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. 1993 Nov;67(11):6439–6446. doi: 10.1128/jvi.67.11.6439-6446.1993

Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs.

P J Bredenbeek 1, I Frolov 1, C M Rice 1, S Schlesinger 1
PMCID: PMC238079  PMID: 8411346

Abstract

Since the recovery of infectious RNA transcripts from full-length cDNA clones, alphavirus genome RNAs have been engineered to allow expression of heterologous RNAs and proteins. The highest levels of expression of heterologous products are achieved when the viral structural genes are replaced by the heterologous coding sequences. Such recombinant RNAs are self-replicating (replicons) and can be introduced into cells as naked RNA, but they require trans complementation to be packaged and released from cells as infectious virion particles. In this report, we describe a series of defective Sindbis virus helper RNAs which can be used for packaging Sindbis virus RNA replicons. The defective helper RNAs contain the cis-acting sequences required for replication as well as the subgenomic RNA promoter which drives expression of the structural protein genes. In cells cotransfected with both the replicon and defective helper RNAs, viral nonstructural proteins translated from the replicon RNA allow replication and transcription of the defective helper RNA to produce the virion structural proteins. A series of defective helper RNAs were compared for the ability to package the replicon RNA as well as for the ability to be replicated and packaged. One defective helper RNA not only packaged the replicon but also was itself encapsidated and would be useful under conditions in which extensive amplification is advantageous. Other defective helper RNAs were able to package the replicon efficiently but were packaged very poorly themselves. These helpers should be useful for applications in which expression of the viral structural proteins or virus spread is not desired.

