Pseudotyped Lentiviruses for Influenza HA

Pseudotyped lentiviruses displaying influenza HAs were produced by transfection of HEK 293 T/17 cells (ECCAC, Public Health England, UK) with 1.0 μg of gag/pol construct, p8.91, 1.5 μg of a luciferase reporter carrying construct, pCSFLW, 250 μg of TMPRSS4-expressing construct and 1.0 μg of HA glycoprotein-expressing construct^{12 (link)}. Transfections were performed in 10 ml of media DMEM 10% FCS, 1% penicillin–streptomycin, 20% L-glutamate and left for 8 hours. One unit of endogenous NA (Sigma, USA) was added to 10 ml of new media to induce virus budding. Media was removed 48 hours post the induction of budding and filtered with a 0.45-μm syringe. The pseudotyped influenza viruses were stored at −80 °C.
The strain accession numbers used for production of the pseudotyped lentiviruses are A/California/04/2009 (AEE69009), A/Solomon Islands/3/2006 (ABU99109), A/USSR/90/1977 (AAA43240), A/Denver/1957 (ABD15258), A/Iowa/1943 (ABO38373), A/PR/8/1934 (CCH23213), A/WSN/1933 (ACF54598) and A/South Carolina/1/1918 (AAC57065).
The p8.91, pCSFLW and TMPRSS4-expressing construct were gifts from Dr Nigel Temperton, whilst the HA-expressing plasmids were either gifts from Dr. Temperton or produced through the cloning of GeneArt Strings (Thermo Fischer Scientific, USA) into the pI.18 expression vector (also a gift from Dr. Temperton). All plasmids are available on request.