African swine fever virus protease, a new viral member of the SUMO-1-specific protease family

African swine fever virus (ASFV) is a complex DNA virus that employs polyprotein processing at Gly-Gly-Xaa sites as a strategy to produce several major core components of the viral particle. The virus gene S273R encodes a 31-kDa protein that contains a "core domain" with the conserved catalytic residues characteristic of SUMO-1-specific proteases and the adenovirus protease. Using a COS cell expression system, it was found that protein pS273R is capable of cleaving the viral polyproteins pp62 and pp220 in a specific way giving rise to the same intermediates and mature products as those produced in ASFV-infected cells. Furthermore, protein pS273R, like adenovirus protease and SUMO-1-specific enzymes, is a cysteine protease, because its activity is abolished by mutation of the predicted catalytic histidine and cysteine residues and is inhibited by sulfhydryl-blocking reagents. Protein pS273R is expressed late after infection and is localized in the cytoplasmic viral factories, where it is found associated with virus precursors and mature virions. In the virions, the protein is present in the core shell, a domain where the products of the viral polyproteins are also located. The identification of the ASFV protease will allow a better understanding of the role of polyprotein processing in virus assembly and may contribute to our knowledge of the emerging family of SUMO-1-specific proteases.     

The African Swine Fever Virus Thymidine Kinase Gene Is Required for Efficient Replication in Swine Macrophages and for Virulence in Swine

African swine fever virus (ASFV) replicates in the cytoplasm of infected cells and contains genes encoding a number of enzymes needed for DNA synthesis, including a thymidine kinase (TK) gene. Recombinant TK gene deletion viruses were produced by using two highly pathogenic isolates of ASFV through homologous recombination with an ASFV p72 promoter–β-glucuronidase indicator cassette (p72GUS) flanked by ASFV sequences targeting the TK region. Attempts to isolate double-crossover TK gene deletion mutants on swine macrophages failed, suggesting a growth deficiency of TK⁻ ASFV on macrophages. Two pathogenic ASFV isolates, ASFV Malawi and ASFV Haiti, partially adapted to Vero cells, were used successfully to construct TK deletion viruses on Vero cells. The selected viruses grew well on Vero cells, but both mutants exhibited a growth defect on swine macrophages at low multiplicities of infection (MOI), yielding 0.1 to 1.0% of wild-type levels. At high MOI, the macrophage growth defect was not apparent. The Malawi TK deletion mutant showed reduced virulence for swine, producing transient fevers, lower viremia titers, and reduced mortality. In contrast, 100% mortality was observed for swine inoculated with the TK⁺ revertant virus. Swine surviving TK⁻ ASFV infection remained free of clinical signs of African swine fever following subsequent challenge with the parental pathogenic ASFV. The data indicate that the TK gene of ASFV is important for growth in swine macrophages in vitro and is a virus virulence factor in swine.     

Proteolytic processing in African swine fever virus: evidence for a new structural polyprotein, pp62.

We have identified an open reading frame (ORF), CP530R, within the EcoRI C' fragment of the African swine fever virus (ASFV) genome that encodes a polyprotein of 62 kDa (pp62). Antisera raised against different regions of ORF CP530R recognized a polypeptide of 62 kDa in ASFV-infected cells during the late phase of virus replication, after the onset of viral DNA synthesis. Pulse-chase experiments showed that polyprotein pp62 is posttranslationally processed to give rise to two proteins of 35 kDa (p35) and 15 kDa (p15). This proteolytic processing was found to take place at the consensus sequence Gly-Gly-X through an ordered cascade of proteolytic cleavages like that which also occurs with ASFV polyprotein pp220 (C. Simón-Mateo, G. Andrés, and E. Viñuela, EMBO J. 12:2977-2987, 1993). Immunofluorescence studies showed that polyprotein pp62 is localized in the viral factories. In addition, immunoprecipitation analysis of purified virus particles showed that mature products p35 and p15 are major structural proteins. According to these results, polyprotein processing represents an essential strategy for the maturation of ASFV structural proteins.     

Deletion of a CD2-Like Gene, 8-DR, from African Swine Fever Virus Affects Viral Infection in Domestic Swine

An African swine fever virus (ASFV) gene with similarity to the T-lymphocyte surface antigen CD2 has been found in the pathogenic African isolate Malawi Lil-20/1 (open reading frame [ORF] 8-DR) and a cell culture-adapted European virus, BA71V (ORF EP402R) and has been shown to be responsible for the hemadsorption phenomenon observed for ASFV-infected cells. The structural and functional similarities of the ASFV gene product to CD2, a cellular protein involved in cell-cell adhesion and T-cell-mediated immune responses, suggested a possible role for this gene in tissue tropism and/or immune evasion in the swine host. In this study, we constructed an ASFV 8-DR gene deletion mutant (Δ8-DR) and its revertant (8-DR.R) from the Malawi Lil-20/1 isolate to examine gene function in vivo. In vitro, Δ8-DR, 8-DR.R, and the parental virus exhibited indistinguishable growth characteristics on primary porcine macrophage cell cultures. In vivo, 8-DR had no obvious effect on viral virulence in domestic pigs; disease onset, disease course, and mortality were similar for the mutant Δ8-DR, its revertant 8-DR.R, and the parental virus. Altered viral infection was, however, observed for pigs infected with Δ8-DR. A delay in spread to and/or replication of Δ8-DR in the draining lymph node, a delay in generalization of infection, and a 100- to 1,000-fold reduction in virus titers in lymphoid tissue and bone marrow were observed. Onset of viremia for Δ8-DR-infected animals was significantly delayed (by 2 to 5 days), and mean viremia titers were reduced approximately 10,000-fold at 5 days postinfection and 30- to 100-fold at later times; moreover, unlike in 8-DR.R-infected animals, the viremia was no longer predominantly erythrocyte associated but rather was equally distributed among erythrocyte, leukocyte, and plasma fractions. Mitogen-dependent lymphocyte proliferation of swine peripheral blood mononuclear cells in vitro was reduced by 90 to 95% following infection with 8-DR.R but remained unaltered following infection with Δ8-DR, suggesting that 8-DR has immunosuppressive activity in vitro. Together, these results suggest an immunosuppressive role for 8-DR in the swine host which facilitates early events in viral infection. This may be of most significance for ASFV infection of its highly adapted natural host, the warthog.     

African swine fever virus protein MGF-505-7R promotes virulence and pathogenesis by inhibiting JAK1- and JAK2-mediated signaling

African swine fever virus (ASFV) is a large DNA virus that is highly contagious and pathogenic in domestic pigs with a mortality rate up to 100%. However, how ASFV suppresses JAK-STAT1 signaling to evade the immune response remains unclear. In this study, we found that the ASFV-encoded protein MGF-505-7R inhibited proinflammatory IFN-γ-mediated JAK-STAT1 signaling. Mechanistically, MGF-505-7R was found to interact with JAK1 and JAK2 and mediate their degradation. Further study indicated that MGF-505-7R promoted degradation of JAK1 and JAK2 by upregulating the E3 ubiquitin ligase RNF125 expression and inhibiting expression of Hes5, respectively. Consistently, MGF-505-7R-deficient ASFV induced high levels of IRF1 expression and displayed compromised replication both in primary porcine alveolar macrophages and pigs compared with wild-type ASFV. Furthermore, MGF-505-7R deficiency attenuated the virulence of the ASFV and pathogenesis of ASF in pigs. These findings suggest that the JAK-STAT1 axis mediates the innate immune response to the ASFV and that MGF-505-7R plays a critical role in the virulence of the ASFV and pathogenesis of ASF by antagonizing this axis. Thus, we conclude that deletion of MGF-505-7R may serve as a strategy to develop attenuated vaccines against the ASFV.     

African Swine Fever virus proteinase is essential for core maturation and infectivity

African swine fever virus (ASFV) encodes two polyprotein precursors named pp220 and pp62 that are sequentially processed during viral infection, giving rise to six major structural proteins. These reside at the core shell, a matrix domain located between the endoplasmic reticulum-derived inner envelope and the DNA-containing nucleoid. Proteolytic processing of the polyprotein precursors is catalyzed by the viral proteinase pS273R, a cysteine proteinase that shares sequence similarity with the SUMO1-processing peptidases. We describe here the construction and characterization of an ASFV recombinant, vS273Ri, that inducibly expresses the ASFV proteinase. Using vS273Ri, we show that repression of proteinase expression inhibits polyprotein processing and strongly impairs infective virus production. Electron microscopic examination of vS273Ri-infected cells showed that inhibition of proteolytic processing leads to the assembly of defective icosahedral particles containing a noncentered electron-dense nucleoid surrounded by an abnormal core shell of irregular thickness. The analysis of purified extracellular defective particles revealed that they contain the unprocessed pp220 and pp62 precursors, as well as the major DNA-binding nucleoid proteins p10 and pA104R. Altogether, these results indicate that the proteolytic processing of the polyproteins is not required for their incorporation into the assembling particles nor for the incorporation of the DNA-containing nucleoid. Instead, the ASFV proteinase is involved in a late maturational step that is essential for proper core assembly and infectivity.     

Solution structures of 2 : 1 and 1 : 1 DNA polymerase-DNA complexes probed by ultracentrifugation and small-angle X-ray scattering

We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme–DNA complexes of rat DNA polymerase β (Pol β) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol β–DNA complex, whereas only 1 : 1 Pol X–DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol β–DNA and 1 : 1 Pol X–DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDa 5′-dRP lyase domain of the second Pol β molecule with the active site of the 1 : 1 Pol β–DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5′-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5′-dRP lyase reaction in the crystal structures of the closed ternary enzyme–DNA–dNTP complexes, we propose that the asymmetric 2 : 1 Pol β–DNA complex enhances the function of Pol β.     

非洲猪瘟病毒的非必需基因：真的可有可无吗？

非洲猪瘟（African swine fever,ASF）是由非洲猪瘟病毒（African swine fever virus,ASFV）引起的一种出血性、致死性的猪烈性传染病。ASF在全球广泛传播,给养猪业造成重大的经济损失。ASFV基因组庞大,可编码150多种蛋白,一些非必需基因编码的蛋白与调控病毒毒力、复制和免疫逃逸等相关。通过删除ASFV毒力相关的非必需基因所构建的减毒株是当前比较有前景的疫苗,然而其安全性有待提高。系统地鉴定ASFV非必需基因及其功能,不仅有助于ASF基因缺失疫苗的研发,也有益于ASFV致病机制研究。本文对目前已鉴定的ASFV非必需基因及其功能研究进行了总结分析,着重讨论了影响ASFV毒力、调控病毒复制、参与免疫逃逸的非必需基因及其编码蛋白的功能,旨在加深对ASFV病原学的认识,为新的ASFV非必需基因的鉴定和功能研究提供参考。     

