Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1995 Oct;69(10):6228–6238. doi: 10.1128/jvi.69.10.6228-6238.1995

Replication of avian leukosis viruses with mutations at the primer binding site: use of alternative tRNAs as primers.

J M Whitcomb 1, B A Ortiz-Conde 1, S H Hughes 1
PMCID: PMC189520  PMID: 7545245

Abstract

We have tested whether avian leukosis viruses (ALVs) can use tRNAs other than tRNATrp to initiate reverse transcription. The primer binding site (PBS) of a wild-type ALV provirus, which is complementary to the 3' end of tRNA(Trp), was replaced with sequences homologous to the 3' ends of six different chicken tRNAs (tRN(APro), tRNA(Lys), tRNA(Met), tRNA(Ile), tRNA(Phe), and tRNA(Ser)). Transfection of these proviruses into chicken embryo fibroblasts resulted in the production of infectious viruses, all of which apparently used the tRNA specified by the mutated PBS to replicate. However, growth of these viruses resulted in reversion to the wild-type (tRNA(Trp)) PBS. Some of the viruses revert quite quickly, while others are more stable. The relative stability of a given PBS correlated with the concentration of the corresponding tRNA in the virion. We determined the percentage of viral RNA that had a tRNA bound to the PBS and found that the occupancy rate is lower in the mutants than in the wild-type virus. We conclude that many different tRNAs can be used as primers to initiate reverse transcription in ALV. However, ALVs that use tRNA(Trp) have a growth advantage over ALVs that use other tRNAs.

