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. 1995 Oct;69(10):6273–6279. doi: 10.1128/jvi.69.10.6273-6279.1995

Feline immunodeficiency virus reverse transcriptase: expression, functional characterization, and reconstitution of the 66- and 51-kilodalton subunits.

M Amacker 1, M Hottiger 1, U Hübscher 1
PMCID: PMC189525  PMID: 7545246

Abstract

The two subunits of the feline immunodeficiency virus (FIV) reverse transcriptase (RT) were cloned and functionally expressed in Escherichia coli. The recombinant proteins are enzymatically active as homodimers (p66 and p51) as well as a heterodimer p66/p51. The biochemical properties of the FIV RT are very similar to those of the counterpart of the human immunodeficiency virus type 1 in being an RNA-dependent and DNA-dependent DNA polymerase. When a double-stranded DNA containing a small gap of 26 nucleotides was tested, we found a new activity of the FIV RT p66/p51 heterodimer--the cat viral enzyme could perform strand displacement DNA synthesis of approximately 300 bases. The FIV RT homodimer p66 alone could carry out limited strand displacement DNA synthesis, but this activity was stimulated by the p51 subunit at a molar ratio of one molecule of p66 to five molecules of p51. On the other hand, the homodimeric p51 itself was unable to fill a small gap of 26 nucleotides in a double-stranded DNA substrate and was not active by itself in strand displacement DNA synthesis. These data are in agreement with an earlier finding of strand displacement DNA synthesis by human immunodeficiency virus type 1 RT (M. Hottiger, V.N. Podust, R.L. Thimmig, C.S. McHenry, and U. Hübscher. J. Biol. Chem. 269:986-991, 1994). Our data therefore suggest a general and important function of lentiviral p51 subunits in strand displacement DNA synthesis which appears to be required in later stages of the lentiviral replication cycle, when DNA-dependent DNA synthesis occurs on double-stranded DNA.

