A retroviral vector that contained all cis elements required for replication, regulatory and accessory virus genes, reporter genes tk and hyg, but lacking the gag, pol and env genes was constructed and used to score mutations appearing during one cell infection cycle by co-transfecting the vector with helper plasmids encoding the missing genes (2). The hyg gene confers resistance to hygromycin, whereas the tk gene confers sensitivity to bromouridine. Hygromycin was used for selecting cells carrying the vector and bromouridine to score null mutations in the tk gene (996 nucleotides). In total, 349 / 15930 clones were mutant, giving f / c = 0.022. Sequencing of 43 mutants indicated that 13 / 43 mutations were indels. Hence m_i/n/c = 0.022 ´ 13 / 43 / 996 = 9.7 ´ 10^-6. The fraction of nucleotide substitutions that produced the tk null phenotype was unknown and it was not indicated which mutations produced stop codons. The number of possible mutations to stop codons in this gene is 76 and, using information from previous studies (1, 3), it is expected that approximately 1 / 7 of the observed nucleotide substitutions produced such mutations (see MLV (iii) estimate). Hence m_s/n/c = 3 ´ 0.022 ´ 30 / 43 / 7 / 76 = 8.7 ´ 10^-5, and d = 0.072. Mutations arising during transfection were not controlled, potentially introducing false positives.

1. Drake, J. W., B. Charlesworth, D. Charlesworth, and J. F. Crow. 1998. Rates of spontaneous mutation. Genetics 148:1667-1686.

2. Huang, K. J. and D. P. Wooley. 2005. A new cell-based assay for measuring the forward mutation rate of HIV-1. J. Virol. Methods. 124:95-104.

3. Parthasarathi, S., A. Varela-Echavarria, Y. Ron, B. D. Preston, and J. P. Dougherty. 1995. Genetic rearrangements occurring during a single cycle of murine leukemia virus vector replication: characterization and implications. J. Virol. 69:7991-8000.