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Asfivirus

African swine fever virus (ASFV) is a large DNA virus that can cause a fatal hemorrhagic disease in pigs, resulting in economic losses of hundreds of billions in China.

Through cryo-electron microscopy structure research, we have found that African swine fever virus shows significant differences from other nucleocytoplasmic large DNA viruses in terms of multi - layer structures and icosahedral morphology. It has an extremely special five - layer structure. From the inside out, they are the nucleoid, the nucleocapsid, the inner membrane, the capsid, and the outer membrane. By using the "block-based reconstruction algorithm", the near - atomic-resolution structure of the virus capsid was obtained, and several crucial capsid proteins and structural elements were identified. The outer shell layer is composed of 8,280 major capsid proteins p72 and 60 penton proteins. The p72 trimer proteins form most of the outer shell, while the penton proteins form the vertices. Below the outer shell layer is a network formed by at least 8,340 minor capsid proteins. Currently, three different types have been identified (named P1, P2, and P3 respectively). Similar to other nucleocytoplasmic large DNA viruses, the minor capsid proteins are located at the interfaces between adjacent capsomeres and may play a role in stabilizing the entire capsid. Our research results have revealed the complete molecular model of the African swine fever virus capsid structure, which will contribute to the development of various antiviral strategies in the future（Cell host & Microbe, 2019）.

Overall Structure of ASFV

 

In addition, among the numerous proteins encoded by the African swine fever virus, A137R is also a key structural protein associated with the virulence of the virus. Our research team has resolved the structure of A137R using the cryo-EM single-particle reconstruction technique. The structure reveals that A137R self-oligomerizes to form a unique dodecahedral cage-like structure composed of 60 polymers. The A137R cage-like structure is a core component of the African swine fever virus particles. Meanwhile, it provides a promising molecular scaffold for nanotechnology applications, opening up new avenues for the development of vaccines or treatment methods against African swine fever (J Virol, 2024).

A137R forms a dodecahedron cage

 

ASFV replication primarily occurs in viral factories within host cells, involving numerous viral replication protein components. Our team first focused on the ASFV replication protein pA104R, a nucleic acid-binding protein, and elucidated its mechanism of compressing the viral genome and facilitating genome assembly through repeated binding and bending of viral nucleic acids. Building on this, we targeted the interaction interface between pA104R and the viral genome, screened and identified two viral inhibitors that achieved over 90% viral inhibition rate at the cellular level. These findings provide critical insights for designing anti-ASFV drugs targeting the replication stage and advancing epidemic control strategies(Proc Natl Acad Sci U S A，2020).

Subsequently, our team investigated type II DNA topoisomerase pP1192R, another key enzyme in ASFV replication. DNA topoisomerases play pivotal roles in fundamental biological processes including DNA replication, transcription, recombination, and repair, by relaxing DNA supercoiling through introduction of single- or double-strand breaks. Type II DNA topoisomerase pP1192R exhibits high activity during the middle-late stages of infection and serves as a crucial viral replication enzyme(Nucleic Acids Res，2024).

Using multiple structural biology techniques, our team resolved the molecular conformations of pP1192R in various enzymatic stages. After thoroughly evaluating the targeting efficacy of Topo II inhibitors on pP1192R, we found that only the eukaryotic Topo II inhibitor m-AMSA specifically blocked pP1192R enzymatic activity and significantly inhibited ASFV replication in porcine alveolar macrophages (PAMs). Subsequent resolution of the pP1192R-DNA-m-AMSA ternary complex structure revealed difunctional inhibition mechanism of m-AMSA on pP1192R from both biochemical and structural perspectives. In addition to the traditional mechanism of trapping Topo II-DNA covalent complexes, this study also demonstrates for the first time that Topo II inhibitors can suppress enzymatic activity by preventing DNA cleavage and stabilizing non-covalent Topo II-DNA complexes. Furthermore, by resolving the molecular conformation of pP1192R in pre-cleavage state with DNA, we discovered metal ions (A²⁺) bridging the catalytic amino acids of Topo II and the DNA phosphate backbone. This provides direct evidence for the two-metal-ion-dependent DNA cleavage mechanism in Topo II (Nucleic Acids Res，2024).

