Molecular Basis of Adaptation of Enteroviruses to Different Cancer Cell Lines

Viral oncolytic therapy, a novel treatment for cancer using specially designed viruses to kill malignant cells while leaving normal cells unharmed, is currently under intense investigation. Several receptors are up-regulated in cancer cells, including decay-accelerating factor (DAF; CD55) and integrins (αvβ3, αvβ6) and viruses which recognise these receptors could be useful for therapy. Several echoviruses, including Echovirus 11 (E11), bind to DAF; coxsackievirus A9 (CVA9) utilizes an RGD motif to bind to integrins, particularly αvβ6. Some isolates of CVA9 also bind to heparan sulfate proteoglycans (HSPG). This thesis describes work designed to improve our understanding of CVA9 and echovirus cell/receptor tropism. Several echoviruses and CVA9 variants were tested in a panel of 9 cell lines. Distinct patterns of infection were seen, but did not fully correlate with receptor expression, suggesting that other determinants also help to define tropism. To investigate this further, E11 was adapted by passaging on two cell lines, A549 and HeLa. Two mutations were seen in A549-adapted virus, and both mapped to the DAF-binding footprint, suggesting changes to E11/DAF interactions. A single mutation in VP4 was seen in HeLa-adapted virus, and may affect a later stage in cell entry. To investigate CVA9 binding to HSPG, 3 isolates were propagated on A549 cells and heparin-blocked mutants were isolated. Although the isolates are diverse, the same mutation (VP3 Q59R) was seen in two isolates and probably gives a positively-charged cluster with adjacent amino acids. Other mutations were seen close to the RGD motif, where there is already a highly basic sequence. The results suggest multiple potential mechanisms for HSPG-binding. Combinations of some of the adapting mutations discovered could significantly enhance the tropism of these viruses to specific cancer cells and optimise them as oncolytic agents.