RESUMO
Natural killer enhancing factor (NKEF) belongs to the peroxiredoxin family of proteins, a group of antioxidants that has been extensively studied in mammals. Recently, we identified NKEF in the immunoprecipitated proteome of rainbow trout red blood cells (RBCs) exposed to viral hemorrhagic septicemia virus (VHSV). In the present study, we evaluated the role of NKEF in the antiviral response of rainbow trout against VHSV by examining the expression profile of NKEF in VHSV-exposed RBCs and rainbow trout gonad-2 (RTG-2) cell line. We found an in vitro correlation between decreased VHSV replication and increased NKEF expression after RBCs were exposed to VHSV, however this was not found in RTG-2 cells where the infection highly increased and nkef transcripts remained almost unchanged. In addition, siRNA silencing of the nkef gene in rainbow trout RBCs and RTG-2 cells resulted in increased VHSV replication. We also found a correlation between nkef gene silencing and a decrease in the expression of genes related to type 1 interferon (IFN1) pathway. These findings indicated that NKEF is involved in the antiviral mechanisms of rainbow trout RBCs against VHSV and thus support its antiviral role and implication in the modulation of their immune response. Finally, overexpression of NKEF in an EPC cell line significantly reduced VHSV infectivity and was coupled to an increment in IFN1-related genes. In conclusion, NKEF may be a potential target for new therapeutic strategies against viral infections.
RESUMO
Viral hemorrhagic septicemia virus (VHSV) infection appears to be halted in rainbow trout nucleated red blood cells (RBCs). Diverse mechanisms are thought to be related to the antiviral immune response of rainbow trout RBCs to VHSV. However, the specific rainbow trout RBC proteins that interact directly with VHSV are still unknown. In an attempt to identify VHSV-RBC protein interactions, we characterized the immunoprecipitated (IP) proteome of RBCs exposed to VHSV using an antibody against the N protein of VHSV. The IP proteomic characterization identified 31 proteins by mass spectrometry analysis. Among them, we identified interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), a protein belonging to a family of proteins that are induced after the production of type I interferon. Importantly, IFIT5 has been implicated in the antiviral immune response. We confirmed the participation of IFIT5 in the rainbow trout RBC antiviral response by examining the expression profile of IFIT5 in RBCs after VHSV exposure at transcriptional and protein levels. We detected a correlation between the highest IFIT5 expression levels and the decline in VHSV replication at 6 h post-exposure. In addition, silencing ifit5 resulted in a significant increase in VHSV replication in RBCs. Moreover, an increase in VHSV replication was observed in RBCs when the IFIT5 RNA-binding pocket cavity was modulated by using a natural compound from the SuperNatural II database. We performed a proximity ligation assay and detected a significant increase in positive cells among VHSV-exposed RBCs compared to unexposed RBCs, indicating protein-protein colocalization between IFIT5 and the glycoprotein G of VHSV. In summary, these results suggest a possible role of IFIT5 in the antiviral response of RBCs against VHSV.