Quantification of Viral RNA and DNA

One aliquot of nucleic acids extracted from virions was saved to quantitate gRNA. To this end, RNA were incubated with RQ1 DNase (Promega) in presence of RNaseOUT (Invitrogen) during 25 min at 37 °C and extracted with phenol-chloroform then chloroform and finally precipitated with ethanol 100% and washed with ethanol 70%. RNA pellets were dissolved in water and quantitated by measuring optical absorption. Intravirion RNA were reverse transcribed using an oligo(dT) primer with the Expand RT (Roche). A control experiment was systematically performed without RT to control the absence of DNA contamination as previously described66 (link). To monitor viral RNA and MS cDNA, quantitative PCR assay was achieved with 125 ng of tRNA-equivalent virions or 125 ng of cellular DNA samples extracted from cells transfected with either wt or mutant pNL4-3 plasmids, or with empty plasmid as controls (mock). The qPCR was achieved with the SYBR Green kit (Roche) using a RotorGene (Labgene) system. A standard curve was generated from 10² to 10⁶ copies of pNL4-3 plasmid. Nucleic acid level in virions and producer cells was normalized with respect to CAp24 protein (determined by ELISA) and GAPDH gene, respectively25 (link)26 (link). Sequences of primers and detailed PCR conditions were the same as previously used25 (link)26 (link) and will be provided on request.