Unsuccessful attempts to mutate Aq.GFP led to the investigation of appropriate substitutions. FPs which arose from different species than the jellyfish GFP would be expected to be more suitable due to their low sequence homology. Therefore Ptilosarcus GFP and mCherry from the sea pen and mushroom coral (Peelle et al., 2001; Shaner et al., 2004), respectively, were chosen for subsequent replicon studies.
5.14.2.1 Ptilosarcus GFP replicon expression
Replicons encoding Pt.GFP exhibited enhanced fluorescence in comparison to those encoding Aq.GFP. Both GFP count and fluorescence intensity data was substantially higher than data obtained with replicons expressing Aq.GFP. This increase in fluorescence can be attributed to the presence of ribozymes within the Pt.GFP replicons which accounts for ~10-fold of the ~30-fold increase observed. Since further experiments investigating the protein expression profile and stability of Pt.GFP showed these attributes to be comparable to Aq.GFP (discussed below), this suggests the enhanced signal is due to intrinsic characteristics of Pt.GFP.
The best figures of merit to express the ‘brightness’ of a FP are the molar extinction coefficient (EC) and the quantum yield (QY). The EC is a measurement that determines the efficiency of light absorption by the FP and the QY determines the efficiency of the fluorescence process (number of photons emitted in relation to the number absorbed; Chudakov et al., 2010). It is possible that the enhanced fluorescence could be due to Pt.GFP possessing a higher QY and EC in comparison to Aq.GFP; however these measurements have not been calculated for Pt.GFP and are as yet unknown. Nevertheless, these measurements aren’t the only factors that should be taken into account as transcription and translation efficiency, mRNA and protein stability, and chromophore formation can all affect the resulting signal produced by a FP (Chudakov et al., 2010). It should also be noted that Schulte et al., (2006) reported the formation of tetramers for Pt.GFP and it is possible this could lead to increased fluorescence. It is known that oligomeric FPs often exhibit enhanced spectral and biochemical properties in comparison to monomeric FPs (Chudakov et al., 2010).
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The pH stability of a FP can also affect the fluorescent signal obtained. Schulte et al., (2006) reported that Pt.GFP was much more robust at low pH when compared to pH-sensitive variants of Aq.GFP (pHluorins). Cells transfected with replicon RNA will undergo cell death due to the process of viral replication and cell protein synthesis shut-off. The pH of the cells will therefore decrease and become acidic (Lagadic-Gossmann et al., 2004), which can quench the chromophore leading to a loss in fluorescence. Aq.GFP is quenched by 50% at pH 5.5 (Tsien, 1998). Apoptotic cells reach pH levels near to (and lower) than this (Lagadic-Gossmann et al., 2004). Schulte and colleagues reported a pH range of 3.8-8.2 for Pt.GFP which was broader than the range of pH 4.8-7.6 for Aq.GFP, thus the robust pH stability of Pt.GFP could attribute to the higher signal produced by this FP.
5.14.2.1.1 Ptilosarcus GFP structure prediction
Experiments performed by Peelle et al., (2001) to determine the structure of Pt.GFP using CD indicated a β-can formation almost identical to Aq.GFP. Phyre2 structure prediction (Kelley & Sternberg, 2009) carried out during this study is in agreement with this finding and eliminates the possibility that structural differences account for the variation in fluorescent signal generated by Pt.GFP and Aq.GFP.
5.14.2.1.2 Ptilosarcus GFP half-life
GFP expression was monitored in cells treated with CHX to measure the rate of GFP degradation. Fluorescence data generated indicates that both GFPs have similar half-lives of ~36 hr. Aq.GFP has been reported to have a half-life of 26-30 hr based on previous studies (Corish & Tyler-Smith, 1999; Li et al., 1998). The differences between studies could be due to the measurement techniques (image analyses of radiolabelled protein, western blotting and flow cytometry versus live cell imaging) , cell lines used (human cell lines versus rodent cells) the number of time points taken and the duration of the experiments throughout (3 and 12 hr versus 24 hr). The half-life measured during fluorescence experiments correlates well with the N-end rule, where the terminal amino acid determines the half-life of a protein; proteins with a Met N-terminal residue have half-lives of ~30 hr (Gonda et al., 1989), which is the resulting terminal amino acid of these FPs.