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Selected References

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

  1. Ball L. A., Li Y. cis-acting requirements for the replication of flock house virus RNA 2. J Virol. 1993 Jun;67(6):3544–3551. doi: 10.1128/jvi.67.6.3544-3551.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. French R., Ahlquist P. Intercistronic as well as terminal sequences are required for efficient amplification of brome mosaic virus RNA3. J Virol. 1987 May;61(5):1457–1465. doi: 10.1128/jvi.61.5.1457-1465.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Geigenmüller-Gnirke U., Weiss B., Wright R., Schlesinger S. Complementation between Sindbis viral RNAs produces infectious particles with a bipartite genome. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3253–3257. doi: 10.1073/pnas.88.8.3253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hahn C. S., Hahn Y. S., Braciale T. J., Rice C. M. Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2679–2683. doi: 10.1073/pnas.89.7.2679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Levis R., Schlesinger S., Huang H. V. Promoter for Sindbis virus RNA-dependent subgenomic RNA transcription. J Virol. 1990 Apr;64(4):1726–1733. doi: 10.1128/jvi.64.4.1726-1733.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Levis R., Weiss B. G., Tsiang M., Huang H., Schlesinger S. Deletion mapping of Sindbis virus DI RNAs derived from cDNAs defines the sequences essential for replication and packaging. Cell. 1986 Jan 17;44(1):137–145. doi: 10.1016/0092-8674(86)90492-7. [DOI] [PubMed] [Google Scholar]
  8. Liljeström P., Garoff H. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (N Y) 1991 Dec;9(12):1356–1361. doi: 10.1038/nbt1291-1356. [DOI] [PubMed] [Google Scholar]
  9. Liljeström P., Lusa S., Huylebroeck D., Garoff H. In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release. J Virol. 1991 Aug;65(8):4107–4113. doi: 10.1128/jvi.65.8.4107-4113.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lustig S., Jackson A. C., Hahn C. S., Griffin D. E., Strauss E. G., Strauss J. H. Molecular basis of Sindbis virus neurovirulence in mice. J Virol. 1988 Jul;62(7):2329–2336. doi: 10.1128/jvi.62.7.2329-2336.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Meyer F., Weber H., Weissmann C. Interactions of Q beta replicase with Q beta RNA. J Mol Biol. 1981 Dec 15;153(3):631–660. doi: 10.1016/0022-2836(81)90411-3. [DOI] [PubMed] [Google Scholar]
  12. Mills D. R., Priano C., Merz P. A., Binderow B. D. Q beta RNA bacteriophage: mapping cis-acting elements within an RNA genome. J Virol. 1990 Aug;64(8):3872–3881. doi: 10.1128/jvi.64.8.3872-3881.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Monroe S. S., Schlesinger S. Common and distinct regions of defective-interfering RNAs of Sindbis virus. J Virol. 1984 Mar;49(3):865–872. doi: 10.1128/jvi.49.3.865-872.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Monroe S. S., Schlesinger S. RNAs from two independently isolated defective interfering particles of Sindbis virus contain a cellular tRNA sequence at their 5' ends. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3279–3283. doi: 10.1073/pnas.80.11.3279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Piper R. C., Tai C., Slot J. W., Hahn C. S., Rice C. M., Huang H., James D. E. The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT-4) is conferred by the NH2 terminus. J Cell Biol. 1992 May;117(4):729–743. doi: 10.1083/jcb.117.4.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rice C. M., Levis R., Strauss J. H., Huang H. V. Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J Virol. 1987 Dec;61(12):3809–3819. doi: 10.1128/jvi.61.12.3809-3819.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schlesinger S. Alphaviruses--vectors for the expression of heterologous genes. Trends Biotechnol. 1993 Jan;11(1):18–22. doi: 10.1016/0167-7799(93)90070-P. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ulmanen I., Söderlund H., Käriäinen L. Role of protein synthesis in the assembly of Semliki forest virus nucleocapsid. Virology. 1979 Dec;99(2):265–276. doi: 10.1016/0042-6822(79)90006-0. [DOI] [PubMed] [Google Scholar]
  19. Ulmanen I., Söderlund H., Käriäinen L. Semliki Forest virus capsid protein associates with the 60S ribosomal subunit in infected cells. J Virol. 1976 Oct;20(1):203–210. doi: 10.1128/jvi.20.1.203-210.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wei N., Hacker D. L., Morris T. J. Characterization of an internal element in turnip crinkle virus RNA involved in both coat protein binding and replication. Virology. 1992 Sep;190(1):346–355. doi: 10.1016/0042-6822(92)91221-f. [DOI] [PubMed] [Google Scholar]
  21. Weiss B. G., Schlesinger S. Recombination between Sindbis virus RNAs. J Virol. 1991 Aug;65(8):4017–4025. doi: 10.1128/jvi.65.8.4017-4025.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Weiss B., Nitschko H., Ghattas I., Wright R., Schlesinger S. Evidence for specificity in the encapsidation of Sindbis virus RNAs. J Virol. 1989 Dec;63(12):5310–5318. doi: 10.1128/jvi.63.12.5310-5318.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wengler G., Wengler G. Identification of a transfer of viral core protein to cellular ribosomes during the early stages of alphavirus infection. Virology. 1984 Apr 30;134(2):435–442. doi: 10.1016/0042-6822(84)90310-6. [DOI] [PubMed] [Google Scholar]
  24. Xiong C., Levis R., Shen P., Schlesinger S., Rice C. M., Huang H. V. Sindbis virus: an efficient, broad host range vector for gene expression in animal cells. Science. 1989 Mar 3;243(4895):1188–1191. doi: 10.1126/science.2922607. [DOI] [PubMed] [Google Scholar]
  25. van Steeg H., Kasperaitis M., Voorma H. O., Benne R. Infection of neuroblastoma cells by Semliki Forest virus. The interference of viral capsid protein with the binding of host messenger RNAs into initiation complexes is the cause of the shut-off of host protein synthesis. Eur J Biochem. 1984 Feb 1;138(3):473–478. doi: 10.1111/j.1432-1033.1984.tb07940.x. [DOI] [PubMed] [Google Scholar]

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