Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Sep 27;91(20):9490–9494. doi: 10.1073/pnas.91.20.9490

High rate of mismatch extension during reverse transcription in a single round of retrovirus replication.

G A Pulsinelli 1, H M Temin 1
PMCID: PMC44838  PMID: 7524077

Abstract

We made spleen necrosis virus-based retroviral vectors with mutations at the 3' end of the primer binding site region to observe the effects of terminal mismatches on retroviral replication. These vectors, when compared to a vector with the wild-type primer binding sequence, allowed us to assay the effects of the mutations on the viral titer during a single cycle of replication. The mutant vectors had titers that were comparable to the wild-type vector, indicating that reverse transcriptase has no trouble extending mismatches of as many as 3 bases under normal in vivo conditions. These results confirm and extend previous in vitro studies [Yu, H. & Goodman, M. (1992) J. Biol. Chem. 15, 10888-10896] that showed that such mismatch extension could occur in a cell-free system at high concentrations of incorrect nucleotides and in the absence of correct nucleotides. We now show that mismatch extension can occur during normal retroviral replication in cells and at normal physiological nucleotide concentrations.

Full text

PDF
9490

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bakhanashvili M., Hizi A. The fidelity of the reverse transcriptases of human immunodeficiency viruses and murine leukemia virus, exhibited by the mispair extension frequencies, is sequence dependent and enzyme related. FEBS Lett. 1993 Mar 15;319(1-2):201–205. doi: 10.1016/0014-5793(93)80067-5. [DOI] [PubMed] [Google Scholar]
  2. Berwin B., Barklis E. Retrovirus-mediated insertion of expressed and non-expressed genes at identical chromosomal locations. Nucleic Acids Res. 1993 May 25;21(10):2399–2407. doi: 10.1093/nar/21.10.2399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Creighton S., Huang M. M., Cai H., Arnheim N., Goodman M. F. Base mispair extension kinetics. Binding of avian myeloblastosis reverse transcriptase to matched and mismatched base pair termini. J Biol Chem. 1992 Feb 5;267(4):2633–2639. [PubMed] [Google Scholar]
  4. Dougherty J. P., Temin H. M. High mutation rate of a spleen necrosis virus-based retrovirus vector. Mol Cell Biol. 1986 Dec;6(12):4387–4395. doi: 10.1128/mcb.6.12.4387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dougherty J. P., Wisniewski R., Yang S. L., Rhode B. W., Temin H. M. New retrovirus helper cells with almost no nucleotide sequence homology to retrovirus vectors. J Virol. 1989 Jul;63(7):3209–3212. doi: 10.1128/jvi.63.7.3209-3212.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Holmes J., Jr, Clark S., Modrich P. Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5837–5841. doi: 10.1073/pnas.87.15.5837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hu W. S., Temin H. M. Genetic consequences of packaging two RNA genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1556–1560. doi: 10.1073/pnas.87.4.1556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kawai S., Nishizawa M. New procedure for DNA transfection with polycation and dimethyl sulfoxide. Mol Cell Biol. 1984 Jun;4(6):1172–1174. doi: 10.1128/mcb.4.6.1172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kusukawa N., Uemori T., Asada K., Kato I. Rapid and reliable protocol for direct sequencing of material amplified by the polymerase chain reaction. Biotechniques. 1990 Jul;9(1):66-8, 70, 72. [PubMed] [Google Scholar]
  10. Marck C. 'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 1988 Mar 11;16(5):1829–1836. doi: 10.1093/nar/16.5.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. McClary J. A., Witney F., Geisselsoder J. Efficient site-directed in vitro mutagenesis using phagemid vectors. Biotechniques. 1989 Mar;7(3):282–289. [PubMed] [Google Scholar]
  12. Mendelman L. V., Petruska J., Goodman M. F. Base mispair extension kinetics. Comparison of DNA polymerase alpha and reverse transcriptase. J Biol Chem. 1990 Feb 5;265(4):2338–2346. [PubMed] [Google Scholar]
  13. Modrich P. Mechanisms and biological effects of mismatch repair. Annu Rev Genet. 1991;25:229–253. doi: 10.1146/annurev.ge.25.120191.001305. [DOI] [PubMed] [Google Scholar]
  14. Pathak V. K., Temin H. M. Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: deletions and deletions with insertions. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6024–6028. doi: 10.1073/pnas.87.16.6024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Pathak V. K., Temin H. M. Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: substitutions, frameshifts, and hypermutations. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6019–6023. doi: 10.1073/pnas.87.16.6019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Perrino F. W., Preston B. D., Sandell L. L., Loeb L. A. Extension of mismatched 3' termini of DNA is a major determinant of the infidelity of human immunodeficiency virus type 1 reverse transcriptase. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8343–8347. doi: 10.1073/pnas.86.21.8343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pulsinelli G. A., Temin H. M. Characterization of large deletions occurring during a single round of retrovirus vector replication: novel deletion mechanism involving errors in strand transfer. J Virol. 1991 Sep;65(9):4786–4797. doi: 10.1128/jvi.65.9.4786-4797.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Simonsen C. C., Levinson A. D. Isolation and expression of an altered mouse dihydrofolate reductase cDNA. Proc Natl Acad Sci U S A. 1983 May;80(9):2495–2499. doi: 10.1073/pnas.80.9.2495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  20. Temin H. M. Retrovirus variation and reverse transcription: abnormal strand transfers result in retrovirus genetic variation. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6900–6903. doi: 10.1073/pnas.90.15.6900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Thomas D. C., Roberts J. D., Kunkel T. A. Heteroduplex repair in extracts of human HeLa cells. J Biol Chem. 1991 Feb 25;266(6):3744–3751. [PubMed] [Google Scholar]
  22. Watanabe S., Temin H. M. Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors. Mol Cell Biol. 1983 Dec;3(12):2241–2249. doi: 10.1128/mcb.3.12.2241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Whitcomb J. M., Hughes S. H. Retroviral reverse transcription and integration: progress and problems. Annu Rev Cell Biol. 1992;8:275–306. doi: 10.1146/annurev.cb.08.110192.001423. [DOI] [PubMed] [Google Scholar]
  24. Yu H., Goodman M. F. Comparison of HIV-1 and avian myeloblastosis virus reverse transcriptase fidelity on RNA and DNA templates. J Biol Chem. 1992 May 25;267(15):10888–10896. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES