Fugene six transfection reagent

Lentiviruses were produced in HEK293T cells (catalog number CRL-1573; ATCC, Manassas, VA, US) by contransfection of pFUGW transfer vectors and three packaging plasmids (pCMV-VSV-G, pMDLg/pRRE, pRSV-Rev) using Fugene six transfection reagent (catalog number E2692; Promega, Madison, WI, US). The supernatants of the cultures were collected 72 hr after the transfection and clarified by centrifugation (2000 rpm 15 min), and subsequently used for infection of DIV four hippocampal neurons. All experiments were performed on 16–20 DIV cultures when synapses were mature and lentiviral expression of constructs of interest was optimal (Mozhayeva et al., 2002 (link); Deák et al., 2006 (link)). All experiments were performed following protocols approved by the UT Southwestern Institutional Animal Care and Use Committee.

Huh7 ‘donor’ cells (2.5x10⁵ cells/well seeded in six-well tissue culture dishes 24 hr prior to transfection) were co-transfected using FuGENE six transfection reagent (Promega) with 3 μg of pT7HB2.7- wt or mutated glycoproteins and 50 ng of pLTR-luc reporter plasmid. For a positive control, cells were co-transfected with 3 μg of either pCAGGS-GP/wt-M, expressing CCHFV GPs, or 1 μg of phCMV-VSV-G and with 50 ng of the pLTR-luc plasmid. For negative controls, cells were co-transfected with 2 μg of an empty phCMV plasmid and 50 ng of the pLTR-luc plasmid. 12 hr later, transfected cells were detached with Versene (0.53 mM ethylenediaminetetraacetic acid (EDTA); Gibco), counted, and reseeded at the same concentration (10⁵ cells/well) in 12-well plates. Huh7-tat or Huh7-NTCP-tat indicator cells, detached with EDTA and washed, were then added to the transfected cells (3x10⁵ cells per well). After 24 hr of cocultivation, the cells were washed with phosphate-buffered saline (PBS), incubated for 3 min in fusion buffer (130 mM NaCl, 15 mM sodium citrate, 10 mM MES [2-(N-morpholino)ethanesulfonic acid], 5 mM HEPES) at pH 4, pH 5, or pH 7, and then washed three times with normal medium. The luciferase activity was measured 24 hr later using a luciferase assay kit according to the manufacturer’s instructions (Promega).

Drosophila S2 cells (S2- DGRC) were obtained directly from the Drosophila Genomics Resource Center (DGRC) and regularly confirmed to be free of contamination (e.g. mycoplasma) through PCR-based tests as recommended by the NIH. The S2 cells were cultured in Drosophila Schneider's medium supplemented with 10% of fetal bovine serum (Omega Scientific) and 1% Penicillin-Streptomycin-Glutamine (Thermo Fisher) at 25°C in a humidified incubator. Transfection was performed with FuGENE six transfection reagent (Promega). Expression constructs of GFP, mCherry, HhN, Hh, Ihog, and Ihog-YFP used in Drosophila cell culture were cloned into pAcSV plasmid as previously described (Wu et al., 2019 (link)).