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.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) was recently verified as a precise and robust strategy for targeted genome editing [20][21][22]. Most commonly, Cas9 endonuclease and a single-guide RNA (sgRNA) are utilized to target a 20-bp-long DNA region that is complementary to the sgRNA [21,23]. CRISPR/Cas9 technology enables loss-offunction genetic analysis of regulatory elements in the coding or non-coding region of a gene [24,25] and robust potential for genetic modification. The CRISPR/Cas9 system has been applied to develop various antiviral strategies [26,27], including against human herpesvirus (HHV) [28,29]. This strategy is more effective than other antivirus methods, particularly for viruses that integrate into human chromosomes, such as human immunodeficiency virus (HIV) and human papillomavirus (HPV) [30]. It was also demonstrated to effectively eliminate EBV genomic episomes from latent cells [31,32].
The present study aimed to evaluate the effect of CRISPR/Cas9-mediated knockout of LMP1 or LMP2A on CNE-2 cell proliferation.

Reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR)
Total mRNA was extracted from CNE-2 cells with a magnetic mRNA Isolation Kit (New England Biolabs, Ipswich, MA USA) according to the manufacturer's protocol. Equivalent amount of mRNA sample was transcribed into cDNA with a GoScript™ Reverse Transcriptase (Promega, Madison, WI, USA). EBV viral DNA was purified with genomic DNA isolation kits (Qiagen, Venlo, Netherlands) under the guidance of the kit's instruction. The cDNA was examined by RT-qPCR (PrimeScript™ RT Reagent Kit, Takara, Tokyo, Japan) to determine the relative mRNA levels of LMP1, LMP2A, and eIF4E using β-actin for gene expression normalization with LightCycler 2.0 (Roche Diagnostics, Risch-Rotkreuz, Switzerland). To quantify EBV genomic DNA in CNE cells after EBV infection, the levels of EBNA1 and OriP were quantified with RT-qPCR. The EBNA1 or OriP DNA level is presented as a relative value to each DNA level at 0-h post-EBV infection.
Colony-forming assay and cell growth assay CNE-2 cells, with LMP1-or LMP2A-overexpression or with LMP1-and/or LMP2A-knock out, were seeded and incubated in six-well plates for three days. Colony formation was observed under an Olympus BX60 microscope (Olympus, Tokyo, Japan). Cell growth was assessed with CCK-8 assays (DOJINDO, Tokyo, Japan). Briefly, CNE-2 cells were suspended (100 μl/well) in a 96-well plate and incubated in a humidified incubator (at 37°C with 5% CO 2 ) for 24, 48, or 72 h. Next, 10 μl CCK-8 solution was added to each well for 2-h's incubation at 37°C, and then absorbance at 450 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA).
All experiments were approved by the Ethics Committee of Chongqing Medical University (20170121).

Statistical analysis
Quantitative results are presented as mean ± standard deviation and were analyzed with two-tailed Student's ttests. Differences were considered significant at p < 0.05.

