FIG 3.
L-NP interaction is not supported by in vitro-transcribed MG RNA. (A) Effect of in vitro-transcribed virus-specific RNA on LCMV NP-L interaction. A DNA fragment containing the LCMV MG construct with enhanced green fluorescent protein (EGFP) and CAT genes in GP and NP loci, respectively, was amplified using a forward primer containing the T7 promoter sequence (5′-GAAATTAATACGACTCACTATAGGGCGCACCGG-3′), a reverse primer (5′-CGCACAGTGGATCCTAGGCATTTGATTGCG-3′), and pMG/S-CAT/GFP (14) as a template by PCR. Using this DNA fragment as a template, LCMV MG RNA in the genome polarity was transcribed using the T7 polymerase system (MAXIscript, Ambion) (MG-RNA). 293T cells (4.0 × 106 cells/10-cm dish) were cultured overnight and transfected with 3 μg of either pC-NP (WT NP) or pC-Strep-NP (Strep-NP) and 10 μg of pC-FLAG-L (FLAG-L), together with 3 μg of pCAGGS-T7pol (T7-pol) and 3 μg of pT7-MG using PEI Max. “+” or “−” indicates the presence or absence, respectively, of plasmid in the transfection mixture. Total amount of DNA was adjusted using empty pCAGGS. At 72 h posttransfection, cells were washed with PBS and lysed with 1 ml of CLB supplemented with a protease and phosphatase inhibitor cocktail. Cell lysates were clarified by centrifugation (15,000 rpm at 4°C for 10 min) and incubated with (+) or without (−) 500 ng of MG RNA at 4°C for 16 h, prior to PD with Strep-Tactin resin (Strep-Tactin Super flow Plus, Qiagen) at 4°C for 2 h. Beads were washed three times with 1 ml of CLB and the associated protein complex was eluted using CLB containing 2.5 mM desthiobiotin. Protein levels present in cleared cell lysate (Input) and eluate (PD: Strep) were determined by Western blotting. Values for input and PD-analyzed samples correspond to 0.5% and 10%, respectively, of values for whole-cell lysate. (B) The quality of RNA transcript (500 ng) used for panel A was examined by agarose gel electrophoresis.