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J Virol. Mar 1996; 70(3): 1687–1694.
PMCID: PMC189992

Efficient initiation and strand transfer of polypurine tract-primed plus-strand DNA prevent strand transfer of internally initiated plus-strand DNA.


A critical step in retroviral reverse transcription is the initiation of plus-strand DNA synthesis at the polypurine tract (PPT) and strand transfer of the PPT-primed strong-stop DNA to the 5' end of the viral DNA. An attachment site (att) immediately 3' to the PPT is essential for proper integration of proviral DNA into the host chromosome. Plus-strand DNA synthesis is discontinuous in many retroviruses, indicating that sequences upstream of the PPT are also used to initiate plus-strand DNA synthesis (internally initiated DNA). Strand transfer of internally initiated DNA would result in "dead" viral DNA that lacks the att site needed for integration. Strand transfer of the internally initiated DNA could occur if DNA synthesis failed to initiate at the PPT or if the PPT-primed DNA was displaced before strand transfer. We sought to determine the efficiency of DNA synthesis initiating at the PPT and the proportions of PPT-primed DNA and internally initiated DNAs that are utilized for strand transfer. We constructed spleen necrosis virus-based retroviral vectors containing an internal PPT and an att site 5' of the normal PPT and att site. After one replication cycle of the retroviral vectors, the structures of the resulting proviruses were determined by Southern blotting. The analysis suggested that the PPT is an efficient and rapid initiator of plus-strand DNA synthesis and that internally initiated DNAs are rarely utilized for strand transfer. We hypothesize that efficient synthesis and strand transfer of PPT-primed DNA evolved to prevent lethal strand transfers of internally initiated DNAs.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Baltimore D. RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature. 1970 Jun 27;226(5252):1209–1211. [PubMed]
  • Benz EW, Jr, Dina D. Moloney murine sarcoma virions synthesize full-genome-length double-stranded DNA in vitro. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3294–3298. [PMC free article] [PubMed]
  • Blum HE, Harris JD, Ventura P, Walker D, Staskus K, Retzel E, Haase AT. Synthesis in cell culture of the gapped linear duplex DNA of the slow virus visna. Virology. 1985 Apr 30;142(2):270–277. [PubMed]
  • Boone LR, Skalka AM. Viral DNA synthesized in vitro by avian retrovirus particles permeabilized with melittin. I. Kinetics of synthesis and size of minus- and plus-strand transcripts. J Virol. 1981 Jan;37(1):109–116. [PMC free article] [PubMed]
  • Boone LR, Skalka AM. Viral DNA synthesized in vitro by avian retrovirus particles permeabilized with melittin. II. Evidence for a strand displacement mechanism in plus-strand synthesis. J Virol. 1981 Jan;37(1):117–126. [PMC free article] [PubMed]
  • Bosselman RA, Verma IM. Genome organization of retroviruses. V. In vitro-synthesized Moloney murine leukemia viral DNA has long terminal redundancy. J Virol. 1980 Jan;33(1):487–493. [PMC free article] [PubMed]
  • Champoux JJ, Gilboa E, Baltimore D. Mechanism of RNA primer removal by the RNase H activity of avian myeloblastosis virus reverse transcriptase. J Virol. 1984 Mar;49(3):686–691. [PMC free article] [PubMed]
  • Charneau P, Alizon M, Clavel F. A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication. J Virol. 1992 May;66(5):2814–2820. [PMC free article] [PubMed]
  • Charneau P, Clavel F. A single-stranded gap in human immunodeficiency virus unintegrated linear DNA defined by a central copy of the polypurine tract. J Virol. 1991 May;65(5):2415–2421. [PMC free article] [PubMed]