Full Text

The Full Text of this article is available as a PDF (658.6 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. Astrin S. M., Buss E. G., Haywards W. S. Endogenous viral genes are non-essential in the chicken. Nature. 1979 Nov 15;282(5736):339–341. doi: 10.1038/282339a0. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bender W., Chien Y. H., Chattopadhyay S., Vogt P. K., Gardner M. B., Davidson N. High-molecular-weight RNAs of AKR, NZB, and wild mouse viruses and avian reticuloendotheliosis virus all have similar dimer structures. J Virol. 1978 Mar;25(3):888–896. doi: 10.1128/jvi.25.3.888-896.1978. [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. Boyce-Jacino M. T., O'Donoghue K., Faras A. J. Multiple complex families of endogenous retroviruses are highly conserved in the genus Gallus. J Virol. 1992 Aug;66(8):4919–4929. doi: 10.1128/jvi.66.8.4919-4929.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boyce-Jacino M. T., Resnick R., Faras A. J. Structural and functional characterization of the unusually short long terminal repeats and their adjacent regions of a novel endogenous avian retrovirus. Virology. 1989 Nov;173(1):157–166. doi: 10.1016/0042-6822(89)90231-6. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Cordell B., Swanstrom R., Goodman H. M., Bishop J. M. tRNATrp as primer for RNA-directed DNA polymerase: structural determinants of function. J Biol Chem. 1979 Mar 25;254(6):1866–1874. [PubMed] [Google Scholar]
  10. Davis J., Scherer M., Tsai W. P., Long C. Low-molecular- weight Rauscher leukemia virus protein with preferential binding for single-stranded RNA and DNA. J Virol. 1976 May;18(2):709–718. doi: 10.1128/jvi.18.2.709-718.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Harada F., Sawyer R. C., Dahlberg J. E. A primer ribonucleic acid for initiation of in vitro Rous sarcarcoma virus deoxyribonucleic acid synthesis. J Biol Chem. 1975 May 10;250(9):3487–3497. [PubMed] [Google Scholar]
  12. Haseltine W. A., Kleid D. G., Panet A., Rothenberg E., Baltimore D. Ordered transcription of RNA tumor virus genomes. J Mol Biol. 1976 Sep 5;106(1):109–131. doi: 10.1016/0022-2836(76)90303-x. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Hughes S., Kosik E. Mutagenesis of the region between env and src of the SR-A strain of Rous sarcoma virus for the purpose of constructing helper-independent vectors. Virology. 1984 Jul 15;136(1):89–99. doi: 10.1016/0042-6822(84)90250-2. [DOI] [PubMed] [Google Scholar]
  15. Isel C., Marquet R., Keith G., Ehresmann C., Ehresmann B. Modified nucleotides of tRNA(3Lys) modulate primer/template loop-loop interaction in the initiation complex of HIV-1 reverse transcription. J Biol Chem. 1993 Dec 5;268(34):25269–25272. [PubMed] [Google Scholar]
  16. Jiang M., Mak J., Ladha A., Cohen E., Klein M., Rovinski B., Kleiman L. Identification of tRNAs incorporated into wild-type and mutant human immunodeficiency virus type 1. J Virol. 1993 Jun;67(6):3246–3253. doi: 10.1128/jvi.67.6.3246-3253.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jones M. D., Foulkes N. S. Reverse transcription of mRNA by Thermus aquaticus DNA polymerase. Nucleic Acids Res. 1989 Oct 25;17(20):8387–8388. doi: 10.1093/nar/17.20.8387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Karpel R. L., Henderson L. E., Oroszlan S. Interactions of retroviral structural proteins with single-stranded nucleic acids. J Biol Chem. 1987 Apr 15;262(11):4961–4967. [PubMed] [Google Scholar]
  19. Kempler G., Freitag B., Berwin B., Nanassy O., Barklis E. Characterization of the Moloney murine leukemia virus stem cell-specific repressor binding site. Virology. 1993 Apr;193(2):690–699. doi: 10.1006/viro.1993.1177. [DOI] [PubMed] [Google Scholar]
  20. Khan R., Giedroc D. P. Recombinant human immunodeficiency virus type 1 nucleocapsid (NCp7) protein unwinds tRNA. J Biol Chem. 1992 Apr 5;267(10):6689–6695. [PubMed] [Google Scholar]
  21. Levin J. G., Seidman J. G. Selective packaging of host tRNA's by murine leukemia virus particles does not require genomic RNA. J Virol. 1979 Jan;29(1):328–335. doi: 10.1128/jvi.29.1.328-335.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Mak J., Jiang M., Wainberg M. A., Hammarskjöld M. L., Rekosh D., Kleiman L. Role of Pr160gag-pol in mediating the selective incorporation of tRNA(Lys) into human immunodeficiency virus type 1 particles. J Virol. 1994 Apr;68(4):2065–2072. doi: 10.1128/jvi.68.4.2065-2072.1994. [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. 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]
  28. 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]
  29. Petropoulos C. J., Hughes S. H. Replication-competent retrovirus vectors for the transfer and expression of gene cassettes in avian cells. J Virol. 1991 Jul;65(7):3728–3737. doi: 10.1128/jvi.65.7.3728-3737.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Prats A. C., Roy C., Wang P. A., Erard M., Housset V., Gabus C., Paoletti C., Darlix J. L. cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA. J Virol. 1990 Feb;64(2):774–783. doi: 10.1128/jvi.64.2.774-783.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Prats A. C., Sarih L., Gabus C., Litvak S., Keith G., Darlix J. L. Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA. EMBO J. 1988 Jun;7(6):1777–1783. doi: 10.1002/j.1460-2075.1988.tb03008.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pulsinelli G. A., Temin H. M. High rate of mismatch extension during reverse transcription in a single round of retrovirus replication. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9490–9494. doi: 10.1073/pnas.91.20.9490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
  34. Rein A. Retroviral RNA packaging: a review. Arch Virol Suppl. 1994;9:513–522. doi: 10.1007/978-3-7091-9326-6_49. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Sallafranque-Andreola M. L., Robert D., Barr P. J., Fournier M., Litvak S., Sarih-Cottin L., Tarrago-Litvak L. Human immunodeficiency virus reverse transcriptase expressed in transformed yeast cells. Biochemical properties and interactions with bovine tRNALys. Eur J Biochem. 1989 Sep 15;184(2):367–374. doi: 10.1111/j.1432-1033.1989.tb15028.x. [DOI] [PubMed] [Google Scholar]
  37. Sarkar G., Sommer S. S. The "megaprimer" method of site-directed mutagenesis. Biotechniques. 1990 Apr;8(4):404–407. [PubMed] [Google Scholar]
  38. Sawyer R. C., Hanafusa H. Comparison of the small RNAs of polymerase-deficient and polymerase-positive Rous sarcoma virus and another species of avian retrovirus. J Virol. 1979 Mar;29(3):863–871. doi: 10.1128/jvi.29.3.863-871.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shank P. R., Linial M. Avian oncovirus mutant (SE21Q1b) deficient in genomic RNA: characterization of a deletion in the provirus. J Virol. 1980 Nov;36(2):450–456. doi: 10.1128/jvi.36.2.450-456.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Tse W. T., Forget B. G. Reverse transcription and direct amplification of cellular RNA transcripts by Taq polymerase. Gene. 1990 Apr 16;88(2):293–296. doi: 10.1016/0378-1119(90)90047-u. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Wakefield J. K., Rhim H., Morrow C. D. Minimal sequence requirements of a functional human immunodeficiency virus type 1 primer binding site. J Virol. 1994 Mar;68(3):1605–1614. doi: 10.1128/jvi.68.3.1605-1614.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Waters L. C., Mullin B. C. Transfer RNA into RNA tumor viruses. Prog Nucleic Acid Res Mol Biol. 1977;20:131–160. doi: 10.1016/s0079-6603(08)60471-7. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Whitcomb J. M., Kumar R., Hughes S. H. Sequence of the circle junction of human immunodeficiency virus type 1: implications for reverse transcription and integration. J Virol. 1990 Oct;64(10):4903–4906. doi: 10.1128/jvi.64.10.4903-4906.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES