Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Feb;71(2):1191–1195. doi: 10.1128/jvi.71.2.1191-1195.1997

Complementation of a primer binding site-impaired murine leukemia virus-derived retroviral vector by a genetically engineered tRNA-like primer.

A H Lund 1, M Duch 1, J Lovmand 1, P Jørgensen 1, F S Pedersen 1
PMCID: PMC191172  PMID: 8995641

Abstract

Reverse transcription of retroviral genomes is primed by a tRNA annealed to an 18-nucleotide primer binding site. Here, we present a primer complementation system to study molecular interaction of the replication machinery with the primer and primer binding site in vivo. Introduction of eight base substitutions into the primer binding site of a murine leukemia virus-based vector allowed efficient RNA encapsidation but resulted in severely reduced vector replication capacity. Replication was restored upon complementation with a synthetic gene designed to encode a complementary tRNA-like primer, but not with a noncomplementary tRNA-like molecule. The engineered primer was shown to be involved in both the initiation of first-strand synthesis and second-strand transfer. These results provide an in vivo demonstration that the retroviral replication machinery may recognize sequence complementarity rather than actual primer binding site and 3' primer sequences. Use of mutated primer binding site vectors replicating via engineered primers may add additional control features to retroviral gene transfer technology.

Full Text

The Full Text of this article is available as a PDF (234.8 KB).

