A retroviral vector containing the gag and pol genes and two reporter genes, bsd and eYFP, but defective for env, regulatory, and accessory genes was used to score mutations appearing during one cell infection cycle (1). The eYFP gene encodes the yellow fluorescent protein and was used to count the total number of cells carrying the vector. 293T cells stably expressing the vector were transfected with a helper plasmid to yield pseudotyped viruses, which were used to transduce fresh cells. The bsd gene encoded resistance to basticidin, but had a premature ochre stop codon such that only cells receiving a revertant virus would be resistant to blasticidin. This gave f / c = (2.0 - 4.0) ´ 10^-6, and sequencing of 16 revertants showed nine single nucleotide substitutions (to the wild-type or three other codons), five apparent G®A hypermutations, and two deletions. Here, T is unknown for indels and hypermutations, but for single nucleotide substitutions, T_s = 7. Using the latter and taking f / c = 3.0 ´ 10^-6, m_s/n/c = 3 ´ 3.0 ´ 10^-6 ´ 9 / 16 / 7 = 7.3 ´ 10^-7. Additional assays were carried out with HIV-1 and other retroviruses by directly co-transfecting cells with the vector and the helper plasmid instead of using stable eYFP producers, but it was shown that transfection was a significant source of mutation and hence these data do not provide a reliable estimate of the absolute mutation rate.

1. Laakso, M. M. and R. E. Sutton. 2006. Replicative fidelity of lentiviral vectors produced by transient transfection. Virology 348:406-417.