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Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1985 Sep;5(9):2341–2348. doi: 10.1128/mcb.5.9.2341

Gene expression from both intronless and intron-containing Rous sarcoma virus clones is specifically inhibited by anti-sense RNA.

L J Chang, C M Stoltzfus
PMCID: PMC366961  PMID: 2426579

Abstract

To distinguish the inhibitory effect of anti-sense RNA on translation from the effect on splicing, a plasmid (pLC32) was constructed from a cDNA clone of the Rous sarcoma virus (RSV) envelope gene (env) mRNA. Transcription of this plasmid results in the synthesis of RNA identical to the RSV env gene mRNA which does not require splicing to be expressed. Plasmids derived from pLC32 were also constructed in which the env gene coding sequence and 5' noncoding leader sequences were inserted in the opposite orientation relative to the RSV long terminal repeats (LTRs). pLC32 DNA transfected by the calcium phosphate coprecipitation technique efficiently rescued infectious virus from quail cells infected with an RSV mutant deleted in the env gene [R(-)Q cells], indicating that the intron sequences are dispensable in env gene expression. When the inverted constructs were cotransfected with pLC32, significantly less infectious virus was produced. The extent of the inhibition depended upon the concentration ratio of the two plasmids. The maximum inhibition (80%) occurred when the ratio of inverted constructs to pLC32 was 12:1. The inhibition is specific for the inverted orientation since cotransfection of pLC32 with several other plasmids containing viral LTRs and defective src and env genes at similar concentrations did not inhibit the production of infectious virus. In addition, the inverted constructs did not interfere with the expression of an LTR-driven chloramphenicol acetyltransferase gene. When cotransfected with a wild-type Prague A RSV DNA plasmid (pJD100), the inverted constructs also greatly inhibited expression and replication of virus in R(-)Q quail cells. These data suggest that the specific inhibition is caused by hybridization of complementary RNA transcribed from the inverted constructs to the env mRNA, thereby blocking its expression. The fact that expression of both intron-containing and intronless clones are inhibited to the same extent suggest that inhibition by anti-sense RNA from the env exon regions does not act at the level of RNA splicing.

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Selected References

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

  1. Chang L. J., Stoltzfus C. M. Cloning and nucleotide sequences of cDNAs spanning the splice junctions of Rous sarcoma virus mRNAs. J Virol. 1985 Mar;53(3):969–972. doi: 10.1128/jvi.53.3.969-972.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Coleman J., Green P. J., Inouye M. The use of RNAs complementary to specific mRNAs to regulate the expression of individual bacterial genes. Cell. 1984 Jun;37(2):429–436. doi: 10.1016/0092-8674(84)90373-8. [DOI] [PubMed] [Google Scholar]
  3. Cullen B. R., Raymond K., Ju G. Functional analysis of the transcription control region located within the avian retroviral long terminal repeat. Mol Cell Biol. 1985 Mar;5(3):438–447. doi: 10.1128/mcb.5.3.438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cullen B. R., Skalka A. M., Ju G. Endogenous avian retroviruses contain deficient promoter and leader sequences. Proc Natl Acad Sci U S A. 1983 May;80(10):2946–2950. doi: 10.1073/pnas.80.10.2946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Darlix J. L., Zuker M., Spahr P. F. Structure-function relationship of Rous sarcoma virus leader RNA. Nucleic Acids Res. 1982 Sep 11;10(17):5183–5196. doi: 10.1093/nar/10.17.5183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ficht T. A., Chang L. J., Stoltzfus C. M. Avian sarcoma virus gag and env gene structural protein precursors contain a common amino-terminal sequence. Proc Natl Acad Sci U S A. 1984 Jan;81(2):362–366. doi: 10.1073/pnas.81.2.362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gorman C. M., Merlino G. T., Willingham M. C., Pastan I., Howard B. H. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6777–6781. doi: 10.1073/pnas.79.22.6777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gruss P., Lai C. J., Dhar R., Khoury G. Splicing as a requirement for biogenesis of functional 16S mRNA of simian virus 40. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4317–4321. doi: 10.1073/pnas.76.9.4317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. HANAFUSA H., HANAFUSA T., RUBIN H. ANALYSIS OF THE DEFECTIVENESS OF ROUS SARCOMA VIRUS, II. SPECIFICATION OF RSV ANTIGENICITY BY HELPER VIRUS. Proc Natl Acad Sci U S A. 1964 Jan;51:41–48. doi: 10.1073/pnas.51.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hamer D. H., Leder P. Splicing and the formation of stable RNA. Cell. 1979 Dec;18(4):1299–1302. doi: 10.1016/0092-8674(79)90240-x. [DOI] [PubMed] [Google Scholar]
  11. Izant J. G., Weintraub H. Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis. Cell. 1984 Apr;36(4):1007–1015. doi: 10.1016/0092-8674(84)90050-3. [DOI] [PubMed] [Google Scholar]
  12. Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. doi: 10.1128/mr.47.1.1-45.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lerner T. L., Hanafusa H. DNA sequence of the Bryan high-titer strain of Rous sarcoma virus: extent of env deletion and possible genealogical relationship with other viral strains. J Virol. 1984 Feb;49(2):549–556. doi: 10.1128/jvi.49.2.549-556.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lopata M. A., Cleveland D. W., Sollner-Webb B. High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res. 1984 Jul 25;12(14):5707–5717. doi: 10.1093/nar/12.14.5707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Melton D. A. Injected anti-sense RNAs specifically block messenger RNA translation in vivo. Proc Natl Acad Sci U S A. 1985 Jan;82(1):144–148. doi: 10.1073/pnas.82.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mizuno T., Chou M. Y., Inouye M. A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (micRNA). Proc Natl Acad Sci U S A. 1984 Apr;81(7):1966–1970. doi: 10.1073/pnas.81.7.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Paterson B. M., Roberts B. E., Kuff E. L. Structural gene identification and mapping by DNA-mRNA hybrid-arrested cell-free translation. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4370–4374. doi: 10.1073/pnas.74.10.4370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pestka S., Daugherty B. L., Jung V., Hotta K., Pestka R. K. Anti-mRNA: specific inhibition of translation of single mRNA molecules. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7525–7528. doi: 10.1073/pnas.81.23.7525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Schwartz D. E., Tizard R., Gilbert W. Nucleotide sequence of Rous sarcoma virus. Cell. 1983 Mar;32(3):853–869. doi: 10.1016/0092-8674(83)90071-5. [DOI] [PubMed] [Google Scholar]
  20. Schöler H. R., Gruss P. Specific interaction between enhancer-containing molecules and cellular components. Cell. 1984 Feb;36(2):403–411. doi: 10.1016/0092-8674(84)90233-2. [DOI] [PubMed] [Google Scholar]
  21. Sealy L., Privalsky M. L., Moscovici G., Moscovici C., Bishop J. M. Site-specific mutagenesis of avian erythroblastosis virus: erb-B is required for oncogenicity. Virology. 1983 Oct 15;130(1):155–178. doi: 10.1016/0042-6822(83)90125-3. [DOI] [PubMed] [Google Scholar]
  22. Simons R. W., Kleckner N. Translational control of IS10 transposition. Cell. 1983 Sep;34(2):683–691. doi: 10.1016/0092-8674(83)90401-4. [DOI] [PubMed] [Google Scholar]
  23. Stacey D. W., Allfrey V. G., Hanafusa H. Microinjection analysis of envelope-glycoprotein messenger activities of avian leukosis viral RNAs. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1614–1618. doi: 10.1073/pnas.74.4.1614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Stephenson M. L., Zamecnik P. C. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc Natl Acad Sci U S A. 1978 Jan;75(1):285–288. doi: 10.1073/pnas.75.1.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Swanstrom R., Parker R. C., Varmus H. E., Bishop J. M. Transduction of a cellular oncogene: the genesis of Rous sarcoma virus. Proc Natl Acad Sci U S A. 1983 May;80(9):2519–2523. doi: 10.1073/pnas.80.9.2519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Swanstrom R., Varmus H. E., Bishop J. M. Nucleotide sequence of the 5' noncoding region and part of the gag gene of Rous sarcoma virus. J Virol. 1982 Feb;41(2):535–541. doi: 10.1128/jvi.41.2.535-541.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wyke J. A., Linial M. Temperature-sensitive avian sarcoma viruses: a physiological comparison of twenty mutants. Virology. 1973 May;53(1):152–161. doi: 10.1016/0042-6822(73)90474-1. [DOI] [PubMed] [Google Scholar]
  28. Zamecnik P. C., Stephenson M. L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci U S A. 1978 Jan;75(1):280–284. doi: 10.1073/pnas.75.1.280. [DOI] [PMC free article] [PubMed] [Google Scholar]

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