Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 May;70(5):3290–3297. doi: 10.1128/jvi.70.5.3290-3297.1996

Binding of intracellular anti-Rev single chain variable fragments to different epitopes of human immunodeficiency virus type 1 rev: variations in viral inhibition.

Y Wu 1, L Duan 1, M Zhu 1, B Hu 1, S Kubota 1, O Bagasra 1, R J Pomerantz 1
PMCID: PMC190196  PMID: 8627813

Abstract

Intracellular immunization to target the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev has been explored as a genetic therapy for AIDS. Efficient intracellular expression of rearranged immunoglobulin heavy and light chain variable regions of anti-Rev monoclonal antibodies, with various vectors, and subsequent inhibition of HIV-1 replication have been previously reported by our laboratories. To further understand the molecular mechanisms and effects that intracellular anti-Rev single chain variable fragments (SFvs) have against HIV-1, via blocking of Rev function, two anti-Rev SFvs which specifically bind to differing epitopes of the Rev protein have been cloned. One SFv binds to the Rev activation domain, and the second SFv binds to the distal C terminus of Rev in the nonactivation region. Further studies now demonstrate that both anti-Rev SFvs lead to variable resistance to HIV-1 infection. Although binding affinity assays demonstrated that the SFv which specifically recognizes the Rev activation domain (D8) had an extracellular binding affinity significantly lower than that of the SFv specific to the nonactivation region (D1O), the SFv D8 demonstrated more potent activity in inhibiting virus production in human T-cell lines and peripheral blood mononuclear cells than did SFv D10. Thus, extracellular binding affinities of an SFv for a target viral protein cannot be used to directly predict its activity as an intracellular immunization moiety. These data demonstrate potential approaches for intracellular immunization against HIV-1 infection, by efficiently blocking specific motifs of Rev to after the function of this retroviral regulatory protein. These studies extend the understanding of the effects, on a molecular level, of SFvs binding to critical epitopes of Rev and further suggest that rational design of SFvs, with interactions involving specific viral moieties which mediate HIV-1 expression, may hold promise for the clinical application of genetic therapies to combat AIDS.

Full Text

The Full Text of this article is available as a PDF (446.4 KB).

