Molecular mechanisms of cap and poly(A) independent translation of barley yellow dwarf virus RNA

Barley yellow dwarf virus RNA contains a translation element (3 \u27TE) that confers efficient cap-independent initiation at the 5 proximal AUG. Direct end-labeling of RNAs verified the absence of a 5 \u27 modification on virion RNA. Thus BYDV differs from related viruses by having neither a genome-linked protein nor a 5 \u27 cap. To function in the 3\u27 UTR, the 3 \u27TE must recruit ribosomes and associated translation factors, and communicate with the 5\u27 end of the mRNA where translation initiates. The communication function is mediated by direct base pairing between the 3\u27TE and the 5\u27 UTR. We propose that protein factors interact with the 3\u27TE to facilitate initiation and ribosome recruitment. The 3\u27TE binds to initiation factor eIF4F, demonstrated by using the 3\u27TE to purify this complex from wheat germ extract. eIF4F is a complex of two factors, cap binding protein eIF4E and adaptor protein eIF4G. Using purified initiation factors, both eIF4E and eIFiso4E bound directly and specifically to the TE, with a Kd≈2*10-7M. Non-functional TE mutants did not interact with eIF4E/iso4E. The binding of initiation factor 4E follows the genetic evidence that the 3\u27 TE can functionally substitute for a cap both in vitro and in vivo. Because the TE is located in the 3\u27 UTR of BYDV, we speculate that formation of the initiation complex may not be required at the 5 \u27 end, but may be delivered there by additional mechanisms. In plant cells dependent on both a cap and poly(A) tail, additional viral sequence (at most 869 nt) beginning five bases upstream of the 3\u27TE is required for translation. Previously, we found that the function of this additional sequence can be at least partially replaced by a poly(A) tail, suggesting a similar role. This downstream element, bases 5010 to 5677, is dubbed the poly(A) mimic (PAM). The PAM can also function in conjunction with a 5 \u27 cap. Analysis of PAM mutants defines three interacting sequences with differing roles in translation. These data show that independent elements in BYDV have evolved to functionally replace the roles of a cap and poly(A) tail in translation of the viral mRNA