Table 1. Requirements for optimal viral IRES-driven translation in ribosome depleted RRL.
| Virus | Optimal salt conditions | Maximal stimulation by poly(A) in depleted RRL | |||||
|---|---|---|---|---|---|---|---|
| KCl (mM) | MgCl2 (mM) | ||||||
| –Ac | +Ad | –Ac | +Ad | Factor | KCl (mM)e | MgCl2 (mM)e | |
| PVa | 97 (80) | 110 | 0.5 (0.2) | 0.5 | 5–10 × | 120 | 1.0 |
| HRV | <50 (65) | 50 | <0.3 (0.7) | <0.3 | 5–10 × | 80 | 0.5 |
| HAV | <75 (65) | <75 | <0.3 (0.7) | <0.3 | 20–40 × | 105 | 0.9 |
| EMCV | >130 (120) | >130 | <0.3 (0.7) | 0.6 | 2–4 × | >130 | >0.3 |
| FMDV | 85 (125) | 85 | 0.5 (0.7) | 0.7 | 2 × | 106 | 1.0 |
| HCV | >130 (110) | >130 | 0.5 (0.6) | 0.5 | 2–4 ×f | –g | –g |
| Op24b | 110 | 125 | <0.3 | 0.75 | 10–20 × | >130 | 0.9 |
aTranslation reactions were programmed with 5 µg/ml of IRESp24-derived mRNAs which had been synthesised in uncapped, non-polyadenylated (–A) or uncapped, polyadenylated form (+A).
bCapped pOp24-derived mRNAs with or without a poly(A) tail were translated at 3 µg/ml.
c,dThe values for optimal concentrations of added KCl and MgCl2 were adjusted to take into account differences in endogenous salt inherent to the batch of RRL used as compared to that used in Borman et al. (22). Values in parenthesesc are those reported in Borman et al. (22).
eThe values cited are the concentrations of KCl and MgCl2 allowing maximal stimulation of each type of mRNA by polyadenylation.
fHCV IRES activity is not stimulated by polyadenylation but is affected by the presence of the viral X region. Stimulation factor is given for translation with the viral X region.
gStimulation of HCV IRES activity by poly(A) is not evidenced under any salt concentrations tested. Conversely, stimulation mediated by the viral X region is insensitive to changes in salt concentrations within the ranges tested (50–140 mM KCl and 0.2–1.2 mM MgCl2).