Growth of Semliki Forest Virus
Semliki Forest virus (SFV) is an enveloped RNA virus, with a genome of positive polarity that belongs to the Alphavirus group of the family Togaviridae. It readily infects a variety of mammalian and insect cells and can be grown to high titres in tissue culture. Upon infection, host cell-specific synthesis of macromolecules is suppressed within a few hours, structural viral components are made, and new virus particles bud out from the plasma membrane of the infected cell. The SFV particle is spherical with a diameter of approximately 65 nm (molecular mass ≈ 42 × 103kDa). It consists of a nucleocapsid (NC), a single copy of the RNA genome packed together with 240 copies of a capsid (C) protein (33kDa), that is surrounded by a lipid membrane in which 80 glycoprotein complexes, the viral spikes, are anchored. The viral spikes are trimeric associations of a protein complex: two membranespanning proteins, E1 and E2 (49 and 52kDa), and a peripheral protein, E3 (10kDa) (Garoff et al., 1982; Strauss and Strauss, 1994). This article provides protocols to grow SFV in intermediate scale (up to ≈1.5 mg; protocol A) and small scale (35S-methionine labelled; protocol B).
II. MATERIALS AND INSTRUMENTATION
Culture medium Glasgow minimum essential medium (MEM) (BHK-21) (Cat. No. 21710), foetal bovine serum (FBS) (Cat. No. 10106), tryptose phosphate broth (Cat. No. 18050), 1M HEPES (Cat. No. 15630), L-glutamine 200mM (100X) (Cat. No. 25030), penicillin-streptomycin (Cat. No. 15140), MEM (Cat. No. 21090-022), bovine albumin fraction V solution 7.5% (BSA; Cat. No. 15260-037), and phosphate-buffered saline (PBS) Dulbecco's with Ca2+ and Mg2+ (Cat. No. 14040) are from GIBCO BRL. Sea-plaque agarose (Cat. No. 50100) is from FMC Bio Products. Redivue-[35S]methionine (Cat. No. AG 1094) is from Amersham Biosciences. Sucrose (Cat. No. 0335) is from Amresco. Tris (Cat. No. 146861) is from Angus. Sodium chloride (NaCl) (Cat. No. 106404), HCl (Cat. No. M317), and Titriplex III (EDTA; Cat. No. 108418) are from Merck. NaOH (Cat. No. 05- 400201) is from EKA Nobel AB (Tamro). Cholesterol (Cat. No. C3045), neutral red (Cat. No. N6634), and methionine-free MEM (Cat. No. 31900-012) are from Sigma-Aldrich. The density gradient fractionator (Model 185) is from Instrumentation Specialities Company (ISCO). Filter papers No. 1 (Cat. No. 1001 090) are from Whatman. The 75-cm2 flasks (Cat. No. 3375) and 162-cm2 flasks (Cat. No. 3150) are from Corning Life Sciences. Sixty-millimeter tissue culture plates (Cat. No. 50288) are from Nunc. BHK-21 cells C- 13 (Cat. No. CRL-8544) are from American Type Culture Collection. Cotton-tipped applicators are from Solon manufacturing company. Fifty-milliliter Nalgene tubes (Cat. No. 3139-0050) are from Nalge Incorporated. SW 28 tubes (Cat. No. 344058), SW 40 tubes (Cat. No. 331374), and 6ml-scintillation vials (Cat. No. 566831) are from Beckman. Eppendorf tubes, 1.5 ml (Cat. No. 0030 102.002) and 2.0ml (Cat. No. 0030 120.094), are from Eppendorf-Netheler-Hinz GmbH. Emulsifier Safe is from Packard Instrument Co. Inc.
A. Growth of SFV
B. Growth of 35S-Methionine-Labelled SFV
C. Quantitation of Infectious Virus Particles by Plaque Titration
Cells used for SFV infections in this protocol (old BHK cells) are BHK-21 cells that with time in culture have transformed further. In doing so, they have lost the extended form of normal BHK-21 cells (freshly obtained from ATCC) and appear more like penta- or hexagons. The SFV strain used [SFV4 (Liljestr6m et al., 1991)] had undergone an unknown number of passages in the old BHK cells (Glasgow et al., 1991) before it was cloned. At present the specific titre (i.e., the number of infectious virus particles divided by the total number of virus particles produced) is approximately 10 times higher when a virus preparation is titrated on old BHK cells as compared to normal BHK- 21 cells. This is also the case when the virus is produced in normal BHK-21 cells and most likely reflects an adaptive change in SFV4 that facilitates entry into the old BHK cells.
The expected yield of SFV particles is approximately 1 µg/cm2 of confluent BHK cells. When larger amounts (mg) of SFV are desirable, the amount of complete BHK medium used in the production step (Section III,A, step 4) can be reduced down to 20 ml per 162-cm2 bottle.
