Sp6 polymerase

DENV-2 strain 16681 was propagated on C6/36 cells, as described previously [27 (link)]. Recombinant GFP-DENV was generated from the infectious clone pFK-DV-G2A strain 16681 (kind gift from Ralf Bartenschlager University of Heidelberg, [28 (link)]). The pFK-DV-G2A clone was propagated in E. coli strain D5α. Upon plasmid purification, the plasmid was linearized with XbaI (New England Biolabs, Ipswich, Massachusetts, USA) and capped RNA transcripts were synthesized by use of an SP6 polymerase (New England Biolabs). Viruses were harvested at 72 hours post-transfection (hpt) of RNA in BHK-21 cells via electroporation (Biorad Gene Pulser Xcell machine; 850 V, 25 μF, no resistance). Thereafter, GFP-DENV was propagated once by infecting C6/36 cells (MOI 0.1). Progeny virions were harvested, aliquoted and snap-frozen at 120 hours post-infection (hpi). WNV strain NY385-99 was a generous gift from Jaap Goudsmit (Crucell Holland BV) and was propagated on BHK-21 cells, as previously described [29 (link)]. All virus preparations were characterized, as described before [27 (link),30 (link)] by determination of the number of infectious particles by plaque assay on BHK-15 cells and the number of genome-equivalent particles by Q-RT-PCR. UV-inactivated GFP-DENV was obtained by exposure of the virus to UV-light for 4h. Inactivation was confirmed by plaque assay.

All genes of interest were transcribed by means of SP6 polymerase (NEB, England) according to the manufacturer’s instructions. RNA was snap frozen in liquid nitrogen and stored at −80 °C. Translation war performed with the Flexi Retic Kit (Promega, Mannheim, Germany) for 90 min in the presence of ³⁵S Met/Cys (Perkin Elmer, Waltham, MA, USA). Translated preproteins were aliquoted, snap frozen, and stored at –80 °C until further use.

All cells employed in this study, SK6-, PK15- and STE-cells, as wells as primary porcine cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% BVDV-free FCS at 37 °C and 5%CO₂. Virus was rescued from cDNA in vitro transcribed into RNA (SP6 polymerase, New England Biolabs), which subsequently was transfected in 5 × 10⁶ SK6-cells by electroporation (Bio-Rad Gene Pulser). Transfection efficiency was assessed 14 h after transfection by immunohistochemistry specifically staining CSFV E2 with the mouse monoclonal antibody A18. Virus containing supernatant for infection experiments was harvested 36 h after electroporation, clarified by centrifugation for 5 min at 3000 × g and stored in aliquots at −70 °C.
Titer was determined in ffu/ml on SK6-cells employing 10-fold dilution steps. 14 h after infection, cells were stained by immunohistochemistry as described above and foci of infected cells were counted using a Nikon Eclipse TS100 microscope.

SFV-derived plasmids were linearized by digestion with SpeI and transcribed in the presence of cap analog (New England Biolabs, Ipswich, MA) using SP6 polymerase (New England Biolabs)^{17 (link)}. Fifty µg of in vitro transcribed RNA were electroporated into 5 × 10⁶ BHK-21 cells by electroporation as described previously^{13 (link)}. After transfection, cells were allowed to recover for 24–28 h before addition of puromycin at 5 µg/ml (Sigma, St. Louis, MO). To select for puromycin resistant cells, medium was replaced every 2–3 days with fresh puromycin containing medium. Upon selection, cells were always passaged in the presence of puromycin at the indicated concentration.

Synthetic infectious RNA was produced as previously described [24 (link)]. Briefly, 2.5 µg DNA of the plasmids pL588 and pL602 were digested with XhoI and purified using phenol-chloroform extraction. The linearized plasmid DNA was transcribed into genomic recLindaV RNA using SP6 polymerase (NEB). A total volume of 50 µL of the transcription mixture was DNase digested. The RNA was purified with the RNeasy Mini Kit (QIAGEN), eluted in RNase free water, and diluted with water to a final concentration of 0.25 µg/µL. SK-6 cells were transfected with 2.5 µg of the synthetic RNA by electroporation as previously described [25 (link)] and incubated for 24 h until progeny virus was harvested from the supernatant.