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.

James C Romero-Masters,Paul J Farrell,Erik A Ranheim,Shane M Huebner,Makoto Ohashi,Jillian A Bristol,Jenny Gumperz,Nathan M Sherer,Scott E Nelson,Eric C Johannsen,Bayleigh E Benner,Dana C Baiu,Elizabeth A Barlow,Nicholas Van Sciver,Gail L Turk,Shannon C Kenney,Sankar Swaminathan

doi:10.1371/journal.ppat.1008365

James C Romero-Masters, Paul J Farrell + Show 15 more

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https://doi.org/10.1371/journal.ppat.1008365

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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 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.

Highlights

Epstein-Barr virus (EBV) is a herpes virus that infects most of the world’s population and causes infectious mononucleosis
All Type 2 (T2) strains contain the Zp-V3 form of the BZLF1 immediateearly promoter while most Type 1 (T1) strains contain the Zp-P form; the Zp-V3 variant is more responsive to B-cell receptor (BCR) stimulation due to the presence of an NFAT binding site
Using in vitro generated lymphoblastoid cell lines (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

Summary

Introduction

Epstein-Barr virus (EBV) is a herpes virus that infects most of the world’s population and causes infectious mononucleosis. EBV infection of primary B cells in vitro is sufficient to transform these cells into long-term lymphoblastoid cell lines (LCLs) that proliferate indefinitely and form tumors when injected into immune deficient mice. EBV-infected lymphomas occurring in immunocompetent patients often do not express LMP1 and/or EBNA2 and EBNA3C [2,3]. The ability of EBV to transform B cells into long-term LCLs in vitro may not adequately model certain aspects of EBV-associated B-cell lymphomas in humans. We have recently established a humanized mouse model that better models many aspects of the human disease, including a role for lytic infection [4], and the development of EBV-induced lymphomas in the absence of LMP1 or EBNA3C [5,6]

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