Tobacco plants constitutively expressing the TMV movement protein (MP) were inoculated with TMV (1).  Since the viral MP gene was complemented in trans, selection on this gene was probably absent or weak (a » 1).  Viruses were extracted three days post-inoculation and individual particles were isolated by infecting MP transgenic plants which form local necrotic lesions.  These individual clones were assayed for loss of function of the MP gene, which is essential, by inoculating plants not expressing the MP transgene, and mutants were sequenced.  The relevant parameters are f = 0.038, c = 5.7, and L = 804.  Twenty-four out of 35 mutations were indels.  Assuming that all mutations inactivated the gene and using Equation 3, m_i/n/c = 0.038 ´ 24 / 35 / 5.7 / 804 = 5.7 ´ 10^-6.  For substitutions, the fraction of total mutations that produce the null phenotype was unknown, and nonsense mutations were not observed.  The authors used a correction factor of 4.78 derived from DNA-based microbes.  According to this and using Equation 2, m_s/n/c = 0.038 ´ 4.78 ´ 11 / 35 / 5.7 / 804 = 1.2 ´ 10^-5.  Alternatively, one can use the fraction of lethal substitutions estimated in other viruses, p_L = 0.2-0.4.  Using this alternate information to estimate the fraction of substitutions that inactivate the TMV MP protein, and taking p_L = 0.3, T_s = 3 ´ 804 ´ 0.3 = 723, hence m_s/n/c = 3 ´ 0.038 ´ 11 / 35 / 5.7 / 723 = 8.7 ´ 10^-6.  The two approaches yield similar results (we use the latter).  The corresponding indel fraction is d = 0.40.

    1.    Malpica, J. M., A. Fraile, I. Moreno, C. I. Obies, J. W. Drake, and F. Garcia-Arenal. 2002. The rate and character of spontaneous mutation in an RNA virus. Genetics 162:1505-1511.