Roles of nucleic acid substrates and cofactors in the vhs protein activity of pseudorabies virus.

Ya-Fen Liu,Fong-Yuan Lin,Pei-Yun Tsai,Kuan-Hsun Lin,Hui-Wen Lin,Tien-Jye Chang,Wei-Li Hsu,Songkhla Chulakasian

doi:10.1186/s13567-015-0284-y

Ya-Fen Liu, Fong-Yuan Lin + Show 6 more

Open Access

https://doi.org/10.1186/s13567-015-0284-y

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Abstract

Pseudorabies virus (PrV) belongs to the α-herpesvirinae of which human simplex virus (HSV) is the prototype virus. One of the hallmarks of HSV infection is shutoff of protein synthesis that is mediated by various viral proteins including vhs (virion host shutoff), which is encoded by the UL41 gene. However, the function of PrV vhs is poorly understood. Due to the low sequence similarity (39.3%) between the HSV and PrV UL41 proteins, vhs might not share the same biochemistry characteristics. The purpose of this study was to characterize the nuclease activity of the PrV vhs protein with respect to substrate specificity, its requirements in terms of cofactors, and the protein regions, as well as key amino acids, which contribute to vhs activity. Our results indicated that, similar to HSV vhs, PrV vhs is able to degrade ssRNA and mRNA. However, PrV vhs also targeted rRNA for degradation, which is novel compared to the HSV-1 vhs. Activity assays indicated that Mg2+ alone enhances RNA degradation mediated by PrV vhs, while K+ and ATP are not sufficient to induce activity. Finally, we demonstrated that each of the four highly conserved functional boxes of PrV vhs contributes to RNA degradation and that, in particular, residues 152, 169, 171, 172, 173 343, 345, 352 and 356, which are conserved among α-herpesviruses, are key amino acids needed for PrV vhs ribonuclease activity.

Highlights

Pseudorabies virus (PrV), an enveloped dsDNA virus belonging to the α-herpesvirinae, causes Aujeszky’s disease of swine [1]
human simplex virus (HSV) vhs has ribonuclease activity that produces a reduction in overall protein synthesis via mRNA degradation; this effect on the cellular translational apparatus is beneficial in terms of viral protein synthesis [3]
In vitro assay of ribonuclease activity mediated by PrV vhs Ribonuclease activity of a thioredoxin-PrV vhs fusion protein has been previously described; the vhs assay used involved the degradation of RNA markers in reaction buffer at high concentrations of NaCl (0.3 M) and imidazole (0.25 M) [16]

Summary

Introduction

Pseudorabies virus (PrV), an enveloped dsDNA virus belonging to the α-herpesvirinae, causes Aujeszky’s disease of swine [1]. HSV vhs has ribonuclease activity that produces a reduction in overall protein synthesis via mRNA degradation; this effect on the cellular translational apparatus is beneficial in terms of viral protein synthesis [3]. In addition to cellular mRNA, vhs degrades viral RNAs, which facilitates a sharpening of the transitions between the successive phases of viral protein synthesis [3]. The protein’s endonuclease activity was explored using vhs protein that had been translated using a variety of different systems, including rabbit reticulocyte lysate (RRL) [8] and the budding yeast Saccharomyces cerevisiae [9]. Vhs translated using RRL induced degradation of a variety of RNAs including ssRNA and mRNA with a 5′ cap and a 3′poly (A) tail [8].

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