Novel swine virulence determinant in the left variable region of the African swine fever virus genome

Previously we have shown that the African swine fever virus (ASFV) NL gene deletion mutant E70DeltaNL is attenuated in pigs. Our recent observations that NL gene deletion mutants of two additional pathogenic ASFV isolates, Malawi Lil-20/1 and Pr4, remained highly virulent in swine (100% mortality) suggested that these isolates encoded an additional virulence determinant(s) that was absent from E70. To map this putative virulence determinant, in vivo marker rescue experiments were performed by inoculating swine with infection-transfection lysates containing E70 NL deletion mutant virus (E70DeltaNL) and cosmid DNA clones from the Malawi NL gene deletion mutant (MalDeltaNL). A cosmid clone representing the left-hand 38-kb region (map units 0.05 to 0.26) of the MalDeltaNL genome was capable of restoring full virulence to E70DeltaNL. Southern blot analysis of recovered virulent viruses confirmed that they were recombinant E70DeltaNL genomes containing a 23- to 28-kb DNA fragment of the Malawi genome. These recombinants exhibited an unaltered MalDeltaNL disease and virulence phenotype when inoculated into swine. Additional in vivo marker rescue experiments identified a 20-kb fragment, encoding members of multigene families (MGF) 360 and 530, as being capable of fully restoring virulence to E70DeltaNL. Comparative nucleotide sequence analysis of the left variable region of the E70DeltaNL and Malawi Lil-20/1 genomes identified an 8-kb deletion in the E70DeltaNL isolate which resulted in the deletion and/or truncation of three MGF 360 genes and four MGF 530 genes. A recombinant MalDeltaNL deletion mutant lacking three members of each MGF gene family was constructed and evaluated for virulence in swine. The mutant virus replicated normally in macrophage cell culture but was avirulent in swine. Together, these results indicate that a region within the left variable region of the ASFV genome containing the MGF 360 and 530 genes represents a previously unrecognized virulence determinant for domestic swine.     

Hairpin loop structure of African swine fever virus DNA.

The ends of African swine fever virus genome are formed by a 37 nucleotide-long hairpin loop composed, almost entirely, of incompletely paired A and T residues. The loops at each DNA end were present in two equimolar forms that, when compared in opposite polarities, were inverted and complementary (flip-flop), as in the case of poxvirus DNA. The hairpin loops of African swine fever and vaccinia virus DNAs had no homology, but both DNAs had a 16 nucleotide-long sequence, close to the hairpin loops, with an homology of about 80%. An analysis of African swine fever virus replicating DNA showed head-to-head and tail-to-tail linked molecules that may be replicative intermediates.     

An African swine fever virus gene with homology to DNA ligases.

Sequence analysis of the SalI g region of the genome of a virulent isolate of ASFV (Malawi Lil 20/1) has revealed an open reading frame with the potential to encode a 48 kilodalton (kD) polypeptide which has significant homology with eukaryotic and prokaryotic DNA ligases. This ASFV encoded gene also contains the putative active site region of DNA ligases including the lysine residue which is necessary for enzyme-adenylate adduct formation, but lacks the C-terminal basic region conserved in other eukaryotic DNA ligases. A novel [32P]-labelled potential DNA ligase-adenylate adduct of M(r) 45 kD was observed upon incubation of ASFV infected cell cytoplasmic extracts with alpha-[32P]-ATP and subsequent analysis of products by SDS/PAGE. These data together suggest that ASFV encodes its own DNA ligase.     