Selected References

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

  1. Adachi A., Gendelman H. E., Koenig S., Folks T., Willey R., Rabson A., Martin M. A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986 Aug;59(2):284–291. doi: 10.1128/jvi.59.2.284-291.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bagasra O., Wright S. D., Seshamma T., Oakes J. W., Pomerantz R. J. CD14 is involved in control of human immunodeficiency virus type 1 expression in latently infected cells by lipopolysaccharide. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6285–6289. doi: 10.1073/pnas.89.14.6285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bahner I., Zhou C., Yu X. J., Hao Q. L., Guatelli J. C., Kohn D. B. Comparison of trans-dominant inhibitory mutant human immunodeficiency virus type 1 genes expressed by retroviral vectors in human T lymphocytes. J Virol. 1993 Jun;67(6):3199–3207. doi: 10.1128/jvi.67.6.3199-3207.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baltimore D. Gene therapy. Intracellular immunization. Nature. 1988 Sep 29;335(6189):395–396. doi: 10.1038/335395a0. [DOI] [PubMed] [Google Scholar]
  5. Biocca S., Ruberti F., Tafani M., Pierandrei-Amaldi P., Cattaneo A. Redox state of single chain Fv fragments targeted to the endoplasmic reticulum, cytosol and mitochondria. Biotechnology (N Y) 1995 Oct;13(10):1110–1115. doi: 10.1038/nbt1095-1110. [DOI] [PubMed] [Google Scholar]
  6. Bogerd H. P., Fridell R. A., Madore S., Cullen B. R. Identification of a novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins. Cell. 1995 Aug 11;82(3):485–494. doi: 10.1016/0092-8674(95)90437-9. [DOI] [PubMed] [Google Scholar]
  7. Chang D. D., Sharp P. A. Regulation by HIV Rev depends upon recognition of splice sites. Cell. 1989 Dec 1;59(5):789–795. doi: 10.1016/0092-8674(89)90602-8. [DOI] [PubMed] [Google Scholar]
  8. Chen S. Y., Bagley J., Marasco W. A. Intracellular antibodies as a new class of therapeutic molecules for gene therapy. Hum Gene Ther. 1994 May;5(5):595–601. doi: 10.1089/hum.1994.5.5-595. [DOI] [PubMed] [Google Scholar]
  9. Duan L., Bagasra O., Laughlin M. A., Oakes J. W., Pomerantz R. J. Potent inhibition of human immunodeficiency virus type 1 replication by an intracellular anti-Rev single-chain antibody. Proc Natl Acad Sci U S A. 1994 May 24;91(11):5075–5079. doi: 10.1073/pnas.91.11.5075. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  10. Duan L., Oakes J. W., Ferraro A., Bagasra O., Pomerantz R. J. Tat and rev differentially affect restricted replication of human immunodeficiency virus type 1 in various cells. Virology. 1994 Mar;199(2):474–478. doi: 10.1006/viro.1994.1148. [DOI] [PubMed] [Google Scholar]
  11. Duan L., Pomerantz R. J. Elimination of endogenous aberrant kappa chain transcripts from sp2/0-derived hybridoma cells by specific ribozyme cleavage: utility in genetic therapy of HIV-1 infections. Nucleic Acids Res. 1994 Dec 11;22(24):5433–5438. doi: 10.1093/nar/22.24.5433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Duan L., Zhang H., Oakes J. W., Bagasra O., Pomerantz R. J. Molecular and virological effects of intracellular anti-Rev single-chain variable fragments on the expression of various human immunodeficiency virus-1 strains. Hum Gene Ther. 1994 Nov;5(11):1315–1324. doi: 10.1089/hum.1994.5.11-1315. [DOI] [PubMed] [Google Scholar]
  13. Duan L., Zhu M., Bagasra O., Pomerantz R. J. Intracellular immunization against HIV-1 infection of human T lymphocytes: utility of anti-rev single-chain variable fragments. Hum Gene Ther. 1995 Dec;6(12):1561–1573. doi: 10.1089/hum.1995.6.12-1561. [DOI] [PubMed] [Google Scholar]
  14. Feitelson M. A., Zhu M., Duan L. X., London W. T. Hepatitis B x antigen and p53 are associated in vitro and in liver tissues from patients with primary hepatocellular carcinoma. Oncogene. 1993 May;8(5):1109–1117. [PubMed] [Google Scholar]
  15. 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]
  16. Fritz C. C., Zapp M. L., Green M. R. A human nucleoporin-like protein that specifically interacts with HIV Rev. Nature. 1995 Aug 10;376(6540):530–533. doi: 10.1038/376530a0. [DOI] [PubMed] [Google Scholar]
  17. Furuta R. A., Kubota S., Maki M., Miyazaki Y., Hattori T., Hatanaka M. Use of a human immunodeficiency virus type 1 Rev mutant without nucleolar dysfunction as a candidate for potential AIDS therapy. J Virol. 1995 Mar;69(3):1591–1599. doi: 10.1128/jvi.69.3.1591-1599.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Iversen A. K., Shpaer E. G., Rodrigo A. G., Hirsch M. S., Walker B. D., Sheppard H. W., Merigan T. C., Mullins J. I. Persistence of attenuated rev genes in a human immunodeficiency virus type 1-infected asymptomatic individual. J Virol. 1995 Sep;69(9):5743–5753. doi: 10.1128/jvi.69.9.5743-5753.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kubota S., Duan L., Furuta R. A., Hatanaka M., Pomerantz R. J. Nuclear preservation and cytoplasmic degradation of human immunodeficiency virus type 1 Rev protein. J Virol. 1996 Feb;70(2):1282–1287. doi: 10.1128/jvi.70.2.1282-1287.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Leavitt M. C., Yu M., Yamada O., Kraus G., Looney D., Poeschla E., Wong-Staal F. Transfer of an anti-HIV-1 ribozyme gene into primary human lymphocytes. Hum Gene Ther. 1994 Sep;5(9):1115–1120. doi: 10.1089/hum.1994.5.9-1115. [DOI] [PubMed] [Google Scholar]
