We thank Dr. David Szymkowski for reading and comments on the manuscript

pressed from the Second Cistron in the Bicistronic mRNAs Studies to identify internal initiation in isolated viral or cellular RNA regions utilizing the transfection of bicistronic reporter plasmids as described above have seen strong criticism; a commonly cited caveat to the experimental approach being falsepositive IRES activity attributable to alternative splicing or cryptic promoter activity. In view of the sequence disparity between the natural variants and the reference pNL4.3-IRES, we undertook to verify the presence of the full length bicistronic RNA in cells transfected with the dl VAR plasmids. VAR IRESes that exhibited activity similar to or below the reference pNL4.3-IRES, were not included in this analysis. Total RNA was extracted from cells previously transfected with dl VAR1, dl VAR2, dl VAR3, dl VAR4, or dl VAR12, and analyzed by RTPCR as described by us and others . Our results indeed confirmed the presence of full length bicistronic mRNA for the dl DEMCV, the dl HIV-1 IRES, and all tested dl VAR constructs. Even though results are not quantitative nor do they rule out the presence of other RNA species, they confirm, in all cases, the expression of the full-length bicistronic mRNA. No product was observed when the PCR reaction was conducted without a previous step of reverse transcription confirming the absence of DNA contamination in the RNA preparation. Next we quantified the amount of bicistronic mRNA by individually amplifying RLuc or FLuc using a quantitative RT-qPCR assay. For this total RNA extracted from cells transfected with dl DEMCV, dl HIV-1 IRES, dl VAR1, dl VAR2, dl VAR3, dl VAR4, or dl VAR12, was amplified in parallel reactions using primers specifically designed to recognize RLuc or FLuc. The RNA concentration obtained by quantifying the FLuc cistron was divided by the RNA concentration obtained by quantifying RLuc cistron. Results show that for the different RNAs the RNA-FLuc/RNA-RLuc ratio was close to one; dl DEMCV, dl HIV-1 IRES, dl VAR1, dl VAR2, dl VAR3 , dl VAR4, or dl VAR12 . One exception could however be VAR4 as the results revealed a slight, yet PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189787 not significant, increase in bicistronic RNA when RLuc cistron was quantified. In despite of this observation, in general and when taking together, results presented in figures 2B and 2D suggest that only one RNA species expressing RLuc and FLuc is generated. Next we evaluated if this bicistronic mRNA could indeed drive cap- and IRES dependent translation initiation. After several unsuccessful results using RNA transfection assays in cells, we decided to address this question in Xenopus laevis oocytes, an experimental system that has proven useful for the study of viral IRESes. In vitro synthesized, capped and polyadenylated RNAs corresponding to the dl DEMCV, dl HIV-1 IRES, and the dl VAR vectors were microinjected into the cytoplasm of Xenopus laevis oocytes. RLuc and FLuc activities were Rocaglamide measured 24 h post-injection as standardized in previous studies. All values were expressed relative to the RLuc or FLuc activity obtained with the control dl HIV-1 IRES RNA, which was arbitrarily set to 1. The RLuc levels from the dl HIV-1 IRES, and the dl VAR mRNAs were comparable, suggesting not only that cap-dependent translation initiation was functional in oocytes, but also confirming that similar amounts of RNA were microinjected in all cases. As described in previous studies, a low level of expression from the second cistron of the dl DEMCV reporter RNA, con

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