A retroviral vector containing the lacZ a-complementation sequence (neutral mutational target) was used to score mutations appearing during a single cell infection cycle in several related studies (2^-4). In the first study (4), f / c = 70 / 15424 (66 mutant clones, four of them carrying two mutations). The mutational spectrum was constituted by 46 nucleotide substitutions (six nonsense mutations (1)) and 24 indels (17 frameshifts). Given that T_s = 219 (see SNV (i) estimate), m_s/n/c = 3 ´ 46 / 15424 / 219 = 4.1 ´ 10^-5. Using nonsense mutations only, (see SNV (i) estimate), T_s = 20 and m_s/n/c = 3 ´ 6 / 15424 / 20 = 5.8 ´ 10^-5 (the latter value is used). For indels, using the T_i values given in the SNV (i) estimate, m_i/n/c = 17 / 15424 / 150+ 7 / 15424 / 280 = 9.0 ´ 10^-6, and d = 0.13. In a second study (3), the same method was used to score mutations in vpr null mutants and vpr null mutants complemented in trans by virus producer cells. This showed that vpr reduces the viral mutation rate by approximately threefold and that its mechanism of action consists of recruiting the host uracil DNA glycosylase into viral particles. In the presence of a functional vpr protein provided in trans, f / c = 0.006. The mutational spectrum was unknown, but assuming that it was similar to the one reported in the previous study (4), nucleotide substitutions should constitute approximately 2 / 3 of all the observed mutations. Given that T_s = 219, m_{s/n/c s} = 3 ´ 0.006 ´ 2 / 3 / 219 = 5.5 ´ 10^-5. In a third study (2), the same method was used to score mutations in the absence or presence of the antiretroviral drugs AZT or 3TC, as well as in viruses encoding reverse transcriptase variants resistant to these drugs (note: viruses were vpr deficient). This study demonstrated that these drugs increase the mutation rate of the virus in a dose-dependent manner (up to sevenfold for the concentrations used) and that some resistance mutations can also increase the viral mutation rate (up to threefold). The average mutation frequency per cycle from three independent experiments for the wild-type virus was f / c = 0.005 (0.004, 0,005, and 0.006) in the absence of drugs. Sequencing of 40 mutant clones showed that 22 carried nucleotide substitutions (there were three additional G ® A hypermutants, but these are not counted here because the numbers of substitutions carried by each hypermutant were not provided), six carried frameshifts and two carried other indels. Taking T_s = 219 for substitutions, T_i = 150 for frameshifts, and T_i = 280 for other indels, m_s/n/c = 3 ´ 0.005 ´ 22 / 40 / 219 = 3.7 ´ 10^-5, m_i/n/c = 0.005 ´ 6 / 20 /150 + 0.005 ´ 2 / 20 / 280 = 1.2 ´ 10^-5, and d = 0.22. The geometric mean of the three m_s/n/c values is 4.9 ´ 10^-5. The average of the two indel fractions is d = 0.18.

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

2. Mansky, L. M. and L. C. Bernard. 2000. 3'-Azido-3'-deoxythymidine (AZT) and AZT-resistant reverse transcriptase can increase the in vivo mutation rate of human immunodeficiency virus type 1. J. Virol. 74:9532-9539.

3. Mansky, L. M., S. Preveral, L. Selig, R. Benarous, and S. Benichou. 2000. The interaction of vpr with uracil DNA glycosylase modulates the human immunodeficiency virus type 1 In vivo mutation rate. J. Virol. 74:7039-7047.

4. Mansky, L. M. and H. M. Temin. 1995. Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase. J. Virol. 69:5087-5094.