Tobacco plants constitutively expressing the TEV polymerase gene NIb were inoculated with TEV (1). Since the viral NIb gene was complemented in trans, selection on this gene was probably absent or weak (a » 1). Samples from 20 plants were taken at different time points ranging 5-60 days post inoculation used for RT-PCR, cloning, and sequencing. In total, 52 mutations (36 substitutions and 6 indels) were identified in 472 NIb clones (L = 1536). Since the viral genomic RNA is translated as a polyprotein, indels that modify the reading frame or nonsense mutations in the NIb gene prevent the correct expression of downstream genes (here, the capsid gene). As a first approach, we can focus on presumably lethal mutations. Of the 36 substitutions, two produced premature stop codons. The number of possible such mutations in the NIb gene is T_s = 251. Hence, m_s/n/c = 2 / 251 / 472 = 1.7 ´ 10^-5. For indels, m_i/n/c = 6 / 1536 / 472 = 8.2 ´ 10^-6 and thus d = 0.32. Immediately after stop codon mutant appears in a cell, it can be replicated, transcribed, and packaged normally by the non-mutant proteins present in the cell, but the mutant should be unable to initiate a second infection cycle. Hence, the estimate is in per cell infection units. However, suppression of stop codons or complementation between viruses at high moi could allow a subset of mutants to complete several infection cycles, leading to an overestimation of the mutation rate. RT-PCR errors constitute another source of overestimation. As an alternative approach we can focus on presumably neutral mutations, which are all except nonsense mutations and indels because NIb was trans complemented (T_s = 1536 ´ 3 - 251 = 4357). The viral yield per cell was B = 1555 as determined in vitro using transfected protoplasts and it was estimated that c = 3.16 per day, hence c varied from 16 to 190 (5-60 days). According to a regression analysis of the number of mutations on the number of cell infection cycles done in the original publication, m_s/n/c = 4.8 ´ 10^-6. The latter value is used. However, taking into account that the first approach was expected to produce an overestimation, the two estimates are reasonably consistent.

1. Tromas, N. and S. F. Elena. 2010. The Rate and Spectrum of Spontaneous Mutations in a Plant RNA Virus. Genetics.