Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Feb;11(2):1092–1098. doi: 10.1128/mcb.11.2.1092

Regulation of the extent of splicing of influenza virus NS1 mRNA: role of the rates of splicing and of the nucleocytoplasmic transport of NS1 mRNA.

F V Alonso-Caplen 1, R M Krug 1
PMCID: PMC359785  PMID: 1824958

Abstract

Influenza virus NS1 mRNA is spliced by host nuclear enzymes to form NS2 mRNA, and this splicing is regulated in infected cells such that the steady-state amount of spliced NS2 mRNA is only about 10% of that of unspliced NS1 mRNA. This regulation would be expected to result from a suppression in the rate of splicing coupled with the efficient transport of unspliced NS1 mRNA from the nucleus. To determine whether the rate of splicing of NS1 mRNA was controlled by trans factors in influenza virus-infected cells, the NS1 gene was inserted into an adenovirus vector. The rates of splicing of NS1 mRNA in cells infected with this vector and in influenza virus-infected cells were measured by pulse-labeling with [3H]uridine. The rates of splicing of NS1 mRNA in the two systems were not significantly different, strongly suggesting that the rate of splicing of NS1 mRNA in influenza virus-infected cells is controlled solely by cis-acting sequences in NS1 mRNA itself. In contrast to the rate of splicing, the extent of splicing of NS1 mRNA in the cells infected by the adenovirus recombinant was dramatically increased relative to that occurring in influenza virus-infected cells. This could be attributed largely, if not totally, to a block in the nucleocytoplasmic transport of unspliced NS1 mRNA in the recombinant-infected cells. Most of the unspliced NS1 mRNA was in the nuclear fraction, and no detectable NS1 protein was synthesized. When the 3' splice site of NS1 mRNA was inactivated by mutation, NS1 mRNA was transported and translated, indicating that the transport block occurred because NS1 rRNA was committed to the splicing pathway. This transport block is apparently obviated in influenza virus-infected cells. These experiments demonstrate the important role of the nucleocytoplasmic transport of unspliced NS1 mRNA in regulating the extent of splicing of NS1 mRNA.

