Abstract
E165R, a highly specific dUTP nucleotidohydrolase (dUTPase) encoded by the African swine fever virus (ASFV) genome, is required for productive replication of ASFV in swine macrophages. Here, we solved the high-resolution crystal structures of E165R in its apo state and in complex with its product dUMP. Structural analysis explicitly defined the architecture of the active site of the enzyme as well as the interaction between the active site and the dUMP ligand. By comparing the ASFV E165R structure with dUTPase structures from other species, we found that the active site of E165R is highly similar to those of dUTPases from Mycobacterium tuberculosis and Plasmodium falciparum, against which small-molecule chemicals have been developed, which could be the potential drug or lead compound candidates for ASFV. Our results provide important basis for anti-ASFV drug design by targeting E165R.IMPORTANCE African swine fever virus (ASFV), an Asfivirus affecting pigs and wild boars with up to 100% case fatality rate, is currently rampaging throughout China and some other countries in Asia. There is an urgent need to develop therapeutic and preventive reagents against the virus. Our crystallographic and biochemical studies reveal that ASFV E165R is a member of trimeric dUTP nucleotidohydrolase (dUTPase) family that catalyzes the hydrolysis of dUTP into dUMP. Our apo-E165R and E165R-dUMP structures reveal the constitutive residues and the configuration of the active center of this enzyme in rich detail and give evidence that the active center of E165R is very similar to that of dUTPases from Plasmodium falciparum and Mycobacterium tuberculosis, which have already been used as targets for designing drugs. Therefore, our high-resolution structures of E165R provide useful structural information for chemotherapeutic drug design.
Highlights
E165R, a highly specific dUTP nucleotidohydrolase encoded by the African swine fever virus (ASFV) genome, is required for productive replication of ASFV in swine macrophages
We further determined the value of Michaelis constant (Km), which was 3.788 Ϯ 0.191 m⌴ (Fig. 1D). These results suggested that the purified ASFV E165R protein is a homotrimeric dUTP nucleotidohydrolase (dUTPase) with strict substrate specificity
By comparing the primary sequences of the five motifs from E165R and other trimeric dUTPases, we found that S72 from motif II, D91 from motif III, and Q120 from motif IV, which are responsible for forming hydrogen bonds with dUMP, are highly conserved among dUTPases from different species (Fig. S2)
Summary
E165R, a highly specific dUTP nucleotidohydrolase (dUTPase) encoded by the African swine fever virus (ASFV) genome, is required for productive replication of ASFV in swine macrophages. African swine fever virus (ASFV), the causative agent of African swine fever (ASF), is a large enveloped double-stranded DNA virus that belongs to the Asfarviridae family [1]. The enzyme degrades dUTP in the cytoplasm and minimizes misincorporation of uracil into viral DNA, which plays an essential role in maintaining the fidelity of genome replication It is indispensable for reproductive infection of ASFV in swine macrophages, the natural host cell for the virus [7]. Inhibition of the dUTPase activity can be detrimental to ASFV replication and an effective measure of treating ASFV infection
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