Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Jun;70(6):3636–3644. doi: 10.1128/jvi.70.6.3636-3644.1996

Selection and characterization of replication-competent revertants of a Rous sarcoma virus src gene oversplicing mutant.

L Zhang 1, S B Simpson 1, C M Stoltzfus 1
PMCID: PMC190239  PMID: 8648698

Abstract

All retroviruses require both unspliced and spliced RNA for a productive infection. One mechanism by which Rous sarcoma virus achieves incomplete splicing involves suboptimal env and src 3' splice sites. We have previously shown that mutagenesis of the nonconsensus src polypyrimidine tract to a 14-nucleotide uninterrupted polypyrimidine tract results in an oversplicing phenotype and a concomitant defective replication in permissive chicken embryo fibroblasts. In this report, we show that splicing at the src 3' splice site (3'ss) is further negatively regulated by the suppressor of src splicing cis element which is located approximately 100 nucleotides upstream of the src 3'ss. The increase in splicing at the src 3'ss results in a corresponding increase in splicing at a cryptic 5'ss within the env gene. Two classes of replication-competent revertants of the src oversplicing mutant (pSAP1) were produced after infection, and these mutants were characterized by molecular cloning and sequence analysis. Class I revertants are transformation-defective revertants in which the src 3'ss and the src gene are deleted by homologous recombination at several different sites within the imperfect direct repeat sequences that flank the src gene. Cells infected with these transformation-defective revertants produce lower levels of virus particles than cells infected with the wild-type virus. Class II revertants bear small deletions in the region containing the branchpoint sequence or polypyrimidine tract of the src 3'ss. Insertion of these mutated sequences into pSAP1 restored inefficient splicing at the src 3'ss and efficient replication in chicken embryo fibroblasts. All of these mutations caused reduced splicing at the src 3'ss when they were tested in an in vitro splicing system. These results indicate that maintenance of a weak src 3'ss is necessary for efficient Rous sarcoma virus replication.

Full Text

The Full Text of this article is available as a PDF (513.0 KB).

