Encephalomyocarditis virus (EMCV), a Picornavirus, encodes two primary host antagonists, the Leader (L) and 2A proteins. In this thesis, we sought to further define the biochemical mechanism of host cell shutdown induced by these two viral proteins. L (67 amino acids) binds cellular RanGTPase and induces hyperphosphorylation of nuclear pore proteins, which shuts down active nucleocytoplasmic trafficking of critical antiviral signaling proteins. We showed that L and Ran bind tightly with a low KD (equilibrium dissociation constant) of 3 nM. RCC1 facilitated this binding and overcame GDP and GTP inhibition of recombinant L:Ran binding. The in vitro requirement of RCC1, an exclusively nuclear protein, suggests an active localization of L to the nucleus, despite L lacking a nuclear localization signal (NLS). We showed that 2A (143 amino acids), which encodes an NLS, binds L with a KD=1.5 µM, far less so than L:Ran, suggesting a more transient interaction. This binding is unaffected by the phosphorylation status of L, but requires several residues in the L hinge region and the first fifty amino acids of 2A. L:2A binding may explain previously observed phenotypes of 2A mutant viruses, instead impacting L localization to the pore or proper processing of the L-P1-2A precursor by the viral 3C protease. 2A induces a general shutdown of translation through an unresolved mechanism. Addition of recombinant 2A to translation extracts with translation reporter constructs favored viral IRES-driven over host 5' cap-driven translation. The first fifty amino acids of 2A were sufficient for inducing this IRES:cap shift, but not for the general translation shutdown observed with full 2A. 2A contains a C-terminal eIF4E binding site and disruption of eIF4E:4G interactions enhanced 2A-induced cap-dependent translation shutdown. 2A also induces the formation of salt-sensitive 80S ribosomes, suggesting that 2A may be assembling translation-deficient initiation complexes. Taken together, these studies have further defined the biochemical mechanism of L, the only known direct inhibitor of Ran, and protein 2A, a potent viral inhibitor of cellular translation. We have also shown that L and 2A activities should not necessarily be considered independent of one another, but instead cooperative.