  21. Li G., Lisziewicz J., Sun D., Zon G., Daefler S., Wong-Staal F., Gallo R. C., Klotman M. E. Inhibition of Rev activity and human immunodeficiency virus type 1 replication by antisense oligodeoxynucleotide phosphorothioate analogs directed against the Rev-responsive element. J Virol. 1993 Nov;67(11):6882–6888. doi: 10.1128/jvi.67.11.6882-6888.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Malim M. H., Böhnlein S., Hauber J., Cullen B. R. Functional dissection of the HIV-1 Rev trans-activator--derivation of a trans-dominant repressor of Rev function. Cell. 1989 Jul 14;58(1):205–214. doi: 10.1016/0092-8674(89)90416-9. [DOI] [PubMed] [Google Scholar]
  23. Malim M. H., Hauber J., Le S. Y., Maizel J. V., Cullen B. R. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature. 1989 Mar 16;338(6212):254–257. doi: 10.1038/338254a0. [DOI] [PubMed] [Google Scholar]
  24. Malim M. H., McCarn D. F., Tiley L. S., Cullen B. R. Mutational definition of the human immunodeficiency virus type 1 Rev activation domain. J Virol. 1991 Aug;65(8):4248–4254. doi: 10.1128/jvi.65.8.4248-4254.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Malim M. H., Tiley L. S., McCarn D. F., Rusche J. R., Hauber J., Cullen B. R. HIV-1 structural gene expression requires binding of the Rev trans-activator to its RNA target sequence. Cell. 1990 Feb 23;60(4):675–683. doi: 10.1016/0092-8674(90)90670-a. [DOI] [PubMed] [Google Scholar]
  26. Marasco W. A., Haseltine W. A., Chen S. Y. Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7889–7893. doi: 10.1073/pnas.90.16.7889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mhashilkar A. M., Bagley J., Chen S. Y., Szilvay A. M., Helland D. G., Marasco W. A. Inhibition of HIV-1 Tat-mediated LTR transactivation and HIV-1 infection by anti-Tat single chain intrabodies. EMBO J. 1995 Apr 3;14(7):1542–1551. doi: 10.1002/j.1460-2075.1995.tb07140.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  29. Minton A. P. Holobiochemistry: the effect of local environment upon the equilibria and rates of biochemical reactions. Int J Biochem. 1990;22(10):1063–1067. doi: 10.1016/0020-711x(90)90102-9. [DOI] [PubMed] [Google Scholar]
  30. Parnas H., Parnas I., Ravin R., Yudelevitch B. Glutamate and N-methyl-D-aspartate affect release from crayfish axon terminals in a voltage-dependent manner. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11586–11590. doi: 10.1073/pnas.91.24.11586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pomerantz R. J., Seshamma T., Trono D. Efficient replication of human immunodeficiency virus type 1 requires a threshold level of Rev: potential implications for latency. J Virol. 1992 Mar;66(3):1809–1813. doi: 10.1128/jvi.66.3.1809-1813.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pomerantz R. J., Trono D. Genetic therapies for HIV infections: promise for the future. AIDS. 1995 Sep;9(9):985–993. doi: 10.1097/00002030-199509000-00002. [DOI] [PubMed] [Google Scholar]
  33. Sarver N., Cantin E. M., Chang P. S., Zaia J. A., Ladne P. A., Stephens D. A., Rossi J. J. Ribozymes as potential anti-HIV-1 therapeutic agents. Science. 1990 Mar 9;247(4947):1222–1225. doi: 10.1126/science.2107573. [DOI] [PubMed] [Google Scholar]
  34. Scharfmann R., Axelrod J. H., Verma I. M. Long-term in vivo expression of retrovirus-mediated gene transfer in mouse fibroblast implants. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4626–4630. doi: 10.1073/pnas.88.11.4626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Strohal R., Kroemer G., Wick G., Kofler R. Complete variable region sequence of a nonfunctionally rearranged kappa light chain transcribed in the nonsecretor P3-X63-Ag8.653 myeloma cell line. Nucleic Acids Res. 1987 Mar 25;15(6):2771–2771. doi: 10.1093/nar/15.6.2771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sullenger B. A., Gallardo H. F., Ungers G. E., Gilboa E. Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication. Cell. 1990 Nov 2;63(3):601–608. doi: 10.1016/0092-8674(90)90455-n. [DOI] [PubMed] [Google Scholar]
  37. Yu M., Poeschla E., Wong-Staal F. Progress towards gene therapy for HIV infection. Gene Ther. 1994 Jan;1(1):13–26. [PubMed] [Google Scholar]
  38. Zhang H., Bagasra O., Niikura M., Poiesz B. J., Pomerantz R. J. Intravirion reverse transcripts in the peripheral blood plasma on human immunodeficiency virus type 1-infected individuals. J Virol. 1994 Nov;68(11):7591–7597. doi: 10.1128/jvi.68.11.7591-7597.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES