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

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 Mg(2+) 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.

Figure 1

Sequence alignment of the vhs coding regions. The vhs sequences of HSV-1, HSV-2, and PRV were analysed by DNA Star MegaAlign software. Identical residues are denoted as “.” and deleted amino acids are indicate as “-”. Four conserved region, designated as Boxes I–IV (reported in Berthomme et al. [15]) are marked. In addition, several deduced residues, numbered according to the PrV vhs gene, that have been reported as being responsible for ribonuclease activities are indicated with arrowheads.

Figure 2

Expression and purification of recombinant PrV vhs protein using E. coli . The coding region of PrV vhs was inserted downstream of the NUS gene in pET44 vector (A). In this expression system, the recombinant vhs protein was fused with NUS tag protein and a his tag at the N and C terminus, respectively. Under IPTG induction, NUS-vhs-his with a predicted molecular weight of ~110 kDa was expressed (B, lane 2) and then purified using Ni–NTA beads (lane 3). Subsequently, the N-terminal NUS tag was removed by thrombin treatment (lane 4). M protein marker; Lane 1 non-induction lysate. The identity of the PrV vhs was initially confirmed by Western blot analysis using antibodies against the his tag (C). The arrow indicates the uncleaved NUS-vhs-his recombinant protein, and the thin arrowheads indicate the NUS tag and vhs-his after thrombin digestion. The ribonuclease activity of PrV vhs was tested using single stranded RNA (D). RNA substrates generated by in vitro transcription were incubated with assay buffer (mock), NUS protein (as a negative control), or recombinant PrV vhs protein for the indicated times (0, 10, 20, 40, and 60 min). The RNA reaction products were then analyzed by 1.3% agarose-formaldehyde gel electrophoresis and the amount of RNA remaining was measured by imageJ system. The experiment was repeated three times and the RNA content at the time point of 0 h was set to 100% (E).

Figure 3

An in vitro assay of RNase activity mediated by PrV vhs using various substrates. (A) Different types of RNA, including single stranded RNA without cap (ssRNA), with cap and polyA tail (mRNA) and cellular total rRNA, were incubated with assay buffer (mock), recombinant vhs and the appropriate tag proteins, either NUS or Thioredoxin (THX) (negative control), for various times (as indicated above the gel). The degradation pattern was resolved by agarose electrophoresis. In addition to RNA, various DNA substrates (B) and a DNA-RNA hybrid (C) were also tested.

Figure 4

The catalytic factor requirements for RNase activity mediated by PrV vhs. To increase the sensitivity, PrV vhs was synthesized using the TNT^® T7 Quick coupled Translation system (RRL) and simultaneously labeled with S³⁵ (A). (B) Rabbit reticulocyte (RRL) translated PrV vhs protein displays ribonuclease activity in vitro. RNA internally labeled with α-[P³²]-ATP was incubated with only assay buffer (mock control), RRL (as a vhs negative control), or in vitro translated PrV vhs at 37 °C for the indicated times (0, 15, and 30 min). The RNA reaction products were resolved by agarose gel electrophoresis followed by autoradiography. (C) Contribution of positive ions (e.g. Mg²⁺, K⁺) and ATP to PrV vhs mediated RNase activity. To deplete the residual ions in RRL, desalted plain RRL or RRL translated vhs, as indicated by asterisks, were prepared using Sephadex G-25 spin columns. The RNase activities of the desalted lysates were analyzed in assay buffer D or buffer F containing all, or none of the three factors (Mg²⁺, K⁺, ATP), respectively. To identify the requirements in terms of individual co-factor for vhs-dependent RNase activity, the RNA degradation activity of desalted PrV vhs was further analyzed in two systems: either using buffers missing one of the three co-factors, namely buffers B, E, and C without Mg²⁺, K⁺, or ATP, respectively (D), or in buffers containing only one co-factors (buffer F supplemented with Mg²⁺, K⁺, or ATP) (E). To confirm the contribution of Mg²⁺, the divalent chelator EDTA was added to the reaction containing Mg²⁺. Nuclease activity was measured using a Kodak image analyzer system and the amounts of RNA remaining (%) of three independent assays were plotted (F).

Figure 5

The contribution of conserved functional domains to vhs-dependent RNase activity. The locations of the four boxes (I–IV) in PRV vhs are presented (A). Constructs with deletions of one of the PrV vhs boxes were generated and co-transfected with plasmid expressing luciferase in human 293T cells. Cells transfected with empty vector (pcDNA3.1) or HSV-1 vhs (HSV-1) served as negative and positive controls, respectively. The effects of the individual boxes were evaluated using this in vivo system (luciferase reporter assay). The luciferase activities of three independent assays were plotted (B). In addition, the RNA level of the reporter gene was also monitored by Northern blot analysis (upper panel in C). A loading of total RNA of 10 μg per gel lane was used.

Figure 6

Contribution of the fourteen conserved residues present in PrV vhs to RNase activity in vivo. Reporter plasmid, pRluc (encoding Renilla luciferase) was co-transfected with each one of the vhs constructs into human 293T cells. The effect of each vhs protein on reporter gene expression was evaluated based on luciferase activity. HSV-1 vhs (HSV) served as a positive control for PrV vhs-dependent RNase activity. The luciferase activities of three independent assays were plotted (A). A structure indicating the key residues of PrV vhs surrounding the Mg²⁺ in the protein was created by simulation (B).