During 5'-cap-dependent translation, methylated 5'-cap and 3'-poly(A) tail work synergistically in a poly(A) binding protein (PABP)-dependent manner to facilitate translation via promoting the formation of a closed mRNA loop. On the other hand, during internal translation initiation, the requirement for and the roles of 3'-poly(A) tail and PABP vary depending on specific characteristics of each internal ribosomal entry site (IRES). In this study, we analyzed the effect of 3'-poly(A) tail and phylogenetically divergent PABPs on a polypurine tract-containing IRES element derived from the coat protein gene of crucifer-infecting tobamovirus (CrTMV IRES(CP)). We find that mutations in the internal polypurine tract decrease IRES activity in a heterologous (mammalian) system in vivo. Moreover, these mutations decrease the high-affinity binding of all phylogenetically divergent PABPs derived from Arabidopsis and yeast in electro mobility gel shift assays in vitro. Partial PABP depletion and reconstitution assays using Arabidopsis-derived PABP2, 3, 5, 8 and yeast Pab1p provide further evidence that CrTMV IRES(CP) requires PABP for maximal activity. Furthermore, stronger enhancement in the presence of 3'-poly(A) and the absence of 5'-methylated cap suggests a potential joint interaction between PABP, the CrTMV IRES(CP) and the 3'-poly(A).