Alphaviruses: Semliki Forest Virus and Sindbis Virus Vectors for Gene Transfer into Neurons

Markus U. Ehrengruber1, Sondra Schlesinger2, Kenneth Lundstrom3

1 Department of Biology, Kantonsschule Hohe Promenade, Zurich, Switzerland, 2 Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, 3 PanTherapeutics, Lutry, Switzerland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.22
DOI:  10.1002/0471142301.ns0422s57
Online Posting Date:  October, 2011
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Alphaviral vectors based on Semliki Forest virus and Sindbis virus infect many host cell types, causing rapid and high‐level transgene expression. In the CNS, Semliki Forest virus and Sindbis virus exhibit an outstanding preference for neurons rather than glial cells, compared to other viruses. Generation of high‐titer virus stocks is rapid (less than two days) and typically requires biosafety level 1 or 2 containment. Wild‐type vectors are cytotoxic, permitting short‐term transgene expression. However, mutant vectors with decreased cytotoxicity, to prolong host cell survival, have been developed. They also increase transgene expression and cellular co‐infection, permitting heteromeric protein expression in individual cells. In addition, mutants with temperature‐dependent control of transgene expression and altered host cell preference to target interneurons and astrocytes rather than principal neurons are available. Other alphavirus vectors based on Venezuelan equine encephalitis and Eastern equine encephalitis virus replicons have been engineered, too. Alphavirus vectors have been successfully used not only in neuroscience, but also for other applications including drug discovery, structural biology, vaccine development, and cancer therapy. Curr. Protoc. Neurosci. 57:4.22.1‐4.22.27. © 2011 by John Wiley & Sons, Inc.

Keywords: Semliki Forest virus; Sindbis virus; baby hamster kidney 21 (BHK) cell; hippocampal neuron; in vitro transcription

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Packaged SFV and SIN Replicons
  • Basic Protocol 2: Activation of Packaged SFV Replicons
  • Basic Protocol 3: Infection of Hippocampal Slices
  • Basic Protocol 4: Infection of Dispersed Neurons
  • Alternate Protocol 1: Lipid‐Mediated Cotransfection of RNA
  • Support Protocol 1: Titer Determination for the Number of Infectious SFV or SIN Particles
  • Support Protocol 2: Metabolic Labeling
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Preparation of Packaged SFV and SIN Replicons

  • Vector plasmid pSFV2gen (cf. GenBank EF535150) or any mutant thereof [see Table 4.22.2; available from Markus U. Ehrengruber ( ) or Kenneth Lundstrom ( )]
    Table 4.2.2   MaterialsTroubleshooting Guide for Alphavirus‐Mediated Transfection into Neurons

    Problem Possible cause Solution
    Smearing of RNA bands RNA degradation Use RNase‐free conditions for in vitro transcription
    Agarose gel electrophoresis was too long Run RNA 2‐3 cm into the gel
    Low RNA yields Partially linearized plasmid Check the linearized plasmid on an agarose gel to verify complete digestion
    In vitro transcription reaction not optimal Use reagents as specified in these protocols. To obtain higher RNA yields one may increase the total amount of SP6 RNA polymerase to 100 U
    Low transfection efficiency Insufficient RNA Optimize the RNA amount used for the cotransfection
    Not using optimal electroporation conditions or amount of DMRIE‐C reagent Optimize transfection conditions using vector RNA expressing GFP or β‐galactosidase
    Cell density too high or too low Use cells that are ∼80% confluent at time of transfection
    Poor cell viability following cotransfection of RNA Suboptimal electroporation conditions or excessive DMRIE‐C reagent Optimize transfection conditions
    Cell density too low Use cells that are ∼80% confluent at time of DMRIE‐C‐mediated transfection
    Low titer of the viral stock RNA degradation Use RNase‐free conditions for in vitro transcription and cotransfection
    Capping reaction (incorporation of cap analog m7G(5′)ppp(5′)G) not working Try different source or new stock of cap
    Adjust the amount of SP6 RNA polymerase. Only a fraction of the RNA will be capped when too much SP6 RNA polymerase is used.
    Insufficient RNA or uneven concentrations of recombinant vector and helper RNA Optimize packaging reaction by using vector RNA expressing GFP or β‐galactosidase
    Estimate relative RNA concentrations by agarose gel electrophoresis. Use vector to helper RNA ratios of 1‐2 to 1 µg.
    Did not use BHK‐21 cells All packaging procedures have been optimized for BHK‐21 cells
    Low recombinant protein expression in infected cells Did not use an optimal concentration of infectious replicons Construct a dose‐response curve with the infectious replicons to determine the amount required for optimal expression
    Did not activate the packaged SFV replicons Prior to infection activate the SFV stock with α‐chymotrypsin
    Use a new preparation of α‐chymotrypsin
    Recombinant protein is unstable Remove any protein degradation signals and/or use protease inhibitors
    Poor cell viability following infection Used too much of the viral stock Optimize infection conditions
    Cell density is too low Use cells that are ∼80% confluent at the time of infection
    BHK‐21 cell supernatant is toxic for neurons Transfer the viral particles into neuronal medium before infecting the neurons
    Limited infection of slice cultures Clogging of the glass micropipet during virus injection During the injection procedure verify repeatedly that virus solution exits from the pipet tip by lifting the micropipet out of the cutting medium and applying pressure from the syringe
    To prevent or remove salt crystal‐induced clogging of the tip, lower the micropipet tip into cutting medium
    Dilution of virus in micropipet by capillary action Use micropipets with a smaller tip opening
    Slice cultures show many dead, non‐infected cells around injection sites Excessive pressure used for virus injection Use micropipets with a smaller tip opening; apply less pressure

