Determining new functions of Adenovirus VA-RNAI and its use in cancer therapy

Virus-Associated RNA I (VA-RNAI; VAI) of adenovirus is a nontranslated RNA molecule known to interact with several dsRNA-binding molecules in host cells. Adenovirus lacking VA-RNAI shows reduced replication and toxicity in normal tissues but replicates with high efficiency in some cancer cell lines. In this work, efficient replication of VA-RNAI-deleted adenovirus dl331 was confirmed to be independent of cancer cells’ k-ras mutation status, leaving the basis of its selectivity unknown. VA-RNAI expression was necessary to prevent dsRNA-dependent phosphorylation of eIF2α in Suit-2 and HCT116 cells, with a smaller effect in PANC-1 and none in MiaPaCa2. VA-RNAI was shown to suppress virus-induced autophagy in two cell lines, whether expressed from the virus or a plasmid. Unexpectedly, the viral protein E1A was observed to colocalise with autophagic vesicles. VARNAI was also shown to prevent starvation-induced autophagy in susceptible cell lines. RNA was collected from normal NHBE cells infected with VA-RNAIdeleted dl331 or VA-RNAI-intact control dl309 viruses. Microarray analysis of these samples showed a significant change in gene expression as VA-RNAI accumulated in the cells. Most notably, several genes involved in cell cycle progression were upregulated during infection with dl331 when compared to dl309. RT-qPCR showed a similar pattern of gene regulation in Suit-2 cells responding to infection. Cell cycle analysis of infected Suit-2 cells showed a transient accumulation of cells in S-phase in response to dl309 but not dl331 at the approximate time that VA-RNAI reached its highest level. This work has demonstrated that VA-RNAI's ability to block eIF2α phosphorylation inhibits the induction of autophagy after infection or starvation. Given the newly understood importance of autophagy in cancer pathogenesis and the responses of malignant cells to radiation and chemotherapeutic drugs, this new function is potentially important for the vi design of future oncolytic adenoviruses. However, this activity is not sufficient to explain support for replication of VA-RNAI-deleted viruses in cancer cell lines. Data from the microarray and cell cycle analyses suggest that VA-RNAI may play a further role in modulating the host cell’s replication cycle, although the mechanism for this is currently unclear.EThOS - Electronic Theses Online ServiceGBUnited Kingdo