Study On the Mechanism of LMP2A Maintaining Epstein-barr Virus Latency Infection Through Interaction With CXCR4

28 Epstein-Barr virus (EBV) belongs to the γ-herpesvirus subfamily and is the first human tumor 29 virus to be discovered. The global adult infection rate exceeds 90%. EBV can participate in the 30 regulation of multiple genes and multiple signal pathways through its latent genes. Many studies 31 have reported that CXCR4 is involved in the development of gastric cancer, but there are few studies 32 on the specific mechanism of its role in EBV-associated gastric cancer (EBVaGC). In this study, we 33 explored the mechanism by which EBV-encoded products maintain EBV latent infection through 34 interaction with CXCR4, and the role of CXCR4 in EBV positive cells. The results show that there 35 is a positive feedback between the EBV-encoded products and CXCR4, and LMP2A can activate 36 CXCR4 through the NF-κB pathway. In addition, CXCR4 can be fed back to LMP2A and EBNA1 37 through the ERK signaling pathway. At the same time, CXCR4 can promote the proliferation and 38 migration of EBV-positive cells, reduce the expression of the immediate early protein BZLF1, and 39 play an important role in maintaining the incubation period of EBV infection. These findings are 40 conducive to the further targeted therapy of EBVaGC. 41


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C-X-C motif chemokine receptor 4 (CXCR4), a 352 amino acid rhodopsin like GPCR, was 67 first studied as a chemokine receptor associated with breast cancer metastasis to lung tissue [30].

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CXCR4 is the most widely expressed chemokine receptor in more than 23 human cancers, including 69 breast cancer, ovarian cancer, melanoma, etc., and is involved in tumor growth, angiogenesis, 70 metastasis, etc. [4]. Several studies have reported that CXCR4 is involved in the proliferation and 71 metastasis of gastric cancer and is closely related to the clinical prognosis of gastric cancer [31,32].

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EBVaGC is a unique molecular subtype of gastric cancer that accounts for 8.7% of the total number 73 of gastric cancers according to big data [20]. Based on the high incidence and mortality of gastric 74 cancer worldwide, the pathogenesis of EBVaGC is important.

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EBV can participate in the regulation of multiple signaling pathways through its encoded latent 76 genes, such as NF-κB, JNK, PI3K/AKT, etc., and then regulate the expression of downstream target 77 genes to promote the occurrence and development of tumors. Longnecker      In SNU719-CXCR4 cells that stably expressed CXCR4, the protein expressions of LMP2A 190 and EBNA1 were increased. It was further found that CXCR4 overexpression induced the 191 expression of ERK and phosphorylated ERK(p-ERK) (Fig.3a). Meanwhile, after CXCR4 siRNA 192 treatment, the expressions of ERK and p-ERK in GT38 and SNU719 cells were significantly 193 decreased (Fig.3b, Fig.3c). In order to further understand the mechanism of CXCR4 overexpression 194 leading to the up-regulation of LMP2 and EBNA1, the ERK1/2 inhibitor PD0325901 at 2 μM and 195 4 μM concentrations was cultured in six-well plates inoculated with GT38 cells for 48h, respectively.

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Western-blot results showed that p-ERK was significantly reduced in the inhibitor treatment group,

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and LMP2A and EBNA1 were also reduced to different degrees in a dose-dependent trend (Fig.4a).
In addition, we found that knockdown ERK1/2 had 200 a strong inhibitory effect on the expression of LMP2A and EBNA1 (Fig.4c, Fig.4d). In conclusion,

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these results suggest that LMP2A and EBNA1 expression in EBVaGC is partially dependent on the 202 CXCR4-ERK pathway.

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Our previous studies have demonstrated that LMP2A, the EBV coding product, induces NF- 288 and EBNA1 in SNU719-CXCR4 cells were significantly higher than those in SNU719-NC cells.

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CXCR4 was confirmed to be involved in maintaining latent EBV infection by inhibiting BZLF1 and 290 subsequently inducing potential EBV products LMP2A and EBNA1.

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In conclusion, there is a positive feedback between EBV latency coding products and CXCR4,     The effect of CXCR4 on ERK signal pathway. (a) The protein level of ERK, p-ERK and CXCR4 in SNU719-CXCR4 cell was detected by Western blot (*p < 0.05). (b) The effects on ERK, p-ERK and CXCR4 were analyzed by Western blot after interference of CXCR4 in GT38 cell line (**p < 0.01, *p < 0.05). (c) The effects on ERK, p-ERK and CXCR4 were analyzed by Western blot after interference of CXCR4 in SNU719 cell line (**p < 0.01, *p < 0.05). Data are representative of three independent experiments.   Graphic summary. EBV encoded latent membrane protein 2A activates seven transmembrane proteins CXCR4 through the NF-κB signaling pathway, and CXCR4 upregulates EBV encoded latent membrane protein 2A and EBV nuclear antigen 1 through the ERK signaling pathway.