  • Helper plasmid pSFV‐Helper2 [available from Markus U. Ehrengruber ( ) or Kenneth Lundstrom ( )]
  • Restriction endonucleases SpeI, NruI, and SapI (including corresponding buffers)
  • Vector plasmid pSINRep5 [Frolov and Schlesinger, , ; available from Sondra Schlesinger ( ) or Charles Rice ( )]
  • Linearized/nonlinearized helper plasmid DH‐BB or DH(26S)5′SIN [available from Sondra Schlesinger ( ) or Charles Rice ( )]; for neuronal cells, helper plasmids containing the glycoproteins derived from TE12 virus should be used (Kim et al., ; also available from Sondra Schlesinger or Charles Rice).
  • Restriction endonuclease XhoI (if the cDNA of interest contains an XhoI site, use either NotI or PacI to linearize pSINRep5), including corresponding buffer
  • 0.8% (w/v) agarose gel
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol ( appendix 2A)
  • 3 M sodium acetate ( appendix 2A)
  • 95% and 70% ethanol
  • 10× transcription buffer (SFV system; see recipe) or commercial 5× transcription buffer provided with the SP6 polymerase (SIN system)
  • 10 mM m7G(5′)ppp(5′)G (sodium salt; Pharmacia or New England Biolabs)
  • 50 mM dithiothreitol (DTT)
  • rNTP mix (10 mM rATP, 10 mM rCTP, 10 mM rUTP, 5 mM rGTP; Roche Molecular Biochemicals)
  • 10 to 50 U/µl RNase inhibitor (Roche Molecular Biochemicals)
  • 10 to 20 U/µl SP6 RNA polymerase (Roche Molecular Biochemicals, Invitrogen, Epicentre Technologies, or Promega)
  • Gel loading buffer ( appendix 2A)
  • Molecular weight marker (e.g., digested λ DNA)
  • BHK‐21 cells (ATCC #CRL‐6281, ∼80% confluent)
  • Phosphate‐buffered saline (PBS; appendix 2A), DEPC‐treated to remove RNase, 37°C
  • Trypsin‐EDTA (0.5 mg/ml trypsin and 0.2 mg/ml EDTA in PBS)
  • Complete BHK‐21 cell medium (see recipe)
  • Cells appropriate for testing of transgene expression, growing in 6‐ or , 12‐, or 24‐well plates or 35‐mm dishes, and appropriate culture medium
  • 1.5‐ml microcentrifuge tubes
  • Two heating blocks or water baths (37°C and 80° to 90°C)
  • Sterile electroporation gap cuvettes (e.g., 0.2‐cm gap, Bio‐Rad)
  • Electroporator (e.g., Gene Pulser, Bio‐Rad)
  • Tissue culture flasks or dishes (24‐, 35‐, 60‐, or 100‐mm)
  • Plastic syringes (10‐ or 20‐ml) with attached 0.22‐µm sterile filters
  • Additional reagents and equipment for subcloning (Struhl, ), preparation of plasmid DNA ( appendix 1J), restriction endonuclease digestion ( appendix 1M), phenol/chloroform extraction and ethanol precipitation of DNA ( appendix 1G), spectrophotometric quantitation of RNA and DNA ( appendix 1K), agarose gel electrophoresis ( appendix 1N), tissue culture ( appendix 3B), SDS‐PAGE for protein analysis (Gallagher, ), and autoradiography (Voytas and Ke, )

Basic Protocol 2: Activation of Packaged SFV Replicons

  • 20 mg/ml α‐chymotrypsin (Sigma)
  • Packaged SFV4 replicons (see protocol 1)
  • 10 mg/ml aprotinin (Sigma or Roche Molecular Biochemicals)

