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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Abstract

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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Materials

Basic Protocol 1: Preparation of Packaged SFV and SIN Replicons

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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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Figures

  •   FigureFigure 4.22.1 Generation of recombinant SFV and SIN particles. (A) Vector RNA encoding non‐structural proteins 1‐4 ( nsP1‐4) and the transgene (gray) under the control of the subgenomic RNA promoter (broad arrow), and defective helper RNA (encoding the structural alphavirus proteins downstream of the subgenomic RNA promoter) are obtained by in vitro transcription from their respective cDNAs and cotransfected into BHK‐21 cells. Only the vector RNA contains the packaging signal required for encapsidation into the nucleocapsid. (B) Within the cytoplasm of BHK‐21 cells, vector RNA replication occurs through the action of nsP1‐4. In parallel, the defective helper RNA is also replicated and transcribed by nsP1‐4, and the capsid protein, spike protein E1, and precursor protein p62 (which is later split into the E2 and E3 spike proteins) are translated from its subgenomic RNA. Capsid proteins package only vector RNA containing the transgene (gray) while spike proteins are incorporated into the BHK‐21 cell membrane. Nucleocapsids dock to the cell membrane where spike proteins have been incorporated, thus allowing the budding of SFV and SIN particles. (C) Upon infection of a neuron, nucleocapsids are released into the cytoplasm and the vector RNA is liberated. The replicase complex composed of nsP1‐4 amplifies the vector RNA, and the transgene is translated into the recombinant protein.
  •   FigureFigure 4.22.2 Map of the pSFV2gen vector plasmid. Numbering starts with the first nucleotide of the cDNA derived from the genomic RNA of the wild‐type SFV4 strain. Upstream from the SFV4 cDNA is the SP6 promoter (gray box and arrow) for in vitro transcription by SP6 RNA polymerase. The regions encoding the nonstructural proteins are shown ( nsp1‐4). Unique restriction sites and their positions to insert the cDNA of interest (multiple cloning site) and linearize for in vitro transcription are given. Regions corresponding to the subgenomic RNA promoter (SG; arrow indicating the start of the subgenomic RNA), the 3′‐untranslated region (3′UTR), poly(A) sequence, origin of replication ( ori), and ampicillin resistance gene ( amp) are also indicated.
  •   FigureFigure 4.22.3 Map of the pSINRep5 vector plasmid. Numbering starts with the first nucleotide of the cDNA derived from SIN genomic RNA. Upstream from the SIN cDNA is the SP6 promoter (gray box and arrow) for in vitro transcription by SP6 RNA polymerase. Unique restriction sites and their positions, including those for insertion of the cDNA of interest (cloning) and linearization for in vitro transcription, are indicated. The figure also indicates the subgenomic RNA promoter (SG; the arrow at position 7598 shows the start of the subgenomic RNA), 3′ untranslated region (3′UTR), poly(A) sequence, ampicillin resistance gene ( Amp; nt 8227‐9076), and origin of replication ( ori). See Bredenbeek et al. () for further details
  •   FigureFigure 4.22.4 Quality control of linearized plasmid DNA and in vitro–transcribed RNA on an agarose gel. Lane 1, NruI‐linearized DNA for pSFV4(PD)‐mRFP1, encoding monomeric red fluorescent protein 1 (mRFP1; Ehrengruber and Goldin, ); lane 2, 2 µl in vitro‐transcribed RNA for pSFV4(PD)‐mRFP1; lane 3, SpeI‐digested DNA for pSFV‐Helper2; lane 4, 2 µl in vitro‐transcribed RNA for pSFV‐Helper2; lane 5, 1‐kb DNA ladder (New England Biolabs). The agarose gel was stained with ethidium bromide, and the bands were visualized with UV illumination. Note that in lanes 2 and 4, which contain in vitro–transcribed RNA, faint bands for the linearized DNA templates are also detectable (corresponding to lanes 1 and 3, respectively).
  •   FigureFigure 4.22.5 Injection of viral particles into hippocampal slice cultures. (A) Virus injection setup comprised of a Narishige micromanipulator mounted onto a metal base with an acrylic glass stand holding a 35‐mm plastic petri dish in which the roller‐tube slice culture is submerged in ∼2 ml cutting medium. The glass pipet for the viral solution is attached via an air‐tight fitting (plastic O ring) to an acrylic glass holder to which a 1‐ml syringe is attached via a plastic tubing. The whole virus injection setup is standing in a biosafety level 2 hood. Photo courtesy of Edith M. Schneider Gasser. (B) Schematic illustration for injecting viral solution into a hippocampal slice where axons (i.e., Schaffer collaterals) from CA3 pyramidal cells connect to dendrites from CA1 pyramidal cells (as indicated by the arrow). By localizing the injection sites to the pyramidal cell layer of the CA3 or CA1 region, respectively, the genetic modification of excitatory neurons is restricted to either pre‐ or post‐synaptic pyramidal cells.
  •   FigureFigure 4.22.6 Recombinant SFV4‐mediated gene transfer into pyramidal cells of cultured rat hippocampal slices. (A, B) Expression of GFP, as revealed by fluorescence microscopy in living slices, at 48 days in culture and 5 days after injection of ∼105 (A) and ∼104 (B) infectious particles into the pyramidal cell layer. Fluorescence illumination of the CA1 region. (C) GFP expression in CA3 pyramidal cells of a living slice at 11 days in culture and 2 days post‐infection. Note the GFP‐positive dendritic spines typical of pyramidal cells (arrowheads). (D) Expression of an N‐terminal GFP fusion of the ionotropic glutamate receptor 1 subunit. Fluorescence illumination from the CA1 region of a living slice at 29 days in culture and 4 days post‐infection. The cDNA for the GFP fusion construct was provided by Volker Mack and Dr. Rolf Sprengel (Max Planck Institute for Medical Research, Heidelberg, Germany). Abbreviations: so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum. Bars: 300 µm (A), 75 µm (B and D), and 25 µm (C).
  •   FigureFigure 4.22.7 Recombinant SFV4‐mediated expression of metabotropic glutamate receptor 2 (mGluR2) in dispersed hippocampal neurons. Neurons from embryonic day 18 rats were cultured on glass coverslips for 5 days and then infected. Confocal images of cells fixed at 16 hr post‐infection, treated with antibodies against mGluR2 and microtubule‐associated protein type 2 (MAP2, a dendritic marker), and detected with secondary antibodies coupled to Texas red (mGluR2) or fluorescein isothiocyanate (MAP2).

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Literature Cited

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   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.
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   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.
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   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.
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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
   http://www.cdc.gov/biosafety/
  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.
  http://www.efbs.admin.ch/en/documentation/recommendations/index.html
  Biosafety classification of the Swiss expert committee SECB for work with genetically modified viral vectors, including SFV and SIN (December 2009).
  http://www.bvl.bund.de/SharedDocs/Downloads/06_Gentechnik/ZKBS/01_Allgemeine_Stellungnahmen_deutsch/09_viren/zkbs_viren_Sindbis_Virus_Semliki_Forest_Virus.pdf?__blob=publicationFile
  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.
  http://www.invitrogen.com/
  This site contains some information on the SFV and SIN system (search the catalog for “Semliki” or “Sindbis”).
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