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.