Expression of Proteins Using Semliki Forest Virus Vectors

Peter Liljeström1, Henrik Garoff1

1 Karolinska Institute, Novum Research Center, Huddinge, Sweden
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.20
DOI:  10.1002/0471142727.mb1620s29
Online Posting Date:  May, 2001
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Abstract

Semliki Forest virus (SFV) vectors have been developed to provide a convenient system to express protein‐encoding sequences in virtually any animal cell. This unit presents two strategies for protein expression using SFV vectors. In both cases the protein‐coding sequence of interest is cloned into a plasmid vector, which is subsequently used to produce recombinant RNA in vitro. This RNA, which is of positive polarity, is transfected into cells and there is amplified by virtue of its self‐encoded RNA replicase. The same replicase also produces a shorter RNA species that encodes the protein of interest. In the first protocol, cells are transfected (either by electroporation or liposome‐mediated transfection) and directly analyzed for expression of the heterologous protein. Accompanying support protocols provide methods for checking expression and transfection through galactosidase assays of transfected cells and cell lysates. The other strategy employs in vivo packaging of the RNA into SFV particles; recombinant RNA is cotransfected with a special helper RNA that codes for the structural proteins needed for virus assembly. SFV particles carrying only recombinant RNA are formed and are used to infect cells for analysis of protein expression. Accompanying support protocols describe methods for titrating and purifying recombinant virus stocks. Although the protocols presented here are designed for use with BHK (baby hamster kidney) cells, the virus has a very broad host range and can be used with many different cell types.

     
 
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Table of Contents

  • Strategic Planning: Choice of SFV Vector
  • Basic Protocol 1: Expression of Proteins from Recombinant SFV RNA Using Electroporative Transfection
  • Alternate Protocol 1: Expression of Proteins from Recombinant SFV RNA Using Liposome‐Mediated Transfection
  • Support Protocol 1: Screening for Gene Expression Using β‐Galactosidase
  • Basic Protocol 2: Expression of Protein from In Vivo–Packaged Recombinant SFV Particles
  • Support Protocol 2: Determination of Recombinant Virus Titer
  • Support Protocol 3: Purification of SFV Particles
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Expression of Proteins from Recombinant SFV RNA Using Electroporative Transfection

  Materials
  • DNA fragment encoding protein of interest
  • pSFV1, pSFV3, or pSFV3‐lacZ expression vector (GIBCO/BRL; see Fig. )
  • SpeI restriction endonuclease and buffer (unit 3.1)
  • recipe10× SP6 RNA polymerase buffer
  • 50 mM dithiothreitol (DTT)
  • 10 mM m7G(5′)ppp(5′)G
  • reciperNTP mix
  • 40 U/µl RNasin (Promega) or other RNase inhibitor
  • 60 U/µl SP6 RNA polymerase (unit 3.8)
  • recipe5× TD solution
  • λ DNA molecular weight markers (e.g., λ digested with EcoRI + HindIII; unit 2.5)
  • BHK‐21 cells (ATCC)
  • recipeComplete BHK‐21 medium
  • recipePhosphate‐buffered saline (PBS; Reagents and Solutions), 37°C, room temperature, and ice cold
  • Trypsin/EDTA solution: 0.5 mg/ml trypsin/0.2 mg/ml EDTA in PBS
  • recipeStarvation medium
  • 15 mCi/ml [35S]methionine (> 1000 Ci/mmol)
  • recipeChase medium
  • recipeNP‐40 lysis buffer
  • 75‐cm2 tissue culture flask
  • Electroporator (e.g., Bio‐Rad)
  • 0.2‐ or 0.4‐cm electroporation cuvette
  • 35‐mm tissue culture plate
  • Additional reagents and equipment for subcloning (unit 3.16), preparation of plasmid DNA (unit 1.7), restriction endonuclease digestion (unit 3.1), phenol extraction and ethanol precipitation of DNA (unit 2.12.1), spectrophotometric quantitation of RNA and DNA ( appendix 3A3), agarose gel electrophoresis (unit 2.5), SDS‐PAGE for protein analysis (unit 10.210.2), and autoradiography ( appendix 3A3)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. All incubations are performed in a humidified, 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: Expression of Proteins from Recombinant SFV RNA Using Liposome‐Mediated Transfection