Selected References

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

  1. Amendt B. A., Hesslein D., Chang L. J., Stoltzfus C. M. Presence of negative and positive cis-acting RNA splicing elements within and flanking the first tat coding exon of human immunodeficiency virus type 1. Mol Cell Biol. 1994 Jun;14(6):3960–3970. doi: 10.1128/mcb.14.6.3960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amendt B. A., Si Z. H., Stoltzfus C. M. Presence of exon splicing silencers within human immunodeficiency virus type 1 tat exon 2 and tat-rev exon 3: evidence for inhibition mediated by cellular factors. Mol Cell Biol. 1995 Aug;15(8):4606–4615. doi: 10.1128/mcb.15.8.4606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Amendt B. A., Simpson S. B., Stoltzfus C. M. Inhibition of RNA splicing at the Rous sarcoma virus src 3' splice site is mediated by an interaction between a negative cis element and a chicken embryo fibroblast nuclear factor. J Virol. 1995 Aug;69(8):5068–5076. doi: 10.1128/jvi.69.8.5068-5076.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arrigo S., Beemon K. Regulation of Rous sarcoma virus RNA splicing and stability. Mol Cell Biol. 1988 Nov;8(11):4858–4867. doi: 10.1128/mcb.8.11.4858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berberich S. L., Stoltzfus C. M. Mutations in the regions of the Rous sarcoma virus 3' splice sites: implications for regulation of alternative splicing. J Virol. 1991 May;65(5):2640–2646. doi: 10.1128/jvi.65.5.2640-2646.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bizub D., Katz R. A., Skalka A. M. Nucleotide sequence of noncoding regions in Rous-associated virus-2: comparisons delineate conserved regions important in replication and oncogenesis. J Virol. 1984 Feb;49(2):557–565. doi: 10.1128/jvi.49.2.557-565.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cullen B. R. Regulation of human immunodeficiency virus replication. Annu Rev Microbiol. 1991;45:219–250. doi: 10.1146/annurev.mi.45.100191.001251. [DOI] [PubMed] [Google Scholar]
  8. Dirksen W. P., Hampson R. K., Sun Q., Rottman F. M. A purine-rich exon sequence enhances alternative splicing of bovine growth hormone pre-mRNA. J Biol Chem. 1994 Mar 4;269(9):6431–6436. [PubMed] [Google Scholar]
  9. Fu X. D., Katz R. A., Skalka A. M., Maniatis T. The role of branchpoint and 3'-exon sequences in the control of balanced splicing of avian retrovirus RNA. Genes Dev. 1991 Feb;5(2):211–220. doi: 10.1101/gad.5.2.211. [DOI] [PubMed] [Google Scholar]
  10. Gontarek R. R., McNally M. T., Beemon K. Mutation of an RSV intronic element abolishes both U11/U12 snRNP binding and negative regulation of splicing. Genes Dev. 1993 Oct;7(10):1926–1936. doi: 10.1101/gad.7.10.1926. [DOI] [PubMed] [Google Scholar]
  11. Green M. R. Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. Annu Rev Cell Biol. 1991;7:559–599. doi: 10.1146/annurev.cb.07.110191.003015. [DOI] [PubMed] [Google Scholar]
  12. Katz R. A., Kotler M., Skalka A. M. cis-acting intron mutations that affect the efficiency of avian retroviral RNA splicing: implication for mechanisms of control. J Virol. 1988 Aug;62(8):2686–2695. doi: 10.1128/jvi.62.8.2686-2695.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Katz R. A., Skalka A. M. Control of retroviral RNA splicing through maintenance of suboptimal processing signals. Mol Cell Biol. 1990 Feb;10(2):696–704. doi: 10.1128/mcb.10.2.696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Maniatis T. Mechanisms of alternative pre-mRNA splicing. Science. 1991 Jan 4;251(4989):33–34. doi: 10.1126/science.1824726. [DOI] [PubMed] [Google Scholar]
  15. McKeown M. Alternative mRNA splicing. Annu Rev Cell Biol. 1992;8:133–155. doi: 10.1146/annurev.cb.08.110192.001025. [DOI] [PubMed] [Google Scholar]
  16. McNally M. T., Beemon K. Intronic sequences and 3' splice sites control Rous sarcoma virus RNA splicing. J Virol. 1992 Jan;66(1):6–11. doi: 10.1128/jvi.66.1.6-11.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McNally M. T., Gontarek R. R., Beemon K. Characterization of Rous sarcoma virus intronic sequences that negatively regulate splicing. Virology. 1991 Nov;185(1):99–108. doi: 10.1016/0042-6822(91)90758-4. [DOI] [PubMed] [Google Scholar]
  18. Nelson K. K., Green M. R. Mammalian U2 snRNP has a sequence-specific RNA-binding activity. Genes Dev. 1989 Oct;3(10):1562–1571. doi: 10.1101/gad.3.10.1562. [DOI] [PubMed] [Google Scholar]
  19. Omer C. A., Pogue-Geile K., Guntaka R., Staskus K. A., Faras A. J. Involvement of directly repeated sequences in the generation of deletions of the avian sarcoma virus src gene. J Virol. 1983 Aug;47(2):380–382. doi: 10.1128/jvi.47.2.380-382.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pathak V. K., Temin H. M. Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: deletions and deletions with insertions. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6024–6028. doi: 10.1073/pnas.87.16.6024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pulsinelli G. A., Temin H. M. 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. doi: 10.1128/jvi.65.9.4786-4797.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Reed R. The organization of 3' splice-site sequences in mammalian introns. Genes Dev. 1989 Dec;3(12B):2113–2123. doi: 10.1101/gad.3.12b.2113. [DOI] [PubMed] [Google Scholar]
