome and the production of virus particles in the cells after knockout of the p300 are reduced;(4)knockout of p300 inhibited the proliferation of KSHV infected BCBL1 cells.These findings indicate that p300 promotes B-lymphoma cell proliferation by inhibiting the reactivation of Kaposi's sarcoma-associated herpesvirus.Purpose: 1.To understand the effects of p300 on the proliferation of KSHV infection BCBL1 cells and regulation of KSHV genes expression.2.To explore the effect of p300 on KSHV virus replication.3.To investigate the effect of p300 on BCBL1 cell proliferation.Method: 1.Construction of BCBL1 cell line with stable knockout p300 a)Construction of BCBL1 cell line with stable knockout p300 In our study,we will use the lenti-CRISPR lentiviral knockout system to design a sg RNA containing the target gene p300.The sg RNA is transfected into 293 T cells with the lentiviral packaging system.The infected BCBL1 cell was collected and screened with puromycin into stable cell lines.b)Identification of the stable cell lines that knockout p300 To confirm the knockout of p300 in the cell line,the expression of p300 in the BCBL1 sg RNA cell line was detected by western blot,and the expression level of p300 m RNA was detected by RT-q PCR.2.Analysis of the stable cell lines that knockout p300 with RNA-seq The high-throughput sequencing of the extracted RNA was conducted in BCBL1-sgctrl and BCBL1-sgp300 cells.3.To explore the effect on KSHV lytic replication in infected BCBL1 cells after knockout the p300 To identify the effect of p300 on KSHV virus replication,BCBL1-sgctrl and BCBL1-sgp300 cells were treated with TPA/SB inducer for 5 days.Cell and supernatant were collected at different time points,and the viral DNA was extracted from cells and supernatants,and the alteration of copy numbers in the KSHV genome was detected by RT-q PCR.4.To detect the effect of p300 on the proliferation of BCBL1 cells Cell numbers and proliferation were measured using trypan blue staining and CFSE reagent.Result: 1.We successfully construct a stable cell line that knockout p300 and obtained RNA-seq data.The result showed that p300 knockout resulted in a significant increase in the KSHV early lytic geges and promote gene expression associated with BCBL1 cell proliferation.2.After activation of KSHV with TPA/SB,it was found that knockout of p300 significantly inhibited the activation of KSHV,in other words,knockout of p300 significantly inhibited the expression of RTA and K8 gene.Additionally,the intracellular KSHV genome copy number and the virion production were reduced.3.We observed that the proliferation of BCBL1 was inhibited by knockout of p300.Conclusion: In conclusion,this study demonstrated that p300 promotes proliferation of B-lymphoma cells by inhibiting the reactivation of Kaposi's sarcoma-associated herpesvirus(KSHV),which further helped elucidate the oncogenic mechanism of PEL caused by KSHV infection.