Pmcherry n1

The Nef proteins from the HIV-1 NL4-3 strain and from three SIV strains (cpz GAB1, mac239 and smm FWR1), tagged with a C-terminal FTS tag, were cloned into a pcDNA5/FRT/TO Dox-inducible plasmid. The human ATG9A, myrlysin (also known as LOH12CR1 and BORCS5), and lyspersin (also known as C17orf59 and BORCS6), and the HIV-1 pNL4-3 Nef cDNAs were cloned into a pcDNA3.1 plasmid encoding a single C-terminal FTS tag. The cDNAs encoding human ATG9A and SERINC5, and Nef from pNL4-3, were cloned into pEGFP-N1 (Clontech). Nef from pNL4-3, was cloned into pmCherry-N1 (Clontech). The full-length infectious HIV-1 molecular clone pNL4-3 [82 (link)], and its Nef-defective (pNL4-3 ΔNef) [83 (link)] and Env-defective (pNL4-3/KFS) [84 (link)] derivatives have been described previously. The plasmid pHCMV-G [85 (link)], encoding the G glycoprotein of vesicular stomatitis virus (VSV) was kindly provided by Dr. Jane Burns (University of California, San Diego, La Jolla, CA). All mutations were generated by site directed mutagenesis (QuikChange, Agilent, or Q5^® Hot Start High-Fidelity 2X, NEB) and confirmed by DNA sequencing.

The gM (GP100) and gN (GP73) ORFs were separately cloned into expression vectors that also tagged the C-terminal domain (GFP for gM, mCherry or FLAG for gN). For GFP tagged gM, the GP100 ORF was PCR cloned as a BamH I (5’) EcoR I (3’) fragment lacking a stop codon into pAcGFP-N1 (Clontech) using primers gMTagF and gMTagR. For mCherry tagged gN, The GP73 ORF minus a stop codon was similarly PCR cloned as a BamH I (5’) EcoR I (3’) fragment into pmCherry-N1 (Clontech) using primers gNTagF and gNTagR. For FLAG tagged gN, both full length (codons 1–132) and a N terminal truncated (codons 40–132) GP73 ORF were PCR cloned separately (lacking a stop codon) into the expression vector pCMV3Tag8 (Agilent technologies Inc.) as BamH I / Hind III fragments to C-terminal epitope tag the ORFs using forward primers FGP73BmShort for truncated GP73 or FGP73BmFull for full length GP73 plus reverse primer RGP73nostopHd (S1 Table). Recombinant plasmids were designated pgN(f)FLAG and pgN(s)FLAG for full length and truncated expression plasmids respectively.

All GFP-tagged constructs were made in the pEGFP-N1 or pEGFP-C1 vectors (Clontech). The N-terminal fragment (AA 1–96) of human Sec61ß was cloned by PCR amplification using primers 5’-CTGGAAGATCTATGCCTGGTCCGACCC-3’ and 5’-CTGGAGGTACCAGCGAACGAGTGTACTTGCCC-3’. The PCR products were then digested by BglII and KpnI (Takara), and ligated into pmCherry-N1 (Clontech) using the T4-DNA Ligase (Roche). The ACC-GFP construct was already described in [30 (link)]. Cloning of the AH of Sar1 or the ALPS motifs of Nup133 and GMAP210 in the expression vectors was done by insertion of the sequences into the appropriate vectors by mutagenesis (Quickchange Lightning Site-directed mutagenesis kit, Agilent technologies) following the protocol described by Geiser and collaborators [51 (link)]. S1 Table summarizes the details of all constructs made in this study. All point mutants were obtained using a site-directed mutagenesis kit (Quickchange Lightning Site-directed mutagenesis kit, Agilent technologies). All constructs were verified by DNA sequencing.

The WNV_Kunjin replicon was constructed based on the WNV_Kunjin sequence (accession number AY274504), and the LGTV replicon was constructed based on the LGTV strain TP21 sequence (accession number NC003690). The constructs are driven by a T7 promoter for in vitro transcription to express the replicons as described in (28 (link)) (Fig. 3A).
The TBEV NS3 and NS5 (Torö-2003, AH013799) genes were amplified by PCR with primers introducing suitable endonuclease restriction sites. Full-length NS5 and NS3 were cloned into the mammalian cell expression pKH3, which were kindly provided by Ian Macara and Ben Margolis, to have a triple HA tag at the N terminus. NS3 mutations were generated by PCR of the HA-NS3 construct or the replicon construct (35 (link)) with 5′ phosphorylated primers introducing mutated sites, followed by a ligation. The product was treated with restriction enzyme DpnI (Thermo Scientific) to remove the methylated plasmid PCR template.
The TBEV_Toro anchored prM-E (Torö-2003, AH013799) genes were amplified by PCR with primers introducing suitable endonuclease restriction sites. Full-length anchored prM-E was cloned into the mammalian cell expression pmCherry N1 (Clontech) with the mCherry tag at the C terminus.
Renilla luciferase expressing construct (pGL4.74, Promega) was used for normalizing transfections of TBEV_Toro DNA replicons.