An EBNA3A-mutated Epstein-Barr virus (EBV) retains the capacity for lymphomagenesis in a cord blood-humanized mouse model

EBV causes B-cell lymphomas and transforms B cells in vitro The EBV protein, EBNA3A, collaborates with EBNA3C to repress p16 expression and is required for efficient transformation in vitro An EBNA3A-deleted EBV mutant was recently reported to establish latency in humanized mice but not cause tumors. Here we compare the phenotypes of an EBNA3A-mutated EBV (Delta3A), versus wild-type (WT) EBV, in a cord blood-humanized (CBH) mouse model. The "hypomorph" Delta3A mutant (in which a stop codon is inserted downstream of the first ATG, and the open reading frame disrupted by a 1 bp insertion) expresses very small amounts of EBNA3A using an alternative ATG at residue 15. Delta3A caused B-cell lymphomas at similar rates as WT EBV, but with delayed onset. Delta3A and WT tumors expressed equivalent levels of EBNA2 and p16, but Delta3A tumors in some cases had reduced LMP1. Like the WT EBV tumors, Delta3A lymphomas were oligoclonal/monoclonal, with typically one dominant IGHV gene being expressed. RNA-seq analysis revealed small but consistent gene expression differences involving multiple cellular genes in the WT EBV- versus Delta3A-infected tumors, and increased expression of genes associated with T cells, suggesting increased T cell infiltration of tumors. Consistent with an impact of EBNA3A on immune function, we found that expression of CLEC2D, a receptor that has previously been shown to influence responses of T and NK cells, was markedly diminished in cells infected with EBNA3A mutant virus. Together these studies suggest that EBNA3A contributes to efficient EBV-induced lymphomagenesis in CBH mice.ImportanceThe EBV protein, EBNA3A, is expressed in latently infected B cells and is important for efficient EBV-induced transformation of B cells in vitro In this study we use a cord blood-humanized mouse model to compare the phenotypes of an EBNA3A "hypomorph" mutant virus (Delta3A) and wild-type EBV. The Delta3A virus caused lymphomas with delayed onset compared to the WT EBV, although tumors occurred at a similar rate. The WT EBV and EBNA3A-mutant tumors expressed similar levels of the EBV protein, EBNA2, and cellular protein, p16, but in some cases Delta3A tumors had less LMP1. Our analysis suggested that Delta3A-infected tumors have elevated T cell infiltrates and decreased expression of the CLEC2D receptor, which may point to potential novel roles of EBNA3A in T cell and NK responses to EBV-infected tumors.