Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway

de Vries, Erik; Tscherne, Donna M.; Wienholts, Marleen J.; Cobos-Jiménez, Viviana; Scholte, Florine; García-Sastre, Adolfo; Rottier, Peter J. M.; de Haan, Cornelis A. M.

The reporter construct pHH-Gluc was derived from plasmid pHH-Fluc [22] by replacing the firefly luciferase coding region with the Gaussia luciferase coding region of pGluc-basic (New England Biolabs). Unique SpeI and XbaI restriction sites were introduced into pHH-Fluc using the Quikchange XL Site-directed mutagenesis kit (Stratagene) and oligonucleotides Spe4262 (5-9GCCTTTCTTTATGTTTTTGGCACTAGTCATTTTACCG-ATGTCACTCAG), Spe4263 (59-CTGAGTGACATCGGTAA-AATGACTAGTGCCAAAAACATAAAGAAAGGC), Xba4260 (59-GTATTTTTCTTTACAATCTAGACTTTCCGCCCTTC-TTGG) and Xba4261 (CCAAGAAGGGCGGAAAGTCTAG-ATTGTAAAGAAAAATAC). A SpeI site was introduced by sitedirected mutagenesis in pGluc-basic directly following the start codon of the Gaussia luciferase coding sequence. The unique SpeI -XbaI fragment of pGluc-basic was subsequently cloned into the SpeI-XbaI site of pHH-Fluc resulting in plasmid pHH-Gluc.