S.O. SUTTER¹, K. TOBLER¹, M. ACKERMANN¹, C. FRAEFEL<sup>1

1</sup>Institute of Virology, University of Zurich, Zurich, Switzerland

parvovirus with a single stranded DNA genome of 4.6 kb, encoding two clusters of genes, rep and cap. In absence of a helper virus, AAV can integrate its genome at a specific site, termed adeno-associated virus preintegration site (AAVS1) on human chromosome 19. In the presence of a helper virus, such as herpes simplex virus type 1 (HSV-1), adenovirus (Ad) or human papilloma virus (HPV), AAV can enter a lytic replication cycle with the production of progeny virus particles. To investigate the global effect of AAV2 infection on a host cell, a global transcriptome analysis by next generation sequencing (NGS) of wild type (wt) AAV2-infected or mock-infected normal human fibroblasts (NHF) was performed. Networking and enrichment analysis of the transcriptome (1930 genes with p < 0.01 and number of reads > 40) revealed several affected biological processes such as chromatin organization, intracellular transport, regulation of the cell cycle, cytoskeleton and organelle organization. To further evaluate the enrichment analysis of the transcriptome, heat maps of the top 50 differentially regulated genes between wtAAV2 and mock-infected cells were created. Further reduction of the NGS data revealed a list of 872 genes, which were either up- or down-regulated at least 1.5-fold in wtAAV2 versus mock-infected cells. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of the 872 genes revealed that the top most affected pathways include cell cycle regulation and p53 signaling.
To confirm the NGS data, 10 genes were selected based on their differential expression profile (down-regulated, close to zero, or up-regulated) in wtAAV2-infected and mock-infected cells and the fold difference in target gene expression was assessed by quantitative RT-PCR (RT-qPCR) resulting in a multiple correlation coefficiency (R2) of 0.897. Moreover, the F-statistics revealed a significant relationship between the variables and the linear regression model (p-value = 0.0003543).
Next, the differential expression of the selected genes will be assessed in mock-infected NHF cells and cells infected with double-stranded self-complementary (sc) AAV2 vectors, rep-negative single stranded AAV2 vectors or inactivated wtAAV2, in order to identify the AAV components responsible for the interference with cellular target genes and the cell cycle progression.