Protein expression rates of gene-rearranged viruses. The stepwise decline in the transcription of genes more distant from the 39-end region promoter highlights how gene order affects gene expression in VSV. Advances in reverse genetics have made it possible to create gene-rearranged virus strains where the transcriptional attenuation mechanism then creates altered levels of gene expression [7, 9, 18, 20] . In one study [18] the three internal genes, P, M, and G, were permuted, and the resulting six possible VSV strains were characterized. Relative rates of viral protein expression in BHK cells were experimentally measured based on their incorporation of [ 35 S]-labeled methionine for a one-hour window at 4 h post-infection [18] . We extended our model to simulate this experiment for mutants representing each geneorder permutation and compared the model prediction with the published results [18] , as shown in Figure 4 . All rates are expressed relative to the synthesis rate of N protein, whose corresponding gene was in position 1, closest to the 39 end of the genome in all strain cases. Expression of gene L, in position 5, was minimal in both simulations and experiments, and the expression of all other genes was above 40%, a feature of the experimental data that the simulation also captured. Most of the points fall close to the parity line, indicating agreement between the simulation and experiment. Noteworthy are two subsets of points. First, the four circled points are exceptions to the general rule that gene order determines the level of gene expression. These were genes in the second position of the genomes that were expressed essentially at the same rate as gene N, in the first position [18] . This result highlights that the expression rate of protein is affected not only by its gene order, or corresponding rate of mRNA production, but also by its length and degradation rate. For a fixed average rate of translational elongation, longer gene products will tend to be produced more slowly. Further, the net rate of protein production will reflect the rates of both protein synthesis and protein degradation. The model accounts for these contributions, and for the circled genes such accounting appears to capture unexpected high translation levels of genes in the second position, which were measured by Ball et al. [18] . The second sets of points, shown in two boxes, indicate mismatches ( Figure 4 ) that, in the most challenging scenario, could reflect unknown strain-specific mechanisms that are not present in our general gene-permutation model. However, one should also note that the experiment is based on labeling and quantifying proteins about 4 h post-infection. This relatively early time point allows one to minimize potentially confounding influences of virion particle assembly and production on cytoplasmic levels of viral proteins, but it also represents a point before the majority of viral proteins have been made ( Figure 1B) .