A retroviral vector containing the lacZ a-complementation gene region as a neutral mutational target was used to score null mutations appearing during a single infection cycle (1). In two different experiments using forward and reverse lacZ orientation, the total number of colonies screened was 328810 (172906 and 155904, respectively). The total number of nucleotide substitutions scored was 706, of which 102 were multiple-site mutations (most G®A hypermutations). The length of the lacZ sequence used was 174 nucleotides and thus the total number of possible mutations is 174 ´ 3 = 522, but the actual mutational target is smaller because many mutations will not produce the null phenotype (white colonies). In a previous study using a similar sequence, it was determined that T_s = 219 (2). Using these figures, the mutation rate including host-mediated hypermutation is m_s/n/c = 3 ´ 706 / 328810 / 219 = 2.9 ´ 10^-5. Alternatively, we can use the method based on scoring stop codons. The number of stop-codon mutations was 82, and there are 17 possible nonsense substitutions in the lacZ sequence used in this study. Therefore, the mutation rate is m_s/n/c = 3 ´ 83 / 328810 / 17 = 4.4 ´ 10^-5. We use the latter value. The total number of indels was 66 (frameshifts and large indels). Hence m_i/n/c = 66 / 328810 / 174 = 1.2 ´ 10^-6, and the indel ratio is d = 0.03.

1. Abram, M. E., A. L. Ferris, W. Shao, W. G. Alvord, and S. H. Hughes. 2010. Nature, position, and frequency of mutations made in a single cycle of HIV-1 replication. J. Virol. 84:9864-9878.

2. Bebenek, K., J. Abbotts, J. D. Roberts, S. H. Wilson, and T. A. Kunkel. 1989. Specificity and mechanism of error-prone replication by human immunodeficiency virus-1 reverse transcriptase. J Biol. Chem. 264:16948-16956.