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. 2002 Feb;76(3):1415-21.
doi: 10.1128/jvi.76.3.1415-1421.2002.

The g5R (D250) gene of African swine fever virus encodes a Nudix hydrolase that preferentially degrades diphosphoinositol polyphosphates

Jared L Cartwright  1 , Stephen T Safrany, Linda K Dixon, Edward Darzynkiewicz, Janusz Stepinski, Richard Burke, Alexander G McLennan
Affiliations

The g5R (D250) gene of African swine fever virus encodes a Nudix hydrolase that preferentially degrades diphosphoinositol polyphosphates

Jared L Cartwright et al. J Virol. 2002 Feb.

Erratum in

  • J Virol 2002 Jul;76(13):6864

Abstract

The African swine fever virus (ASFV) g5R gene encodes a protein containing a Nudix hydrolase motif which in terms of sequence appears most closely related to the mammalian diadenosine tetraphosphate (Ap4A) hydrolases. However, purified recombinant g5R protein (g5Rp) showed a much wider range of nucleotide substrate specificity compared to eukaryotic Ap4A hydrolases, having highest activity with GTP, followed by adenosine 5'-pentaphosphate (p5A) and dGTP. Diadenosine and diguanosine nucleotides were substrates, but the enzyme showed no activity with cap analogues such as 7mGp3A. In common with eukaryotic diadenosine hexaphosphate (Ap6A) hydrolases, which prefer higher-order polyphosphates as substrates, g5Rp also hydrolyzes the diphosphoinositol polyphosphates PP-InsP5 and [PP]2-InsP4. A comparison of the kinetics of substrate utilization showed that the k(cat)/K(m) ratio for PP-InsP5 is 60-fold higher than that for GTP, which allows classification of g5R as a novel diphosphoinositol polyphosphate phosphohydrolase (DIPP). Unlike mammalian DIPP, g5Rp appeared to preferentially remove the 5-beta-phosphate from both PP-InsP5 and [PP]2-InsP4. ASFV infection led to a reduction in the levels of PP-InsP5, ATP and GTP by ca. 50% at late times postinfection. The measured intracellular concentrations of these compounds were comparable to the respective K(m) values of g5Rp, suggesting that one or all of these may be substrates for g5Rp during ASFV infection. Transfection of ASFV-infected Vero cells with a plasmid encoding epitope-tagged g5Rp suggested localization of this protein in the rough endoplasmic reticulum. These results suggest a possible role for g5Rp in regulating a stage of viral morphogenesis involving diphosphoinositol polyphosphate-mediated membrane trafficking.

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Figures

FIG. 1.
FIG. 1.
Partial sequence alignment of the region of virally encoded Nudix hydrolases surrounding the Nudix motif. Alignments were performed with the CLUSTAL X program. Residues that are identical or similar in more than half the sequences are shaded black and gray, respectively. The conserved amino acids of the Nudix motif and the downstream tyrosine residue conserved in Ap4A hydrolases are shown in boldface above the partial human Ap4A hydrolase sequence (U is any hydrophobic aliphatic amino acid). Viral sequences are arranged into three groups: the iridovirus and ASFV sequences, followed by the vaccinia virus D9-related sequences, followed by the vaccinia virus D10-related sequences. Abbreviations: Ap4Ase, Ap4A hydrolase; CIV, Chilo iridescent virus; LDV, lymphocystis disease virus; FPV, fowlpox virus; Yb, Yaba monkey tumor virus; MCV, molluscum contagiosum virus; RFV, rabbit fibroma virus; My, myxoma virus; MSV, Melanoplus sanguinipes entomopoxvirus.
FIG. 2.
FIG. 2.
Expression and purification of recombinant g5R protein in E. coli analyzed by SDS-PAGE. Lane 1, molecular weight markers. Samples of soluble protein were extracted from E. coli transformed with pET-g5R and induced with 0.5 mM IPTG for 0 h (lane 2), 1 h (lane 3), 2 h (lane 4), and 3 h (lane 5). Lane 6 shows a sample of recombinant g5R protein after purification on Ni2+-NTA-agarose.
FIG. 3.
FIG. 3.
Nucleotide substrate utilization by g5Rp. Rates of hydrolysis were determined from a 50-μl assay sample by HPLC at a fixed substrate concentration of 200 μM with 2.1 μg of enzyme protein as described in Materials and Methods and are expressed relative to the rate of GTP hydrolysis. The 100% value corresponds to 0.2 μmol/min/mg of protein. Values are the means of duplicate determinations with a difference between each determination and the mean of ≤5%.
FIG. 4.
FIG. 4.
Identification of the products of hydrolysis of a selection of substrates by g5Rp. Rates of hydrolysis were determined by HPLC at a fixed substrate concentration of 200 μM as described in Materials and Methods. The substrates were ATP (a), p4A (b), p5A (c), Gp3G (d), Gp4G (e), and Gp5G (f). The absorbance profiles of control incubations without enzyme are offset by −0.02 for clarity. Curves: with enzyme (solid line), without enzyme (dotted line), and gradient (dashed line).
FIG. 5.
FIG. 5.
Activity of g5Rp toward [PP]2-InsP4 in vitro. Activity was measured by incubation of g5Rp with [3H][PP]2-InsP4 for 30 min and analysis of the products by HPLC as described in Materials and Methods (•). The corresponding HPLC profile from a zero time control is also shown (○), as are the relative elution profiles of authentic InsP6 and 5-PP-InsP5 standards (dotted line).
FIG. 6.
FIG. 6.
Localization of an HA epitope-tagged g5R protein in ASFV-infected cells. A plasmid containing the g5R gene fused to a C-terminal sequence encoding the HA epitope tag and expressed under the control of its own ASFV promoter was transfected into Vero cells that were infected with the BA71V isolate of ASFV. Cells were fixed in paraformaldehyde at 10 h postinfection, permeabilized with 0.2% Triton X-100, stained with rat anti-HA monoclonal antibody (1 in 800 dilution) and goat anti-rat Alexa Fluor 568 conjugate secondary antibody (1 in 800 dilution), and then visualized by confocal microscopy. The two panels show different representative cells. Bars, 10 μm.
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