| Summary: | Coxsackievirus B3 (CVB3)-induced myocarditis is a common heart disease in children and young adults. This viral infection can cause severe cardiac injury and patients may experience progressive heart failure, which may ultimately lead to dilated cardiomyopathy (DCM). Since the development of viral myocarditis involves complex interactions between host cells and the virus, the mechanisms by which CVB3 causes myocarditis and progression to DCM are still not well understood. The small CVB3 genome contains 11 genes, of which the proteases 2A (2A[sup pro]) and 3C (3C[sup pro]) are known to play critical roles in the virus life cycle by processing the viral polyprotein and in viral pathogenesis by cleaving the host proteins. Recent studies have demonstrated that overexpression of picornaviral protease 2A[sup pro] or 3C[sup pro] leads to caspase-3 activation and apoptosis. However, the molecular mechanisms that link these viral proteases to the induction of host cell death remain unclear. My dissertation addresses this issue by identifying cellular proteins that are cleaved and/or activated by these CVB3 proteases and the subsequent apoptotic pathways induced by these cleavages. In order to achieve this goal, full length CVB3 protease genes 2A and 3C were cloned into a eukaryotic expression vector using a PCR-based strategy. The resulting plasmids were then transiently transfected into HeLa cells. The transfected samples were subjected to Western blot analyses to detect cleavage (activation) of various cellular proteins. Upon protease expression, cell morphological alterations and reduction in cell viability are observed in both 2A[sup pro]- and 3C[sup pro]-transfected cells. Functional evaluation of 2A[sup pro] or 3C[sup pro] expression has also shown that caspase-3 and -8 are activated and their substrates, poly(ADP-ribose) polymerase (PARP) and Bid, are cleaved in these transfected cells. These results indicate that the expression of CVB3 2A[sup pro] or 3C[sup pro] in mammalian cells is sufficient to induce cell apoptosis through a caspase-dependent pathway. When other apoptotic pathways were explored, Western blot analyses of Bcl-2 family members demonstrated that only 3C[sup pro] but not 2A[sup pro] can up-regulate expression of Bax, a pro-apoptotic protein. However, expression of Bcl-2 protein, an anti-apoptotic member of Bcl-2 family protein, remains unchanged in both 2A[sup pro]- and 3C[sup pro]-transfected cells. Up-regulation of Bax and the presence of truncated Bid (tBid) further contribute to the release of cytochrome c from the mitochondria and activation of caspase-9. Finally, cleavage of host transcription factor, cyclic AMP responsive element binding protein (CREB) and translation initiation factors, eukaryotic translation initiation factor 4GI (elF4GI) and NAT1, by 2A[sup pro] or 3C[sup pro] are also observed. These proteolytic activities are accompanied by a severe inhibition of host mRNA and protein synthesis, which also contributes to the viral protease-induced cell death. In conclusion, the data suggest that the mechanism of apoptosis induced in CVB3 2A[sup pro]- or 3C[sup pro]-transfected HeLa cells is likely through multiple converging pathways. Firstly, both 2A[sup pro] and 3C[sup pro] can induce HeLa cell apoptosis through a caspase-dependent pathway. Secondly, 2A[sup pro] and 3C[sup pro] can also induce intrinsic mitochondria-mediated pathway through up-regulation of Bax and release of cytochrome c from mitochondria. Finally, cleavage of host transcription and translation initiation factors by 2A[sup pro] or 3C[sup pro] results in the inhibition of host protein expression, which further enhances apoptotic cell death.
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