African Swine Fever Virus dUTPase Is a Highly Specific Enzyme Required for Efficient Replication in Swine Macrophages

Abstract

The African swine fever virus (ASFV) gene E165R, which is homologous to dUTPases, has been characterized. A multiple alignment of dUTPases showed the conservation in ASFV dUTPase of the motifs that define this protein family. A biochemical analysis of the purified recombinant enzyme showed that the virus dUTPase is a trimeric, highly specific enzyme that requires a divalent cation for activity. The enzyme is most probably complexed with Mg2+, the preferred cation, and has an apparent Km for dUTP of 1 μM. Northern and Western blotting, as well as immunofluorescence analyses, indicated that the enzyme is expressed at early and late times of infection and is localized in the cytoplasm of the infected cells. On the other hand, an ASFV dUTPase-deletion mutant (vΔE165R) has been obtained. Growth kinetics showed that vΔE165R replicates as efficiently as parental virus in Vero cells but only to 10% or less of parental virus in swine macrophages. Our results suggest that the dUTPase activity is dispensable for virus replication in dividing cells but is required for productive infection in nondividing swine macrophages, the natural host cell for the virus. The viral dUTPase may play a role in lowering the dUTP concentration in natural infections to minimize misincorporation of deoxyuridine into the viral DNA and ensure the fidelity of genome replication.

Complete sequence determination of a novel reptile iridovirus isolated from soft-shelled turtle and evolutionary analysis of Iridoviridae

“…dUTPase is an essential enzyme and plays multiple cellular roles [ 35 ]. In cells infected with Epstein-Barr virus, virally encoded dUTPase homologs function as highly specific enzymes for efficient replication, or serve to upregulate several proinflammatory cytokines [ 36 , 37 ].…”

A Proteomic Atlas of the African Swine Fever Virus Particle

“…Another enzyme that can contribute to ensure the fidelity of genome replication is the viral dUTPase (pE165R), which seems to play a role in lowering the dUTP concentration to minimize misincorporation of deoxyuridine into the viral DNA (59). The viral dUTPase activity has been shown to be dispensable for virus replication in dividing Vero cells but not in nondividing swine macrophages.…”

The Molecular Biology of Frog Virus 3 and other Iridoviruses Infecting Cold-Blooded Vertebrates

“…As with vaccinia virus [76][77][78], iridoviruses that infect ectothermic animals encode a series of proteins whose function is to ensure an adequate supply of nucleotides for viral DNA synthesis. These enzymes include the two subunits of ribonucleotide reductase [79,80], thymidine kinase [30], thymidylate synthase, dUTPase [81], purine nucleoside phosphorylase (PNP), and a dihydrofolate reductase homolog [12,82]. Not all of these putative enzymes are found within each viral species, and it is postulated that specific enzymes might be needed in certain hosts.…”