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Crystal Structure of African Swine Fever Virus dUTPase Reveals a Potential Drug Target.

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.

SUBMITTER: Li C

PROVIDER: S-EPMC6819664 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Crystal Structure of African Swine Fever Virus dUTPase Reveals a Potential Drug Target.

Li Changyao C Chai Yan Y Song Hao H Weng Changjiang C Qi Jianxun J Sun Yeping Y Gao George F GF

mBio 20191029 5

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 structu ...[more]

PMID: 31662460

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Project description:The African swine fever virus (ASFV) is the deadly pathogen of African swine fever (ASF) that induces high mortality, approaching 100% in domestic pigs, causes enormous losses to the global pig industry, and threatens food security. Currently, there is no effective treatment or preventive countermeasure. dUTPases (deoxyuridine 5'-triphosphate pyrophosphatases) are ubiquitous enzymes that are essential for the hydrolysis of dUTP and prevent the misincorporation of dUTP into newly synthesized DNA. Here, we present the crystal structures of the ASFV dUTPase in complex with the product dUMP and cofactor Mg2+ at a resolution of 2.2 Å. We observed that a unique "turning point" at G125 plays an unexpected critical role in the swapping region of the C-terminal segment, which is further stabilized by the interactions of the last C-terminal ? strand with the ?1 and ?2 strands, thereby positioning the catalytic motif 5 into the active site of its own subunit instead of into a third subunit. Therefore, the ASFV dUTPase employs a novel two-subunit active site that is different than the classic trimeric dUTPase active site, which is composed of all three subunits. Meanwhile, further results confirmed that the configuration of motifs 1 to 5 has high structural homology with and a catalytic mechanism similar to that of the known trimeric dUTPases. In general, our study expands the information not only on the structural diversity of the conserved dUTPase family but also on the details needed to utilize this dUTPase as a novel target in the treatment of ASF.IMPORTANCE African swine fever virus (AFSV), a large enveloped double-stranded DNA virus, causes a deadly infection in domestic pigs. In addition to Africa, Europe, and South America, countries in Asia, such as China, Vietnam, and Mongolia, have suffered the hazards posed by ASFV outbreaks in recent years. Until now, there has been no vaccine for protection from ASFV infection or effective treatments to cure ASF. Here, we solved the crystal structure of the ASFV dUTPase-dUMP-Mg2+ complex. The ASFV dUTPase displays a noncanonical folding pattern that differs from that of the classic homotrimeric dUTPase, in which the active site is composed of two subunits. In addition, several nonconserved residues within the 3-fold axis channel play a vital role in ASFV dUTPase homotrimer stability. Our finding on these unique structural features of the ASFV dUTPase could be explored for the design of potential specific inhibitors that target this unique enzyme.

Project description:African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs that is responsible for serious economic and production losses. It is caused by the African swine fever virus (ASFV), a large and complex icosahedral DNA virus of the Asfarviridae family. Currently, there is no effective treatment or approved vaccine against the ASFV. pS273R, a specific SUMO-1 cysteine protease, catalyzes the maturation of the pp220 and pp62 polyprotein precursors into core-shell proteins. Here, we present the crystal structure of the ASFV pS273R protease at a resolution of 2.3 Å. The overall structure of the pS273R protease is represented by two domains named the "core domain" and the N-terminal "arm domain." The "arm domain" contains the residues from M1 to N83, and the "core domain" contains the residues from N84 to A273. A structure analysis reveals that the "core domain" shares a high degree of structural similarity with chlamydial deubiquitinating enzyme, sentrin-specific protease, and adenovirus protease, while the "arm domain" is unique to ASFV. Further, experiments indicated that the "arm domain" plays an important role in maintaining the enzyme activity of ASFV pS273R. Moreover, based on the structural information of pS273R, we designed and synthesized several peptidomimetic aldehyde compounds at a submolar 50% inhibitory concentration, which paves the way for the design of inhibitors to target this severe pathogen.IMPORTANCE African swine fever virus, a large and complex icosahedral DNA virus, causes a deadly infection in domestic pigs. In addition to Africa and Europe, countries in Asia, including China, Vietnam, and Mongolia, were negatively affected by the hazards posed by ASFV outbreaks in 2018 and 2019, at which time more than 30 million pigs were culled. Until now, there has been no vaccine for protection against ASFV infection or effective treatments to cure ASF. Here, we solved the high-resolution crystal structure of the ASFV pS273R protease. The pS273R protease has a two-domain structure that distinguishes it from other members of the SUMO protease family, while the unique "arm domain" has been proven to be essential for its hydrolytic activity. Moreover, the peptidomimetic aldehyde compounds designed to target the substrate binding pocket exert prominent inhibitory effects and can thus be used in a potential lead for anti-ASFV drug development.

Project description:Recently moderate-virulence classical swine fever virus (CSFV) strains have been proven capable of generating postnatal persistent infection (PI), defined by the maintenance of viremia and the inability to generate CSFV-specific immune responses in animals. These animals also showed a type I interferon blockade in the absence of clinical signs. In this study, we assessed the infection generated in 7-week-old CSFV PI wild boars after infection with the African swine fever virus (ASFV). The wild boars were divided in two groups and were infected with ASFV. Group A comprised boars who were CSFV PI in a subclinical form and Group B comprised pestivirus-free wild boars. Some relevant parameters related to CSFV replication and the immune response of CSFV PI animals were studied. Additionally, serum soluble factors such as IFN-α, TNF-α, IL-6, IL-10, IFN-γ and sCD163 were analysed before and after ASFV infection to assess their role in disease progression.After ASFV infection, only the CSFV PI wild boars showed progressive acute haemorrhagic disease; however, the survival rates following ASFV infection was similar in both experimental groups. Notwithstanding, the CSFV RNA load of CSFV PI animals remained unaltered over the study; likewise, the ASFV DNA load detected after infection was similar between groups. Interestingly, systemic type I FN-α and IL-10 levels in sera were almost undetectable in CSFV PI animals, yet detectable in Group B, while detectable levels of IFN-γ were found in both groups. Finally, the flow cytometry analysis showed an increase in myelomonocytic cells (CD172a+) and a decrease in CD4+ T cells in the PBMCs from CSFV PI animals after ASFV infection.Our results showed that the immune response plays a role in the progression of disease in CSFV subclinically infected wild boars after ASFV infection, and the immune response comprised the systemic type I interferon blockade. ASFV does not produce any interference with CSFV replication, or vice versa. ASFV infection could be a trigger factor for the disease progression in CSFV PI animals, as their survival after ASFV was similar to that of the pestivirus-free ASFV-infected group. This fact suggests a high resistance in CSFV PI animals even against a virus like ASFV; this may mean that there are relevant implications for CSF control in endemic countries. The diagnosis of ASFV and CSFV co-infection in endemic countries cannot be ruled out and need to be studied in greater depth.

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Crystal Structure of African Swine Fever Virus dUTPase Reveals a Potential Drug Target.

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Crystal Structure of African Swine Fever Virus dUTPase Reveals a Potential Drug Target.

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