Recombinant adenoviruses harboring SAG2 (Ad-SAG2) or the Escherichia coli β-galactosidase (Ad-Ctrl) coding sequences were generated as previously described [39] and [42]. Recombinant influenza viruses carrying wild type NA segment (vNA) or NA38-SAG2-recombinant NA segment (vNA38-SAG2

herein named FLU-SAG2) GPCR Compound Library cost were generated by the twelve plasmid-driven genetic reverse technique, as described by Fodor and co-workers with modifications [41]. Briefly, co-cultures of HEK293T cells (4 × 105 cells/well) and MDCK cells (3 × 105 cells/well) were transfected with either of the NA segment transfer plasmids (pPRNA or pPRNA38-SAG2; 0.5 μg), together with expression plasmids pcDNA-PB1, pcDNA-PB2, pcDNA-NP and pcDNA-PA (0.5 μg of each plasmid) and other seven Influenza A/WSN/33 segments transfer plasmids (0.5 μg of each plasmid) using Fugene 6 Reagent® (ROCHE). Transfected cells were incubated at 35 °C and 5% CO2 in complete DMEM with 10% FCS. After 24 h of incubation, culture medium was replaced by complete ABT 888 DMEM with 2% FCS and cells were incubated for additional

48 h. Infectious vNA or FLU-SAG2 particles were recovered from cell culture supernatants and amplified once on MDCK cells in complete DMEM supplemented with 2% FCS. Next, viruses were submitted to two plaque-purification rounds. After being cloned in plaque-purification assays, viruses were amplified three times on MDCK cells (m.o.i. = 0.001) for 72 h at 35 °C in complete DMEM with 2% FCS, to prepare the work stocks. Viral stocks were titrated on MDCK cell monolayers, in standard plaque assays, using agarose overlay in complete DMEM with next 2% FCS. Viral RNA (vRNA) was obtained from cell-free

supernatants of infected MDCK cultures. vRNA extraction and RT-PCR analysis were performed as previously described [27]. Amplicons were analyzed on 1% agarose gel and visualized by ethidium bromide staining. RT-PCR products were purified using QiaEXII® kit (Qiagen). The presence of mutations was determined by sequencing using Dynamic ET Dye Terminator Cycle Sequencing KIT® (AMERSHAM) and a Megabace 1000 automatic sequencer (AMERSHAM). MDCK cells (8 × 105 cells/well) were grown in complete DMEM supplemented with 5% FCS. MDCK cells were mock-infected or infected with vNA or FLU-SAG2 at m.o.i. = 2. Northern blot analysis was performed with total RNA samples extracted 24 h after infection, as previously described [27]. Blotted RNAs were hybridized with SAG2-specific 32P-labeled riboprobe, allowing indistinctly the detection of RNAs of negative (vRNA) and positive (cRNA and mRNA) orientation, as previously described [27]. Detection of radioactive-labeled RNAs was performed by membrane exposure to X-ray film (KODAK). MDCK cells were mock-infected or infected with vNA or FLU-SAG2 at m.o.i. = 2. After 24 h, cell extracts were collected and analyzed by Western blot.