The aim of this research is to understand the mechanism by which poliovirus specifically inactivates translation of cellular mRNA while allowing efficient translation of virus RNA in infected HeLa cells, an event known as host cell shutoff. It is known that poliovirus infection of HeLa cells results in inactivation of an initiation factor, the cap binding protein (CBP) complex, which its believed to facilitate binding of capped mRNAs to ribosomal 40S subunits. It is hypothesized that specific inactivation of CBP complex is sufficient to cause the dramatic shutoff of host cell translation observed in infected cells. Further, it is believed that inactivation of CBP complex results from cleavage of the p220 component of the complex. Poliovirus translation is not thought to be dependent on CPB complex since viral RNAs do not contain a 5' m7GTP cap structure and thus initiate translation by a cap-independent mechanism. Although there is much circumstantial evidence to support this hypothesis, a direct cause and effect correlation between p220 cleavage and host cell shutoff in vivo or in vitro has not been established. the studies proposed in this application, through isolation and functional studies of two different proteinases, will define the biochemical mechanism of the specific inhibition of translation in poliovirus- infected cells.
The first aim i s to isolate the viral activator of p220 cleavage, poliovirus proteinase 2A, and produce antibodies to this protein. Then proteinase 2A will be expressed in vitro and in vivo in systems where inhibitory effects on capped mRNA translation can be directly measured and compared with cleavage of p220. Likewise, several poliovirus genes will be expressed in vitro in the absence of functional 2A proteinase to test if other viral genes contribute to the shutoff mechanism. In other experiments, site specific mutagenesis and expression of 2A in vitro will be performed to separate p220 cleavage from host cell shutoff functions and to define functional domains within the proteinase. Together, these studies will define the role of proteinase 2A in the mechanism of host cell shutoff. Secondly, biochemical techniques and affinity chromatography will be used to identify and purify the cellular p220-specific proteinase from HeLa cells. Once this important enzyme has been identified, experiments will be performed to characterize its functions and the role of p220 cleavage in the inhibition of capped mRNA translation.
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