  • Colicelli J, Goff SP. Structure of a cloned circular retroviral DNA containing a tRNA sequence between the terminal repeats. J Virol. 1986 Feb;57(2):674–677. [PMC free article] [PubMed]
  • Colicelli J, Goff SP. Sequence and spacing requirements of a retrovirus integration site. J Mol Biol. 1988 Jan 5;199(1):47–59. [PubMed]
  • DesGroseillers L, Rassart E, Zollinger M, Jolicoeur P. Synthesis of murine leukemia viral DNA in vitro: evidence for plus-strand DNA synthesis at both ends of the genome. J Virol. 1982 Apr;42(1):326–330. [PMC free article] [PubMed]
  • Dougherty JP, Temin HM. High mutation rate of a spleen necrosis virus-based retrovirus vector. Mol Cell Biol. 1986 Dec;6(12):4387–4395. [PMC free article] [PubMed]
  • Dougherty JP, Wisniewski R, Yang SL, Rhode BW, Temin HM. New retrovirus helper cells with almost no nucleotide sequence homology to retrovirus vectors. J Virol. 1989 Jul;63(7):3209–3212. [PMC free article] [PubMed]
  • Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. [PubMed]
  • Finston WI, Champoux JJ. RNA-primed initiation of Moloney murine leukemia virus plus strands by reverse transcriptase in vitro. J Virol. 1984 Jul;51(1):26–33. [PMC free article] [PubMed]
  • Furfine ES, Reardon JE. Human immunodeficiency virus reverse transcriptase ribonuclease H: specificity of tRNA(Lys3)-primer excision. Biochemistry. 1991 Jul 23;30(29):7041–7046. [PubMed]
  • Gilboa E, Mitra SW, Goff S, Baltimore D. A detailed model of reverse transcription and tests of crucial aspects. Cell. 1979 Sep;18(1):93–100. [PubMed]
  • Harris JD, Scott JV, Traynor B, Brahic M, Stowring L, Ventura P, Haase AT, Peluso R. Visna virus DNA: discovery of a novel gapped structure. Virology. 1981 Sep;113(2):573–583. [PubMed]
  • Hsu TW, Taylor JM. Single-stranded regions on unintegrated avian retrovirus DNA. J Virol. 1982 Oct;44(1):47–53. [PMC free article] [PubMed]
  • Hu WS, Temin HM. Retroviral recombination and reverse transcription. Science. 1990 Nov 30;250(4985):1227–1233. [PubMed]
  • Huber HE, McCoy JM, Seehra JS, Richardson CC. Human immunodeficiency virus 1 reverse transcriptase. Template binding, processivity, strand displacement synthesis, and template switching. J Biol Chem. 1989 Mar 15;264(8):4669–4678. [PubMed]
  • Hungnes O, Tjotta E, Grinde B. The plus strand is discontinuous in a subpopulation of unintegrated HIV-1 DNA. Arch Virol. 1991;116(1-4):133–141. [PubMed]
  • Hungnes O, Tjøtta E, Grinde B. Mutations in the central polypurine tract of HIV-1 result in delayed replication. Virology. 1992 Sep;190(1):440–442. [PubMed]
  • Jorgensen RA, Rothstein SJ, Reznikoff WS. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. [PubMed]
  • Kawai S, Nishizawa M. New procedure for DNA transfection with polycation and dimethyl sulfoxide. Mol Cell Biol. 1984 Jun;4(6):1172–1174. [PMC free article] [PubMed]
  • Kung HJ, Fung YK, Majors JE, Bishop JM, Varmus HE. Synthesis of plus strands of retroviral DNA in cells infected with avian sarcoma virus and mouse mammary tumor virus. J Virol. 1981 Jan;37(1):127–138. [PMC free article] [PubMed]
  • Kupiec JJ, Tobaly-Tapiero J, Canivet M, Santillana-Hayat M, Flügel RM, Périès J, Emanoil-Ravier R. Evidence for a gapped linear duplex DNA intermediate in the replicative cycle of human and simian spumaviruses. Nucleic Acids Res. 1988 Oct 25;16(20):9557–9565. [PMC free article] [PubMed]
  • Luo GX, Sharmeen L, Taylor J. Specificities involved in the initiation of retroviral plus-strand DNA. J Virol. 1990 Feb;64(2):592–597. [PMC free article] [PubMed]
  • Mitra SW, Chow M, Champoux J, Baltimore D. Synthesis of murine leukemia virus plus strong stop DNA initiates at a unique site. J Biol Chem. 1982 Jun 10;257(11):5983–5986. [PubMed]
