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. 1996 Jun;70(6):3440–3448. doi: 10.1128/jvi.70.6.3440-3448.1996

Mutations in the Ty3 major homology region affect multiple steps in Ty3 retrotransposition.

K J Orlinsky 1, J Gu 1, M Hoyt 1, S Sandmeyer 1, T M Menees 1
PMCID: PMC190217  PMID: 8648676

Abstract

The Saccharomyces cerevisiae retroviruslike element Ty3 encodes the major structural proteins capsid (CA) and nucleocapsid in the GAG3 open reading frame. The Ty3 CA protein contains a sequence (QGX2EX5FX3LX3H, where H is a hydrophobic residue) which has not been observed in other retrotransposons but which is similar to the major homology region (MHR) described for retrovirus CA. In this study the effects of mutations in the Ty3 MHR on particle formation, processing, DNA synthesis, and transposition were examined. Each of the mutations tested resulted in severe defects in transposition, with disruption occurring prior to or at particle formation, subsequent to particle formation and prior to completion of DNA synthesis, and subsequent to DNA synthesis. Changing the Q in the motif to R had relatively little effect on particle formation but decreased transposition to about 13% of that of a wild-type element. Changing G to A or V almost completely eliminated the formation of intracellular particles, possibly by disruption of CA-CA interactions. Changes introduced at the position of E resulted in blocked processing, blocked DNA synthesis, or a block at some post-reverse transcription step, depending on the nature of the mutation introduced. These results showed that the integrity of the Ty3 MHR is required for multiple aspects of Ty3 replication involving CA. These functions are independent of extracellular budding and of infection, aspects of the retroviral life cycle which are not recapitulated in replication of the Ty3 retrotransposon.