African Swine Fever Virus Uses Macropinocytosis to Enter Host Cells

African swine fever (ASF) is caused by a large and highly pathogenic DNA virus, African swine fever virus (ASFV), which provokes severe economic losses and expansion threats. Presently, no specific protection or vaccine against ASF is available, despite the high hazard that the continued occurrence of the disease in sub-Saharan Africa, the recent outbreak in the Caucasus in 2007, and the potential dissemination to neighboring countries, represents. Although virus entry is a remarkable target for the development of protection tools, knowledge of the ASFV entry mechanism is still very limited. Whereas early studies have proposed that the virus enters cells through receptor-mediated endocytosis, the specific mechanism used by ASFV remains uncertain. Here we used the ASFV virulent isolate Ba71, adapted to grow in Vero cells (Ba71V), and the virulent strain E70 to demonstrate that entry and internalization of ASFV includes most of the features of macropinocytosis. By a combination of optical and electron microscopy, we show that the virus causes cytoplasm membrane perturbation, blebbing and ruffles. We have also found that internalization of the virions depends on actin reorganization, activity of Na⁺/H⁺ exchangers, and signaling events typical of the macropinocytic mechanism of endocytosis. The entry of virus into cells appears to directly stimulate dextran uptake, actin polarization and EGFR, PI3K-Akt, Pak1 and Rac1 activation. Inhibition of these key regulators of macropinocytosis, as well as treatment with the drug EIPA, results in a considerable decrease in ASFV entry and infection. In conclusion, this study identifies for the first time the whole pathway for ASFV entry, including the key cellular factors required for the uptake of the virus and the cell signaling involved.     

Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion

Inflammasome is an intracellular signaling complex of the innate immune system. Activation of inflammasomes promotes the secretion of interleukin 1β (IL-1β) and IL-18 and triggers pyroptosis. Caspase-1 and -11 (or -4/5 in human) in the canonical and non-canonical inflammasome pathways, respectively, are crucial for inflammasome-mediated inflammatory responses. Here we report that gasdermin D (GSDMD) is another crucial component of inflammasomes. We discovered the presence of GSDMD protein in nigericin-induced NLRP3 inflammasomes by a quantitative mass spectrometry-based analysis. Gene deletion of GSDMD demonstrated that GSDMD is required for pyroptosis and for the secretion but not proteolytic maturation of IL-1β in both canonical and non-canonical inflammasome responses. It was known that GSDMD is a substrate of caspase-1 and we showed its cleavage at the predicted site during inflammasome activation and that this cleavage was required for pyroptosis and IL-1β secretion. Expression of the N-terminal proteolytic fragment of GSDMD can trigger cell death and N-terminal modification such as tagging with Flag sequence disrupted the function of GSDMD. We also found that pro-caspase-1 is capable of processing GSDMD and ASC is not essential for GSDMD to function. Further analyses of LPS plus nigericin- or Salmonella typhimurium-treated macrophage cell lines and primary cells showed that apoptosis became apparent in Gsdmd^−/− cells, indicating a suppression of apoptosis by pyroptosis. The induction of apoptosis required NLRP3 or other inflammasome receptors and ASC, and caspase-1 may partially contribute to the activation of apoptotic caspases in Gsdmd^−/− cells. These data provide new insights into the molecular mechanisms of pyroptosis and reveal an unexpected interplay between apoptosis and pyroptosis.     

African swine fever virus IAP homologue inhibits caspase activation and promotes cell survival in mammalian cells

African swine fever virus (ASFV) A224L is a member of the inhibitor of apoptosis protein (IAP) family. We have investigated the antiapoptotic function of the viral IAP both in stably transfected cells and in ASFV-infected cells. A224L was able to substantially inhibit caspase activity and cell death induced by treatment with tumor necrosis factor alpha and cycloheximide or staurosporine when overexpressed in Vero cells by gene transfection. We have also observed that ASFV infection induces caspase activation and apoptosis in Vero cells. Furthermore, using a deletion mutant of ASFV lacking the A224L gene, we have shown that the viral IAP modulates the proteolytic processing of the effector cell death protease caspase-3 and the apoptosis which are induced in the infected cells. Our findings indicate that A224L interacts with the proteolytic fragment of caspase-3 and inhibits the activity of this protease during ASFV infection. These observations could indicate a conserved mechanism of action for ASFV IAP and other IAP family members to suppress apoptosis.     

非洲猪瘟病毒的免疫逃逸策略

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)引起的一种猪烈性传染病.目前无商品化的ASF疫苗,一旦发病,仅能依靠快速扑杀进行防控,严重威胁我国养猪及相关行业的健康发展.ASF疫苗研发面临的主要困难是对ASFV的毒力相关基因、致...     

