Bovine viral diarrhea virus E2 targets SLC3A2 to modulate lipid peroxidation for replication advantage

Abstract

The eukaryotic expression plasmids were constructed in this study including pCAGGS-N-HA-SLC3A2, pCAGGS-N-HA-SLC7A11, pCAGGS-SLC3A2(1-146 aa), pCAGGS-N-HA-SLC3A2(147-573 aa), pCAGGS-C-FLAG-E2, pCAGGS-C-FLAG-E2-Dom1, pCAGGS-C-FLAG-E2-Dom2, pCAGGS-C-FLAG-E2-Dom3, pCAGGS-C-FLAG-E2-Dom3-I, pCAGGS-C-FLAG-E2-Dom3-II, pCAGGS-C-FLAG-E2-Dom3-III, pCAGGS-C-FLAG-CSFV-E2, pCAGGS-C-FLAG-BDV-E2. The construction procedures were briefly described as follows: Total RNA was extracted from BVDV-infected MDBK cells with the TransZol Up Kit (ET111-01-V2, TransGen Biotech, Shenzhen, China) and reverse-transcribed into cDNA, and then the BVDV E2 gene was amplified by PCR, while the SLC3A2, CSFV-E2, and BDV-E2 genes were commercially synthesized from TsingKe Co., Ltd. (Wuhan, China). The primers are listed in Table 2.|||2.8. Small interfering RNA (siRNA) transfection MDBK cells were cultured in 12-well plates until cells reached 80 % to 90 % confluence and then were transiently transfected with specific siRNA targeting SLC3A2 (SLC3A2i) and SLC7A11 (SLC7A11i), or NC designed by TsingKe Co., Ltd. (Wuhan, China). Lipofectamine 3000 reagent (Invitrogen, USA) was used for transfection, and Opti-MEM (Thermo Fisher Scientific, USA) was used as medium. In parallel, the NC (siControl nontargeting siRNA) (TsingKe, China) was used as a negative control. At 36 h after transfection, the mRNA expression levels of SLC3A2 and SLC7A11 were measured by RT-qPCR, and at 48 h post-transfection, the protein levels of SLC3A2 and SLC7A11 were determined by Western blot.

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