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Functional organization of the late transcriptional unit of canine adenovirus type 2

Ecole Nationale Veterinaire d'Alfort

¹ E-mail: bklonjkowski{at}vet-alfort.fr

Vectors derived from canine adenovirus type 2 (CAV-2) are attractive for gene therapy and as live recombinant vaccines. Construction of the CAV-2 vectors described thus far has been achieved by modification of different CAV-2 genes, such as early regions 1 and 3 or the fiber gene. Modification of these genes was underpinned by previous description of their mRNA and protein-coding sequences. Similarly, the construction of new CAV-2 vectors bearing changes in other genomic regions, and in particular many of those expressed late in the viral cycle, will require prior characterization of the corresponding transcriptional units. In this study we provide a detailed description of the late transcriptional organization of the CAV-2 genome. We delimited the major late transcription unit (MLTU) and its six families of mRNAs controlled by the putative major late promoter (MLP). All mRNAs expressed from the MLTU had a common non-coding tripartite leader, 222 nucleotides in length, at their 5' end. In transient transfection assays, the predicted MLP sequence was able to direct luciferase gene expression and the TPL sequence gave rise to a higher amount of transgene product. Identification of viral transcriptional products following in vitro infection confirmed most of the predicted protein-coding regions deduced from computer analysis of the CAV-2 genome. These findings contribute to a better understanding of gene expression in CAV-2 and lay the foundation required for genetic modifications aimed at vector optimisation.

Received 6 October 2008; accepted 16 January 2009.