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FIG. 1. Schematic of the region of the Raji genome showing the E3C deletion, and the two B958 cosmids SalIC and EcoRIB, which overlap at the region of EBNA3C and EBNA1. The coordinates for the cosmids are based on the B958 genome (3, 4).

to B lymphocytes. P3HR-1 cells are a suitable packaging cell lines since the endogenous EBV genome is transformation-incompetent and replication-competent. Although the assembly and transfection of very large amplicons into a latently infected lymphoblast packaging cell lines is inefficient, successful transfectants can be selected by using an amplicon-based positive selection marker and the transfected cells can be expanded in large amounts and fully characterized (1, 5). Such cell lines tend to be stable over several months of continuous positive selection. The induction of lytic replication results in amplification and packaging of linear concatemers of the amplicon. The endogenous EBV genome is also packaged into virus. Although the host range of EBV and of the amplicon packaged virus, in culture, is limited to B lymphocytes, EBV-transformed B lymphocytes are derived from genetically deficient humans to retain immortal cell lines with the specific genetic deficiency. Amplicon-based vectors can, therefore, be used for experimental reconstitution of genetic deficiencies in these cells, in vitro. EBV amplicons have recently been used to correct the TAP1 or 2 deficiency in B lymphoblasts derived from patients with type 1 diabetes (8). In a substantial fraction of cases, restoration of TAP1 expression corrected the abnormally low class I MHC expression that is characteristic of type 1 diabetes. An amplicon vector has also been used to correct an enzymatic defect (9).

The principal limitations of EBV-amplicon-based systems are their restricted host range, the production of replication competent endogenous EBV by the packaging cell line, and the possible effect of EBNA1 on cell growth. EBNA1 is a sequence-specific DNA binding protein that has transcriptional transactivating effects; an effect on transcription of cell genes could have important biological consequences. EBV amplicons packaged in P3HR-1 cells are mixed with P3HR-1 EBV. Although the P3HR-1 EBV cannot immortalize primary human lymphocytes, P3HR-1 is replication-competent and encodes all proteins that are important in EBV-mediated cell growth transformation except for EBNA2 and EBNALP. The EBV host range could likely be expanded by modification of the glycoprotein composition of the EBV outer envelope encoded by the packaging cell line. Gp350 is a highly specific ligand for CD21 that is abundantly expressed only on B lymphocytes. Modification of the ligand determinant or inclusion of other glycoproteins could broaden the host range. Gp350 can for example be incorporated into envelopes along with glycoproteins of varicella zoster virus (10).

Recombinant EBV-Based Vectors: Positive Selection Markers. Recent strategies for evaluating the effects of site-specific mutations in the viral genome have enabled a focused assessment of the role of specific genes and intragenic elements in primary B-lymphocyte growth transformation and in lytic EBV infection. The favored approach has been to introduce an EBV DNA fragment with a selectable marker into latently infected cells along with an expression vector for the Z immediate early transactivator of lytic EBV replication (1). Z induces lytic infection and the replicating EBV genome can recombine with the transfected EBV DNA fragment. The progeny virus could then be harvested and plated onto lymphocytes at a low multiplicity, and recombinant infected cells could be identified by genetic selection or marker-specific PCR (1113). One strategy is to transfect a cell carrying a wild-type latent EBV genome with a mutated EBV DNA fragment carrying an expression vector for a gene that encodes an enzyme that inactivates a toxic drug (11, 12, 1416). Progeny virus can then be used to infect primary B lymphocytes or a non-EBV-infected continuous B-cell line such as an EBV-negative Burkitt lymphoma (BL) cell line. Cells infected with the recombinant EBV can then be positively selected by growing the infected cells in the presence of the toxic drug (11, 12, 1416). An advantage of this strategy is that mutations can be made in a gene that is essential for primary B-lymphocyte transformation and the mutant progeny genome can be positively identified by infection of BL cells and by plating of the infected cells under selective conditions (11, 12, 16). The weakness of the BL-cell approach is that BL cells are less susceptible to EBV infection than primary B lymphocytes. Further, reactivation of lytic infection from BL cells is more difficult than from primary B lymphocytes (11, 12, 16).

Recombinant EBV-Based Vectors: Marker Rescue for Transformation. The favored approach for most recombinant EBV molecular genetic analyses has been to use cells infected with the replication-competent but transformation-incompetent P3HR-1 EBV strain as the parent for positive selection by marker rescue of transformation. The P3HR-1 EBV genome is deleted for a DNA segment that includes the last two exons of EBNALP and the EBNA2 exon (16, 17). Recombination of P3HR-1 EBV with a transfected wild-type EBV DNA fragment spanning the EBNALP and EBNA2 deletion was, therefore, readily identified by the unique ability of the recombinant progeny virus to transform primary B lymphocytes into long-term lymphoblastoid cell lines (LCLs). The recombinant viral DNA can be characterized in the resultant LCL clones. Virus replication can be induced in these clones and the properties of the recombinant virus can be assayed on infection of new primary B lymphocytes. This has been a simple and relatively reproducible marker rescue strategy so that the efficiency of marker rescue of various specifically mutated EBNALP/ EBNA2 DNA fragments can be compared with wild-type DNA after the initial transfection into P3HR-1 cells. Null mutations in EBNALP/EBNA2 can be inferred by the consistent inability of several isogenic DNA fragments to marker rescue transforming virus in controlled experiments.

Recombinant EBV-Based Vectors: Second Site Homologous Recombination to Construct Specific Mutations at any Site in the EBV Genome Other Than EBNALP/EBNA2. Surprisingly, the same marker rescue of transformation strategy could be used to select for recombination at any other site in the EBV genome. A nonlinked second cotransfected EBV DNA fragment was incorporated into at least 10% of the P3HR-1 genomes that had recombined with the EBNA2 and EBNALP DNA fragment (18). EBV second-site recombinants that had acquired a mutation that did not interfere with transformation were readily derived since a total of 103 marker-rescued recombinants could be obtained from a single transfection and

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