For many members of the Picornaviridae family, infection of cells results in a shutoff of host protein synthesis. For rhinoviruses and enteroviruses, the shutoff has been explained in part by the cleavage of eukaryotic initiation factor 4GI (eIF4GI), a component of the cap-binding protein complex eIF4F. The cleavage of eIF4GI is mediated by the virus-specific proteinase 2Apro and results in inhibition of cap-dependent, but not cap-independent, translation. The inhibition of host protein synthesis after infection with human rhinovirus 14 (HRV-14) lags behind the cleavage of eIF4GI. Recently, we discovered a functional homolog of eIF4GI, termed eIF4GII, and showed that cleavage of eIF4GII coincides with the shutoff of host cell protein synthesis after poliovirus infection (Gradi et al., Proc. Natl. Acad. Sci. USA 95:11089-11094, 1998). We wished to determine whether eIF4GII cleavage kinetics could also explain the lack of correlation between the kinetics of eIF4GI cleavage and the shutoff of host protein synthesis after rhinovirus infection. In this study, we examined the correlation between human rhinovirus-induced shutoff of host protein synthesis and cleavage of eIF4GI and eIF4GII. In HRV-14-infected HeLa cells, almost no intact eIF4GI could be detected by 4 h postinfection, while only 4% of eIF4GII was cleaved at this time. By 6 h, however, 67% of eIF4GII was cleaved, and this cleavage coincided with a significant (60%) decline of host translation. These results suggest that cleavage of both eIF4GI and eIF4GII is required for HRV-mediated inhibition of host cell protein synthesis and that the cleavage of eIF4GII is the rate-limiting step in the shutoff of host cell protein synthesis after rhinovirus infection.