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Production of single-round pseudoviruses

SA Shamim Ahmed
DP Durgadevi Parthasarathy
RN Rachael Newhall
TP Tashina Picard
MA Morgainne Aback
SR Sneha Ratnapriya
WA William Arndt
WV Widaliz Vega-Rodriguez
NK Natalie M. Kirk
YL Yuying Liang
AH Alon Herschhorn
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We produced pseudoviruses as we previously described9,15,34–36. Briefly, a packaging plasmid (psPAX2), a reporter plasmid (pHIVec2.luc), and an Env-expressing plasmid were co-transfected into 293 T cells using effectene transfection reagent (Qiagen). After a 48-h incubation, the cell supernatant was collected and centrifuged for 5 min at 600–900 × g at 4 °C. The amount of p24 in the supernatant was measured using the HIV-1 p24 antigen capture assay (Cat# 5421, Advanced BioScience Laboratories), and the virus-containing supernatant was frozen in single-use aliquots at −80 °C. In some cases, the Env-expressing plasmid was replaced by Env-expressing mRNA that was transfected using Trans-IT (Mirus Bio LLC, Madison, WI) or by the direct addition of mRNA-LNPs, both 24 h post-transfection of the packaging and reporter DNA plasmids.

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