Molecular Characterization of Viral Responsive Protein 15 and Its Possible Role in Nuclear Export of Virus in Black Tiger Shrimp Penaeus monodon

Krisadaporn Jaturontakul,Tomoyuki Mori,Thapanan Jatuyosporn,Premruethai Supungul,Sun-Yong Kim,Pasunee Laohawutthichai,Kuakarun Krusong,Toshio Hakoshima,Anchalee Tassanakajon

doi:10.1038/s41598-017-06653-7

Abstract

A viral responsive protein 15 from Penaeus monodon (PmVRP15) has been reported to be important for white spot syndrome virus (WSSV) infection in vivo. This work aims to characterize PmVRP15 and investigate its possible role in nuclear import/export of the virus. Circular dichroism spectra showed that PmVRP15 contains high helical contents (82%). Analytical ultracentrifugation suggested that PmVRP15 could possibly form oligomers in solution. A subcellular fractionation study showed that PmVRP15 was found in heavy and light membrane fractions, indicating that PmVRP15 may be associated with endoplasmic reticulum. Double-stranded RNAi-mediated knockdown of PmVRP15 gene expression in vitro showed no effect on WSSV copy number in whole hemocyte cells. However, PmVRP15 silencing resulted in an accumulation of WSSV DNA in the nucleus of PmVRP15-silenced hemocytes. Immunofluorescence confocal microscopy showed that PmVRP15 knockdown hemocytes had a much lower level of VP28 (WSSV envelope protein), in comparison to that in the control. It is likely that PmVRP15 may play a role in viral nuclear egress.

Highlights

Despite intensive investigations of white spot syndrome virus (WSSV) infection, the mechanisms of WSSV entry and propagation in shrimp have not yet been fully understood
MALDI-TOF MS analysis showed that the molecular mass of recombinant PmVRP15 was 15,899.9 Da, which corresponded to the calculated MW of PmVRP15 protein (15,859.5 Da), using ExPASy server[23]
Output from the CDSSTR/SMP180 suggested that rPmVRP15 is an α-helical protein containing 82% α-helix, 4% β-strand, 8% turn and 6% unordered

Summary

Introduction

Despite intensive investigations of WSSV infection, the mechanisms of WSSV entry and propagation in shrimp have not yet been fully understood. Several methods including expressed sequenced tag (EST)[7,8,9], DNA microarray[10,11,12,13,14] and proteomic[15,16,17,18,19], have been used to analyse molecular changes during WSSV infection. Suppression subtractive hybridization (SSH) of WSSV-challenged P. monodon hemocytes identified the novel viral responsive protein 15 (PmVRP15) as one of the most highly up-regulated genes in the acute phase of WSSV infection[20]. PmVRP15 knockdown resulted in a significant decrease in viral gene expression and cumulative mortality rate of WSSV-infected shrimp, indicating that PmVRP15 is crucial for WSSV propagation. An endoplasmic reticulum (ER) stress-induced protein, which was reported to be important for the survival of WSSV infected shrimps[21]. A possible role of PmVRP15 in nuclear import/export has been investigated by measuring WSSV copy number in nuclear and cytoplasmic fractions

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Conclusion