To score mutations appearing during a single cell
infection cycle, pseudotyped viruses were obtained by
co-transfecting 293T cells with a viral vector defective for env gene and a helper
plasmid (1). HeLa
cells were infected with these viruses, selected for antibiotic resistance,
cloned, and used for DNA amplification and sublcloning
using a phage l library. Sequencing of six nearly full-length viral
genomes (9072 nucleotides on average, Ts
= 27216) yielded four nucleotide substitutions and no indels. Hence, ms/n/c = 3 ´ 4 / 6 / 27216 = 7.3 ´ 10-5. In
another assay, lymphocytes were infected with the pseudotyped
viruses, sorted by fluorescence using flow cytometry,
cloned, and used for DNA amplification by long-range PCR and direct sequencing
of PCR products. Sequencing of seven
large portions of viral genomes (7791 nucleotides on average, Ts = 23373) yielded eight
nucleotide substitutions and three indels.
Hence, ms/n/c =
3 ´ 8 / 7 / 23373 = 1.5 ´ 10-4. Taking
the geometric mean of the two estimates, ms/n/c
= 1.0 ´ 10-4. The average
Ts is 25295. For indels, mi/n/c = 3 / 7 / 7791 = 5.5 ´ 10-5, and d = 0.35. The fraction of stop codon mutations was
unusually high (4 / 12) and no synonymous substitutions were observed,
suggesting that cell clones receiving inactive viruses were favored,
and selection acting on the virus during the cell infection cycle was not
controlled for. Also, the fraction of
mutations resulting from transfection was unknown. Finally, the pseudotyped
viruses lacked vpr,
which may have an effect on the mutation rate (2). These factors could have led to a high number
of false positives and thus this estimate should be taken with caution.
1. Gao, F., Y. Chen, D. N. Levy, J. A. Conway, T.
B. Kepler, and H. Hui.
2004. Unselected mutations in the human immunodeficiency virus type 1 genome
are mostly nonsynonymous and often deleterious. J. Virol. 78:2426-2433.
2. 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.