Once activated, the membrane-associated Arf-GTP induces a curvature in the lipid bilayer, which in turn, facilitates the formation of secretory vesicles. Membrane-bound Arf1 can also recruit a diverse array of effectors, including COPI, clathrin, SIRT6-IN-1 inhibitor cytoskeletal regulators, and lipid-modifying enzymes. Arf1 has been shown to colocalize with the enteroviral replication machinery. Poliovirus 3A and 3CD protein synthesis was found to induce the translocation of Arf1 to membranes. The substitution of the F441 residue to S441 in the viral 3CD protein caused a loss of function in Arf1 translocation activity, and it was lethal for the virus. In addition, cell-free poliovirus replication was inhibited by adding peptides from the N-terminus of Arf1; this result suggested that Arf1 activity played a significant role in viral replication. Arf1, Arf3, and Arf5 were reported to be activated upon poliovirus infection. Our RP6530 results also demonstrated that Arf1 and Arf3 were upregulated and activated in EV71 infections. However, our knockdown results showed that a single knockdown of either Arf1 or any other Arf had no effect on EV71 replication. This was consistent with Lanke��s result that Arf1 was dispensable for coxsackievirus B3 replication. This might be explained by the high homology of the Arf proteins, which may enable Arfs to compensate for one another to preserve cellular functions. In the Class I Arfs, Arf1 and Arf3 are 96% identical; the Class II Arfs, Arf4 and Arf5, are 90% identical; the Class I and II Arfs exhibit about 80% identity, and Arf6 is only about 70% identical to the other Arfs. When we tested combinations of double Arf knockdowns, we found that knocking down both Arf1 and Arf3 inhibited EV71 replication in cells. Volpicelli-Daley et al showed that Arf1 and Arf3 were important for protein transport between the endoplasmic reticulum and the Golgi network, and double knockdowns of Arf1 and Arf3 led to a redistribution of COPI proteins from the Golgi membranes to the cytosol. This was consistent with our previous findings that COPI activity was required for EV71 replication. In contrast, other combined double knockdowns did not affect viral replication. Although many studies point to the involvement of different Arf proteins in picornavirus replication, our observations, together with other reports, showed that Arf overexpression could not rescue virus replication from BFA exposure.