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Selected References

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  1. Bendinelli M., Pistello M., Lombardi S., Poli A., Garzelli C., Matteucci D., Ceccherini-Nelli L., Malvaldi G., Tozzini F. Feline immunodeficiency virus: an interesting model for AIDS studies and an important cat pathogen. Clin Microbiol Rev. 1995 Jan;8(1):87–112. doi: 10.1128/cmr.8.1.87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blanar M. A., Rutter W. J. Interaction cloning: identification of a helix-loop-helix zipper protein that interacts with c-Fos. Science. 1992 May 15;256(5059):1014–1018. doi: 10.1126/science.1589769. [DOI] [PubMed] [Google Scholar]
  3. Blanco L., Bernad A., Lázaro J. M., Martín G., Garmendia C., Salas M. Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. J Biol Chem. 1989 May 25;264(15):8935–8940. [PubMed] [Google Scholar]
  4. Boone L. R., Skalka A. M. 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. doi: 10.1128/jvi.37.1.117-126.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Charneau P., Mirambeau G., Roux P., Paulous S., Buc H., Clavel F. HIV-1 reverse transcription. A termination step at the center of the genome. J Mol Biol. 1994 Sep 2;241(5):651–662. doi: 10.1006/jmbi.1994.1542. [DOI] [PubMed] [Google Scholar]
  7. DeStefano J. J., Buiser R. G., Mallaber L. M., Myers T. W., Bambara R. A., Fay P. J. Polymerization and RNase H activities of the reverse transcriptases from avian myeloblastosis, human immunodeficiency, and Moloney murine leukemia viruses are functionally uncoupled. J Biol Chem. 1991 Apr 25;266(12):7423–7431. [PubMed] [Google Scholar]
  8. Elder J. H., Schnölzer M., Hasselkus-Light C. S., Henson M., Lerner D. A., Phillips T. R., Wagaman P. C., Kent S. B. Identification of proteolytic processing sites within the Gag and Pol polyproteins of feline immunodeficiency virus. J Virol. 1993 Apr;67(4):1869–1876. doi: 10.1128/jvi.67.4.1869-1876.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hottiger M., Gramatikoff K., Georgiev O., Chaponnier C., Schaffner W., Hübscher U. The large subunit of HIV-1 reverse transcriptase interacts with beta-actin. Nucleic Acids Res. 1995 Mar 11;23(5):736–741. doi: 10.1093/nar/23.5.736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hottiger M., Podust V. N., Thimmig R. L., McHenry C., Hübscher U. Strand displacement activity of the human immunodeficiency virus type 1 reverse transcriptase heterodimer and its individual subunits. J Biol Chem. 1994 Jan 14;269(2):986–991. [PubMed] [Google Scholar]
  11. Hsu T. W., Taylor J. M. Single-stranded regions on unintegrated avian retrovirus DNA. J Virol. 1982 Oct;44(1):47–53. doi: 10.1128/jvi.44.1.47-53.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Junghans R. P., Boone L. R., Skalka A. M. Products of reverse transcription in avian retrovirus analyzed by electron microscopy. J Virol. 1982 Aug;43(2):544–554. doi: 10.1128/jvi.43.2.544-554.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Katz R. A., Skalka A. M. The retroviral enzymes. Annu Rev Biochem. 1994;63:133–173. doi: 10.1146/annurev.bi.63.070194.001025. [DOI] [PubMed] [Google Scholar]
  14. Kesti T., Frantti H., Syväoja J. E. Molecular cloning of the cDNA for the catalytic subunit of human DNA polymerase epsilon. J Biol Chem. 1993 May 15;268(14):10238–10245. [PubMed] [Google Scholar]
  15. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Matson S. W., Fay P. J., Bambara R. A. Mechanism of inhibition of the avian myeloblastosis virus deoxyribonucleic acid polymerase by adriamycin. Biochemistry. 1980 May 13;19(10):2089–2096. doi: 10.1021/bi00551a014. [DOI] [PubMed] [Google Scholar]
  18. Miyazawa T., Tomonaga K., Kawaguchi Y., Mikami T. The genome of feline immunodeficiency virus. Arch Virol. 1994;134(3-4):221–234. doi: 10.1007/BF01310563. [DOI] [PubMed] [Google Scholar]
  19. North T. W., Cronn R. C., Remington K. M., Tandberg R. T., Judd R. C. Characterization of reverse transcriptase from feline immunodeficiency virus. J Biol Chem. 1990 Mar 25;265(9):5121–5128. [PubMed] [Google Scholar]
  20. North T. W., Hansen G. L., Zhu Y., Griffin J. A., Shih C. K. Expression of reverse transcriptase from feline immunodeficiency virus in Escherichia coli. Antimicrob Agents Chemother. 1994 Feb;38(2):388–391. doi: 10.1128/aac.38.2.388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Olmsted R. A., Hirsch V. M., Purcell R. H., Johnson P. R. Nucleotide sequence analysis of feline immunodeficiency virus: genome organization and relationship to other lentiviruses. Proc Natl Acad Sci U S A. 1989 Oct;86(20):8088–8092. doi: 10.1073/pnas.86.20.8088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pedersen N. C., Ho E. W., Brown M. L., Yamamoto J. K. Isolation of a T-lymphotropic virus from domestic cats with an immunodeficiency-like syndrome. Science. 1987 Feb 13;235(4790):790–793. doi: 10.1126/science.3643650. [DOI] [PubMed] [Google Scholar]
  23. Podust V. N., Hübscher U. Lagging strand DNA synthesis by calf thymus DNA polymerases alpha, beta, delta and epsilon in the presence of auxiliary proteins. Nucleic Acids Res. 1993 Feb 25;21(4):841–846. doi: 10.1093/nar/21.4.841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Podust V., Mikhailov V., Georgaki A., Hübscher U. DNA polymerase delta and epsilon holoenzymes from calf thymus. Chromosoma. 1992;102(1 Suppl):S133–S141. doi: 10.1007/BF02451797. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Remington K. M., Zhu Y. Q., Phillips T. R., North T. W. Rapid phenotypic reversion of zidovudine-resistant feline immunodeficiency virus without loss of drug-resistant reverse transcriptase. J Virol. 1994 Feb;68(2):632–637. doi: 10.1128/jvi.68.2.632-637.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  28. Talbott R. L., Sparger E. E., Lovelace K. M., Fitch W. M., Pedersen N. C., Luciw P. A., Elder J. H. Nucleotide sequence and genomic organization of feline immunodeficiency virus. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5743–5747. doi: 10.1073/pnas.86.15.5743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Taylor J. M., Cywinski A., Smith J. K. Discontinuities in the DNA synthesized by an avian retrovirus. J Virol. 1983 Dec;48(3):654–659. doi: 10.1128/jvi.48.3.654-659.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thimmig R. L., McHenry C. S. Human immunodeficiency virus reverse transcriptase. Expression in Escherichia coli, purification, and characterization of a functionally and structurally asymmetric dimeric polymerase. J Biol Chem. 1993 Aug 5;268(22):16528–16536. [PubMed] [Google Scholar]
  31. Thömmes P., Ferrari E., Jessberger R., Hübscher U. Four different DNA helicases from calf thymus. J Biol Chem. 1992 Mar 25;267(9):6063–6073. [PubMed] [Google Scholar]
  32. Weiser T., Gassmann M., Thömmes P., Ferrari E., Hafkemeyer P., Hübscher U. Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus. J Biol Chem. 1991 Jun 5;266(16):10420–10428. [PubMed] [Google Scholar]
  33. 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]
  34. Whiting S. H., Champoux J. J. Strand displacement synthesis capability of Moloney murine leukemia virus reverse transcriptase. J Virol. 1994 Aug;68(8):4747–4758. doi: 10.1128/jvi.68.8.4747-4758.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wlodawer A., Gustchina A., Reshetnikova L., Lubkowski J., Zdanov A., Hui K. Y., Angleton E. L., Farmerie W. G., Goodenow M. M., Bhatt D. Structure of an inhibitor complex of the proteinase from feline immunodeficiency virus. Nat Struct Biol. 1995 Jun;2(6):480–488. doi: 10.1038/nsb0695-480. [DOI] [PubMed] [Google Scholar]
  36. Yamamoto J. K., Ackley C. D., Zochlinski H., Louie H., Pembroke E., Torten M., Hansen H., Munn R., Okuda T. Development of IL-2-independent feline lymphoid cell lines chronically infected with feline immunodeficiency virus: importance for diagnostic reagents and vaccines. Intervirology. 1991;32(6):361–375. doi: 10.1159/000150220. [DOI] [PubMed] [Google Scholar]

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