Basic Protocol 3: Infection of Hippocampal Slices

  • Cutting medium (see recipe), prewarmed to 37°C
  • Infectious SFV or SIN replicon stock
  • 95% ethanol
  • Hippocampal slice cultures (roller‐tube type; unit 6.11)
  • Roller tube culture medium (unit 6.11)
  • Glass capillaries (i.d. 1.5 mm, o.d. 1.17 mm; e.g., Clark capillaries, Harvard Apparatus)
  • Electrode puller
  • Autoclavable electrode holder
  • Micromanipulator (Narishige)
  • Metal plate containing a base for a 35‐mm petri dish
  • 3‐way valve
  • 1‐ml syringe
  • Plastic tubing (i.d. 1 mm, o.d. 3 mm)
  • 35‐mm plastic petri dishes
  • Dissection microscope
  • Microloader pipet tips (autoclaved; Eppendorf)
  • Forceps

Basic Protocol 4: Infection of Dispersed Neurons

  • Infectious SFV or SIN replicons
  • Complete BHK‐21 cell medium (see recipe)
  • 50% and 20% (w/v) sucrose solution
  • Ultracentrifuge tubes for Beckman SW 40 or SW 41 rotor (e.g., Beckman or Contron)
  • Ultracentrifuge with Beckman SW 40 or SW 41 rotor
  • Centriprep‐50 or Centriprep‐100 centrifugal concentrators (Amicon)
  • 50‐ml disposable plastic tubes (Falcon)
  • Low‐speed centrifuge (500 to 3000 × g), accommodating the 50‐ml plastic tubes
  • 1.5‐ml microcentrifuge tubes
  • 1‐ml disposable plastic pipet
  • Additional reagents and equipment for producing primary cultures of neurons in plastic petri dishes, multi‐well plates, or on coverslips, and preparation of the corresponding neuronal culture medium (unit 3.2)

Alternate Protocol 1: Lipid‐Mediated Cotransfection of RNA

  • Opti‐MEM I reduced‐serum medium (Invitrogen)
  • DMRIE‐C reagent (Invitrogen)
  • Vortex

Support Protocol 1: Titer Determination for the Number of Infectious SFV or SIN Particles

  • 100% methanol
  • 0.2% (w/v) gelatin in PBS (blocking solution)
  • Primary antibody directed against the recombinant protein
  • Secondary antibody coupled to detection system
  • 2.5% (w/v) DABCO (reduces fading of FITC) in Mowiol 4‐88 (Calbiochem)
  • Glass slides

Support Protocol 2: Metabolic Labeling

  • Infected or transfected cells (see Basic Protocols protocol 11, protocol 22, protocol 44, and protocol 5 for cell lines and neurons)
  • Starvation medium (see recipe)
  • >1000 Ci/mmol [35S]methionine (1 mCi/0.1 ml), added to starvation medium
  • Chase medium (see recipe)
  • Lysis buffer (see recipe)
  • 2× SDS‐PAGE sample buffer (see recipe)
  • Precast 10% (w/v) Tris‐glycine polyacrylamide gels (Novex/Invitrogen)
  • Fixing solution (see recipe)
  • Amplify solution (Amersham Pharmacia Biotech)
  • Hyperfilm‐MP (Amersham Pharmacia Biotech)
  • SDS‐PAGE apparatus
  • Power unit
  • Bio‐Rad gel dryer
  • Additional reagents and equipment for SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE; see Gallagher, and appendix 1A)
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Literature Cited

