CRISPR/Cas9-mediated LMP1 knockout inhibits Epstein-Barr virus infection and nasopharyngeal carcinoma cell growth

BACKGROUND: A strong association between Epstein-Barr virus (EBV) infection and nasopharyngeal carcinoma (NPC) has been widely recognized in recent decades. The aim of the present study was to investigate latent membrane protein 1 (LMP1) regulation of nasopharyngeal carcinoma (NPC) CNE-2 cell growth and then examine the effects of LMP1-knockout with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9on Epstein-Barr virus (EBV) infection and CNE-2 cell growth. METHODS: Human NPC CNE-2 cells were infected with the recombinant LMP1- and LMP2A-carrying lentivirus, and then examined for cell growth with the colony forming assay as well as for the activation of transcription of eukaryotic translation initiation factor 4E (eIF4E) with reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and western blot. CRISPR/Cas9-mediated knockout of LMP1 or LMP2A was performed with a single-guide RNA (sgRNA) targeting sequences within LMP1 or LMP2A. The knockout effect and the EBV proliferation were examined with RT-qPCR, western blot and cell growth assay. RESULTS: LMP1 overexpression promoted CNE-2 cell growth, compared to LMP2A overexpression. Loss-of-function experiments confirmed that eukaryotic translation initiation factor 4E (eIF4E) upregulation mediated this effect. LMP1 knockout significantly inhibited EBV proliferation in CNE-2 cells and markedly inhibited LMP1-mediated promotion of cell growth. The knockout of either LMP1 or LMP2A blocked the eIF4E activation, which is induced either by the EBV infection or by the overexpression of LMP1 or LMP2A. CONCLUSION: We confirmed the LMP1-mediated promotion of NPC cell growth. Such promotion can be effectively blocked by CRISPR/Cas9-mediated LMP1 knockout. Precise LMP1 knockout might be a promising method for targeted inhibition of EBV infection and NPC cell growth.