Quantitative Proteomic Analysis of A549 Cells Infected with Human Respiratory Syncytial Virus

Munday, Diane C.; Emmott, Edward; Surtees, Rebecca; Lardeau, Charles-Hugues; Wu, Weining; Duprex, W. Paul; Dove, Brian K.; Barr, John N.; Hiscox, Julian A.

Trafficking Analysis of P and M2-1 Proteins-The P and the M2-1 genes were amplified by reverse transcription-PCR from total RNA extracted from HRSV A2-infected cells. Complementary DNA served as a template for PCR amplification and was subcloned into TOPO TA vector pCR2.1 (Invitrogen). The P gene was then cloned into the cyan fluorescent vector pECFP-C1 (Clontech), and the M2-1 gene was cloned into pTri-ExNeo vector (Novagen). The orientation of the insert was confirmed by sequencing (data not shown). A549 cell monolayers were grown on glass coverslips in 12-well dishes, transfected with 2 g/well plasmid using Lipofectamine 2000 in Opti-MEM (Invitrogen) according to the manufacturer's instructions, and added to antibioticfree media. After 24 h at 37°C, the transfection mixture was removed. The plates were then incubated as described above for a further 24 h. Control wells of transfection only and media only were included to assess any background reactivity. Nuclei were stained with propidium iodide (Invitrogen), and coverslips were mounted onto glass slides with glycerol.