   Agapov, E.V., Frolov, I., Lindenbach, B.D., Pragai, B.M., Schlesinger, S., and Rice, C.M. 1998. Non‐cytopathogenic Sindbis RNA vectors for heterologous gene expression. Proc. Natl. Acad. Sci. U.S.A. 95:12989‐12994.
   Berglund, P., Sjöberg, M., Garoff, H., Atkins, G.J., Sheahan, B.J., and Liljeström, P. 1993. Semliki Forest virus expression system: Production of conditionally infectious recombinant particles. Biotechnology 11:916‐920.
   Bredenbeek, P.J., Frolov, I., Rice, C.M., and Schlesinger, S. 1993. Sindbis virus expression vectors: Packaging of RNA replicons by using defective helper RNAs. J. Virol. 67:6439‐6446.
   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.
   Ehrengruber, M.U. 2002a. Alphaviral gene transfer in neurobiology. Brain Res. Bull 59:13‐22.
   Ehrengruber, M.U. 2002b. Alphaviral vectors for gene transfer into neurons. Mol. Neurobiol. 26:183‐201.
   Ehrengruber, M.U. and Goldin, A.L. 2007. Semliki Forest virus vectors with mutations in the nonstructural protein 2 gene permit extended super‐infection in neuronal and non‐neuronal cells. J. Neurovirol. 13:353‐363.
   Ehrengruber, M.U., Lundstrom, K., Schweitzer, C., Heuss, C., Schlesinger, S., and Gähwiler, B.H. 1999. Recombinant Semliki Forest virus and Sindbis virus efficiently infect neurons in hippocampal slice cultures. Proc. Natl. Acad. Sci. U.S.A. 96:7041‐7046.
   Ehrengruber, M.U., Hennou, S., Büeler, H., Naim, H.Y., Déglon, N., and Lundstrom, K. 2001. Gene transfer into neurons from hippocampal slices: Comparison of recombinant Semliki Forest virus, adenovirus, adeno‐associated virus, lentivirus, and measles virus. Mol. Cell. Neurosci. 17:855‐871.
   Ehrengruber, M.U., Ehler, E., Billeter, M.A., and Naim, H.Y. 2002. Measles virus spreads in rat hippocampal neurons by cell‐to‐cell contact and in a polarized fashion. J. Virol. 76:5720‐5728.
   Ehrengruber, M.U., Renggli, M., Raineteau, O., Hennou, S., Vähä‐Koskela, M.J.V., Hinkkanen, A.E., and Lundstrom, K. 2003. Semliki Forest virus A7(74) transduces hippocampal neurons and glial cells in a temperature‐dependent dual manner. J. Neurovirol. 9:16‐28.
   Fazakerley, J.K., Pathak, S., Scallan, M., Amor, S., and Dyson, H. 1993. Replication of the A7(74) strain of Semliki Forest virus is restricted in neurons. Virology 195:627‐637.
   Fazakerley, J.K., Cotterill, C.L., Lee, G., and Graham, A. 2006. Virus tropism, distribution, persistence and pathology in the corpus callosum of the Semliki Forest virus‐infected mouse brain: A novel system to study virus‐oligodendrocyte interactions. Neuropathol. Appl. Neurobiol. 32:397‐409.
   Fragkoudis, R., Tamberg, N., Siu, R., Kiiver, K., Kohl, A., Merits, A., and Fazakerley, J.K. 2009. Neurons and oligodendrocytes in the mouse brain differ in their ability to replicate Semliki Forest virus. J. Neurovirol. 15:57‐70.
   Frolov, I. and Schlesinger, S. 1994. Translation of Sindbis virus mRNA: Effects of sequences downstream of the initiating codon. J. Virol. 68:8111‐8117.
   Frolov, I. and Schlesinger, S. 1996. Translation of Sindbis virus mRNA: Analysis of sequences downstream of the initiating AUG codon that enhance translation. J. Virol. 70:1182‐1190.
   Frolov, I., Frolova, E., and Schlesinger, S. 1997. Sindbis virus replicons and Sindbis virus: Assembly of chimeras and of particles deficient in virus RNA. J. Virol. 71:2819‐2829.
   Gähwiler, B.H. 1981. Organotypic monolayer cultures of nervous tissue. J. Neurosci. Methods 4:329‐342.
   Gallagher, S.R. 2006. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Mol. Biol. 75:10.2A.1‐10.2A.37.
   Griffin, D.E. 1998. A review of alphavirus replication in neurons. Neurosci. Biobehav. Rev. 22:721‐723.
   Gwag, B.J., Kim, E.Y., Ryu, B.R., Won, S.J., Ko, H.W., Oh, Y.J., Cho, Y.‐G., Ha, S.J., and Sung, Y.C. 1998. A neuron‐specific gene transfer by a recombinant defective Sindbis virus. Mol. Brain Res. 63:53‐61.
   Heilig, J.S., Elbing, K.L., and Brent, R. 1998. Large‐scale preparation of plasmid DNA. Curr. Protoc. Mol. Biol. 41:1.7.1‐1.7.16.
   Hennou, S., Kato, A., Schneider, E.M., Lundstrom, K., Gähwiler, B.H., Inokuchi, K., Gerber, U., and Ehrengruber, M.U. 2003. Homer‐1a/Vesl‐1S enhances hippocampal synaptic transmission. Eur. J. Neurosci. 18:811‐819.