Contributions of an endonuclease IV homologue to DNA repair in the African swine fever virus

We recently demonstrated that African swine fever virus DNA polymerase X (Pol X) is extremely error-prone during single-nucleotide gap-filling and that the downstream ASFV DNA ligase seals 3' mismatched nicks with high efficiency. To further assess the credence of our hypothesis that these proteins may promote viral diversification by functioning within the context of an aberrant DNA repair pathway, herein we characterize the third protein expected to function in this system, a putative AP endonuclease (APE). Assays of the purified protein using oligonucleotide substrates unequivocally establish canonical APE activity, 3'-phosphatase and 3'-phosphodiesterase activities (in the context of a single-nucleotide gap), 3' --> 5' exonuclease activity (in the context of a nick), and nucleotide incision repair activity against 5,6-dihydrothymine. The 3' --> 5' exonuclease activity is shown to be highly dependent upon the identity of the nascent 3' base pair and to be inhibited when 2-deoxyribose-5-phosphate, rather than phosphate, constitutes the 5' moiety of the nick. ASFV APE retains activity when assayed in the presence of EDTA but is inactivated by incubation with 1,10-phenanthroline in the absence of a substrate, suggesting that it is an endonuclease IV homologue possessing intrinsic metal cofactors. The activities of ASFV APE, when considered alongside those of Pol X and ASFV DNA ligase, provide an enhanced understanding of (i) the types of damage that are likely to be sustained by the viral genome and (ii) the mechanisms by which the minimalist ASFV DNA repair pathway, consisting of just these three proteins, contributes to the fitness of the virus.     

African swine fever virus encodes a gene with extensive homology to type II DNA topoisomerases.

Nucleotide sequencing of a virulent African swine fever virus (ASFV) isolate (Malawi LIL20/1) identified an open reading frame of 1191 amino acid residues encoding a protein of 134.9 kDa. This gene mapped to the SalI i and j restriction endonuclease fragments of the ASFV genome. The predicted polypeptide was found to share 21.1% identity over a 1077 amino acid region with the human type II DNA topoisomerase. The sequence is compared to other type II DNA topoisomerases and the possible roles in ASFV replication are discussed.     

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.     

Use of damaged DNA and dNTP substrates by the error-prone DNA polymerase X from African swine fever virus

The structural specificity that translesion DNA polymerases often show for a particular class of lesions suggests that the predominant criterion of selection during their evolution has been the capacity for lesion tolerance and that the error-proneness they display when copying undamaged templates may simply be a byproduct of this adaptation. Regardless of selection criteria/evolutionary history, at present both of these properties coexist in these enzymes, and both properties confer a fitness advantage. The repair polymerase, Pol X, encoded by the African swine fever virus (ASFV) is one of the most error-prone polymerases known, leading us to previously hypothesize that it may work in tandem with the exceptionally error-tolerant ASFV DNA ligase to effect viral mutagenesis. Here, for the first time, we test whether the error-proneness of Pol X is coupled with a capacity for lesion tolerance by examining its ability to utilize the types of damaged DNA and dNTP substrates that are expected to be relevant to ASFV. We (i) test Pol X's ability to both incorporate opposite to and extend from ubiquitous oxidative purine (7,8-dihydro-8-oxoguanine), oxidative pyrimidine (5,6-dihydroxy-5,6-dihydrothymine), and noncoding (AP site) lesions, in addition to 5,6-dihydrothymine, (ii) determine the catalytic efficiency and dNTP specificity of Pol X when catalyzing incorporation opposite to, and when extending from, 7,8-dihydro-8-oxoguanine in a template/primer context, and (iii) quantitate Pol X-catalyzed incorporation of the damaged nucleotide 8-oxo-dGTP opposite to undamaged templates in the context of both template/primer and a single-nucleotide gap. Our findings are discussed in light of ASFV biology and the mutagenic DNA repair hypothesis described above.     

Intra-epidemic genome variation in highly pathogenic African swine fever virus (ASFV) from the country of Georgia

Background

African swine fever virus (ASFV) causes an acute hemorrhagic infection in suids with a mortality rate of up to 100%. No vaccine is available and the potential for catastrophic disease in Europe remains elevated due to the ongoing ASF epidemic in Russia and Baltic countries. To date, intra-epidemic whole-genome variation for ASFV has not been reported. To provide a more comprehensive baseline for genetic variation early in the ASF outbreak, we sequenced two Georgian ASFV samples, G-2008/1 and G-2008/2, derived from domestic porcine blood collected in 2008.