  Additional Materials
  • TE buffer, pH 7.5 ( appendix 22)
  • 3 M sodium acetate, pH 4.8 ( appendix 22)
  • Opti‐MEM transfection medium (GIBCO/BRL)
  • Isopropanol, −20°C
  • 75% (v/v) ethanol
  • Lipofectin (GIBCO/BRL)
  • Nu‐Clean R50 RNA spin columns (IBI)
  • Centrifuge with swinging‐bucket rotor
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. All incubations are performed in a humidified, 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Screening for Gene Expression Using β‐Galactosidase

  Materials
    BHK‐21 cells transfected with pSFV‐lacZ vector (first protocol 1basic protocol, step , or first protocol 2alternate protocol, step )
  • Methanol, −20°C
  • recipeXgal stain solution
  • Stereomicroscope

Basic Protocol 2: Expression of Protein from In Vivo–Packaged Recombinant SFV Particles

  Materials
  • pSFV1, pSFV3, or pSFV3‐lacZ vector (GIBCO/BRL; see Fig. )
  • pSFV‐Helper 2 DNA (GIBCO/BRL; see Fig. )
  • BHK‐21 cells (ATCC)
  • recipeMinimum essential medium (MEM), supplemented
  • recipePhosphate‐buffered saline (PBS; Reagents and Solutions)
  • recipeα‐chymotrypsin solution
  • 2 mg/ml aprotinin (Sigma)
  • 35‐mm tissue culture plate
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. All incubations are performed in a humidified, 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 2: Determination of Recombinant Virus Titer

  Materials
  • Recombinant virus stock (second protocol 4basic protocol)
  • recipePBS‐Eisen
  • Methanol, −20°C
  • Blocking buffer: 0.5% (w/v) gelatin/0.2% (w/v) BSA in PBS‐Eisen
  • Primary antibody in blocking buffer
  • Secondary antibody in blocking buffer
  • Moviol 4‐88 solution containing 2.5% DABCO (1,4‐diazobicyclo‐[2.2.2]‐octane)
  • Glass coverslips
  • Additional reagents and equipment for immunofluorescent labeling of monolayer cells (unit 14.6)

Support Protocol 3: Purification of SFV Particles

  Additional Materials
  • Transfected BHK‐21 cells (first protocol 1basic protocol, step )
  • 20% (w/v) and 55% (w/v) sucrose in recipeTNE buffer
  • recipeTNE buffer
  • Beckman SW‐40 or SW‐41 centrifuge tubes and rotor, or equivalent
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Figures

