Diez nuevos clones de H brasiliensis promisorios para la

The translation profile of the pBEV 2A replicon was initially investigated in comparison to pGFP-PAC and pLa-GFP-PAC replicons. Preliminary results indicated that up- and down-stream protein production was altered within the pBEV 2A replicon. Both the pLa-GFP-PAC and pGFP-PAC replicons generated translation profiles indicative of polyproteins encoding a ribosome skipping sequence. The decrease in upstream protein synthesis within the pBEV 2A replicon (i.e. GFP-PAC) could be attributed to the lack of FMDV 2A which mediates the production of a molar excess of proteins encoded upstream. These initial findings can be used to investigate the hypothesis outlined above for 2A- mediated regulation of FMDV translation and prompts further experimentation. Further studies using the replicons described within this chapter will be designed to examine (i) the ratios of proteins up- and down-stream of 2A synthesised throughout replication, (ii) host-cell shut-off by viral proteinases (Lpro & BEV 2Apro), (iii) particle yield derived from infectious copies, and (iv) interactions of Lpro (or 2Apro) with the host-cell immune response. Protein ratios will be difficult to quantify due to the multiple precursors synthesised by the FMDV genome. In an attempt to measure differences in protein synthesis between replicons containing BEV 2Apro and FMDV 2A, pulse-labelling experiments will be performed involving immunoprecipitation of both cellular and in vitro translation extracts with anti-GFP and anti-3D antibodies. Protein levels will be quantified and differences between each replicon calculated. This will allow de novo synthesis of both upstream and downstream to be measured, rather than accumulated protein levels. The levels of eEF2 and phosphorylated-eEF2 will also be monitored within both WT and BEV replicon transfected cells. This will be correlated with protein levels synthesised using the differing 2A’s to examine whether this translation factor has a role in regulating FMDV 2A ribosome-skipping.

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Before such experiments can commence, polyprotein processing within these replicons must be improved through modification of the C-terminus of BEV 2Apro with varying 3Cpro dipeptides (following removal of TaV 2A) and passaging RNA from replicon transfected cells, or, by virus rescue and passage (Pirbright Institute): genomes would be sequenced throughout the process of passage and the properties of this new form of picornavirus compared with FMDV.

The final results chapter of this thesis describes the influence of dinucleotide frequency on Echovirus 7 growth kinetics and the potential application of this strategy to the FMDV replicon system for vaccine development.

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Chapter 7: RNA Virus Attenuation by Codon-pair

De-Optimisation is an Artefact of Increases in

CpG/UpA Dinucleotide Frequencies.

7.1 Introduction

There are a number of factors which contribute to translation regulation within eukaryotes, bacteria and viruses. These comprise codon usage, relative aminoacyl-tRNA abundancies, elongation rates (ribosome ‘processivity’) and translation regulation (Gingold & Pilpel, 2011). Biases exist toward certain synonymous codons (the “codon bias”) and codon-pairs (the “codon-pair bias”). Codon-pair bias refers to the preferential pairing of certain codons over others (e.g. in human genes the codon-pair GCC-GAA encodes the adjacent amino acids alanine-glutamic acid less often than GCA-GAG, even though GCC and GAA are the most used codons; (Luke et al., 2013). These biases are thought to influence gene expression by altering translation efficiency.

Both codon and codon-pair de-optimisation have been adopted as strategies for attenuating viral replication within RNA viruses (Burns et al., 2006; Coleman et al., 2008; Martrus et al., 2013; Mueller et al., 2006, 2010; Ni et al., 2014). This approach involves replacement of codons within the viral coding region for synonymous non-preferred codons or codon-pairs. It is thought that by altering codon usage, translation efficiency is altered resulting in reduced viral replication. The design of such viruses has the potential for the production of safer, non- reverting, live-attenuated vaccines, since attenuation arises from the incorporation of literally 100s of mutations – each of which only very slightly reduces replicative fitness, but taken together produce significant attenuation. Traditional methods of producing live, attenuated, vaccines involve attenuation of viral genomes by serial passage in tissue-culture, such as the OPV Sabin vaccine which had huge impacts on human health. This method of attenuation is time consuming and relies upon chance alone to generate attenuating mutations which then must undergo another lengthy process of selection to produce the required phenotype. However, using this method often a small number of key (attenuating) mutations may back mutate leading to reversion to virulence. The introduction of a large number of synonymous changes within the genome

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reduces, therefore, the risk of back-mutation increasing the genetic stability of the vaccine strain.

Another compositional feature which can affect the replication phenotype is the suppression of CpG and UpA dinucleotide frequencies among RNA and small DNA viruses (Rima & Mcferran, 1997; Simmonds et al., 2013). Artificially increasing the dinucleotide frequencies has been shown to impair replication kinetics of Echovirus 7 (E7) and PV (Atkinson et al., 2014; Burns et al., 2009). The authors of these studies speculate that the selection of disfavoured codon- pairs which attenuate PV and other viruses alters the CpG/UpA frequency within the genome, and it is this which accounts for, the most part (if not all!), the attenuating effects observed. Indeed, Burns et al., (2009) demonstrated that their previous strategy using codon de-optimised PV mutants to attenuate viral replication was due to greatly enhanced frequencies of CpG and UpA dinucleotides, rather than alterations in codon or codon-pair bias.

The study described in this chapter sought to disentangle the relationship between codon-pair bias (CPB) and dinucleotide frequencies in a re-examination of their effects on the replication of an RNA virus, E7. To resolve this functionally, comparisons were made of the replication kinetics and relative fitness of native E7 with a series of novel mutants of E7 in which dinucleotide frequencies and CPB were independently manipulated.