pSFV1

Among the proteins encoded by hepatitis C virus (HCV), the highly conserved core protein is thought to be involved in the immunomodulatory properties of the hepatitis virus. Recombinant viral vectors that express the HCV core protein and allow its transduction and thus expression of the protein into dendritic cells (DCs) may be useful tools for analyzing core protein properties. Vaccinia viruses and retroviruses have been used to transduce human DCs. Likewise, the use of Semliki Forest Virus to transfer genes into DCs has also been reported. This study aims to use SFV vector to express HCV Core protein in DCs derived from human monocytes, in which the subgenomic RNA encoding the structural protein is replaced by the HCV Core sequence, and then analyze the effect of its expression on DC function. This study demonstrates the use of rSFV to transduce human monocyte-derived DCs. However, even at an MOI of 20, no expression of HCV Core protein was detected. On the other hand, transduction induces activation of DCs, leading to IFN-induced upregulation of MxA protein, which is involved in blocking SFV replication but reducing heterologous protein expression.

In order to express the HCV core protein into human monocyte-derived DCs using recombinant SFV and thus evaluate the impact of this protein on DC function, the genomic region encoding HCV Core protein (aa 1-191) was cloned into SFV plasmid vector pSFV1 under the control of the subgenomic SFV promoter. The researchers first examined HCV Core protein expression after transfection of pSFV1-Core-derived in vitro-transcribed RNA into BHK-21 cells. Forty-eight hours after transfection, cells were tested for Core expression by indirect immunofluorescence using anti-HCV Core mAb B12.F8. Subsequently, HCV core protein expression of recombinant SFV particles was studied in BHK-21 cells. Figure 1b shows immunostaining analysis of infected cells with anti-HCV Core mAb B12.F8 48 hours after transduction. Additionally, cells were harvested at 12, 24, 36, 48, and 72 hours post-infection. The expression of HCV core protein at each time point was analyzed by Western blotting.

Figure 1. Indirect immunofluorescence method was used to detect hepatitis C virus core protein in BHK-21 cells. (a) BHK-21 cells transfected with pSFV1-Core-derived in vitro transcribed RNA, (b) BHK-21 cells infected with rSFV1-Core recombinant particles at MOI 0.1, and (c) BHK-21 control cells. (Navas, Maria-Cristina, et al., 2019)