Transit mrna transfection kit

PLC/PRF/5, HuH-7 and HuH-7-Lunet BLR cells were seeded at a density of 7*10⁵ cells/T25 flask. PLC/PRF/5 and HuH-7-Lunet BLR were maintained in MEM supplemented with 10 % FCS, 2 mM L-glutamine, 1 % NEAA and 1 % penicillin/streptomycin. HuH-7 cells were cultured in DMEM supplemented as above. All cells were incubated at 37 °C and 5 % CO₂. On the next day, cells showed confluency of 70 % and were transfected with in vitro-transcribed and capped RNA from the genomic plasmids using the TransIT®-mRNA Transfection kit (Mirus Bio LLC, Madison, WI, USA). Equal amounts of transfection reagents and 7.5 µg RNA were mixed in 750 µl OptiMEM and added dropwise to flask with culture medium. One day later, the medium was completely removed and replaced by 5 ml fresh culture medium supplemented with 2.5 µg/ml amphotericin B and 30 mM MgCl₂. Subsequently, the cells were incubated at 34.5 °C as described previously (Schemmerer et al., 2019 (link)). The media were completely substituted with 5 ml fresh media twice weekly and supernatants were stored at −80 °C.

Using the TransIT-mRNA Transfection Kit (Mirus, Madison, WI), 1 × 10⁷ 293T cells were transiently transfected with mRNA (20 µg). The supernatant was collected 48 hours after transfection, centrifuged (500 × g) for 5 minutes, layered on a 25% glycerol/Tris/NaCl/EDTA cushion, and centrifuged (110,000 × g) for 2 hours at 4 °C. Supernatants were aspirated and VLP-containing pellets were resuspended in phosphate-buffered saline. Isolated VLPs were analyzed by electron microscopy or Western blot. The corresponding 293T cells were lysed with RIPA buffer 48 hours after transfection. Protein concentrations were measured using a BCA assay kit (ThermoFisher, Waltham, MA). Western blot, ELISA, and electron microscopy experiments were performed using standard methodologies. For Western blots presented in Figs. 1–3, the molecular weight was estimated using Precision Plus Dual Color Standards (Bio-Rad, cat. no. 1610374, Philadelphia, PA). For Western blots presented in Fig. 6, both MagicMark XP Western Protein Standard (Invitrogen, cat. no. LC5602, Waltham, MA) and Precision Plus Dual Color Standards (Bio-Rad, cat. no. 1610374) were used to obtain reference bands approximately every 5 to 10 kDa to determine the molecular weights of the Western blot bands (Supplementary Figure).

Synthetic mRNAs were generated using mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Invitrogen) using PCR product templates, which were produced by incorporating a T7 promoter sequence into the forward primers. Following ARCA-capped transcription and poly(A) tailing, synthetic RNA was purified and recovered using a QuickRNA miniprep kit (Zymo Research). Transfection of cells with mRNAs was achieved using a TransIT-mRNA transfection kit (Mirus Bio) according to the manufacturer’s instructions.

IAV, PR8/delNS1 (NS1 deletion mutant) was a gift from A. Garcıa-Sastre and P. Palese. ZIKV strain (SZ01) was provided from Shanghai Public Health Clinical Center, Fudan University. ZIKV stocks were propagated in Vero cells after inoculating at a multiplicity of infection (MOI) of 0.01 and harvesting supernatants after 5 days. Sindbis virus (SINV) were rescued from the plasmid of pSVN1, which was gift from Dr. C.M. Rice. Briefly, the plasmids were linearized with XhoI and then SINV genomic RNAs were transcribed in vitro using an SP6 mMESSAGE mMACHINE kit (Ambion). Purified SINV genomic RNAs were transfected into BHK cells by TransIT®-mRNA Transfection Kit (Mirus Bio, WI). Viruses were harvested and tittered as previously described [30 (link)]. PR8/delNS1, ZIKV, SINV titre were 5 × 10⁵, 2 × 10⁶, 1 × 10⁷ plaque-forming units (PFU)/mL respectively.

SINV_GFP, SINV_mC, SINV_B2, SINV_NS1, SINV_NS4A, SINV_NS4B and SINV_capsid viruses were rescued separately from the plasmids of pTE/5′2J/GFP, pTE/5′2J/mC, pTE/5′2J/B2, pTE/5′2J/NS1, pTE/5′2J/NS4A, pTE/5′2J/NS4B and pTE/5′2J/capsid. The pTE/5′2J/GFP (SINV expression EGFP) and pTE/5′2J were gifts from Dr C.M. Rice. In pTE/5′2J, the gene of interest can be inserted at a multiple cloning site (MCS) downstream of the duplicated subgenomic promotor sequence. These plasmids were constructed by ligating PCR products of ZIKV genome RNA (1-376 nt), NoV-B2, IAV-NS1 and ZIKV-NS4A, NS4B, Capsid flanked by XbaI sites at 5′ and ApaI sites at 3′ into the MCS of pTE/5′2J. Briefly, these plasmids were linearized with XhoI and then SINV genomic RNAs were transcribed in vitro using an SP6 mMESSAGE mMACHINE kit (Ambion). Purified SINV genomic RNAs were transfected into BHK cells by TransIT®-mRNA Transfection Kit (Mirus Bio, WI). Viruses were harvested and titred as previously described [30 (link)]. SINV_GFP, SINV_mC, SINV_B2, SINV_NS1, SINV_capsid, SINV_NS4A and SINV_NS4B viruses titre were 5×10⁶, 2×10⁶, 1×10⁶, 1.2×10⁷, 1×10⁷, 2×10⁶, 1×10⁶ PFU/mL, respectively.