Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location
The combined data reiterate the importance of the native EMCV IRES configuration for protein optimum expression in bicistronic contexts. Virus mapping experiments have suggested that ribosome recognition may actually be enhanced by including the first few codons of the polyprotein ORF (preferred IRES; Figure 1A) (22). However, since most IRES users prefer to avoid amino-terminal fusion sequences, very good expression (as shown here) can usually be achieved by linking the heterologous ORF directly to the native AUG in its native IRES context (5,17). A caveat to this linkage is that rare or unusual codons for bulky, charged amino acids (e.g., Pro, Trp, Cys, Arg, Lys) should probably be avoided within the first 2–3 codons of the cistron.
The 5′ side of the minimum IRES needs to avoid disruption of stem H (4) defining a total viral fragment of about 430 bases, including the A6 bifurcation loop in the JK segment. All p#CITE vectors, as well as the original pE5LVP0, extend 5′ for an additional 120–130 viral bases into an upstream oligo(C) tract (e.g., to base 273). The extra length is not required for ribosome recognition, but it can provide a useful spacer between the A and B cistrons, allowing the IRES to fold without undue structural constraints. If the IRES is placed too close to the A cistron or immediately adjacent to a 5′ cap (i.e., preceding the A cistron of a monocistronic DNA vector), it is likely to fail or interfere with translation from the upstream ORF. As a general rule, attention to the IRES origin and preferred sequence are required for optimum activity. Methods sections describing comparative IRES expression experiments should always be careful to cite this information.
AcknowledgmentsThis work was supported by National Institutes of Health (NIH) grant no. AI-17331 to A.C.P. The authors thank Dr. Aleksey Aminev for the generous gift ofplasmid pIRES-luc and Ms. Rachel Groppo for plasmid pF/R-wt.
Competing Interests Statement A.C.P. is the senior author on the original, as well as subsequent papers describing the EMCV IRES technology, which is wholly owned by the Wisconsin Alumni Research Foundation (WARF) and who also administers the U.S. patent covering this technology. A.C.P. receives a fractional royalty. Y.A.B. declares no competing interests.
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