Abstract
During viral infections, some viruses subvert the host proteins to promote the translation or RNA replication with their protease-mediated cleavage. Poly (A)-binding protein (PABP) is a target for several RNA viruses; however, the impact of duck hepatitis A virus (DHAV) on PABP remains unknown. In this study, we demonstrated for the first time that DHAV infection stimulates a decrease in endogenous PABP and generates two cleavage fragments. On the basis of in vitro cleavage assays, an accumulation of PABP cleavage fragments was detected in duck embryo fibroblast (DEF) cell extracts incubated with functional DHAV 3C protease. In addition, DHAV 3C protease was sufficient for the cleavage of recombinant PABP without the assistance of other eukaryotic cellular cofactors. Furthermore, using site-directed mutagenesis, our data demonstrated a 3C protease cleavage site located between Q367 and G368 in duck PABP. Moreover, the knockdown of PABP inhibited the production of viral RNA, and the C-terminal domain of PABP caused a reduction in viral replication compared to the N-terminal domain. Taken together, these findings suggested that DHAV 3C protease mediates the cleavage of PABP, which may be a strategy to manipulate viral replication.
Highlights
In eukaryotes, the 5′-terminal cap structure is necessary to initiate translation
Experiments were performed in duck embryo fibroblast (DEF) cells to investigate whether there were any detectable changes in native Poly (A)-binding protein (PABP) or any other detectable changes during duck hepatitis A virus (DHAV) infection, as translational control pathways have been reported to be changed or dysregulated in some established cell lines[32]
The expression level of 3C protease increased over time in DEF cells infected with DHAV, while the β-actin levels remained relatively stable during infection (Fig. 2a and b)
Summary
The 5′-terminal cap structure is necessary to initiate translation. During translation initiation, the m7G (5′) ppp (5′) N structure is first recognized by translation eukaryotic initiation factor 4 F (eIF4F). The band intensities representing PABP protein expression levels were quantitated using the β-actin control bands as a reference (for each time point) using the Image J software. These proteases in different picornaviruses exhibit varying effects on the same cellular protein. For duck hepatitis A virus (DHAV), intact eIF4G was reported to be essential for the internal initiation of translation[12]. As another central regulator, the multifunctional PABP is a target for RNA viruses. The relationship between PABP and DHAV is completely unknown
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