The g5R (D250) Gene of African Swine Fever Virus Encodes a Nudix Hydrolase That Preferentially Degrades Diphosphoinositol Polyphosphates

Abstract

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 (Ap 4 A) hydrolases. However, purified recombinant g5R protein (g5Rp) showed a much wider range of nucleotide substrate specificity compared to eukaryotic Ap 4 A hydrolases, having highest activity with GTP, followed by adenosine 5′-pentaphosphate (p 5 A) and dGTP. Diadenosine and diguanosine nucleotides were substrates, but the enzyme showed no activity with cap analogues such as 7mGp 3 A. In common with eukaryotic diadenosine hexaphosphate (Ap 6 A) hydrolases, which prefer higher-order polyphosphates as substrates, g5Rp also hydrolyzes the diphosphoinositol polyphosphates PP-InsP 5 and [PP] 2 -InsP 4 . A comparison of the kinetics of substrate utilization showed that the k cat / K m ratio for PP-InsP 5 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-β-phosphate from both PP-InsP 5 and [PP] 2 -InsP 4 . ASFV infection led to a reduction in the levels of PP-InsP 5 , 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.