Polyprotein processing in African swine fever virus: a novel gene expression strategy for a DNA virus.

This report shows that African swine fever virus (ASFV)--a large DNA-containing virus--synthesizes a polyprotein to produce several of its structural proteins. By immunoprecipitation analysis, we have found that ASFV polyprotein is a 220 kDa myristoylated polypeptide (pp220) which, after proteolytic processing, gives rise to four major structural proteins: p150, p37, p34 and p14. Processing of the ASFV polyprotein takes place at the consensus sequence Gly-Gly-X and occurs through an ordered cascade of proteolytic cleavages. So far, polyprotein processing as a mechanism of gene expression had been found only in positive-strand RNA viruses and retroviruses. According to the results presented here, ASFV is the first example of a DNA virus that synthesizes a polyprotein as a strategy of gene expression.     

Polypeptides of African swine fever virus

The published data on the characteristics and properties of structural and nonstructural polypeptides of the African porcine virus are reviewed. Localization of the viral proteins in virions and infected cells, kinetics of biosynthesis, glycosylation, phosphorylation and the antigenicity of the proteins are discussed.     

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.     

Nanopore sequencing of African swine fever virus

正Dear Editor,African swine fever (ASF) is one of the most pathogenic viral diseases in pigs caused by African swine fever virus(ASFV). The fatality rate is almost 100%, which brings huge economic losses to the hog industry in countries with epi-     

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、致病及其免疫逃逸机制知之甚少。本文对ASFV的免疫逃逸研究进行了总结,探讨了ASFV免疫逃逸基因及其编码蛋白的功能,以便加深对ASFV及其免疫逃逸策略的认知,为致病机制研究和疫苗研发提供借鉴。     

Purification and properties of African swine fever virus

We describe a method for African swine fever (ASF) virus purification based on equilibrium centrifugation in Percoll density gradients of extracellular virions produced in infected VERO cells that yielded about 15 +/- 9% recovery of the starting infectious virus particles. The purified virus preparations were essentially free of a host membrane fraction (vesicles) that could not be separated from the virus by previously described purification methods. The purified virus sedimented as a single component in sucrose velocity gradients with a sedimentation coefficient of 3,500 +/- 300S, showed a DNA-protein ratio of 0.18 +/- 0.02 and a specific infectivity of 2.7 X 10(7) PFU/micrograms of protein, and remained fully infectious after storage at -70 degrees C for at least 7 months. The relative molecular weights of the 34 polypeptides detected in purified virus particles ranged from 10,000 to 150,000. Some of these proteins were probably cellular components that might account for the reactivity of purified virus with antiserum against VERO cells.     

Extracellular protease of Natrialba magadii: purification and biochemical characterization

A serine protease secreted by the haloalkaliphilic archaeon Natrialba magadii at the end of the exponential growth phase was isolated. This enzyme was purified 83 fold with a total yield of 25% by ethanol precipitation, affinity chromatography, and gel filtration. The native molecular mass of the enzyme determined by gel filtration was 45 kDa. Na. magadii extracellular protease was dependent on high salt concentrations for activity and stability, and it had an optimum temperature of 60°C in the presence of 1.5 M NaCl. The enzyme was stable and had a broad pH profile (6–12) with an optimum pH of 8–10 for azocasein hydrolysis. The protease was strongly inhibited by diisopropyl fluorophosphate (DFP), phenylmethyl sulfonylfluoride (PMSF), and chymostatin, indicating that it is a serine protease. It was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and activated by thiol-containing reducing agents such as dithiotreitol (DTT) and 2-mercaptoethanol. This protease degraded casein and gelatin and showed substrate specificity for synthetic peptides containing Phe, Tyr, and Leu at the carboxyl terminus, showing that it has chymotrypsin-like activity. Na. magadii protease presented no cross-reactivity with polyclonal antibodies raised against the extracellular protease of Natronococcus occultus, suggesting that although these proteases share several biochemical traits, they might be antigenically unrelated. Received: October 1, 1999 / Accepted: February 1, 2000     

African swine fever virus interaction with microtubules

The role of microtubules in intracellular transport of African swine fever virus (ASFV) and virus-induced inclusions was studied by immunofluorescence using anti-ASFV and anti-tubulin antibodies, by electron microscopy of infected Vero cells and by in vitro binding of virions to purified microtubules. MTC, a reversible colchicine analogue, was used to depolymerize microtubules. In cells treated with MTC multiple large inclusions containing ASFV antigens and particles were observed in the cytoplasm. Removal of the drug lead to migration and fusion of the inclusions at a perinuclear location. To study the effect of microtubule repolymerization on virus particle distribution, the particles were counted in thin sections of MTC treated cells and at different times after removal of the drug. In cells treated with MTC 6.8% and 3.6% of the virus particles were found respectively in the cytoplasm and at the cell membrane while 38% of the particles were located around the virosome. With reversal of the drug effect the number of virus particles around the virosomes progressively decreased to 10% at 2 h while the number of particles in the cytoplasm and at the cell membrane increased. At 2 h after removal of the drug 33.5% of the particles were found budding from the cell membrane. Virus particles were found closely associated with microtubules in cytoskeletons obtained by Triton X-100 extraction of taxol treated cells. The association of virus particles with microtubules was also observed in vitro using purified microtubules and virus particles. The results show that microtubules are involved in the transport of African swine fever virus particles from the assembly site to the cell surface and in the movement and fusion of the virus inclusions.     

African swine fever virus attachment protein.