  23. Roscigno R. F., Weiner M., Garcia-Blanco M. A. A mutational analysis of the polypyrimidine tract of introns. Effects of sequence differences in pyrimidine tracts on splicing. J Biol Chem. 1993 May 25;268(15):11222–11229. [PubMed] [Google Scholar]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Smith C. W., Patton J. G., Nadal-Ginard B. Alternative splicing in the control of gene expression. Annu Rev Genet. 1989;23:527–577. doi: 10.1146/annurev.ge.23.120189.002523. [DOI] [PubMed] [Google Scholar]
  26. Sorge J., Ricci W., Hughes S. H. cis-Acting RNA packaging locus in the 115-nucleotide direct repeat of Rous sarcoma virus. J Virol. 1983 Dec;48(3):667–675. doi: 10.1128/jvi.48.3.667-675.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Staffa A., Cochrane A. The tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3' splice site. J Virol. 1994 May;68(5):3071–3079. doi: 10.1128/jvi.68.5.3071-3079.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stoltzfus C. M., Fogarty S. J. Multiple regions in the Rous sarcoma virus src gene intron act in cis to affect the accumulation of unspliced RNA. J Virol. 1989 Apr;63(4):1669–1676. doi: 10.1128/jvi.63.4.1669-1676.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stoltzfus C. M. Synthesis and processing of avian sarcoma retrovirus RNA. Adv Virus Res. 1988;35:1–38. doi: 10.1016/s0065-3527(08)60707-1. [DOI] [PubMed] [Google Scholar]
  30. Strohman R. C., Moss P. S., Micou-Eastwood J., Spector D., Przybyla A., Paterson B. Messenger RNA for myosin polypeptides: isolation from single myogenic cell cultures. Cell. 1977 Feb;10(2):265–273. doi: 10.1016/0092-8674(77)90220-3. [DOI] [PubMed] [Google Scholar]
  31. Tanaka K., Watakabe A., Shimura Y. Polypurine sequences within a downstream exon function as a splicing enhancer. Mol Cell Biol. 1994 Feb;14(2):1347–1354. doi: 10.1128/mcb.14.2.1347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Temin H. M. Retrovirus variation and reverse transcription: abnormal strand transfers result in retrovirus genetic variation. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6900–6903. doi: 10.1073/pnas.90.15.6900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tsichlis P. N., Donehower L., Hager G., Zeller N., Malavarca R., Astrin S., Skalka A. M. Sequence comparison in the crossover region of an oncogenic avian retrovirus recombinant and its nononcogenic parent: genetic regions that control growth rate and oncogenic potential. Mol Cell Biol. 1982 Nov;2(11):1331–1338. doi: 10.1128/mcb.2.11.1331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Voynow S. L., Coffin J. M. Evolutionary variants of Rous sarcoma virus: large deletion mutants do not result from homologous recombination. J Virol. 1985 Jul;55(1):67–78. doi: 10.1128/jvi.55.1.67-78.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wigler M., Pellicer A., Silverstein S., Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 1978 Jul;14(3):725–731. doi: 10.1016/0092-8674(78)90254-4. [DOI] [PubMed] [Google Scholar]
  36. Wu J., Manley J. L. Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. Genes Dev. 1989 Oct;3(10):1553–1561. doi: 10.1101/gad.3.10.1553. [DOI] [PubMed] [Google Scholar]
  37. Xu R., Teng J., Cooper T. A. The cardiac troponin T alternative exon contains a novel purine-rich positive splicing element. Mol Cell Biol. 1993 Jun;13(6):3660–3674. doi: 10.1128/mcb.13.6.3660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yamamoto T., Tyagi J. S., Fagan J. B., Jay G., deCrombrugghe B., Pastan I. Molecular mechanism for the capture and excision of the transforming gene of avian sarcoma virus as suggested by analysis of recombinant clones. J Virol. 1980 Aug;35(2):436–443. doi: 10.1128/jvi.35.2.436-443.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yeakley J. M., Hedjran F., Morfin J. P., Merillat N., Rosenfeld M. G., Emeson R. B. Control of calcitonin/calcitonin gene-related peptide pre-mRNA processing by constitutive intron and exon elements. Mol Cell Biol. 1993 Oct;13(10):5999–6011. doi: 10.1128/mcb.13.10.5999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zamore P. D., Patton J. G., Green M. R. Cloning and domain structure of the mammalian splicing factor U2AF. Nature. 1992 Feb 13;355(6361):609–614. doi: 10.1038/355609a0. [DOI] [PubMed] [Google Scholar]
  41. Zhang L., Stoltzfus C. M. A suboptimal src 3' splice site is necessary for efficient replication of Rous sarcoma virus. Virology. 1995 Feb 1;206(2):1099–1107. doi: 10.1006/viro.1995.1033. [DOI] [PubMed] [Google Scholar]
  42. Zhuang Y. A., Goldstein A. M., Weiner A. M. UACUAAC is the preferred branch site for mammalian mRNA splicing. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2752–2756. doi: 10.1073/pnas.86.8.2752. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES