Abstract
Betanodavirus infection induces viral nervous necrosis (VNN) in fish. However, the role of cell death and autophagy in the pathogenesis of VNN remains unknown. This study aimed to investigate the effect of red-spotted grouper nervous necrosis virus (RGNNV) infection on Bcl2 downregulation and overexpression on asymmetric interaction between cell death and autophagy. The mRFP-LC3 reporter system was used to identify autophagosome formation in GF-1 (Grouper fin-1) fish cells. We found that the RGNNV could strongly induce autophagosome formation 36 h post-infection (hpi) after autophagy inhibitor 3-MA had downregulated anti-apoptotic genes such as Bcl2 and Bcl2L1 (Bcl-xL). We proposed that the overexpression of Bcl2 and Bcl2L1 can modulate both cell death and autophagy. Then, we found that it can also reduce either type III cell death or autophagy, which are mildly correlated with reduced viral replication. Our data suggest that RGNNV-induced Bcl2 downregulation correlates with the asymmetrical interaction between cell death induction and the autophagy process, which resembles viral replication.
Highlights
Betanodaviruses are the causative agents of viral nervous necrosis (VNN), an infectious neuropathological condition characterized by necrosis of the brain and retina in fish [1]
Results from the ANXA5/annexin A5 and Propidium Iodide (PI) staining analysis indicated that Bcl2 and Bcl2L1 overexpression significantly abolished red-spotted grouper nervous necrosis virus (RGNNV)-induced cell death (Figure 4)
Bcl2 and Bcl2L1 overexpression in cells significantly inhibited RGNNVinduced the expression of viral death factors (B2 and protein α) by about 25% (Figure 3C) at 48 h and decreased viral titers (Figure 5) at 24 h (~0.5-fold) and 48 hpi (~0.3-fold)
Summary
Betanodaviruses are the causative agents of viral nervous necrosis (VNN), an infectious neuropathological condition characterized by necrosis of the brain and retina in fish [1]. RNA1 encodes a 110 kDa protein acting on an RNA-dependent RNA polymerase (protein A) for replication of the viral genome. RNA2 encodes a 42 kDa capsid protein [4,6] that can trigger post-apoptotic necrotic cell death through a cytochrome c release-mediated pathway in GF-1 cells [7]. In the middle–late replication stage, betanodaviruses were shown to synthesize a sub-genomic RNA3 from the 3 terminus of RNA1 during genome replication, which encodes two non-structural proteins, B1 (absent in some strains) [8] and B2 (as a death gene) [1,9]
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