Selected References

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

  1. Aiyar A., Cobrinik D., Ge Z., Kung H. J., Leis J. Interaction between retroviral U5 RNA and the T psi C loop of the tRNA(Trp) primer is required for efficient initiation of reverse transcription. J Virol. 1992 Apr;66(4):2464–2472. doi: 10.1128/jvi.66.4.2464-2472.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barat C., Lullien V., Schatz O., Keith G., Nugeyre M. T., Grüninger-Leitch F., Barré-Sinoussi F., LeGrice S. F., Darlix J. L. HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. EMBO J. 1989 Nov;8(11):3279–3285. doi: 10.1002/j.1460-2075.1989.tb08488.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barat C., Schatz O., Le Grice S., Darlix J. L. Analysis of the interactions of HIV1 replication primer tRNA(Lys,3) with nucleocapsid protein and reverse transcriptase. J Mol Biol. 1993 May 20;231(2):185–190. doi: 10.1006/jmbi.1993.1273. [DOI] [PubMed] [Google Scholar]
  4. Baum C., Hegewisch-Becker S., Eckert H. G., Stocking C., Ostertag W. Novel retroviral vectors for efficient expression of the multidrug resistance (mdr-1) gene in early hematopoietic cells. J Virol. 1995 Dec;69(12):7541–7547. doi: 10.1128/jvi.69.12.7541-7547.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Blain S. W., Goff S. P. Nuclease activities of Moloney murine leukemia virus reverse transcriptase. Mutants with altered substrate specificities. J Biol Chem. 1993 Nov 5;268(31):23585–23592. [PubMed] [Google Scholar]
  7. Challita P. M., Skelton D., el-Khoueiry A., Yu X. J., Weinberg K., Kohn D. B. Multiple modifications in cis elements of the long terminal repeat of retroviral vectors lead to increased expression and decreased DNA methylation in embryonic carcinoma cells. J Virol. 1995 Feb;69(2):748–755. doi: 10.1128/jvi.69.2.748-755.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chapman K. B., Byström A. S., Boeke J. D. Initiator methionine tRNA is essential for Ty1 transposition in yeast. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3236–3240. doi: 10.1073/pnas.89.8.3236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Colicelli J., Goff S. P. Isolation of a recombinant murine leukemia virus utilizing a new primer tRNA. J Virol. 1986 Jan;57(1):37–45. doi: 10.1128/jvi.57.1.37-45.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Das A. T., Klaver B., Berkhout B. Reduced replication of human immunodeficiency virus type 1 mutants that use reverse transcription primers other than the natural tRNA(3Lys). J Virol. 1995 May;69(5):3090–3097. doi: 10.1128/jvi.69.5.3090-3097.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Duch M., Paludan K., Lovmand J., Pedersen L., Jørgensen P., Pedersen F. S. A correlation between dexamethasone inducibility and basal expression levels of retroviral vector proviruses. Nucleic Acids Res. 1993 Oct 11;21(20):4777–4782. doi: 10.1093/nar/21.20.4777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Götte M., Fackler S., Hermann T., Perola E., Cellai L., Gross H. J., Le Grice S. F., Heumann H. HIV-1 reverse transcriptase-associated RNase H cleaves RNA/RNA in arrested complexes: implications for the mechanism by which RNase H discriminates between RNA/RNA and RNA/DNA. EMBO J. 1995 Feb 15;14(4):833–841. doi: 10.1002/j.1460-2075.1995.tb07061.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harada F., Peters G. G., Dahlberg J. E. The primer tRNA for Moloney murine leukemia virus DNA synthesis. Nucleotide sequence and aminoacylation of tRNAPro. J Biol Chem. 1979 Nov 10;254(21):10979–10985. [PubMed] [Google Scholar]
  14. Haseltine W. A., Panet A., Smoler D., Baltimore D., Peters G., Harada F., Dahlberg J. E. Interaction of tryptophan tRNA and avian myeloblastosis virus reverse transcriptase: further characterization of the binding reaction. Biochemistry. 1977 Aug 9;16(16):3625–3632. doi: 10.1021/bi00635a019. [DOI] [PubMed] [Google Scholar]
  15. Isel C., Ehresmann C., Keith G., Ehresmann B., Marquet R. Initiation of reverse transcription of HIV-1: secondary structure of the HIV-1 RNA/tRNA(3Lys) (template/primer). J Mol Biol. 1995 Mar 24;247(2):236–250. doi: 10.1006/jmbi.1994.0136. [DOI] [PubMed] [Google Scholar]
  16. Kaleko M., Garcia J. V., Osborne W. R., Miller A. D. Expression of human adenosine deaminase in mice after transplantation of genetically-modified bone marrow. Blood. 1990 Apr 15;75(8):1733–1741. [PubMed] [Google Scholar]
  17. Li X., Mak J., Arts E. J., Gu Z., Kleiman L., Wainberg M. A., Parniak M. A. Effects of alterations of primer-binding site sequences on human immunodeficiency virus type 1 replication. J Virol. 1994 Oct;68(10):6198–6206. doi: 10.1128/jvi.68.10.6198-6206.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lund A. H., Duch M., Lovmand J., Jørgensen P., Pedersen F. S. Mutated primer binding sites interacting with different tRNAs allow efficient murine leukemia virus replication. J Virol. 1993 Dec;67(12):7125–7130. doi: 10.1128/jvi.67.12.7125-7130.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Majors J. E., Varmus H. E. Nucleotide sequencing of an apparent proviral copy of env mRNA defines determinants of expression of the mouse mammary tumor virus env gene. J Virol. 1983 Sep;47(3):495–504. doi: 10.1128/jvi.47.3.495-504.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mikkelsen J. G., Lund A. H., Kristensen K. D., Duch M., Sørensen M. S., Jørgensen P., Pedersen F. S. A preferred region for recombinational patch repair in the 5' untranslated region of primer binding site-impaired murine leukemia virus vectors. J Virol. 1996 Mar;70(3):1439–1447. doi: 10.1128/jvi.70.3.1439-1447.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Miller A. D. Retrovirus packaging cells. Hum Gene Ther. 1990 Spring;1(1):5–14. doi: 10.1089/hum.1990.1.1-5. [DOI] [PubMed] [Google Scholar]
