Epstein-Barr virus nuclear antigen 1 does not induce lymphoma in transgenic FVB mice

Abstract

The lymphoma-inducing potential of Ig heavy-chain enhancer- and promoter-regulated Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) was evaluated in three transgenic FVB mouse lineages. EBNA1 was expressed at a higher level in transgenic B220(+) splenocytes than in EBV-infected lymphoblastoid cell lines. EBNA1 was also expressed in B220(-) transgenic splenocytes and thymocytes. Before killing and assessments at 18-26 months, EBNA1-transgenic mice did not differ from control mice in mortality. At 18-26 months EBNA1-transgenic mice did not differ from littermate control in ultimate body weight, in spleen size or weight, in lymph node, kidney, liver, or spleen histology, in splenocyte fractions positive for cluster of differentiation (CD)3epsilon, CD4, CD8, CD62L, B220, CD5, IgM, IgD, MHC class II, CD11b, or CD25, or in serum IgM, IgG, or total Ig levels. Lymphomas were not found in spleens or other organs of 18- to 26-month-old EBNA1-transgenic (n=86) or control (n=45) FVB mice. EBNA1-transgenic lineages had a higher pulmonary adenoma prevalence than did littermate controls (39% versus 7%). However, the adenoma prevalence was not higher in EBNA1-transgenic mice than has been described for FVB mice, and EBNA1 was not expressed in normal pulmonary epithelia or adenomas.

Fig. 1.

FLAG-epitope-tagged EBNA1 transgene expression construct, functional test, and PCR detection of transgenes. (A) Transgene expression plasmid pEμPμFE1 is composed of Eμ and Pμ upstream of the FLAG-tagged EBNA1 ORF. (B) EBV-negative BJAB B lymphoblasts were transiently cotransfected with pEμPμFE1, pCDNAFE1, or pcDNA FLAG vector control, reporter plasmid pFL, and control pGKβ-galactosidase DNA. Relative luciferase activity is fold over the luciferase activity of pFL transfected with vector control DNA and was also corrected for transfection efficiency as assayed by β-galactosidase. (C) PCR screening used primer sets 1 and 2 to identify pEμPμFE1 DNA in 58, 68, and 73 founder mice among 25 progeny from embryo microinjections.

Fig. 2.

Comparison of body weight, spleen size, spleen cell types, and serum Ig levels in EBNA1-transgenic and littermate control mice. (A) Comparison of body weight, spleen weight, and spleen length at 18-26 months. (B) Comparison of total nucleated spleen cell numbers. (C) Flow cytometry analysis of splenocyte protein surface marker expression at 18-24 months. Six independent experiments were done, and the values shown are averages with standard deviation based on 26 EBNA1-transgenic and 26 control mice (CD3ε), 32 transgenic and 32 control mice (CD4), 29 transgenic and 29 control mice (CD8), 3 transgenic and 3 control mice (CD62L, MHCII, and CD11b), 19 transgenic and 19 control mice (B220), 15 transgenic and 15 control mice (CD5), 15 transgenic and 15 control mice (IgM), 6 transgenic and 6 control mice (IgD), and 4 transgenic and 4 control mice (CD25). (D) Duplicate ELISA analyses of serum Ig levels from 39 EBNA1 and 34 control 18- to 24-month-old mice. Error bars, 2 SD.

Fig. 3.

EBNA1-transgenic protein expression. (A) EBNA1 protein levels in third-generation lineages 58, 68, and 73 B220 (+) and (-) splenocytes, CD8(+) thymic cells, and total bone marrow cells were compared with levels in equal numbers of EBV-transformed human B lymphocytes, LCLs, or EBV-negative BJAB lymphoblasts (BJ). FV, FVB control cells. The protein loaded in each lane is indicated. Western blots of the same extracts with EBNA1 and tubulin antibody are shown. (B) Western blots of 40-μg protein extracts from BJ cells, BJ cells that had been converted 2-3× EBNA1 overexpression, BJFE1, or from spleens of 12th-generation lineage 73 and littermate control mice. (C) Western blot of protein extracts from kidney (Ki), liver (Li), stomach (St), brain (Br), thymus (Th), and spleen (Sp) of lineage 58 or littermate control with EBNA1-specific antibody. ns, nonspecific reactivity with hepatocyte extracts. (D) In vitro gel shift with nuclear extracts from BJFE1 cells or total cell extract from lineages 73 and 68 or littermate control splenocytes. Extracts were incubated with labeled EBNA1-cognate DNA probe in the presence or absence of EBNA1 (E1) or control (C) antibody, or wild-type (W) or mutant (M) competitor DNA.

Fig. 4.

An EBNA1-transgenic mouse spleen lymphoid follicle (F) and a lung adenoma are shown (Upper and Lower, respectively). Tissues were stained with hematoxylin/eosin, B220 antibody, or EBNA1 antibody. The mouse lung (Lower) has a typical adenoma (AD) in the upper left corner, a peripulmonary lymphoid deposit (LY) in the center, and adjacent normal lung (LU) in the lower right corner. EBNA1 staining of the adenoma and normal lung resulted in light brown background cytoplasm staining and little or no nuclear staining, whereas ≈30% of the lymphoid nuclei stained light to dark brown. Photomicrographs were at ×100 (Upper) and ×400 (Lower), using a Zeiss photomicroscope and digital camera.