Generating Measles Virus Knockout Strains

The Schwarz vaccine strain MeV and MeV-ΔV were previously described [28 (link)]. Briefly, V protein expression from MeV was knock-down following a two-step PCR strategy to generate MeV-ΔV virus. These PCRs introduced a mutation interfering with RNA editing (the native sequence UUAAAAAGGGCACAGA was mutated to UUAAGAAGGGCACAGA) [28 (link)]. MeV with a GFP (MeV-GFP) was previously described [27 (link)] while MeV-ΔV-GFP was performed by cloning the GFP ORF via Sbfi and Tth111I sites from PTM3-egfp plasmid and put the fragment in the MeV-ΔV virus. Unless overwise specified, 10⁶ THP-1 cells were seeded in 12-well plates for infection in 500μL non-supplemented RPMI. 500μL 2X supplemented RPMI was added 2 hours post-infection to restore the appropriate concentration of supplements. THP-1 cells were infected with MeV at MOI 0.1, 0.5, 1 or 3 for 24 hours. As negative control, cells were infected at a MOI 0.5 with MeV, previously inactivated at 70°C for 30 minutes. For both infections, viruses were absorbed in serum-free RPMI for a defined period at 37°C before dilution with complete RPMI. To study the role of the RNA polymerase III, inhibitor was added to the culture medium at final concentrations of 25 or 50 μM for ML-60218 (Merck Millipore) 2 hpi. Monolayers of Vero or THP-1 cells were infected with MeV, MeV-ΔV, MeV-GFP and MeV-ΔV-GFP at MOI 0.1. At various times post infection, cells were scraped into culture medium and froze at -80°C. After medium clarification of cell debris, virus titers were determined. For this purpose, Vero cells were seeded into 96-well plates (7,500 cells/well) and infected with serial 1:10 dilutions of virus sample in DMEM-5% FCS. After incubation for 7 days, cells were stained with crystal violet, and the TCID₅₀ values were calculated by using the Karber method.
One-STrEP Tag Purification-HEK-293T (8.10⁷) cells were either infected with rMeV/STrEP-V or with the negative control rMeV/STrEP-Cherry viruses at MOI 1. At 24 hpi, cells were lysed and tagged protein co-complexes were purified and analyzed by Western Blot as previously described [30 (link)].