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The pPt.GFPΔ1D-JC3 plasmid exhibited a higher fluorescent signal than pPt.GFP-JC3. Peelle et al., (2001) investigated the effect of the addition of a 14 amino acid flag-tag to the C-terminus of Pt.GFP upon fluorescence. They observed a 1.4-fold increase in Pt.GFP expression, which explains observations reported in this study where insertion of the FMDV Δ1D capsid sequence (~ 40 aa) enhanced fluorescence ~ 10-fold.
The addition of ~40 aa of Δ1D increased fluorescence 10-fold whereas a combination of both Δ1A and Δ1D caused a 1.5-fold reduction. It is interesting to note that the N-terminal residue of both Aq.GFP and Pt.GFP will be the terminal glycine from Δ1A (produced by Lpro cleavage). The N-terminal region of picornavirus 1A proteins comprises a myristolation signal: it may well be the case that this [Δ1A-Pt.GFP-Δ1D] fusion protein also becomes myristolated and this could affect the half-life or fluorescent properties of Pt.GFP.
PAC C-terminal fusions also increased Pt.GFP fluorescence but, interestingly, not to the same extent as the much shorter Δ1D C-terminal extension. It appears, therefore, that different C-terminal extensions have different affects upon the fluorescent properties of Pt.GFP.
As a general observation, cells expressing plasmid constructs encoding PAC displayed a rounded-up morphology in comparison to cells transfected with constructs lacking the PAC gene (data not shown). Acetyl-coenzyme A (acetyl- CoA) is very important for cellular metabolism (Vara et al., 1985). Since acetyl- CoA is used during the biosynthesis of PAC, it’s depletion may produce cytopathic effects.
Plasmid pJC3 displayed fluorescence ~3-fold higher than pPt.GFP Δ1D-JC3. This was unexpected due to the large differences observed between replicons expressing Aq.GFP or Pt.GFP. The main differences between experiments carried out with replicon RNA versus plasmid DNA is cytoplasmic replication versus nuclear transcription. It is known that Aq.GFP expression was limited when used within plants due to the presence of a cryptic splice site. Expression was restored following alterations to the codon usage within Aq.GFP (Haseloff et al., 1997). Hence, it is possible such a site exists within Pt.GFP and this could explain the observations during these experiments.
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5.14.2.2 mCherry replicon systems
Initial fluorescence from mCherry replicons was reduced in comparison to Aq.GFP and Pt.GFP replicons. The QY of mCherry is 0.22 in contrast to 0.60 of Aq.GFP with the relative brightness of mCherry 47% that of Aq.GFP (Chudakov et al., 2010; Shaner et al., 2004); therefore it is unsurprising mCherry fluorescence was ~15-fold lower than Aq.GFP at 2 hr p.t. and ~2-fold overall. Differences between attenuated forms can clearly be detected by the use of mCherry FP. ‘Leaderless’ replicons displayed reduced fluorescence with the pLL- mCherry replicon also having a 2-3 hr lag in replication. Replicons lacking L and encoding Aq.GFP failed to show such a decrease, which was due to cleavage of this FP by Lpro (Chapter 3; Tulloch et al., 2014a). Replacement with a FP that is not cleaved by the viral protease demonstrates the true potential of this replicon system. Indeed, Pt.GFP replicons of a similar nature have been constructed (undertaken by a senior honours project student) and display identical trends, with ‘leaderless’ replicons showing attenuation (data not shown). This is further evidence that this system can be used to measure attenuated phenotypes and allows us to use ‘leaderless’ replicons within our panel of ‘control’ attenuated genomes as a benchmark for attenuation. The use of FPs with different spectral properties will also be very useful for competition and trans-complementation studies using replicons.
5.14.3 GFP expression derived from replicon forms encoding polymerase