Full text

PDF
1092

Images in this article

1095Image
on p.1095
1096Image
on p.1096
1096Image
on p.1096
1097Image
on p.1097

Selected References

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

  1. Agris C. H., Nemeroff M. E., Krug R. M. A block in mammalian splicing occurring after formation of large complexes containing U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins. Mol Cell Biol. 1989 Jan;9(1):259–267. doi: 10.1128/mcb.9.1.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alonso-Caplen F. V., Katze M. G., Krug R. M. Efficient transcription, not translation, is dependent on adenovirus tripartite leader sequences at late times of infection. J Virol. 1988 May;62(5):1606–1616. doi: 10.1128/jvi.62.5.1606-1616.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Babich A., Feldman L. T., Nevins J. R., Darnell J. E., Jr, Weinberger C. Effect of adenovirus on metabolism of specific host mRNAs: transport control and specific translational discrimination. Mol Cell Biol. 1983 Jul;3(7):1212–1221. doi: 10.1128/mcb.3.7.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berkner K. L., Sharp P. A. Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant. Nucleic Acids Res. 1985 Feb 11;13(3):841–857. doi: 10.1093/nar/13.3.841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berkner K. L., Sharp P. A. Expression of dihydrofolate reductase, and of the adjacent EIb region, in an Ad5-dihydrofolate reductase recombinant virus. Nucleic Acids Res. 1984 Feb 24;12(4):1925–1941. doi: 10.1093/nar/12.4.1925. [DOI] [PMC free article] [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. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  9. Greenberg M. E., Ziff E. B. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature. 1984 Oct 4;311(5985):433–438. doi: 10.1038/311433a0. [DOI] [PubMed] [Google Scholar]
  10. Greenspan D., Krystal M., Nakada S., Arnheiter H., Lyles D. S., Palese P. Expression of influenza virus NS2 nonstructural protein in bacteria and localization of NS2 in infected eucaryotic cells. J Virol. 1985 Jun;54(3):833–843. doi: 10.1128/jvi.54.3.833-843.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Inglis S. C., Barrett T., Brown C. M., Almond J. W. The smallest genome RNA segment of influenza virus contains two genes that may overlap. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3790–3794. doi: 10.1073/pnas.76.8.3790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Inglis S. C., Brown C. M. Differences in the control of virus mRNA splicing during permissive or abortive infection with influenza A (fowl plague) virus. J Gen Virol. 1984 Jan;65(Pt 1):153–164. doi: 10.1099/0022-1317-65-1-153. [DOI] [PubMed] [Google Scholar]
  13. Inglis S. C., Brown C. M. Spliced and unspliced RNAs encoded by virion RNA segment 7 of influenza virus. Nucleic Acids Res. 1981 Jun 25;9(12):2727–2740. doi: 10.1093/nar/9.12.2727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Lamb R. A., Choppin P. W., Chanock R. M., Lai C. J. Mapping of the two overlapping genes for polypeptides NS1 and NS2 on RNA segment 8 of influenza virus genome. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1857–1861. doi: 10.1073/pnas.77.4.1857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lamb R. A., Choppin P. W. Segment 8 of the influenza virus genome is unique in coding for two polypeptides. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4908–4912. doi: 10.1073/pnas.76.10.4908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lamb R. A., Lai C. J., Choppin P. W. Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: colinear and interrupted mRNAs code for overlapping proteins. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4170–4174. doi: 10.1073/pnas.78.7.4170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lamb R. A., Lai C. J. Expression of unspliced NS1 mRNA, spliced NS2 mRNA, and a spliced chimera mRNA from cloned influenza virus NS DNA in an SV40 vector. Virology. 1984 May;135(1):139–147. doi: 10.1016/0042-6822(84)90124-7. [DOI] [PubMed] [Google Scholar]
  19. Lamb R. A., Lai C. J. Sequence of interrupted and uninterrupted mRNAs and cloned DNA coding for the two overlapping nonstructural proteins of influenza virus. Cell. 1980 Sep;21(2):475–485. doi: 10.1016/0092-8674(80)90484-5. [DOI] [PubMed] [Google Scholar]
  20. Lamb R. A., Lai C. J. Spliced and unspliced messenger RNAs synthesized from cloned influenza virus M DNA in an SV40 vector: expression of the influenza virus membrane protein (M1). Virology. 1982 Dec;123(2):237–256. doi: 10.1016/0042-6822(82)90258-6. [DOI] [PubMed] [Google Scholar]
  21. Legrain P., Rosbash M. Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm. Cell. 1989 May 19;57(4):573–583. doi: 10.1016/0092-8674(89)90127-x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Mansour S. L., Grodzicker T., Tjian R. An adenovirus vector system used to express polyoma virus tumor antigens. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1359–1363. doi: 10.1073/pnas.82.5.1359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pikielny C. W., Rosbash M. mRNA splicing efficiency in yeast and the contribution of nonconserved sequences. Cell. 1985 May;41(1):119–126. doi: 10.1016/0092-8674(85)90066-2. [DOI] [PubMed] [Google Scholar]
  25. Plotch S. J., Krug R. M. In vitro splicing of influenza viral NS1 mRNA and NS1-beta-globin chimeras: possible mechanisms for the control of viral mRNA splicing. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5444–5448. doi: 10.1073/pnas.83.15.5444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shapiro G. I., Gurney T., Jr, Krug R. M. Influenza virus gene expression: control mechanisms at early and late times of infection and nuclear-cytoplasmic transport of virus-specific RNAs. J Virol. 1987 Mar;61(3):764–773. doi: 10.1128/jvi.61.3.764-773.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Smith D. B., Inglis S. C. Regulated production of an influenza virus spliced mRNA mediated by virus-specific products. EMBO J. 1985 Sep;4(9):2313–2319. doi: 10.1002/j.1460-2075.1985.tb03932.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Young J. F., Desselberger U., Palese P., Ferguson B., Shatzman A. R., Rosenberg M. Efficient expression of influenza virus NS1 nonstructural proteins in Escherichia coli. Proc Natl Acad Sci U S A. 1983 Oct;80(19):6105–6109. doi: 10.1073/pnas.80.19.6105. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES