Pcdna3

The FL coding sequence of the mouse MR (mMR, Nr3c2) was obtained by amplifying the renal cDNA by PCR and inserting it into the pcDNA3.1 vector (Thermo Fisher). Long site-directed mutagenesis of mouse Δ6 MR was performed with the Q5 Site-Directed Mutagenesis kit (New England Biolabs), with specific primers (Supplementary Table S2) designed with the NEBaseChanger website (http://nebasechanger.neb.com/), using the FL mMR-pcDNA3.1 plasmid as the template.

The mammalian expression vector, pcDNA3.1(+) containing tst encoding
gene was purchased from GenRay Biotechnology (China) and the pcDNA3.1(+) (Invitrogen, USA)
plasmid was used in this program as empty plasmid. The recombinant vector
(pcDNA3.1(+)-tst) was digested using the restriction enzymes
NotI and EcoRV (both from New England BioLabs, USA) to
confirm the presence of tst gene in the recombinant plasmid.

To verify the effect of rabbitfish HNF4α on Δ4 Fad promoter activity, over-expression vector pcDNA3.1-HNF4α was constructed with clone vector plasmid pEASY-Blunt-Zero-HNF4α (GenBank: JF502073.1) and expression vector pcDNA3.1 (Invitrogen) double digested by XbaI and EcoRI-HF (New England Bio labs, Ipswich, UK), then with target HNF4α fragments and empty vector pcDNA3.1 ligated by T4 DNA ligase. Constructed vector pcDNA3.1-HNF4α was sequenced (Shanghai Sangon Biotech Co.) for accuracy. Progressive deletion mutants of Δ4 Fad promoter (100 ng/well) and pcDNA3.1-HNF4α (50 ng/well) were co-transfected into HEK293T cell lines seeded in 96-well cell culture plates, while pGL4.75 (0.02 ng/well) was added as an internal control vector. HEK 293T cell was incubated in 100 μl DMEM + 10% FBS and transfected with plasmid complex. Each plasmid complex was transfected in triplicate in three independent experiments. Cell culture medium was replaced with 75 μl DMEM + 10% FBS at 24 h after transfection. Luciferase assays were performed at 48 h after transfection with the Dual-Glo^TM luciferase assay system, and chemical luminescence intensity was detected in duplicate readings using a microplate reader.

Plasmids containing A3H haplotypes I through IV were previously described [16 (link)], and used as templates to generate C-terminally HA-tagged constructs. All constructs used in this study were cloned into pcDNA3.1 (Thermo Fisher, Waltham, MA, USA) using EcoRI/XhoI restriction sites. Mutations were introduced using standard PCR or using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent, 210515, Santa Clara, CA, USA). Double A3H fusion constructs I–I, II–II, I–II, and II–I were previously described [20 (link)]. The double A3H fusion constructs unique to this study, III–III, III–II, and II–III were made through amplification of haplotypes II and III with a 5’ or 3’ primer containing the linker sequence GGT GGT GGT GGT GGC GCC (Gly-Gly-Gly-Gly-Gly-Ala). The 5’ and 3’ haplotype domains were digested using the KasI restriction site in the linker, and EcoRI (for 5’ domains) or XhoI (for 3’ domains). The 5’ domains, 3’ domains, and the EcoRI/XhoI double digested pcDNA3.1 vector were joined using T4 DNA Ligase (New England BioLabs, M0202, Ipswich, MA, USA). Infectivity experiments were performed using the HIV-1 molecular clone pLAIΔenvLuc2Δvif, which was previously described [28 (link)].

The wild-type L-SIGN cDNA with neck 7-tandem repeats (N7) in pcDNA3.1 (Invitrogen, Inc., Carlsbad, CA, USA) was obtained from the original plasmid, pUNO-hDCSIGN2a (InvivoGen, Inc., San Diego, CA, USA). To make an L-SIGN construct with a 9-tandem-repeat allele, the entire exon 4 harboring nine-tandem repeats (N9) was amplified by 3-RACE and nest PCR from complementary DNA from a human carrying the N9 allele. From the 5’ end in exon 1 to the 3’ end of the L-SIGN neck region, the pGlow-TOPO-N9 plasmid was cut and replaced into the pcDNA3.1 plasmid (N9 constructs) using KpnI and XbaI restriction enzymes (New England Biolabs, Inc., Ipswich, MA, USA) and T4 ligase (New England Biolabs, Inc., Ipswich, MA, USA). The plasmid DNA was extracted and subjected to DNA sequencing to ensure that the N7 and N9 constructs had a 2-tandem repeat difference.

The cloning vector containing the gene for the wild-type human dynamin 1 lacking the PRD (UniProt ID: Q05193) was a gift from Katja Fäelber (Structure and Membrane Interaction of G-Proteins, Max-Delbrück-Centrum für Molekulare Medizin) and that for the MBP tag was a gift from Jinwoo Ahn (Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine). The expression vector pcDNA3.1(+) was obtained from Life Technologies (Invitrogen). The dynamin 1 gene and the MBP tag were amplified by polymerase chain reaction (PCR) (Supplementary Table 2) and then subcloned, using the NEBuilder HiFi Assembly kit (New England Biolabs Inc.), into pcDNA3.1(+) that had been linearized by the restriction enzymes Eco RV and Xba I. The resulting insert, designated as MBP-Dyn1ΔPRD, has a leading Kozak sequence, an N-terminal MBP tag, a hexahistidine tag, followed by a human rhinovirus 3 C protease cut site, and the wild-type Dyn1 ΔPRD. Mutations on this construct were introduced by site-directed mutagenesis using overlapping primers. The sequences of the inserts and mutations were confirmed by DNA sequencing (Genewiz Inc.).