B cells infected with Type 2 Epstein-Barr virus (EBV) have increased NFATc1/NFATc2 activity and enhanced lytic gene expression in comparison to Type 1 EBV infection

Abstract

Humans are infected with two distinct strains (Type 1 (T1) and Type 2 (T2)) of Epstein-Barr virus (EBV) that differ substantially in their EBNA2 and EBNA 3A/B/C latency genes and the ability to transform B cells in vitro. While most T1 EBV strains contain the "prototype" form of the BZLF1 immediate-early promoter ("Zp-P"), all T2 strains contain the "Zp-V3" variant, which contains an NFAT binding motif and is activated much more strongly by B-cell receptor signalling. Whether B cells infected with T2 EBV are more lytic than cells infected with T1 EBV is unknown. Here we show that B cells infected with T2 EBV strains (AG876 and BL5) have much more lytic protein expression compared to B cells infected with T1 EBV strains (M81, Akata, and Mutu) in both a cord blood-humanized (CBH) mouse model and EBVtransformed lymphoblastoid cell lines (LCLs). Although T2 LCLs grow more slowly than T1 LCLs, both EBV types induce B-cell lymphomas in CBH mice. T1 EBV strains (M81 and Akata) containing Zp-V3 are less lytic than T2 EBV strains, suggesting that Zp-V3 is not sufficient to confer a lytic phenotype. Instead, we find that T2 LCLs express much higher levels of activated NFATc1 and NFATc2, and that cyclosporine (an NFAT inhibitor) and knockdown of NFATc2 attenuate constitutive lytic infection in T2 LCLs. Both NFATc1 and NFATc2 induce lytic EBV gene expression when combined with activated CAMKIV (which is activated by calcium signaling and activates MEF2D) in Burkitt Akata cells. Together, these results suggest that B cells infected with T2 EBV are more lytic due to increased activity of the cellular NFATc1/c2 transcription factors in addition to the universal presence of the Zp-V3 form of BZLF1 promoter.

EBNA2-deleted Epstein-Barr virus (EBV) isolate, P3HR1, causes Hodgkin-like lymphomas and diffuse large B cell lymphomas with type II and Wp-restricted latency types in humanized mice

“…In our experiments here, in most cases we used P3HR1 virus derived from a P3HR1 BL cell clone that is lacking the W-Zhet genomes, making it unlikely that that these particles are required for P3HR1-mediated lymphomagenesis. Nevertheless, since we found that the P3HR1 induced tumors (particularly the EBNA-LP-high/LMP1-low foci) are highly lytic (consistent with our recent finding that type 2 EBV is much more lytic in B cells compared to type 1 EBV) [47], it is quite possible that lytic infection plays an important role in promoting the ability of P3HR1 to induce lymphomas in CBH mice.…”

“…EBNA2-deleted P3HR1 EBV causes HLs and DLBCLs in humanized mice Similar to our recent report comparing the phenotypes of type 1 versus type 2 EBV infected lymphomas in CBH mice [47], we found that type 1 B95.8 infected lymphomas express much more LMP1 than type 2 AG876 infected lymphomas, while AG876 infected lymphomas express much more of the BZLF1 lytic viral protein than B95.8 infected lymphomas (Fig 8A). In contrast, lymphomas induced by P3HR1 (although it is a type 2 EBV strain) express much more LMP1 compared to the AG876 type 2 control lymphomas, consistent with the IHC results in Fig 3 and confirming that some P3HR1 virus infected cells have type II latency.…”

“…To compare EBV protein expression in lymphomas infected with each type of virus, FFPE tissues isolated from CBH mice infected with P3HR1, AG876 or B95.8 EBV strains were initially stained with antibodies against EBNA1 (a latency protein expressed in cells with any latency type) and EBNA2 (a latency protein expressed only in cells with type III latency). As expected, both B95.8 and AG876 virus-induced lymphomas (which have previously been shown to have type III latency in CBH mice) [43][44][45]47], express both EBNA1 and EBNA2 ( Fig 2). In contrast, tumors derived from the EBNA2-deleted P3HR1 virus express EBNA1 but not EBNA2 (Fig 2).…”

“…As expected, lymphomas infected with the B95.8 virus express both EBNA-LP and LMP1 in a significant number of tumor cells (Fig 3). Lymphomas infected with type 2 EBV AG876 virus, as we recently reported [47], express less LMP1 compared to type 1 EBV B95.8 infected cells but have similar levels of EBNA-LP (Fig 3).…”

“…In contrast, approximately 50 mice injected with uninfected cord blood have not developed B cell lymphomas in this same model. We also recently used the CBH model to show that type 2 EBV, although much less transforming than type 1 EBV in vitro due to reduced LMP1 expression [46]), is fully competent for inducing Bcell lymphomas in vivo [47].…”