In fusion cloning kit

HEK 293 cells (ATCC^® CRL-1573^™) and 293T cells (ATCC^® CRL-3216^™) were grown in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum (FCS). Cells were incubated in a humidified 5% CO₂ incubator at 37°C. The cDNA encoding the mlEFL35 ORF was synthesized and cloned into a pUCFa vector (FASMAC). The cDNA encoding mlEFL35p fused to an HA or FLAG tag at the N terminus was cloned into the mammalian expression vector pCAGGS [55 (link)] using an In-Fusion cloning kit (BD Clontech). In a similar way, the expression plasmids for N-terminally HA- or FLAG-tagged VP35 and NP of an EBOV isolate, Mayinga (species Zaire ebolavirus) or a RESTV isolate, Pennsylvania (species Reston ebolavirus) were constructed. The ORF of the NS1 protein of influenza A virus (strain PR8) with a splice acceptor site mutation [56 (link)] was C-terminally fused with an HA tag and cloned into pCAGGS. The EBOV minigenome plasmid containing the firefly luciferase gene, p3E5E-luc [39 (link)], was also synthesized and cloned into a pUCFa vector (FASMAC). The NP, VP35, VP30, VP24, and L genes of EBOV (Mayinga) were similarly cloned into pCAGGS. Expression vectors used to provide human IPS-1 have been described previously [57 (link)]. The human TBK1 gene was also cloned into pCAGGS.

We used the nucleotide sequences of NPC1 genes derived from SuBK12-08, YubFKT1, BKT1, FBKT1, ZFBK13-76E, ZFBK11-97, ZFBK15-137RA, and DemKT1, which were determined previously (GenBank accession numbers; LC462997, LC462271, LC462998, LC462999, LC462993, LC462994, LC462995, and LC462996, respectively [20 (link)]). Total RNA was extracted from SuBK12-08 cells using ISOGEN (Nippongene) and mRNA was reverse transcribed with Superscript IV (Invitrogen). The NPC1 gene of SuBK12-08 was amplified with KOD One and inserted into a pSP72 (Promega, Madison, WI, USA) plasmid vector. After sequence confirmation, domain C of the NPC1 gene fragment derived from SuBK12-08 cells (amino acid residues 377–624 [373–620 in HEK293T NPC1 numbering]) and the NPC1 gene fragment derived from HEK293T cells (amino acid residues 1–372 and 621–1279) were amplified with KOD One using specific primers with the HA tag sequence. Then these NPC1 gene fragments were inserted into the pMXs-puro retroviral vector (Cell Biolabs. San Diego, CA, USA) to construct pMXs-chimeric HEK293T/SuBK12-08 NPC1. An In-Fusion cloning kit (BD Clontech) was used to construct the retroviral vectors carrying NPC1 genes. The plasmids of mutant NPC1 genes were constructed by site-directed mutagenesis with KOD One. After sequence confirmation, these mutant genes were inserted into the retroviral vector.