Cell Biol Toxicol (2010) 26:1–20 numerous other viral-encoded proteins, is then imported through the nuclear pore into the nucleus where viral-encoded proteins form a complex with the cellular nuclear matrix to facilitate initiation of the primary transcription events. The viral DNA does not under normal circumstances integrate into the host cell genome, but remains episomal within the nucleus. The Ad genome is transcribed and replicated at discrete replication centres in the nucleus of the infected cell. Ad transcription can be defined as a two-phase event, early (E) and late (L), occurring before and after DNA replication, respectively (Fig. 1). Transcription of the four early genes, E1, E2, E3 and E4, is accompanied by numerous splicing events to generate proteins required for transactivating other viral regions or for modifying the host cellular or immunological environ- ment. Following transport into the host cell nucleus, transcription from the viral E1 gene is initiated, producing E1a and E1b proteins. E1a proteins alter cellular metabolism and activate transcription of the other E genes (Russell 2000), whereas the E1b protein prevents apoptosis of the host cell, promoting onco- genesis and transformation. Together, E1a and E1b also inhibit inflammation caused by Ad infection (Schaack et al. 2004). The E2 gene products interact with a number of cellular factors to enable replication of viral DNA and transcription of the L genes. In contrast, E3 gene products are non-secreted proteins 5 that control various host immune responses by inter- fering with host defence mechanisms (Horwitz 2004). As a result, they are not required for viral replication in culture (Russell 2000) and are often deleted from Ad gene therapy vectors. The E4 gene products modulate viral gene expression and replication through interac- tion with host cell systems. They facilitate virus messenger RNA metabolism, provide functions to inhibit host protein synthesis and promote virus DNA replication (Leppard 1997). In addition, wild-type adenoviruses also transcribe a set of untranslated RNAs (the VA RNAs) that combat cellular defence mechanisms by blocking activation of the interferon response (Mathews and Shenk 1991). Following viral DNA replication, the major late promoter in the viral genome becomes fully activated (Nevins 1981), initi- ating transcription and splicing of the major late transcription unit (MLTU) that produces five different transcripts (L1 to L5) that supply the viral capsid proteins (Farley et al. 2004) and aid maturation of virus particles in the nucleus. Initial studies using Ad vectors focused on the respiratory tract (the natural target tissue of this virus). Subsequent studies demonstrated that Ads can infect a great variety of post-mitotic cells, even those associ- ated with highly differentiated tissues such as skeletal muscle, lung, brain and heart. In 1993, Ad vector systems were first demonstrated to be a viable means Promoter of choice Protein of interest Major late transcription unit E1A E1B VA-RNA E3 Fig. 1 Production of first-generation Ad vectors from wild- type adenovirus genome. Wild-type adenoviruses have a double-stranded, linear DNA genome of approximately 36 kb in length. It is divided into early (E) and late (L) gene transcripts. Transcription of the viral genome begins with the E1 gene, producing E1a and E1b proteins. E1a proteins alter cellular metabolism and activate transcription of the other E genes, whereas the E1b protein prevents apoptosis of the host cell, promoting oncogenesis and transformation. The E2 gene products interact with a number of cellular factors to enable replication of viral DNA and transcription of the L genes. E3 gene products control various host immune responses, and the E2 E4 E4 gene products modulate viral gene expression and replica- tion. In addition, wild-type adenoviruses also transcribe a set of untranslated RNAs (the VA RNAs) that combat cellular defence mechanisms by blocking activation of the interferon response and a MLTU that produces the viral capsid proteins. The creation of first-generation replication-deficient Ad vectors involved the removal of the E1 genes. In addition, E3 genes were also removed, as they are not required for viral replication in culture. This enabled up to 8-kbp foreign DNA, encoding promoters and foreign transgenes, to be incorporated into the genome. Figure adapted from Horwood et al. Arthritis Research, 2002