The propagation of herpesviruses has long been viewed as a temporally regulated sequential process that results from the consecutive expression of speci®c viral transactivators. As a key step in this process, lytic viral DNA replication is considered as a checkpoint that controls the expression of the late structural viral genes. In a novel genetic approach, we show that both hypotheses do not hold true for the Epstein±Barr virus (EBV). The study of viral mutants of EBV in which the early genes BZLF1 and BRLF1 are deleted allowed a precise assignment of the function of these proteins. Both transactivators were absolutely essential for viral DNA replication. Both BZLF1 and BRLF1 were required for full expression of the EBV proteins expressed during the lytic program, although the respective in¯uence of these molecules on the expression of various viral target genes varied greatly. In replication-defective viral mutants, neither early gene expression nor DNA replication was a prerequisite for late gene expression. This work shows that BRLF1 and BZLF1 harbor distinct but complementary functions that in¯uence all stages of viral production.

The binding of the viral major glycoprotein BLLF1 (gp350/220) to the CD21 cellular receptor is thought to play an essential role during infection of B lymphocytes by the Epstein-Barr virus (EBV). However, since CD21-negative cells have been reported to be infectible with EBV, additional interactions between viral and cellular molecules seem to be probable. Based on a recombinant genomic EBV plasmid, we deleted the gene that encodes the viral glycoprotein BLLF1. We tested the ability of the viral mutant to infect different lymphoid and epithelial cell lines. Primary human B cells, lymphoid cell lines, and nearly all of the epithelial cell lines that are susceptible to wild-type EBV infection could also be successfully infected with the viral mutant in vitro, although the efficiency of infection with BLLF1-negative virus was clearly lower than the one observed with wild-type EBV. Our studies show that the interaction between BLLF1 and CD21 is not absolutely required for the infection of lymphocytes and epithelial cells, indicating that viral molecules other than BLLF1 can mediate the binding of EBV to its target cells. In this context, our results further suggest the hypothesis that additional cellular molecules, apart from CD21, allow virus entry into these cells.

The Myc oncoprotein is implicated in transcriptional regulation of a variety of genes pertaining to cell cycle and neoplastic transformation. Examples of both positive and negative regulation have been reported that involve E-box and initiator (Inr) promoter elements, respectively. In both cases, Myc is thought to induce changes in transcription initiation. We have previously shown that overexpression of Myc causes down-regulation of the thrombospondin-1 (tsp-1) gene, an important negative modulator of tumor angiogenesis. In this study, we demonstrate that Myc in combination with Max can bind, albeit with low affinity, to an E-box-like element in the tsp-1 promoter. However, the 2.7 kb DNA segment containing both this non-canonical E-box and an Inr-like sequence does not constitute a Myc-responsive element in a transient expression system. Furthermore, Myc does not significantly affect the rate of initiation or elongation of the tsp-1 mRNA. Thus, in this instance Myc does not act as a canonical transcription factor. Instead, as demonstrated by blocking de novo RNA synthesis, down-regulation of the tsp-1 gene by Myc occurs through increased mRNA turnover. To our knowledge, this is the first example of gene regulation by Myc that involves mRNA destabilization.

Viral gene vectors often rely on packaging cell lines, which provide the necessary factors in trans for the formation of virus-like particles. Previously, we reported on a firstgeneration packaging cell line for gene vectors, which are based on the B-lymphotropic Epstein-Barr virus (EBV), a human g-herpesvirus. This 293HEK-derived packaging cell line harbors a helper virus genome with a genetic modification that prevents the release of helper virions, but efficiently packages vector plasmids into virus-like particles with transducing capacity for human B cells. Here, we extended this basic approach towards a non-transforming, virus-free packaging cell line, which harbors an EBV helper virus genome with seven genetic alterations. In addition, we constructed a novel gene vector plasmid, which is devoid of a prokaryotic antibiotic resistance gene, and thus more suitable for in vivo applications in human gene therapy. We demonstrate in this paper that EBV-based gene vectors can be efficiently generated with this much-improved packaging cell line to provide helper virus-free gene vector stocks with transducing capacity for established human B-cell lines and primary B cells.

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