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Selected References

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  1. Bilanchone V. W., Claypool J. A., Kinsey P. T., Sandmeyer S. B. Positive and negative regulatory elements control expression of the yeast retrotransposon Ty3. Genetics. 1993 Jul;134(3):685–700. doi: 10.1093/genetics/134.3.685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boeke J. D., Styles C. A., Fink G. R. Saccharomyces cerevisiae SPT3 gene is required for transposition and transpositional recombination of chromosomal Ty elements. Mol Cell Biol. 1986 Nov;6(11):3575–3581. doi: 10.1128/mcb.6.11.3575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Clare J., Farabaugh P. Nucleotide sequence of a yeast Ty element: evidence for an unusual mechanism of gene expression. Proc Natl Acad Sci U S A. 1985 May;82(9):2829–2833. doi: 10.1073/pnas.82.9.2829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clark D. J., Bilanchone V. W., Haywood L. J., Dildine S. L., Sandmeyer S. B. A yeast sigma composite element, TY3, has properties of a retrotransposon. J Biol Chem. 1988 Jan 25;263(3):1413–1423. [PubMed] [Google Scholar]
  6. Craven R. C., Leure-duPree A. E., Weldon R. A., Jr, Wills J. W. Genetic analysis of the major homology region of the Rous sarcoma virus Gag protein. J Virol. 1995 Jul;69(7):4213–4227. doi: 10.1128/jvi.69.7.4213-4227.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DesGroseillers L., Jolicoeur P. Physical mapping of the Fv-1 tropism host range determinant of BALB/c murine leukemia viruses. J Virol. 1983 Dec;48(3):685–696. doi: 10.1128/jvi.48.3.685-696.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dorfman T., Bukovsky A., Ohagen A., Höglund S., Göttlinger H. G. Functional domains of the capsid protein of human immunodeficiency virus type 1. J Virol. 1994 Dec;68(12):8180–8187. doi: 10.1128/jvi.68.12.8180-8187.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ehrlich L. S., Agresta B. E., Carter C. A. Assembly of recombinant human immunodeficiency virus type 1 capsid protein in vitro. J Virol. 1992 Aug;66(8):4874–4883. doi: 10.1128/jvi.66.8.4874-4883.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eichinger D. J., Boeke J. D. The DNA intermediate in yeast Ty1 element transposition copurifies with virus-like particles: cell-free Ty1 transposition. Cell. 1988 Sep 23;54(7):955–966. doi: 10.1016/0092-8674(88)90110-9. [DOI] [PubMed] [Google Scholar]
  11. Elder R. T., Loh E. Y., Davis R. W. RNA from the yeast transposable element Ty1 has both ends in the direct repeats, a structure similar to retrovirus RNA. Proc Natl Acad Sci U S A. 1983 May;80(9):2432–2436. doi: 10.1073/pnas.80.9.2432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Farabaugh P. J., Zhao H., Vimaladithan A. A novel programed frameshift expresses the POL3 gene of retrotransposon Ty3 of yeast: frameshifting without tRNA slippage. Cell. 1993 Jul 16;74(1):93–103. doi: 10.1016/0092-8674(93)90297-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  14. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  15. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  16. Franke E. K., Yuan H. E., Bossolt K. L., Goff S. P., Luban J. Specificity and sequence requirements for interactions between various retroviral Gag proteins. J Virol. 1994 Aug;68(8):5300–5305. doi: 10.1128/jvi.68.8.5300-5305.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Freed E. O., Orenstein J. M., Buckler-White A. J., Martin M. A. Single amino acid changes in the human immunodeficiency virus type 1 matrix protein block virus particle production. J Virol. 1994 Aug;68(8):5311–5320. doi: 10.1128/jvi.68.8.5311-5320.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fäcke M., Janetzko A., Shoeman R. L., Kräusslich H. G. A large deletion in the matrix domain of the human immunodeficiency virus gag gene redirects virus particle assembly from the plasma membrane to the endoplasmic reticulum. J Virol. 1993 Aug;67(8):4972–4980. doi: 10.1128/jvi.67.8.4972-4980.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gill G., Ptashne M. Mutants of GAL4 protein altered in an activation function. Cell. 1987 Oct 9;51(1):121–126. doi: 10.1016/0092-8674(87)90016-x. [DOI] [PubMed] [Google Scholar]
  20. González S. A., Affranchino J. L., Gelderblom H. R., Burny A. Assembly of the matrix protein of simian immunodeficiency virus into virus-like particles. Virology. 1993 Jun;194(2):548–556. doi: 10.1006/viro.1993.1293. [DOI] [PubMed] [Google Scholar]
  21. Hansen L. J., Chalker D. L., Orlinsky K. J., Sandmeyer S. B. Ty3 GAG3 and POL3 genes encode the components of intracellular particles. J Virol. 1992 Mar;66(3):1414–1424. doi: 10.1128/jvi.66.3.1414-1424.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hansen L. J., Chalker D. L., Sandmeyer S. B. Ty3, a yeast retrotransposon associated with tRNA genes, has homology to animal retroviruses. Mol Cell Biol. 1988 Dec;8(12):5245–5256. doi: 10.1128/mcb.8.12.5245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hansen M., Jelinek L., Whiting S., Barklis E. Transport and assembly of gag proteins into Moloney murine leukemia virus. J Virol. 1990 Nov;64(11):5306–5316. doi: 10.1128/jvi.64.11.5306-5316.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jolicoeur P., Baltimore D. Effect of Fv-1 gene product on proviral DNA formation and integration in cells infected with murine leukemia viruses. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2236–2240. doi: 10.1073/pnas.73.7.2236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kirchner J., Sandmeyer S. B., Forrest D. B. Transposition of a Ty3 GAG3-POL3 fusion mutant is limited by availability of capsid protein. J Virol. 1992 Oct;66(10):6081–6092. doi: 10.1128/jvi.66.10.6081-6092.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kirchner J., Sandmeyer S. Proteolytic processing of Ty3 proteins is required for transposition. J Virol. 1993 Jan;67(1):19–28. doi: 10.1128/jvi.67.1.19-28.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lee P. P., Linial M. L. Efficient particle formation can occur if the matrix domain of human immunodeficiency virus type 1 Gag is substituted by a myristylation signal. J Virol. 1994 Oct;68(10):6644–6654. doi: 10.1128/jvi.68.10.6644-6654.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mammano F., Ohagen A., Höglund S., Göttlinger H. G. Role of the major homology region of human immunodeficiency virus type 1 in virion morphogenesis. J Virol. 1994 Aug;68(8):4927–4936. doi: 10.1128/jvi.68.8.4927-4936.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Matthews S., Barlow P., Boyd J., Barton G., Russell R., Mills H., Cunningham M., Meyers N., Burns N., Clark N. Structural similarity between the p17 matrix protein of HIV-1 and interferon-gamma. Nature. 1994 Aug 25;370(6491):666–668. doi: 10.1038/370666a0. [DOI] [PubMed] [Google Scholar]