Results
EBV LMP1 promotes NPC CNE-2 cell growth in an eIF4Edependent fashion We overexpressed LMP1 or LMP2A in CNE-2 cells with lentiviral vectors, using CAT-carrying lentivirus as the upregulation control (abbreviated as up control). Figure 1a shows dramatically increased mRNA levels of LMP1 and LMP2A in the LMP1 up-regulation (LMP1 up) and LMP2A up-regulation (LMP2A up) groups, respectively, in comparison with the Up-control group (both p < 0.001). Both LMP  < 0.0001, Fig. 1b). Also, higher eIF4E mRNA levels were observed in LMP1-overexpressing CNE-2 cells with or without LMP2A overexpression (p < 0.001 Fig. 1a). Higher eIF4E protein level was also found in CNE-2 cells (Fig. 1b). To evaluate the LMP regulation of NPC cell growth, we performed colony-forming assays in CNE-2 cells followingLMP1-and LMP2A-lentivirus infection. As indicated in Fig. 1c, significantly larger colonies were formed by CNE-2 cells after LMP1lentivirus infection compared to LMP2A-as well as LMP1&LMP2A-lenticirus infection (p < 0.001 for the LMP1 up and LMP1&LMP2A up groups, Fig. 1d).
Besides, growth curves confirmed that LMP1 increases cell growth (p < 0.0001, Fig. 2a). We evaluated whether LMP1mediated promotion of CNE-2 cell growth is dependent on eIF4E. Effective knockdown of eIF4E (p < 0.001, Fig. 2b) significantly inhibited growth promotion in CNE-2 cells (p < 0.05, Fig. 2 c and d). Thus, LMP1-mediated promotion of NPC cell growth was observed in CNE-2 cells and was at least partly dependent on eIF4E.
CRISPR/Cas9-mediated knockout of LMP1 or LMP2A inhibits EBV replication To explore the potential of CRISPR/Cas9-mediated knockout of EBV oncogenic proteins, LMP1-and LMP2A-targeted gRNA sites and the schematic diagram of the constructed LMP1-and LMP2A-editing systems were shown in Fig. 3a. The conservation of the target sites was checked via aligning the target sequences with other viral coding sequences. The successful insertion and effective transfection of either gRNA1 + 2 (LMP1) / gRNA1 + 2 (LMP2A) or the control plasmid were indicated by the expression of green fluorescence protein, at the 12-h post transfection (Fig. 3b). The editing effectiveness of both LMP targets is shown in Fig. 3 c and d under the same transfection efficiency.  Both mRNA (p < 0.0001 respectively, Fig. 4a) and protein (Fig. 4b) levels of LMP1 and LMP2A were markedly knocked down in CNE-2 cells at 24 h after EBV infection. In addition, the regulation by LMP1 or LMP2A editing on virus replication was also examined. The virus growth was evaluated with the viral DNA levels of EBNA1 and OriP. A significant reduction of EBNA1 (Fig. 4c) and of OriP (Fig. 4d) DNA (p < 0.05 or p < 0.01 for 6 or 12-days postinfection) was caused by the knockout of either LMP. These results confirmed that the LMP1 or LMP2A knockout inhibited the EBV replication.
CRISPR/Cas9mediated knockout of LMP1 inhibits CNE2 cell growth and eIF4E activation CRISPR/Cas9-mediated knockout of both LMP1 and LMP2A was performed before measuring CNE-2 cell growth with colony forming assays. LMP1 knockout markedly decreased colony size in LMP1 up cells (p < 0.001, the right column, Fig. 5 a, b). However, LMP2A knockout did not have any effect (the middle column, Fig. 5 a, b). Significant colony size reduction was also observed in LMP1&LMP2A up cells after LMP1 knockout (p < 0.01) rather than LMP2A knockout. Furthermore, knockout of either LMP did not influence colony size in LMP2A up cells. Therefore, CRISPR/Cas9-mediated knockout of LMP1 inhibited CNE-2 cell growth. In addition, we also examined the regulation by the CRISPR/Cas9-mediated knockout of LMP1 or of LMP2A on the eIF4E activation in CNE-2 cells. As indicated in Fig. 6a, the eIF4E mRNA was upregulated by the EBV infection from 3 to 12 days post-infection (p < 0.001). However, the eIF4E mRNA upregulation was blocked by either LMP1 knockout (p < 0.05 or p < 0.01) or by LMP2A knockout (p < 0.05, Fig. 6b). Such regulation was also confirmed at protein level. Not only LMP1 and LMP2A but also eIF4E were downregulated by the knockout of either LMP1 or LMP2A during EBV infection (Fig. 6c). Each value was an average for triple independent results. Statistical significance was showed as * p < 0.05, ** p < 0.01, **** p < 0.0001, ns: no significance Discussion LMP1 upregulation was demonstrated to confer a proliferative advantage and the ability to resist apoptosis to NPC cells, via the interaction of LMP1 with cell cyclerelated molecules such as NF-κB, JNK, and phosphatidylinositide 3-kinases (PI3K) [33]. In the present study, we confirmed in vitro that LMP1 promoted the proliferation of NPC CNE-2 cells at least partly via eIF4E upregulation. Previous studies indicated that LMP1 stimulated eIF4E transcription [17] in LMP1-harboring NPC B95-8 cells. However, other signaling pathways, including HMGB1 [34], DNAzyme [18] and LKB1-AMPK [19] might also be involved. EBV infection was shown to cause a statistically significant overexpression of HMGB1 in NPC tissues, in association, in association with the malignant status of NPC. The HMGB1 upregulation was demonstrated to induce NPC cells proliferation, RAGE-dependently [34]. DNAzyme (DZ) 1 [18] Fig. 5 CRISPR/Cas9-mediated knockout of LMP1 or LMP2A regulates CNE-2 cell growth. a and b: Colony formation (a) and size (b) of CNE-2 cells following the targeted knockout of LMP1 or LMP2A, via transfection with the LMP1-or LMP2A-specific gRNA-Cas9 co-expression plasmid. Each experiment was independently replicated three times. ** p < 0.01; *** p < 0.001; ns, no significance and LKB1-AMPK pathway [19] have also been found involved in such process via regulating cell cycle [18] or via regulating the proliferation and transformation of epithelial cells [19].
Advanced NPC has a high mortality rate [35]. Due to the anatomical complexity of the nasopharynx, surgical resection for NPC is technically challenging. Despite sensitivity to radiotherapy at early stages and encouraging clinical outcomes, it is still a challenge to administer for the same reason [36]. The roles of induction and adjuvant chemotherapy remain to be well defined [37,38]. Given the overwhelming evidence of a robust oncogenic effect of EBV infection, targeting EBV infection might be a potential therapy. The present study and previous reports [39,40] confirm the effective inhibition that anti-EBV infection strategies have on NPC cell growth. Notably, the coding sequence of the two latent viral proteins, LMP1 and LMP2A, integrates into the host genome and pose the ontogenetic effect for a lifelong time. It is a vital strategy to eliminate the two oncogenic genes from contaminated cells.
The CRISPR/Cas9 system derives from a prokaryotic antiviral immune system and can also effectively eliminate HPV genomic episomes from latent cells [28,29]. Prevention of EBV infection by CRISPR/Cas9 mutagenesis was demonstrated in EBV-transformed Blymphoblastoid cells [41,42], and this was directly related to LMP1 [43]. We constructed here LMP1-and LMP2A-targeted CRISPR/Cas9 systems to knockout the expression of either viral protein in CNE-2 cells, and we confirmed the anti-cancer effect of CRISPR/Cas9-mediated LMP1 knockout in CNE-2 cells. Such anti-cancer effect was associated with the blockage of eIF4E activation, Our promising results indicate that CRISPR/Cas9-mediated knockout of either LMP1 or LMP2A can prevent EBV infection of CNE-2 cells. LMP1-mediated promotion to NPC cell growth 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.  were examined by the method of Real-time quantitative PCR analysis (a) or of western blotting (b). Experiments were independently repeated in triplicate. ** p < 0.01; *** p < 0.001; ns, no significance the study and performed the statistical analysis,conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. All contributors who do not meet the criteria for authorship should be listed in an acknowledgements section.

Funding
The present study was supported by the grant from the Nature Fund of the First Affiliated Hospital of Chongqing Medical University (2018pyjj-01).
Availability of data and materials All data in this manuscript were available upon a request.
Ethics approval and consent to participate Participate were approved by Chongqing Medical University thics committee,reference number (CQMU201604752).

Consent for publication
All authors consent for publication.