Treatment of African swine fever virus particles with nonionic detergents released proteins p35, p17, p14, and p12 from the virion. Of these proteins, only p12 bound to virus-sensitive Vero cells but not to virus-resistant L or IBRS2 cells. The binding of p12 was abolished by whole African swine fever virus and not by similar concentrations of subviral particles that lacked the external proteins. A monoclonal antibody (24BB7) specific for p12 precipitated a protein that, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol, showed a molecular mass of 17 kDa (p17*) instead of 12 kDa as found in the presence of 2-mercaptoethanol. The relationship between these two proteins was confirmed by the conversion of p17* to p12 when the former was isolated from polyacrylamide gels in the absence of 2-mercaptoethanol and subsequently treated with the reducing agent. The supernatant obtained after immunoprecipitation with the p12-specific antibody lacked the virus-binding protein.     

Expression and characterization of the thymidine kinase gene of African swine fever virus.

The thymidine kinase (TK) gene of African swine fever virus (ASFV) was located within the viral genome by using two degenerate oligonucleotide probes derived from sequences of the vaccinia virus and cellular TK genes. The TK gene was mapped within a 0.72-kbp BglII-XhoI fragment (0.242 to 0.246 map units) derived from a 23.9-kbp SalI-B fragment of the ASFV genome. Identification of this region as the ASFV TK gene was confirmed by expression of TK in Escherichia coli and by the synthesis of active TK in a cell-free system programmed with RNA synthesized in vitro. The sequenced gene for TK includes an open reading frame of 588 nucleotides encoding a protein of 196 amino acids. The deduced amino acid sequence shows 32.4% identity with the TK of vaccinia virus.     

Cross-links in African swine fever virus DNA.

African swine fever virus DNA sediments in neutral sucrose density gradients as a single component with a sedimentation coefficient of 60S. In alkaline sucrose density gradients, this material shows two components with sedimentation coefficients of 85S and 95S, respectively. The sedimentation rate value of alkali-denatured virus DNA in neutral sucrose density gradients and the renaturation velocity of denatured DNA show that is reassociated much faster than expected from its genetic complexity. This behavior is compatible with the existence of interstrand cross-links in the molecule. We also present results which suggest that there are only a few such cross-links per molecule, that they are sensitive to S1 nuclease digestion, and that they are probably located next to the ends of the DNA.     

Laboratory-scale inactivation of African swine fever virus and swine vesicular disease virus in pig slurry

Two methods were evaluated for the inactivation of African swine fever (ASV) and swine vesicular disease (SVD) viruses in pig slurry: chemical treatment and heat treatment. The addition of NaOH or Ca(OH)2 at different concentration/time combinations at 4 degrees C and 22 degrees C was examined, as was virus stability at different temperature/time combinations. ASF virus (ASFV) was less resistant to both methods than SVD virus (SVDV). In slurry from one source, ASFV was inactivated at 65 degrees C within 1 min, whereas SVDV required at least 2 min at 65 degrees C. However, it was found that thermal inactivation depended on the characteristics of the slurry used. Addition of 1% (w/v) of NaOH or Ca(OH)2 caused the inactivation of ASFV within 150 s at 4 degrees C; 0.5% (w/v) NaOH or Ca(OH)2 required 30 min for inactivation. NaOH or Ca(OH)2 (1% (w/v)) was not effective against SVDV at 22 degrees C after 30 min, and 1.5% (w/v) NaOH or Ca(OH)2 caused inactivation of SVDV at both 4 degrees C and 22 degrees C. At higher chemical concentrations or temperatures, ASFV and SVDV inactivation was faster in slurry than in buffered medium.     

African swine fever virus cysteine protease pS273R inhibits pyroptosis by noncanonically cleaving gasdermin D

African swine fever (ASF) is a viral hemorrhagic disease that affects domestic pigs and wild boar and is caused by the African swine fever virus (ASFV). The ASFV virion contains a long double-stranded DNA genome, which encodes more than 150 proteins. However, the immune escape mechanism and pathogenesis of ASFV remain poorly understood. Here, we report that the pyroptosis execution protein gasdermin D (GSDMD) is a new binding partner of ASFV-encoded protein S273R (pS273R), which belongs to the SUMO-1 cysteine protease family. Further experiments demonstrated that ASFV pS273R-cleaved swine GSDMD in a manner dependent on its protease activity. ASFV pS273R specifically cleaved GSDMD at G107-A108 to produce a shorter N-terminal fragment of GSDMD consisting of residues 1 to 107 (GSDMD-N_1–107). Interestingly, unlike the effect of GSDMD-N_1–279 fragment produced by caspase-1-mediated cleavage, the assay of LDH release, cell viability, and virus replication showed that GSDMD-N_1–107 did not trigger pyroptosis or inhibit ASFV replication. Our findings reveal a previously unrecognized mechanism involved in the inhibition of ASFV infection-induced pyroptosis, which highlights an important function of pS273R in inflammatory responses and ASFV replication.     

Sequence and characterization of the major early phosphoprotein p32 of African swine fever virus.

The gene encoding protein p32, the most abundant and immunogenic protein induced by African swine fever virus at early times of infection, has been mapped in the EcoRI C' fragment of the genome of the Vero cell-adapted virus strain BA71V. Sequencing analysis has shown the existence of an open reading frame, named C'204L, encoding 204 amino acids. The protein is phosphorylated in serine residues located in the 115 N-terminal amino acids and was phosphorylated when expressed in cells infected with a vaccinia virus recombinant. Protein p32 is not glycosylated in spite of the presence of two putative N-glycosylation sites in the deduced amino acid sequence of the polypeptide. Immunofluorescence experiments have shown that the protein is localized in the cytoplasm of infected cells and not in the plasma membrane. In addition, the protein has been found in the soluble fraction and not in microsomes from BA71V-infected Vero cells. Low levels of the protein have been detected in the medium from infected swine macrophages, which probably corresponds to nonspecific release of cytoplasmic proteins. The protein encoded by other virus isolates shows different electrophoretic mobilities, indicating variability of p32.