Videos

Literature Cited

Literature Cited
   Berglund, P., Sjöberg, M., Garoff, H., Atkins, G.J., Sheahan, B.J., and Liljeström, P. 1993. SFV expression system: Production of conditionally infectious recombinant particles. Bio/Technology. 11:916‐920.
   Bredenbeek, P.J. and Rice, C.M. 1992. Animal RNA virus expression systems. Semin. Virol. 3:297‐310.
   Bron, R., Wahlberg, J.M., Garoff, H., and Wilschut, J. 1992. Membrane fusion of Semliki Forest virus in a model system: Correlation between fusion kinetics and structural changes in the envelope glycoprotein. EMBO J. 12:693‐701.
   Davis, N.L., Willis, L.V., Smith, J.F., and Johnston, R.E. 1989. In vitro synthesis of infectious Venezuelan Equine Encephalitis virus RNA from a cDNA clone: Analysis of a viable deletion mutant. Virology 171:189‐204.
   Garoff, H., Huylebroeck, D., Robinson, A., Tillman, U., and Liljeström, P. 1990. The signal sequence of the p62 protein of Semliki Forest virus is involved in initiation but not in completing chain translocation. J. Cell Biol. 111:867‐876.
   Geigenmüller‐Gnirke, U., Weiss, B., Wright, R., and Schlesinger, S. 1991. Complementation between Sindbis viral RNAs produces infectious particles with a bipartite genome. Proc. Natl. Acad. Sci. U.S.A. 88:3253‐3257.
   Kuhn, R.J., Niesters, H.G.M., Hong, Z., and Strauss, J.H. 1991. Infectious RNA transcripts from Ross River virus cDNA clones and the construction and characterization of defined chimeras with Sindbis virus. Virology 182:430‐441.
   Liljeström, P. 1993. Virally based transient expression systems. Curr. Opin. Ther. Pat. 3:375‐402.
   Liljeström, P. and Garoff, H. 1991a. Internally located cleavable signal sequences direct the formation of Semliki Forest virus membrane proteins from a polyprotein precursor. J. Virol. 65:147‐154.
   Liljeström, P. and Garoff, H. 1991b. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Bio/Technology. 9:1356‐1361.
   Liljeström, P., Lusa, S., Huylebroeck, D., and Garoff, H. 1991. In vitro mutagenesis of a full‐length cDNA clone of Semliki Forest virus: The 6000‐molecular‐weight membrane protein modulates virus release. J. Virol. 65:4107‐4113.
   Lobigs, M. and Garoff, H. 1990. Fusion function of the Semliki Forest virus spike is activated by proteolytic cleavage of the envelope glycoprotein p62. J. Virol. 64:1233‐1240.
   Lobigs, M., Wahlberg, J.M., and Garoff, H. 1990a. Spike protein oligomerization control of Semliki Forest virus fusion. J. Virol. 64:5214‐5218.
   Lobigs, M., Zhao, H., and Garoff, H. 1990b. Function of Semliki Forest virus E3 peptide in virus assembly: Replacement of E3 with an artificial signal peptide abolishes spike heterodimerization and surface expression of E1. J. Virol. 64:4346‐4355.
   Rice, C.M. 1992. Examples of expression systems based on animal RNA viruses: Alphaviruses and influenza virus. Curr. Opin. Biotechnol. 3:522‐532.
   Rice, C.M., Levis, R., Strauss, J.H., and Huang, H.V. 1987. 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. 61:3809‐3819.
   Salminen, A., Wahlberg, J.M., Lobigs, M., Liljeström, P., and Garoff, H. 1992. Membrane fusion process of Semliki Forest virus II: Cleavage dependent reorganization of the spike protein complex controls virus entry. J. Cell Biol. 116:349‐357.
   Sanes, J., Rubenstein, J.L.R., and Nicolas, J.‐F. 1986. Use of recombinant retrovirus to study post‐implantation cell lineage in mouse embryos. EMBO J. 5:3133‐3142.
   Schlesinger, S. and Schlesinger, M.J. 1990. Replication of Togaviridae and Flaviviridae. In Virology (B.N. Fields and D.M. Knipe, eds.) pp. 697‐711. Raven Press, N.Y.
   Suomalainen, M., Liljeström, P., and Garoff, H. 1992. Spike protein–nucleocapsid interactions drive the budding of alphaviruses. J. Virol. 66:4737‐4747.
   Wahlberg, J. and Garoff, H. 1992. Membrane fusion process of Semliki Forest virus I: Low pH‐induced rearrangement in spike protein quaternary structure precedes virus penetration into cells. J. Cell Biol. 116:339‐357.
   Wahlberg, J.M., Boere, W.A., and Garoff, H. 1989. The heterodimeric association between the membrane proteins of Semliki Forest virus changes its sensitivity to mildly acidic pH during virus maturation. J. Virol. 63:4991‐4997.
   Wahlberg, J.M., Bron, R., Wilschut, J., and Garoff, H. 1992. Membrane fusion of Semliki Forest virus involves homotrimers of the fusion protein. J. Virol. 66:7309‐7318.
   Weiss, B.G. and Schlesinger, S. 1991. Recombination between Sindbis virus RNAs. J. Virol. 65:4017‐4025.
   Xiong, C., Levis, R., Shen, P., Schlesinger, S., Rice, C.M., and Huang, H.V. 1989. Sindbis virus: An efficient, broad host range vector for gene expression in animal cells. Science (Wash. DC). 243:1188‐1191.
Key References
   Liljeström et al., 1991. See above.
  Describes construction of a cDNA clone of the SFV genome, which was a basis for the SFV expression vectors.
   Liljeström and Garoff, 1991b. See above.
  Describes use of SFV vectors to express various proteins.
   Berglund et al., 1993. See above.
  Describes use of a second‐generation Helper vector for producing conditionally infectious recombinant SFV particles.
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