Epstein-Barr virus integrates frequently into chromosome 4q, 2q, 1q and 7q of Burkitt's lymphoma cell line (Raji)

Abstract

Epstein-Barr virus (EBV) integration into a Burkitt's lymphoma (BL) cell line (Raji) was investigated, using polymerase chain reaction (PCR), Southern hybridization, genomic library screening and fluorescence in situ hybridization (FISH). BaMHIW fragments of the EBV genome and DNA sequences of the viral latent membrane protein (LMP)1 and LMP2 genes were detected in Raji cells. BaMHI-digested high-molecular weight DNA from Raji cells generated 4 and 10 kb, 23 kb fragments that hybridized to Probe-1 (EBV genome 13232–16189) and Probe-2 (EBV genome 5–3271). Genomic library for Raji cells was constructed. Plaques (1 × 10⁵) were screened with Probe-2, and four positive clones were obtained. Chromosomal integration of EBV DNA was detected in the Raji cell. The viral integration sites included 1p, 1q, 2q, 3p, 3q, 4q, 5q, 6q, 7p, 7q, 9q, 11p, 14q and 15q. Despite this multiplicity of integration sites, integration showed high frequency only at the sites 4q, 2q, 1q and 7q; 64% of the total signals were found in these four chromosomal bands. No viral integration occurred in chromosomes 16–22 or the sex chromosomes (X, Y). This study is the first comprehensive FISH analysis of EBV integration into the chromosomes of the Raji cell line. The findings support the notion that EBV integrates into the Raji cell genome non-randomly.

Introduction

Epstein-Barr virus (EBV) is a prototype gamma herpesvirus that infects more than 90% of the population worldwide (Rickinson and Kieff, 2001). The EBV genome is a double stranded DNA of 172 kb. EBV has been implicated in the pathogenesis of several malignancies, primarily of lymphoid and epithelial cell origin, including Burkitt's lymphoma (BL) and nasopharyngeal carcinoma (NPC) (Niedobitek et al., 2001, Raab-Traub, 2002, Alazard et al., 2003, Griffin, 2000). EBV DNA is detected as a linear form in cells at the lytic phase of EBV infection, but during latent infection the viral genome is maintained in episomal form through the fusion of genomic termini. Most BL and NPC tumors harbor the episomal EBV genome, representing persistent latent infection. Chromosomal integration of EBV DNA provides an alternative way of establishing persistent infection and may represent another mechanism of virus–cell interaction. Integration of EBV DNA into the host genome might be a common event in lymphoma cells, but analysis of integrated EBV DNA is complicated (Takakuwa et al., 2004) because highly methylated EBV DNA hinders mapping of EBV genomes, and multiple copies of the viral episomes produce interfering noise. The large size of the EBV genome compared with other viruses also makes it difficult to identify the integration sites and analyze the flanking cellular sequences. Therefore, the biological influence of EBV integration on the host cell is not yet clear. Studies to date have demonstrated EBV integration in biopsy specimens from BL or other lymphomas (Takakuwa et al., 2004, Luo et al., 2004, Daibata et al., 2002), in vitro EBV-converted NPC cell lines (Chang et al., 2002) and EBV-transformed lymphoblastoid cell lines (LCLs) (Takakuwa et al., 2005), but these studies have revealed no common, invariably concomitant chromosomal abnormality associated with EBV integration. Whether EBV integrates into the human genome randomly or non-randomly is still debatable. The present study explored EBV integration into a Burkitt's lymphoma cell line (Raji) and discussed its possible biological effects.