Gene specific primers

Liver and skeletal muscle were collected and stored in RNAlater™ Stabilization Solution (Thermo Fisher Scientific, Waltham, MA, USA) and total RNA was extracted using the Trizol method, as described previously. The transcript levels of target genes and GAPDH were monitored by LightCycler^®480 Real-time PCR (Roche Diagnostics GmbH, Mannheim, Germany) using gene-specific primers (Eurofins, Bangaluru, India) as shown in Table A1. Results are reported as a relative expression normalized with the GAPDH housekeeping gene. The fold variation was determined using the 2^−ΔΔCt method according to previously published protocol [17 (link)].

RNA from uninfected, PR8-infected, and OK11-infected A549 cells was analyzed for interferon (IFN) RNA transcript while using reverse transcription quantitative-PCR with MultiScribe™ Reverse Transcriptase (Thermo Fisher, Waltham, MA) and the Power SYBR™ Green PCR Master Mix (Thermo Fisher), following the manufacturer’s instructions. RT-qPCR product were detected while using an Applied Biosystems 7300 Real-Time cycler with a program of 30 min at 48 °C for reverse transcription, 10 min at 95 °C for DNA polymerase activation, and 40 cycles of 94 °C for 15 s (denaturing), 60 °C for 60 s (annealing and extension). Gene-specific primers (Eurofins Genomics, LLC, Louisville, KY) were as follows: IFN-β F: 5′-GTCTCCTCCAAATTGCTCTC-3′, R: 5′-ACAGGAGCTTCTGACACTGA-3′; IFN-λ1 F: 5′-GGAGTAGGGCTCAGCGCATA-3′, R: 5′-GCCTCCTCACGCGAGACCTC-3′; IFN-λ2 F: 5′-CGTGGGCTGAGGCTGGATAC-3′, R: 5′-TGGCCCTGACGCTGAAGGTT-3′; IL-27 F: 5′-TGGGCTGAGGCTGGATACAG-3′, R: 5′-TCTGGAGGCCACCGCTGACA-3′; IFN-α2 F: 5′-CCTGATGAAGGAGGACTCCATT-3′, R: 5′-AAAAAGGTGAGCTGGCATACG-3′; and 18S rRNA F: 5′-CTTAGAGGGACAAGTGGCG-3′, R: 5′-GGACATCTAAGGGCATCACA-3′. RT-qPCR data were analyzed using the 2^-ΔΔCt method [33 (link)] so that data are normalized to both the uninfected control and a housekeeping gene (18S rRNA) and graphed as relative fold change.

The cloning of NQO1 and the generation of the NQO1 R139W variant as well as the expression and purification was carried out according to the already described procedure from Lienhart and Gudipati et al. [14 (link)]. The wild-type gene of NQO1 in a pET28a vector was modified with the Quick Change II XL Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA) according to the provided manual with gene specific primers from Eurofins (Luxembourg).

The NQO1 gene sequence (UniProtKB/Swiss-Prot: P15559) was codon-optimized for Escherichia coli expression and chemically synthesized (GeneArt, Carlsbad, CA, USA). The NQO1 genes were cloned into the pET28a (Merck, Darmstadt, Germany) vector with Nde1 and Xho1 restriction sites to encode for an N-terminal histidine-tagged fusion protein by the use of gene-specific primers (Eurofins, Luxembourg). NQO1 P187S, NQO1 Δ50 and NQO1 P187S Δ50 were generated with the Quik-Change II XL Site-Directed Mutagenesis Kit (Santa Clara, CA, USA).

Expression of selected genes was quantified in env-reactive CD4⁺ T cells by real-time qRT-PCR. The indicated CD4⁺ T cell populations were purified by cell sorting, and RNA was isolated using the QIAcube (QIAGEN). Synthesis of cDNA was carried out with the High Capacity Reverse Transcription Kit (Applied Biosystems) with an added RNase inhibitor (Promega Biosciences). A final clean-up was performed with the QIAquick PCR Purification Kit (QIAGEN). Purified cDNA was then used as template for the quantitation of the indicated genes using gene-specific primers (Eurofins MWG Operon) (Table S2). Values were normalized and plotted according to the expression of Hprt in the same samples, using a ΔC_T method.