Research on the molecular mechanisms and inhibitors of key proteins in ASFV

 

Our team resolved the crystal structures of the apo-E165R and E165R-dUMP complexes of African swine fever virus (ASFV) dUTP pyrophosphatase (dUTPase). We analyzed and determined the structure of the enzyme's active site and the interaction sites with the ligand dUMP. Through further comparison of the structures of ASFV-E165R and dUTPases from other species, it was found that the active site of E165R is highly similar to those of dUTPases from Mycobacterium tuberculosis and Plasmodium falciparum. The dUTPases of Mycobacterium tuberculosis and Plasmodium falciparum have been proven to be targets for drug design. For example, α,β-imido-dUTP (dUPNPP) has been used to inhibit the activity of the dUTPase of Mycobacterium tuberculosis, and some deoxyuridine triphenylmethane derivatives have been found to possess antimalarial activity. Therefore, small-molecule chemical drugs developed targeting the active site of ASFV-E165R are likely to be effective in preventing and controlling ASFV. Thus, the research results provide an important basis for the design of anti-ASFV drugs targeting E165R(mBio, 2019).

Structures of E165R-dUMP and dUTPases-product/inhibitor

complexes from other species

 

For the first time, we mapped a panoramic gene-expression profile of primary macrophages infected with African swine fever virus (ASFV), revealing the association patterns between viral and host molecules, as well as the host-cell signaling pathways regulated by viral replication. By using primary porcine alveolar macrophages infected with ASFV and applying single-cell RNA-sequencing technology, we discovered that the expression patterns of different ASFV genes exhibit temporal regularity, which provides crucial evidence for the functional annotation of viral genes.In addition, our team also found that the gene-expression patterns of macrophages change significantly in the environment of viral exposure. Specifically, the expressions of interferon-stimulated genes, inflammation-related genes, and cytokine-related genes are all significantly upregulated.This study provides important scientific evidence for explaining the massive exhaustion of immune cells induced by ASFV infection, which leads to the failure to establish an effective antiviral response. It also provides a theoretical basis for further elucidating the pathogenic mechanism of ASFV(Proc Natl Acad Sci U S A，2020).

 

 

References

1. Liu R, Sun Y, Chai Y, Li S, Li S, Wang L, Su J, Yu S, Yan J, Gao F, Zhang G, Qiu HJ, Gao GF, Qi J, Wang H. The structural basis of African swine fever virus pA104R binding to DNA and its inhibition by stilbene derivatives. Proc Natl Acad Sci U S A 2020; 117(20):11000-11009.

2. Liu S, Luo Y, Wang Y, Li S, Zhao Z, Bi Y, Sun J, Peng R, Song H, Zhu D, Sun Y, Li S, Zhang L, Wang W, Sun Y, Qi J, Yan J, Shi Y, Zhang X, Wang P, Qiu HJ, Gao GF. Cryo-EM Structure of the African Swine Fever Virus. Cell Host Microbe 2019; 26(6):836-843 e833.

3. Liu R, Sun J, Li LF, Cheng Y, Li M, Fu L, Li S, Peng G, Wang Y, Liu S, Qu X, Ran J, Li X, Pang E, Qiu HJ, Wang Y, Qi J, Wang H, Gao GF. Structural basis for difunctional mechanism of m-AMSA against African swine fever virus pP1192R. Nucleic Acids Res 2024; 52(18):11301-11316.

4. Li C, Jia M, Hao T, Peng Q, Peng R, Chai Y, Shi Y, Song H, Gao GF. African swine fever virus A137R assembles into a dodecahedron cage. J Virol 2024; 98(3):e0153623.

5. Wang L, Luo Y, Zhao Y, Gao GF, Bi Y, Qiu HJ. Comparative genomic analysis reveals an 'open' pan-genome of African swine fever virus. Transbound Emerg Dis 2020; 67(4):1553-1562.

6. Zheng Y, Li S, Li SH, Yu S, Wang Q, Zhang K, Qu L, Sun Y, Bi Y, Tang F, Qiu HJ, Gao GF. Transcriptome profiling in swine macrophages infected with African swine fever virus at single-cell resolution. Proc Natl Acad Sci U S A 2022; 119(19):e2201288119.

7. Li C, Chai Y, Song H, Weng C, Qi J, Sun Y, Gao GF. Crystal Structure of African Swine Fever Virus dUTPase Reveals a Potential Drug Target. mBio 2019; 10(5).

8. Yue C, Xiang W, Huang X, Sun Y, Xiao J, Liu K, Sun Z, Qiao P, Li H, Gan J, Ba L, Chai Y, Qi J, Liu P, Qi P, Zhao Y, Li Y, Qiu HJ, Gao GF, Gao G, Liu WJ. Mooring Stone-Like Arg(114) Pulls Diverse Bulged Peptides: First Insight into African Swine Fever Virus-Derived T Cell Epitopes Presented by Swine Major Histocompatibility Complex Class I. J Virol 2022; 96(4):e0137821.