An alternative method to estimate the amount of SFV in a preparation is to use CBB-stained SDS-PAGE gels and compare the intensity of the capsid protein band to that of known amounts of BSA ran under reducing conditions on the same gel (five wells with 0.15, 0.3, 0.6, 1.2, and 2.4µg, respectively, is sufficient). The amount of C protein (mc) in a band is half the amount of BSA in a band of equal intensity. The amount of SFV (mSFV) is calculated as mSFV = (3 × mc)/2.
The stability of the produced SFV is improved if the producer cells are supplied with cholesterol in the growth media. This procedure is indicated as optional in the protocol and is not necessary for the production of stock virus intended for infection of new cells.
The crude virus preparation obtained in Section III,A, step 11 can be purified by isopycnic tartrate gradient centrifugation as described by Haag and collegues (2002). To this end, cholesterol should be used during virus production and the TNE used in step 10 should be replaced by TNM (50mM Tris-HCl, 50mM NaCl, 10mM MgCl2, pH 7.4) for improved virus stability.
The E1 and E2 proteins of SFV comigrate upon SDS-PAGE under reducing conditions. Without reduction, E1 and E2 are separated readily, with E2 showing a higher apparent molecular mass than El.
Intact SFV particles contains 88% (w/w) of protein and 12% (w/w) of RNA (Garoff et al., 1982). This is equivalent to an A260/A280 ratio of 1.4, provided that A260/A280 of pure RNA equals 2.0 (Glaser, 1995; Manchester, 1995). Deviations from this figure (A260/A280= 1.4 ± 0.1) imply that the SFV preparation contains impurities and/or defective particles.
To maintain high virus quality in successive SFV preparations, it is important to use a low multiplicity of infection (MOI). The use of MOI - 0.1 (i.e., 0.1 infectious particle per cell) or less ensures that the initial infection is caused by a single virus particle. In this case, virus particles that carry deletions or other deleterious mutations in their genomes cannot be rescued by multiple infection with functional virus particles. If a high MOI is used in a series of successive infections, the number of so-called defective interfering (DI) particles will increase dramatically (Stark and Kennedy, 1978). The presence of high numbers of DI particles may express itself by low specific infectivity of the newly produced virus. In single round infections, such as radiolabelling experiments, a MOI of 5 to 10 can be advantageous as this will produce a synchronised burst of SFV production in the shortest possible time.
Avoid repeated freeze/thaw cycles, as this will reduce virus infectivity.
If SFV infection is carried out in serum-containing medium, e.g., complete BHK medium, the infectivity of the particles is reduced dramatically. Without interference, at maximum 30% complete BHK medium may be present during infection.
Efficient clarification of the virus containing medium (Section III,A, steps 5-7) is important. Cell debris present during virus pelletation (step 8) will glue the virus particles together and make resuspension difficult.
To preserve the three-dimensional structure of the virus particles, it is important to allow sufficient time for resuspension. Do not decrease the time that the virus is left on ice (Section III,A, step 10).
When trace amounts of SFV proteins are separated on SDS-PAGE, the C protein tends to smear over the lane. This can be avoided if a small volume of BHK cell lysate is included in the sample buffer prior to heating (add 1 µl BHK cell lysate for every 10µl of SDS-PAGE sample buffer). To make BHK cell lysate, grow BHK- 21 cells to 100% confluency in a 35-mm tissue culture plate, lyse in 300µl 1× lysis buffer, and remove cell nuclei by low-speed centrifugation. Store the BHK cell lysate at -20°C. The addition of cell lysate is not necessary when the amount of virus protein in the gel is sufficient for Coomassie brilliant blue staining. Silver staining is not recommended.
Garoff, H., Kondor-Koch, C., and Riedel, H. (1982). Structure and assembly of alphaviruses. Curr. Top. Microbiol. Immunol. 99, 1-50.
Glaser, J. A. (1995). Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios. Biotechniques 18, 62-63.
Glasgow, G. M., Sheahan, B. J., Atkins, G. J., Wahlberg, J. M., Salminen, A., and Liljestr6m, P. (1991). Two mutations in the envelope glycoprotein E2 of Semliki Forest virus affecting the maturation and entry patterns of the virus alter pathogenicity for mice. Virology 185, 741-748.
Haag, L., Garoff, H., Xing, L., Hammar, L., Kan, S.-T., and Cheng, R. H. (2002). Acid-induced movements in the glycoprotein shell of an alphavirus turn the spikes into membrane fusion mode. EMBO J. 21, 255-264.
Liljestr6m, 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.
Manchester, K. L. (1995). Value of A260/A280 ratios for measurement of purity of nucleic acid. Biotechniques 19, 208-210.
Stark, C., and Kennedy, S. I. T. (1978). The generation and propagation of defective-interfering particles of Semliki Forest virus in different cell types. Virology 89, 285-299.
Strauss, J. H., and Strauss, E. G. (1994). The alpha viruses: Gene expression, replication and evolution. Microbiol. Rev. 58, 491-562.
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