Basic research in Epstein-Barr virus (EBV) molecular genetics has provided means to maintain episomes in human cells, to efficiently deliver episomes with up to 150 kbp of heterologous DNA to human B lymphocytes, and to immortalize human B lymphocytes with EBV recombinants that can maintain up to 120 kbp of heterologous DNA. Episome maintenance requires an EBV nuclear protein, EBNA1, whereas immortalization of cells with EBV recombinants requires EBNA1, EBNA2, EBNA3A, EBNA3C, EB-NALP, and LMP1. EBV-derived vectors are useful for experimental genetic reconstitution in B lymphocytes, a cell type frequently used in establishing repositories of human genetic deficiencies. The ability of EBV-infected cells to establish a balanced state of persistence in normal humans raises the possibility that cells infected with EBV recombinants could be useful for genetic reconstitution, in vivo.The Epstein-Barr virus (EBV) genome has yielded reagents that have been useful in vector design. Genetic engineering of specifically mutated EBV recombinants has also had a major impact on basic EBV research and is useful for experimental genetic reconstitution in human cells. The purpose of this article is to review previous work, indicate potential utilities and liabilities, and describe recent experiments that should enable intensive investigation of an under explored and important part of the EBV genome.EBV is similar in its replication to other herpes viruses and many of the genetic approaches have substantial precedence in alpha herpes virus research. However, EBV also differs from alpha herpes viruses in that it establishes latency in and alters the growth of human B lymphocytes (for reviews, see refs. 1 and 2). These properties derive in large measure from a unique set of genes that encode nuclear and integral membrane proteins that have been given the acronyms, EBNAs (EBV nuclear antigens) and LMPs (latent membrane proteins). In initiation of latent infection in B lymphocytes, EBV first expresses EBNALP and EBNA2. EBNA2 upregulates the EBNA promoter leading to a longer primary transcript from which EBNALP, EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNA1 transcripts are derived (Fig. 1). EBNA2 also activates the LMP1 and LMP2 promoters. These proteins act in concert to alter B-lymphocyte growth and enable the maintenance of the EBV genome as a multicopy episome in a state of latent infection. Each of these proteins expressed in latently infected cells, with the exception of EBNA1, has epitopes that are presented on the B-cell surface in the context of common class I major histocompatibility complex (MHC) molecules and are recognized by immune human cytotoxic T lymphocytes. This high level of cytotoxic T-cell recognition and the ability of latently infected cells to shift between full latent gene expression with cell proliferation and an EBNA1 only type of latent infection that is immunologically privileged enables latently infected cells to achieve a balanced state of long-term persistence in humans. This state of long-term persis...