  30. Maurer B., Bannert H., Darai G., Flügel R. M. Analysis of the primary structure of the long terminal repeat and the gag and pol genes of the human spumaretrovirus. J Virol. 1988 May;62(5):1590–1597. doi: 10.1128/jvi.62.5.1590-1597.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Menees T. M., Sandmeyer S. B. Transposition of the yeast retroviruslike element Ty3 is dependent on the cell cycle. Mol Cell Biol. 1994 Dec;14(12):8229–8240. doi: 10.1128/mcb.14.12.8229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Morikawa Y., Kishi T., Zhang W. H., Nermut M. V., Hockley D. J., Jones I. M. A molecular determinant of human immunodeficiency virus particle assembly located in matrix antigen p17. J Virol. 1995 Jul;69(7):4519–4523. doi: 10.1128/jvi.69.7.4519-4523.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Orlinsky K. J., Sandmeyer S. B. The Cys-His motif of Ty3 NC can be contributed by Gag3 or Gag3-Pol3 polyproteins. J Virol. 1994 Jul;68(7):4152–4166. doi: 10.1128/jvi.68.7.4152-4166.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ou C. Y., Boone L. R., Koh C. K., Tennant R. W., Yang W. K. Nucleotide sequences of gag-pol regions that determine the Fv-1 host range property of BALB/c N-tropic and B-tropic murine leukemia viruses. J Virol. 1983 Dec;48(3):779–784. doi: 10.1128/jvi.48.3.779-784.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pryciak P. M., Varmus H. E. Fv-1 restriction and its effects on murine leukemia virus integration in vivo and in vitro. J Virol. 1992 Oct;66(10):5959–5966. doi: 10.1128/jvi.66.10.5959-5966.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rao Z., Belyaev A. S., Fry E., Roy P., Jones I. M., Stuart D. I. Crystal structure of SIV matrix antigen and implications for virus assembly. Nature. 1995 Dec 14;378(6558):743–747. doi: 10.1038/378743a0. [DOI] [PubMed] [Google Scholar]
  37. Reicin A. S., Paik S., Berkowitz R. D., Luban J., Lowy I., Goff S. P. Linker insertion mutations in the human immunodeficiency virus type 1 gag gene: effects on virion particle assembly, release, and infectivity. J Virol. 1995 Feb;69(2):642–650. doi: 10.1128/jvi.69.2.642-650.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rhee S. S., Hunter E. A single amino acid substitution within the matrix protein of a type D retrovirus converts its morphogenesis to that of a type C retrovirus. Cell. 1990 Oct 5;63(1):77–86. doi: 10.1016/0092-8674(90)90289-q. [DOI] [PubMed] [Google Scholar]
  39. Rhee S. S., Hunter E. Structural role of the matrix protein of type D retroviruses in gag polyprotein stability and capsid assembly. J Virol. 1990 Sep;64(9):4383–4389. doi: 10.1128/jvi.64.9.4383-4389.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  41. Spearman P., Wang J. J., Vander Heyden N., Ratner L. Identification of human immunodeficiency virus type 1 Gag protein domains essential to membrane binding and particle assembly. J Virol. 1994 May;68(5):3232–3242. doi: 10.1128/jvi.68.5.3232-3242.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Srinivasakumar N., Hammarskjöld M. L., Rekosh D. Characterization of deletion mutations in the capsid region of human immunodeficiency virus type 1 that affect particle formation and Gag-Pol precursor incorporation. J Virol. 1995 Oct;69(10):6106–6114. doi: 10.1128/jvi.69.10.6106-6114.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Strambio-de-Castillia C., Hunter E. Mutational analysis of the major homology region of Mason-Pfizer monkey virus by use of saturation mutagenesis. J Virol. 1992 Dec;66(12):7021–7032. doi: 10.1128/jvi.66.12.7021-7032.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sveda M. M., Soeiro R. Host restriction of Friend leukemia virus: synthesis and integration of the provirus. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2356–2360. doi: 10.1073/pnas.73.7.2356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Towbin H., Gordon J. Immunoblotting and dot immunobinding--current status and outlook. J Immunol Methods. 1984 Sep 4;72(2):313–340. doi: 10.1016/0022-1759(84)90001-2. [DOI] [PubMed] [Google Scholar]
  46. Wang C. T., Barklis E. Assembly, processing, and infectivity of human immunodeficiency virus type 1 gag mutants. J Virol. 1993 Jul;67(7):4264–4273. doi: 10.1128/jvi.67.7.4264-4273.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wang C. T., Zhang Y., McDermott J., Barklis E. Conditional infectivity of a human immunodeficiency virus matrix domain deletion mutant. J Virol. 1993 Dec;67(12):7067–7076. doi: 10.1128/jvi.67.12.7067-7076.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Weldon R. A., Jr, Wills J. W. Characterization of a small (25-kilodalton) derivative of the Rous sarcoma virus Gag protein competent for particle release. J Virol. 1993 Sep;67(9):5550–5561. doi: 10.1128/jvi.67.9.5550-5561.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wills J. W., Craven R. C. Form, function, and use of retroviral gag proteins. AIDS. 1991 Jun;5(6):639–654. doi: 10.1097/00002030-199106000-00002. [DOI] [PubMed] [Google Scholar]
  50. Wills J. W., Craven R. C., Weldon R. A., Jr, Nelle T. D., Erdie C. R. Suppression of retroviral MA deletions by the amino-terminal membrane-binding domain of p60src. J Virol. 1991 Jul;65(7):3804–3812. doi: 10.1128/jvi.65.7.3804-3812.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Winston F., Minehart P. L. Analysis of the yeast SPT3 gene and identification of its product, a positive regulator of Ty transcription. Nucleic Acids Res. 1986 Sep 11;14(17):6885–6900. doi: 10.1093/nar/14.17.6885. [DOI] [PMC free article] [PubMed] [Google Scholar]

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