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

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 EBV-transformed 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. Author summary EBV causes various human B cell lymphomas and infects B cells in latent or lytic forms. The two different EBV strains (Type 1 (T1) and Type 2 (T2)) are divergent in their EBNA2 and EBNA3A/B/C latency genes, and T1 EBV is more transforming than T2 EBV in vitro. Interestingly, all T2 strains contain the Zp-V3 form of the BZLF1 immediate-early promoter (which regulates lytic viral reactivation) while most T1 strains contain the Zp-P form; the Zp-V3 variant is more responsive to BCR stimulation due to the presence of an NFAT binding site. Here we show that T2 and T1 EBV both induce lymphomas in cord blood-humanized mice, but T2 EBV infected mice have increased lytic infection. Using in vitro generated LCLs, we find that both total and activated NFATc1 and NFATc2 are elevated in T2 EBV-infected LCLs compared to T1 LCLs, and demonstrate that enhanced NFAT activity is required for the lytic phenotype in T2 LCLs. Knockdown of BZLF1 gene expression decreases the growth rate of T2 LCLs. These results suggest that an increased ability to enter lytic infection may partially compensate for the decreased transforming phenotype of T2 EBV infection.