  22. Nagashunmugam T., Velpandi A., Goldsmith C. S., Zaki S. R., Kalyanaraman V. S., Srinivasan A. Mutation in the primer binding site of the type 1 human immunodeficiency virus genome affects virus production and infectivity. Proc Natl Acad Sci U S A. 1992 May 1;89(9):4114–4118. doi: 10.1073/pnas.89.9.4114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nikbakht K. N., Ou C. Y., Boone L. R., Glover P. L., Yang W. K. Nucleotide sequence analysis of endogenous murine leukemia virus-related proviral clones reveals primer-binding sites for glutamine tRNA. J Virol. 1985 Jun;54(3):889–893. doi: 10.1128/jvi.54.3.889-893.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Palmer T. D., Rosman G. J., Osborne W. R., Miller A. D. Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1330–1334. doi: 10.1073/pnas.88.4.1330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Panet A., Berliner H. Binding of tRNA to reverse transcriptase of RNA tumor viruses. J Virol. 1978 May;26(2):214–220. doi: 10.1128/jvi.26.2.214-220.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Panet A., Haseltine W. A., Baltimore D., Peters G., Harada F., Dahlberg J. E. Specific binding of tryptophan transfer RNA to avian myeloblastosis virus RNA-dependent DNA polymerase (reverse transcriptase). Proc Natl Acad Sci U S A. 1975 Jul;72(7):2535–2539. doi: 10.1073/pnas.72.7.2535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pear W. S., Nolan G. P., Scott M. L., Baltimore D. Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8392–8396. doi: 10.1073/pnas.90.18.8392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Peters G. G., Hu J. Reverse transcriptase as the major determinant for selective packaging of tRNA's into Avian sarcoma virus particles. J Virol. 1980 Dec;36(3):692–700. doi: 10.1128/jvi.36.3.692-700.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Peters G., Harada F., Dahlberg J. E., Panet A., Haseltine W. A., Baltimore D. Low-molecular-weight RNAs of Moloney murine leukemia virus: identification of the primer for RNA-directed DNA synthesis. J Virol. 1977 Mar;21(3):1031–1041. doi: 10.1128/jvi.21.3.1031-1041.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rhim H., Park J., Morrow C. D. Deletions in the tRNA(Lys) primer-binding site of human immunodeficiency virus type 1 identify essential regions for reverse transcription. J Virol. 1991 Sep;65(9):4555–4564. doi: 10.1128/jvi.65.9.4555-4564.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Russo T., Costanzo F., Oliva A., Ammendola R., Duilio A., Esposito F., Cimino F. Structure and in vitro transcription of tRNA gene clusters containing the primers of MuLV reverse transcriptase. Eur J Biochem. 1986 Aug 1;158(3):437–442. doi: 10.1111/j.1432-1033.1986.tb09772.x. [DOI] [PubMed] [Google Scholar]
  32. Sarih-Cottin L., Bordier B., Musier-Forsyth K., Andreola M. L., Barr P. J., Litvak S. Preferential interaction of human immunodeficiency virus reverse transcriptase with two regions of primer tRNA(Lys) as evidenced by footprinting studies and inhibition with synthetic oligoribonucleotides. J Mol Biol. 1992 Jul 5;226(1):1–6. doi: 10.1016/0022-2836(92)90117-3. [DOI] [PubMed] [Google Scholar]
  33. Sawyer R. C., Dahlberg J. E. Small RNAs of Rous sarcoma virus: characterization by two-dimensional polyacrylamide gel electrophoresis and fingerprint analysis. J Virol. 1973 Dec;12(6):1226–1237. doi: 10.1128/jvi.12.6.1226-1237.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Seiki M., Hattori S., Hirayama Y., Yoshida M. Human adult T-cell leukemia virus: complete nucleotide sequence of the provirus genome integrated in leukemia cell DNA. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3618–3622. doi: 10.1073/pnas.80.12.3618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Steinberg S., Misch A., Sprinzl M. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 1993 Jul 1;21(13):3011–3015. doi: 10.1093/nar/21.13.3011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wain-Hobson S., Sonigo P., Danos O., Cole S., Alizon M. Nucleotide sequence of the AIDS virus, LAV. Cell. 1985 Jan;40(1):9–17. doi: 10.1016/0092-8674(85)90303-4. [DOI] [PubMed] [Google Scholar]
  37. Wakefield J. K., Kang S. M., Morrow C. D. Construction of a type 1 human immunodeficiency virus that maintains a primer binding site complementary to tRNA(His). J Virol. 1996 Feb;70(2):966–975. doi: 10.1128/jvi.70.2.966-975.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wakefield J. K., Wolf A. G., Morrow C. D. Human immunodeficiency virus type 1 can use different tRNAs as primers for reverse transcription but selectively maintains a primer binding site complementary to tRNA(3Lys). J Virol. 1995 Oct;69(10):6021–6029. doi: 10.1128/jvi.69.10.6021-6029.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Whitcomb J. M., Ortiz-Conde B. A., Hughes S. H. Replication of avian leukosis viruses with mutations at the primer binding site: use of alternative tRNAs as primers. J Virol. 1995 Oct;69(10):6228–6238. doi: 10.1128/jvi.69.10.6228-6238.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES