Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1997 Aug;71(8):6003–6010. doi: 10.1128/jvi.71.8.6003-6010.1997

Identification of sequences downstream of the primer binding site that are important for efficient replication of human immunodeficiency virus type 1.

X Li 1, C Liang 1, Y Quan 1, R Chandok 1, M Laughrea 1, M A Parniak 1, L Kleiman 1, M A Wainberg 1
PMCID: PMC191857  PMID: 9223491

Abstract

Reverse transcription of retroviruses is initiated from an 18-nucleotide (nt) primer binding site (PBS), located within the 5' region of viral genomic RNA, to which the host cell-derived tRNA primer is annealed and also involves viral genomic sequences outside the PBS. We constructed proviral DNA clones of human immunodeficiency virus (HIV) that had selective deletions of either a 7-nt segment found immediately downstream of the PBS or an extended nontranslated 54-nt stretch located immediately downstream of the PBS and containing the aforementioned 7-nt segment. Synthesis of minus-strand strong-stop DNA was assessed with MT-4 cells infected with viruses derived from COS-7 cells that had been transfected with these various constructs. We found that similar levels of minus-strand strong-stop DNA as well as DNA produced after template switching were expressed in MT-4 cells infected with COS-7-derived wild-type viruses or with viruses that had the 7-nt segment deleted. In contrast, significantly lower levels of viral DNA were detected in MT-4 cells after infection with viruses that had deletions of the 54-nt stretch. Furthermore, the molecular clone containing the 7-nt deletion was able to replicate with wild-type kinetics, while that containing the 54-nt deletion displayed a significantly diminished capacity in this regard. Further deletion analysis showed that a 16-nt segment at the 3' end of this 54-nt segment was largely responsible for these effects. We also conducted studies to determine levels of viral mRNA in COS-7 cells that had been transfected with equivalent amounts of DNA derived from either a wild-type HIV construct or our various deletion mutants. In the case of transfections performed with the 7-nt deletion mutant and wild-type HIV DNA, high levels of viral mRNA transcripts were detected, which was not the case for the 54 nt-deletion mutant. However, these various mRNAs possessed similar stabilities, as shown through studies in which transcript formation was arrested by treatment of cells with actinomycin D. Thus, the 54-nt segment of 5' nontranslated RNA, located downstream of the PBS, is involved in efficient expression of each of viral DNA, mRNA, and infectious virus.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

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. Aldovini A., Young R. A. Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J Virol. 1990 May;64(5):1920–1926. doi: 10.1128/jvi.64.5.1920-1926.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arts E. J., Li X., Gu Z., Kleiman L., Parniak M. A., Wainberg M. A. Comparison of deoxyoligonucleotide and tRNA(Lys-3) as primers in an endogenous human immunodeficiency virus-1 in vitro reverse transcription/template-switching reaction. J Biol Chem. 1994 May 20;269(20):14672–14680. [PubMed] [Google Scholar]
  4. Arts E. J., Stetor S. R., Li X., Rausch J. W., Howard K. J., Ehresmann B., North T. W., Wöhrl B. M., Goody R. S., Wainberg M. A. Initiation of (-) strand DNA synthesis from tRNA(3Lys) on lentiviral RNAs: implications of specific HIV-1 RNA-tRNA(3Lys) interactions inhibiting primer utilization by retroviral reverse transcriptases. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10063–10068. doi: 10.1073/pnas.93.19.10063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Arts E. J., Wainberg M. A. Preferential incorporation of nucleoside analogs after template switching during human immunodeficiency virus reverse transcription. Antimicrob Agents Chemother. 1994 May;38(5):1008–1016. doi: 10.1128/aac.38.5.1008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baudin F., Marquet R., Isel C., Darlix J. L., Ehresmann B., Ehresmann C. Functional sites in the 5' region of human immunodeficiency virus type 1 RNA form defined structural domains. J Mol Biol. 1993 Jan 20;229(2):382–397. doi: 10.1006/jmbi.1993.1041. [DOI] [PubMed] [Google Scholar]
  7. Berkhout B., Schoneveld I. Secondary structure of the HIV-2 leader RNA comprising the tRNA-primer binding site. Nucleic Acids Res. 1993 Mar 11;21(5):1171–1178. doi: 10.1093/nar/21.5.1171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Berkhout B., Silverman R. H., Jeang K. T. Tat trans-activates the human immunodeficiency virus through a nascent RNA target. Cell. 1989 Oct 20;59(2):273–282. doi: 10.1016/0092-8674(89)90289-4. [DOI] [PubMed] [Google Scholar]
  9. Berlioz C., Darlix J. L. An internal ribosomal entry mechanism promotes translation of murine leukemia virus gag polyprotein precursors. J Virol. 1995 Apr;69(4):2214–2222. doi: 10.1128/jvi.69.4.2214-2222.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Boulerice F., Bour S., Geleziunas R., Lvovich A., Wainberg M. A. High frequency of isolation of defective human immunodeficiency virus type 1 and heterogeneity of viral gene expression in clones of infected U-937 cells. J Virol. 1990 Apr;64(4):1745–1755. doi: 10.1128/jvi.64.4.1745-1755.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Clavel F., Orenstein J. M. A mutant of human immunodeficiency virus with reduced RNA packaging and abnormal particle morphology. J Virol. 1990 Oct;64(10):5230–5234. doi: 10.1128/jvi.64.10.5230-5234.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cobrinik D., Aiyar A., Ge Z., Katzman M., Huang H., Leis J. Overlapping retrovirus U5 sequence elements are required for efficient integration and initiation of reverse transcription. J Virol. 1991 Jul;65(7):3864–3872. doi: 10.1128/jvi.65.7.3864-3872.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cobrinik D., Soskey L., Leis J. A retroviral RNA secondary structure required for efficient initiation of reverse transcription. J Virol. 1988 Oct;62(10):3622–3630. doi: 10.1128/jvi.62.10.3622-3630.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cullen B. R. Regulation of HIV-1 gene expression. FASEB J. 1991 Jul;5(10):2361–2368. doi: 10.1096/fasebj.5.10.1712325. [DOI] [PubMed] [Google Scholar]
  15. Darlix J. L., Gabus C., Nugeyre M. T., Clavel F., Barré-Sinoussi F. Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1. J Mol Biol. 1990 Dec 5;216(3):689–699. doi: 10.1016/0022-2836(90)90392-Y. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Felber B. K., Hadzopoulou-Cladaras M., Cladaras C., Copeland T., Pavlakis G. N. rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1495–1499. doi: 10.1073/pnas.86.5.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gilboa E., Mitra S. W., Goff S., Baltimore D. A detailed model of reverse transcription and tests of crucial aspects. Cell. 1979 Sep;18(1):93–100. doi: 10.1016/0092-8674(79)90357-x. [DOI] [PubMed] [Google Scholar]
  19. Harrich D., Ulich C., Gaynor R. B. A critical role for the TAR element in promoting efficient human immunodeficiency virus type 1 reverse transcription. J Virol. 1996 Jun;70(6):4017–4027. doi: 10.1128/jvi.70.6.4017-4027.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Harrison G. P., Lever A. M. The human immunodeficiency virus type 1 packaging signal and major splice donor region have a conserved stable secondary structure. J Virol. 1992 Jul;66(7):4144–4153. doi: 10.1128/jvi.66.7.4144-4153.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Haseltine W. A. Molecular biology of the human immunodeficiency virus type 1. FASEB J. 1991 Jul;5(10):2349–2360. doi: 10.1096/fasebj.5.10.1829694. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Isel C., Lanchy J. M., Le Grice S. F., Ehresmann C., Ehresmann B., Marquet R. Specific initiation and switch to elongation of human immunodeficiency virus type 1 reverse transcription require the post-transcriptional modifications of primer tRNA3Lys. EMBO J. 1996 Feb 15;15(4):917–924. [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Kang S. M., Wakefield J. K., Morrow C. D. Mutations in both the U5 region and the primer-binding site influence the selection of the tRNA used for the initiation of HIV-1 reverse transcription. Virology. 1996 Aug 15;222(2):401–414. doi: 10.1006/viro.1996.0437. [DOI] [PubMed] [Google Scholar]
  26. Kang S. M., Zhang Z., Morrow C. D. Identification of a sequence within U5 required for human immunodeficiency virus type 1 to stably maintain a primer binding site complementary to tRNA(Met). J Virol. 1997 Jan;71(1):207–217. doi: 10.1128/jvi.71.1.207-217.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Kim H. J., Lee K., O'Rear J. J. A short sequence upstream of the 5' major splice site is important for encapsidation of HIV-1 genomic RNA. Virology. 1994 Jan;198(1):336–340. doi: 10.1006/viro.1994.1037. [DOI] [PubMed] [Google Scholar]
  30. Lever A., Gottlinger H., Haseltine W., Sodroski J. Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions. J Virol. 1989 Sep;63(9):4085–4087. doi: 10.1128/jvi.63.9.4085-4087.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Li X., Quan Y., Arts E. J., Li Z., Preston B. D., de Rocquigny H., Roques B. P., Darlix J. L., Kleiman L., Parniak M. A. Human immunodeficiency virus Type 1 nucleocapsid protein (NCp7) directs specific initiation of minus-strand DNA synthesis primed by human tRNA(Lys3) in vitro: studies of viral RNA molecules mutated in regions that flank the primer binding site. J Virol. 1996 Aug;70(8):4996–5004. doi: 10.1128/jvi.70.8.4996-5004.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Litvak S., Sarih-Cottin L., Fournier M., Andreola M., Tarrago-Litvak L. Priming of HIV replication by tRNA(Lys3): role of reverse transcriptase. Trends Biochem Sci. 1994 Mar;19(3):114–118. doi: 10.1016/0968-0004(94)90203-8. [DOI] [PubMed] [Google Scholar]
  34. Lori F., di Marzo Veronese F., de Vico A. L., Lusso P., Reitz M. S., Jr, Gallo R. C. Viral DNA carried by human immunodeficiency virus type 1 virions. J Virol. 1992 Aug;66(8):5067–5074. doi: 10.1128/jvi.66.8.5067-5074.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Marquet R., Baudin F., Gabus C., Darlix J. L., Mougel M., Ehresmann C., Ehresmann B. Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism. Nucleic Acids Res. 1991 May 11;19(9):2349–2357. doi: 10.1093/nar/19.9.2349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Miele G., Mouland A., Harrison G. P., Cohen E., Lever A. M. The human immunodeficiency virus type 1 5' packaging signal structure affects translation but does not function as an internal ribosome entry site structure. J Virol. 1996 Feb;70(2):944–951. doi: 10.1128/jvi.70.2.944-951.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Muesing M. A., Smith D. H., Capon D. J. Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein. Cell. 1987 Feb 27;48(4):691–701. doi: 10.1016/0092-8674(87)90247-9. [DOI] [PubMed] [Google Scholar]
  38. Murphy J. E., Goff S. P. Construction and analysis of deletion mutations in the U5 region of Moloney murine leukemia virus: effects on RNA packaging and reverse transcription. J Virol. 1989 Jan;63(1):319–327. doi: 10.1128/jvi.63.1.319-327.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Paillart J. C., Marquet R., Skripkin E., Ehresmann B., Ehresmann C. Mutational analysis of the bipartite dimer linkage structure of human immunodeficiency virus type 1 genomic RNA. J Biol Chem. 1994 Nov 4;269(44):27486–27493. [PubMed] [Google Scholar]
  40. Panganiban A. T., Temin H. M. The terminal nucleotides of retrovirus DNA are required for integration but not virus production. Nature. 1983 Nov 10;306(5939):155–160. doi: 10.1038/306155a0. [DOI] [PubMed] [Google Scholar]
  41. Picard V., Ersdal-Badju E., Lu A., Bock S. C. A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase. Nucleic Acids Res. 1994 Jul 11;22(13):2587–2591. doi: 10.1093/nar/22.13.2587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. 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]
  45. Skripkin E., Isel C., Marquet R., Ehresmann B., Ehresmann C. Psoralen crosslinking between human immunodeficiency virus type 1 RNA and primer tRNA3(Lys). Nucleic Acids Res. 1996 Feb 1;24(3):509–514. doi: 10.1093/nar/24.3.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Skripkin E., Paillart J. C., Marquet R., Ehresmann B., Ehresmann C. Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4945–4949. doi: 10.1073/pnas.91.11.4945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Soudeyns H., Geleziunas R., Shyamala G., Hiscott J., Wainberg M. A. Identification of a novel glucocorticoid response element within the genome of the human immunodeficiency virus type 1. Virology. 1993 Jun;194(2):758–768. doi: 10.1006/viro.1993.1317. [DOI] [PubMed] [Google Scholar]
  48. Trono D. Partial reverse transcripts in virions from human immunodeficiency and murine leukemia viruses. J Virol. 1992 Aug;66(8):4893–4900. doi: 10.1128/jvi.66.8.4893-4900.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Van Lint C., Ghysdael J., Paras P., Jr, Burny A., Verdin E. A transcriptional regulatory element is associated with a nuclease-hypersensitive site in the pol gene of human immunodeficiency virus type 1. J Virol. 1994 Apr;68(4):2632–2648. doi: 10.1128/jvi.68.4.2632-2648.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Vicenzi E., Dimitrov D. S., Engelman A., Migone T. S., Purcell D. F., Leonard J., Englund G., Martin M. A. An integration-defective U5 deletion mutant of human immunodeficiency virus type 1 reverts by eliminating additional long terminal repeat sequences. J Virol. 1994 Dec;68(12):7879–7890. doi: 10.1128/jvi.68.12.7879-7890.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. 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]
  53. 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]
  54. Zack J. A., Arrigo S. J., Weitsman S. R., Go A. S., Haislip A., Chen I. S. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell. 1990 Apr 20;61(2):213–222. doi: 10.1016/0092-8674(90)90802-l. [DOI] [PubMed] [Google Scholar]
  55. el Kharroubi A., Martin M. A. cis-acting sequences located downstream of the human immunodeficiency virus type 1 promoter affect its chromatin structure and transcriptional activity. Mol Cell Biol. 1996 Jun;16(6):2958–2966. doi: 10.1128/mcb.16.6.2958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. el Kharroubi A., Verdin E. Protein-DNA interactions within DNase I-hypersensitive sites located downstream of the HIV-1 promoter. J Biol Chem. 1994 Aug 5;269(31):19916–19924. [PubMed] [Google Scholar]

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

RESOURCES