Abstract
Many viral proteins are related to suppressing apoptosis in target cells and are hence beneficial to viral replication. The V protein of Newcastle disease virus (NDV) is one such protein that plays an important role in inhibiting apoptosis in a species-specific manner. However, to date, there have been no reports clarifying the antiapoptotic mechanisms of the V protein. The present study was undertaken to determine the apoptotic potential of the V protein in a chicken embryo fibroblast cell line (DF-1 cell) and to elucidate its molecular mechanisms of action. Here, a yeast two-hybrid system was used to screen the host proteins that interact with the V protein and identified thioredoxin-like protein 1 (TXNL1) as a potential binding partner. Immuno-colocalization of V protein and TXNL1 protein in DF-1 cells further verified the interaction of the two proteins. Through the overexpression of TXNL1 protein and knockdown of TXNL1 protein in DF-1 cells, the effects of NDV replication and cell apoptosis were examined. Cell apoptosis was detected by flow cytometry. The mRNA and protein expression levels of Bax, Bcl-2 and Caspase-3 were detected by quantitative real-time PCR (Q-PCR) and Western blotting. NDV expression was detected by Q-PCR and plaque assay. The results revealed that the TXNL1 protein induced apoptosis and inhibited NDV replication in DF-1 cells. Furthermore, the Western blot and Q-PCR results suggested that TXNL1 induced cell apoptosis through a pathway involving Bcl-2\\Bax and Caspase-3. Finally, this work provides insight into the mechanism by which the V protein inhibits apoptosis.
Highlights
Newcastle disease (ND) is a severe infectious disease in birds
Further thioredoxin-like protein 1 (TXNL1) was cloned into the pGADT7 vector, and the pGADT7-TXNL1 and pGBKT7-V cotransformants were positive on DDO/X/A and QDO/ X/A plates after incubation (Figure 1A), which indicates that the V protein and TXNL1 have an interactive relationship
To explore the correlativity between the V protein and TXNL1, the expression of endogenous TXNL1 at the mRNA and protein levels was detected after the transfection of pCAGEN-Flag-V in DF-1 cells; the results suggest that the expression of endogenous TXNL1 in these cells was dramatically lower than in the control (Figures 1C and D)
Summary
Newcastle disease (ND) is a severe infectious disease in birds. It is a highly pathogenic disease caused by the Newcastle disease virus (NDV). NDV strains have different levels of virulence among different avian species [2] and can be grouped into three pathotypes, namely, the lentogenic strains, the mesogenic strains and the viscerotropic or neurotropic velogenic strains, based upon the severity of the disease [3]. During P gene transcription, the additional nonstructural (V) protein, which shares a common N terminus with the P gene [6], is produced to help with mRNA editing [7]. The V protein is produced at frequencies of approximately 29% [8]
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