Q5 polymerase

Prpk-HA N-terminal fusion was constructed amplifying the coding sequence from genomic DNA using the primers: 5′-CAC​CAT​GTC​CCT​AGA​AA-TCC​TGA​AAC​AAG​G-3′ and 5′-TTA​ACC​AAT​CAT​GGT​TCT​TTT​GCG​TCC-3´. The amplicon was cloned into the p-ENTR/D-TOPO vector (Invitrogen). Afterwards it was subcloned into the pTHW vector through Gateway technology (Invitrogen). A standard germ cell transformation was followed to obtain at least three independent transgenic insertions for each construct (Spradling and Rubin, 1982 (link)).
Wild Type and kinase-dead version of mice PRPK (PRPK and PRPK^KD) were facilitated by the PhD González-Billault group (Villarroel-Campos et al., 2016 (link)). The coding sequences were amplified with the Q5 polymerase (thermofisher) using a forward primer that includes EcoRI cleavage site and a reverse primer that includes a NotI cleavage site.
Fw 5′ GAA​TTC​ACC​ATG​GCT​GGT​GTG​TCC​TCG​GAG​GCG 3’
Rv 5′ GCG​GCC​GCC​TAC​CCG​ACC​ATG​GAC​CGC​TTT​CGC 3’
The amplified fragments were then cloned into the pUAS attB vector, purified and subsequently injected into attP2 strain via the φC31 integrase system by BestGene Inc.

Lentiviral particles encoding the SARS-CoV-2 spike were prepared by transient transfection of HEK293Tn cells using the CaCl2 method. The lentiviral vector pCDH-EF1a-GFP (System Bioscience), the packaging plasmid psPAXII (Addgene), the spike expression vector phCMV-SARS-CoV-2-Spike (a gift from O. Schwartz), and the pRev plasmid (a gift from P. Charneau) were mixed at a 2:2:1:1 ratio and transfected at 252 µg DNA per 175 cm² cell flask. The pQCXIP-Empty plasmid was used as a negative control for spike expression. At 48 h after transfection, supernatants were collected and concentrated by ultracentrifugation at 23,000 × g for 1 h 30 m at 4 °C on a 20% sucrose cushion. Viral particles were resuspended in PBS and frozen in aliquots at −80 °C until use. Gag p24 antigen concentration was measured with the Alliance HIV-1 p24 Antigen ELISA kit (Perkin Elmer).
Point mutations to generate the mutants were introduced by site-directed mutagenesis of phCMV-SARS-CoV-2-Spike using Q5 polymerase (Thermo Scientific) and validated by sanger sequencing. The primers used are listed in the supplementary information.