The Genomic 3′ UTR of Flaviviruses Is a Translation Initiation Enhancer

Figures

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  Figure 1. Cap-independent translation of WNV genome. (a) Diagram of the RNA constructs harbouring the FLuc coding sequence flanked by different 5′ end and the 3′ UTR of the WNV RNA genome, and control constructs. (b) Translation efficiency of different capped and uncapped RNA constructs. Vero cells were cotransfected with the individual RNA constructs indicated in (a) and m7G-RLuc RNA. Translation activity was determined as FLuc protein activity normalized against the value obtained for RLuc. Values refer to that of the capped RNA coding the FLuc fusion to the WNV 3′ UTR (m7G-FLuc-3′UTR). (c) Translation efficiency of the HCV IRES-dependent RNA constructs. Translation activity was determined as described in (b); values are referred to that of the uncapped WNV construct. Values are the mean of at least three independent experiments ± standard deviation.

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  Figure 2. The 5′ end of the WNV genome promotes the internal initiation of translation. (a) Diagram of the dicistronic RNA constructs harbouring the capped RLuc mRNA at the 5′ end of the FLuc coding sequence flanked by the 5′ end, or truncated variants (del and delAUG, respectively) and the 3′ UTR of the WNV RNA genome. (b) Translation efficiency of dicistronic RNA constructs. Vero and C6/36 cells were transfected with an RNA construct indicated in (a); left and right panels respectively). Translation activity was determined as the FLuc protein activity and normalised against the value obtained for RLuc. Values refer to that of the dicistronic RNA containing the 5′ end (nt 1–160) of the WNV m7G-DIC genome.

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  Figure 3. Translation enhancing effect of the 3′ UTR of the WNV genome in different mammalian cell lines. Different cell lines were cotransfected with the capped or uncapped RNA constructs harbouring the 5′ end of the WNV RNA genome fusion at the 5′ end of the FLuc RNA sequence followed, or not, by the WNV 3′ UTR and m7G-RLuc RNA. Translation activity was determined as described in Figure 1. Values refer to that of the corresponding construct lacking the WNV 3′ UTR, and are the mean of three independent experiments ± standard deviation.

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  Figure 4. The genomic 3′ UTR of WNV enhanced translation in trans. Vero cells were cotransfected with the m7G-WNV or m7G-WNVΔ3′UTR RNA constructs, and m7G-RLuc RNA, in the presence of increasing molar excess of the genomic 3′ UTR WNV RNA fragment and decreasing amounts of the RNA-100 to compensate for the total RNA load in the transfection mix. Translation activity was determined as described in Figure 1. Values refer to that of the corresponding experiment without the 3′ UTR RNA fragment added.

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  Figure 5. WNV translation initiation is enhanced by the 3′ UTRs of DENV and YFV. (a) Diagram of the chimeric RNA constructs harbouring the FLuc coding sequence flanked by the 5′ end of the WNV genome and the 3′ UTR of the DENV or YFV RNA genome. Translation efficiency of different capped and uncapped RNA constructs was determined in Vero and C6/36 cell lines. (b) Vero cells were cotransfected with each individual chimeric RNA construct and m7G-RLuc RNA. Translation activity was determined as described in Figure 1. Values are referred to that of the corresponding construct lacking any 3′ UTR fragment. (c) Mosquito C6/36 cells were cotransfected as in (b) and translation activity determined as described in Figure 1. Values are the mean of three independent experiments ± standard deviation. Black and grey bars correspond to the values of the capped and uncapped RNA constructs, respectively.

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  Figure 6. Highly conserved structural elements of the WNV 3′ UTR contribute to the regulation of 5′ end translation initiation. (a) Secondary structure of the WNV subgenomic RNA construct; the secondary structure elements of the 5′ and 3′ ends of the WNV genome are represented. Arrows indicate the first and last nucleotide included in the deletions to generate the 3′ UTR variants dPK1, dPK2, dPK3 and dSL-III (1–4, respectively). A diagram of the deletion RNA construct variants is shown in the lower panel. The key for the different bars is as in Figure 1. (b) The translation activity of different capped and uncapped RNA constructs carrying variants of the WNV 3′ UTR was determined in Vero cells. Cells were cotransfected with each individual 3′ UTR variant RNA construct and m7G-RLuc RNA. Translation activity was determined as described in Figure 1. Values refer to that of the corresponding wild type 3′ UTR construct. (c) The translation activity of different capped RNA constructs carrying variants of the WNV 3′ UTR was determined in C6/36 cells. Mosquito cells were cotransfected as in (b) and translation activity determined as described in Figure 1. Values are the mean of three independent…