Abstract
The molecular functions of betanodavirus non-structural protein B and its role in host cell survival remain unclear. In the present study, we examined the roles of specific nuclear targeting domains in B1 localization as well as the effect of B1 nuclear localization on the cell cycle and host cell survival. The B1 protein of the Red spotted grouper nervous necrosis virus (RGNNV) was detected in GF-1 grouper cells as early as 24 hours post-infection (hpi). Using an EYFP-B1 fusion construct, we observed nuclear localization of the B1 protein (up to 99%) in GF-1 cells at 48 hpi. The nuclear localization of B1 was mediated by two arginine-rich nuclear targeting domains (B domain: 46RRSRR51; C domain: 63RDKRPRR70) and domain C was more important than domain B in this process. B1 nuclear localization correlated with upregulation of p53 and p21(wef1/cip1); downregulation of Cyclin D1, CDK4 and Mdm2; and G1/S cell cycle arrest in GF-1 cells. In conclusion, nuclear targeting of the RGNNV B1 protein via two targeting domains causes cell cycle arrest by up-regulating p53/p21 and down-regulating Mdm2, thereby regulating host cell survival.
Highlights
RNA viruses belonging to the Nodaviridae family are classified as Alphanoviruses, which primarily infect insects and Betanoviruses, which predominantly infect fish[1,2,3]
B1 protein expression in Red spotted grouper nervous necrosis virus (RGNNV)-infected cells at 24 hpi was mainly localized to the cytoplasm (100%) partially to the nucleus, in up to 45% of cells (Fig. 1b, e–h; indicated by white arrows; Fig. 1c), whereas at 48 hpi, B1 expression was mainly detected in the cytoplasm (100%) and targeting to nucleus in up to 95% of cells (Fig. 1b, i–l; indicated by the red arrow; Fig. 1c)
Our present study identified a 7 amino-acid arginine-rich signal peptide (RDKRPRR) that targeted the non-structural RGNNV B1 protein to the nucleus of infected GF-1 cells
Summary
RNA viruses belonging to the Nodaviridae family are classified as Alphanoviruses, which primarily infect insects and Betanoviruses, which predominantly infect fish[1,2,3]. The B1 gene of the Red spotted grouper nervous necrosis virus (RGNNV) betanodavirus strain has recently been shown to have an anti-necrotic function during early replication[8], whereas the B2 gene has been found to either suppress host siRNA silencing[9,10,11] or play a role in necrosis. Many viruses facilitate their own replication by modulating the host cell cycle. We hypothesized that betanodavirus infection may affect the cell cycle in a manner separate from induction of apoptosis
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