African Swine Fever Virus Structural Protein pE120R Is Essential for Virus Transport from Assembly Sites to Plasma Membrane but Not for Infectivity

This report examines the role of African swine fever virus (ASFV) structural protein pE120R in virus replication. Immunoelectron microscopy revealed that protein pE120R localizes at the surface of the intracellular virions. Consistent with this, coimmunoprecipitation assays showed that protein pE120R binds to the major capsid protein p72. Moreover, it was found that, in cells infected with an ASFV recombinant that inducibly expresses protein p72, the incorporation of pE120R into the virus particle is dependent on p72 expression. Protein pE120R was also studied using an ASFV recombinant in which E120R gene expression is regulated by the Escherichia coli lac repressor-operator system. In the absence of inducer, pE120R expression was reduced about 100-fold compared to that obtained with the parental virus or the recombinant virus grown under permissive conditions. One-step virus growth curves showed that, under conditions that repress pE120R expression, the titer of intracellular progeny was similar to the total virus yield obtained under permissive conditions, whereas the extracellular virus yield was about 100-fold lower than in control infections. Immunofluorescence and electron microscopy demonstrated that, under restrictive conditions, intracellular mature virions are properly assembled but remain confined to the replication areas. Altogether, these results indicate that pE120R is necessary for virus dissemination but not for virus infectivity. The data also suggest that protein pE120R might be involved in the microtubule-mediated transport of ASFV particles from the viral factories to the plasma membrane.     

非洲猪瘟病毒j5R膜蛋白的电脑预测和实验证实

蛋白多肽二级结构的电脑预测表明,非洲猪瘟病毒（ Ａｆｒｉｃａｎ ｓｗｉｎｅ ｆｅｖｅｒ ｖｉｒｕｓ , Ａ Ｓ Ｆ Ｖ）ｊ５ Ｒ阅读框编码１２ ．９ ｋ Ｄａ 膜蛋白。该蛋白的 Ｃ 末端含有一个潜在抗原决定簇,针对其合成肽的抗体能在 Ａ Ｓ Ｆ Ｖ 感染细胞和病毒颗粒中检测到２３ 或２５ ｋ Ｄａ（ 取决于不同毒株） 特异蛋白。免疫荧光试验显示,ｊ５ Ｒ 蛋白主要位于感染细胞的病毒复制部位。油水两相分离和细胞分级分离试验结果证明ｊ５ Ｒ 蛋白是膜相关蛋白     

Development of an Accurate and Sensitive Diagnostic System Based on Conventional PCR for Detection of African Swine Fever Virus in Food Waste

African swine fever virus (ASFV), a highly contagious virus, can cause diseases with high mortality rates in pigs, making it a pathogen of social and economic significance. ASFV has been reported to show potential long-term survival in living livestock, such as pigs, but also in leftover cooking meat and undercooked pork meat. Hence, it is possible that there could be direct reinfection or secondary infection through feed produced from household food waste and treatment facilities. Many polymerase chain reaction (PCR)-based molecular diagnostic techniques to detect ASFV in clinical swine samples have been reported. However, those with applicability for food waste samples, which contain relatively low viral copy numbers and may contain various unknown inhibitors of PCR, are still lacking. In this study, we developed a conventional PCR-based diagnostic system that can detect ASFV with high sensitivity from food waste sample types. The technique shows a 10–100 times higher limit of detection compared to that of previously reported methods based on conventional PCR and quantitative real-time PCR. It is also capable of amplifying a sequence that is approximately 751 nucleotides, which is advantageous for similarity analysis and genotyping. Moreover, a ASFV-modified positive material different from ASFV that could synthesize 1400 nucleotide amplicons was developed to identify false-positive cases and thus enhance diagnostic accuracy. The method developed herein may be applicable for future ASFV monitoring, identification, and genotyping in food waste samples.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-022-01007-y.