  • Omer CA, Faras AJ. Mechanism of release of the avian rotavirus tRNATrp primer molecule from viral DNA by ribonuclease H during reverse transcription. Cell. 1982 Oct;30(3):797–805. [PubMed]
  • Omer CA, Resnick R, Faras AJ. Evidence for involvement of an RNA primer in initiation of strong-stop plus DNA synthesis during reverse transcription in vitro. J Virol. 1984 May;50(2):465–470. [PMC free article] [PubMed]
  • Panganiban AT, Temin HM. The terminal nucleotides of retrovirus DNA are required for integration but not virus production. Nature. 1983 Nov 10;306(5939):155–160. [PubMed]
  • Pathak VK, Temin HM. 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. [PMC free article] [PubMed]
  • Pullen KA, Champoux JJ. Plus-strand origin for human immunodeficiency virus type 1: implications for integration. J Virol. 1990 Dec;64(12):6274–6277. [PMC free article] [PubMed]
  • Pullen KA, Ishimoto LK, Champoux JJ. Incomplete removal of the RNA primer for minus-strand DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase. J Virol. 1992 Jan;66(1):367–373. [PMC free article] [PubMed]
  • Pulsinelli GA, Temin HM. 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. [PMC free article] [PubMed]
  • Rattray AJ, Champoux JJ. The role of Moloney murine leukemia virus RNase H activity in the formation of plus-strand primers. J Virol. 1987 Sep;61(9):2843–2851. [PMC free article] [PubMed]
  • Reddy S, DeGregori JV, von Melchner H, Ruley HE. Retrovirus promoter-trap vector to induce lacZ gene fusions in mammalian cells. J Virol. 1991 Mar;65(3):1507–1515. [PMC free article] [PubMed]
  • Resnick R, Omer CA, Faras AJ. Involvement of retrovirus reverse transcriptase-associated RNase H in the initiation of strong-stop (+) DNA synthesis and the generation of the long terminal repeat. J Virol. 1984 Sep;51(3):813–821. [PMC free article] [PubMed]
  • Riggs JL, McAllister RM, Lennette EH. Immunofluorescent studies of RD-114 virus replication in cell culture. J Gen Virol. 1974 Oct;25(1):21–29. [PubMed]
  • Smith JK, Cywinski A, Taylor JM. Specificity of initiation of plus-strand DNA by Rous sarcoma virus. J Virol. 1984 Nov;52(2):314–319. [PMC free article] [PubMed]
  • Taylor JM, Cywinski A, Smith JK. Discontinuities in the DNA synthesized by an avian retrovirus. J Virol. 1983 Dec;48(3):654–659. [PMC free article] [PubMed]
  • Temin HM. The DNA provirus hypothesis. Science. 1976 Jun 11;192(4244):1075–1080. [PubMed]
  • Temin HM. Structure, variation and synthesis of retrovirus long terminal repeat. Cell. 1981 Nov;27(1 Pt 2):1–3. [PubMed]
  • Temin HM, Mizutani S. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature. 1970 Jun 27;226(5252):1211–1213. [PubMed]
  • Tobaly-Tapiero J, Kupiec JJ, Santillana-Hayat M, Canivet M, Peries J, Emanoil-Ravier R. Further characterization of the gapped DNA intermediates of human spumavirus: evidence for a dual initiation of plus-strand DNA synthesis. J Gen Virol. 1991 Mar;72(Pt 3):605–608. [PubMed]
  • Vara JA, Portela A, Ortín J, Jiménez A. Expression in mammalian cells of a gene from Streptomyces alboniger conferring puromycin resistance. Nucleic Acids Res. 1986 Jun 11;14(11):4617–4624. [PMC free article] [PubMed]
  • Varmus H. Retroviruses. Science. 1988 Jun 10;240(4858):1427–1435. [PubMed]
  • Watanabe S, Temin HM. Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors. Mol Cell Biol. 1983 Dec;3(12):2241–2249. [PMC free article] [PubMed]
  • Whitcomb JM, Kumar R, Hughes SH. 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. [PMC free article] [PubMed]
  • Whiting SH, Champoux JJ. Strand displacement synthesis capability of Moloney murine leukemia virus reverse transcriptase. J Virol. 1994 Aug;68(8):4747–4758. [PMC free article] [PubMed]

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