   Kim, J., Dittgen, T., Nimmerjahn, A., Waters, J., Pawlak, V., Helmchen, F., Schlesinger, S., Seeburg, P.H., and Osten, P. 2004. Sindbis vector SINrep(nsP2S726): A tool for rapid heterologous expression with attenuated cytotoxicity in neurons. J. Neurosci. Methods 133:81‐90.
   Liljeström, P. and Garoff, H. 1991. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Bio/Technology 9:1356‐1361.
   Liljeström, P. and Garoff, H. 1994. Expression of proteins using Semliki Forest virus vectors. Curr. Protoc. Mol. Biol. 29:16.20.1‐16.20.16.
   Lundstrom, K., Richards, J.G., Pink, J.R., and Jenck, F. 1999. Efficient in vivo expression of a reporter gene in rat brain after injection of recombinant replication‐deficient Semliki Forest virus. Gene Ther. Mol. Biol. 3:15‐23.
   Lundstrom, K., Rotmann, D., Hermann, D., Schneider, E.M., and Ehrengruber, M.U. 2001. Novel mutant Semliki Forest virus vectors: Gene expression and localization studies in neuronal cells. Histochem. Cell Biol. 115:83‐91.
   Lundstrom, K., Abenavoli, A., Malgaroli, A., and Ehrengruber, M.U. 2003. Novel Semliki Forest virus vectors with reduced cytotoxicity and temperature‐sensitivity: Long‐term enhancement of transgene expression. Mol. Ther. 7:202‐209.
   Lustig, S., Jackson, A.C., Hahn, C.S., Griffin, D.E., Strauss, E.G., and Strauss, J.H. 1988. Molecular basis of Sindbis virus neurovirulence in mice. J. Virol. 62:2329‐2336.
   Olkkonen, V.M., Liljeström, P., Garoff, H., Simons, K., and Dotti, C.G. 1993. Expression of heterologous proteins in cultured rat hippocampal neurons using the Semliki Forest virus vector. J. Neurosci. Res. 35:445‐451.
   Pan, C.‐H., Greer, C.E., Hauer, D., Legg, H.S., Lee, E.‐Y., Bergen, M.J., Lau, B., Adams, R.J., Polo, J.M., and Griffin, D.E. 2010. A chimeric alphavirus replicon particle vaccine expressing the hemagglutinin and fusion proteins protects juvenile and infant rhesus macaques from measles. J. Virol. 84:3798‐3807.
   Petrakova, O., Volkova, E., Gorchakov, R., Paessler, S., Kinney, R.M., and Frolov, I. 2005. Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in mammalian cells. J. Virol. 79:7597‐7608.
   Perri, S., Driver, D.A., Gardner, J.P., Sherrill, S., Belli, B.A., Dubensky, T.W. Jr., and Polo, J.M. 2000. Replicon vectors derived from Sindbis virus and Semliki Forest virus that establish persistent replication in host cells. J. Virol. 74:9802‐9807.
   Rhême, C., Ehrengruber, M.U., and Grandgirard, D. 2005. Alphaviral cytotoxicity and its implication in vector development. Exp. Physiol. 90:45‐52.
   Schlesinger, S. 2000. Alphavirus expression vectors. Adv. Virus Res. 55:565‐577.
   Smerdou, C. and Liljeström, P. 1999. Two‐helper RNA system for production of recombinant Semliki Forest virus particles. J. Virol. 73:1092‐1098.
   Strauss, J.H. and Strauss, E.G., 1994. The alphaviruses: Gene expression, replication, and evolution. Microbiol. Rev. 58:491‐562.
   Struhl, K. 1987. Subcloning of DNA fragments. Curr. Protoc. Mol. Biol. 13:3.16.1‐3.16‐2.
   Voytas, D. and Ke, N. 1999. Detection and quantitation of radiolabeled proteins and DNA in gels and blots. Curr. Protoc. Mol. Biol. A.3A.1‐A.3A.10.
Key References
  Ehrengruber et al., 1999. See above.
  This paper describes infection of cultured rat hippocampal slices by SFV4 and SIN replicons, compares the results, and shows that neurons are preferentially infected by both vectors.
  Ehrengruber, M.U. 2002a,b. See above.
  These reviews summarize the biology of SFV and SIN and their development into viral vectors. SFV and SIN vectors are compared to other viral vectors, and their application in neurobiological studies is described.
Internet Resources
  This site contains guidelines from the National Institutes of Health (NIH) and Centers for Disease Control and prevention (CDC) on handling alphavirus and the different Biosafety Level practices.
  Biosafety classification of the Swiss expert committee SECB for work with genetically modified viral vectors, including SFV and SIN (December 2009).
  Biosafety classification of the SFV and SIN expression systems in Germany (September 2007, in German). This publication also contains a very good, detailed introduction into these vector systems.
  This site contains some information on the SFV and SIN system (search the catalog for “Semliki” or “Sindbis”).
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