Methods

Genomic DNA was extracted directly from low-volume ASFV PCR-positive porcine blood samples and subjected to next generation sequencing on the Illumina Miseq platform. De novo and mapped sequence assemblies were performed using CLCBio software. Genomic illustrations, sequence alignments and assembly figures were generated using Geneious v10.2.4. Sequence repeat architecture was analyzed using DNASTAR GeneQuest 14.1.0.

Results

The G-2008/1 and G-2008/2 genomes were distinguished from each other by coding changes in seven genes, including MGF 110-1?L, X69R, MGF 505-10R, EP364R, H233R, E199L, and MGF 360-21R in addition to eight homopolymer tract variations. The 2008/2 genome possessed a novel allele state at a previously undescribed intergenic repeat locus between genes C315R and C147L. The C315R/C147L locus represents the earliest observed variable repeat sequence polymorphism reported among isolates from this epidemic. No sequence variation was observed in conventional ASFV subtyping markers. The two genomes exhibited complete collinearity and identical gene content with the Georgia 2007/1 reference genome. Approximately 56 unique homopolymer A/T-tract variations were identified that were unique to the Georgia 2007/1 genome. In both 2008 genomes, within-sample sequence read heterogeneity was evident at six homopolymeric G/C-tracts confined to the known hypervariable ~?7?kb region in the left terminal region of the genome.

Conclusions

This is the first intra-epidemic comparative genomic analysis reported for ASFV and provides insight into the intra-epidemic microevolution of ASFV. The genomes reported here, in addition to the G-2007/1 genome, provide an early baseline for future genome-level comparisons and epidemiological tracing efforts.

     

非洲猪瘟病毒内囊膜蛋白p17与宿主互作蛋白的初步鉴定

非洲猪瘟病毒(African swine fever virus,ASFV)的感染导致猪的死亡率高达100%,给养猪业造成毁灭性灾难。因此,开展针对ASFV感染复制的研究有着重大的意义。目前发现ASFV有超过150个开放阅读框,其中D117L基因编码的内囊膜蛋白p17参与病毒二十面体结构的形成,但是对p17调控宿主细胞功能的机制知之甚少。研究通过免疫沉淀技术联合蛋白质谱分析,初步筛选出与ASFV p17潜在的宿主互作蛋白。通过进一步免疫共沉淀技术和激光共聚焦实验确认了p17与线粒体外膜蛋白TOMM70(translocase of outer mitochondrial membrane 70)、热休克蛋白HSPA8(heat shock 70 kDa protein 8)的互作。该研究为进一步探索p17在ASFV感染过程中的功能提供了重要信息。     

African swine fever virus protein pE296R is a DNA repair apurinic/apyrimidinic endonuclease required for virus growth in swine macrophages

We show here that the African swine fever virus (ASFV) protein pE296R, predicted to be a class II apurinic/apyrimidinic (AP) endonuclease, possesses endonucleolytic activity specific for AP sites. Biochemical characterization of the purified recombinant enzyme indicated that the K(m) and catalytic efficiency values for the endonucleolytic reaction are in the range of those reported for Escherichia coli endonuclease IV (endo IV) and human Ape1. In addition to endonuclease activity, the ASFV enzyme has a proofreading 3'-->5' exonuclease activity that is considerably more efficient in the elimination of a mismatch than in that of a correctly paired base. The three-dimensional structure predicted for the pE296R protein underscores the structural similarities between endo IV and the viral protein, supporting a common mechanism for the cleavage reaction. During infection, the protein is expressed at early times and accumulates at later times. The early enzyme is localized in the nucleus and the cytoplasm, while the late protein is found only in the cytoplasm. ASFV carries two other proteins, DNA polymerase X and ligase, that, together with the viral AP endonuclease, could act as a viral base excision repair system to protect the virus genome in the highly oxidative environment of the swine macrophage, the virus host cell. Using an ASFV deletion mutant lacking the E296R gene, we have determined that the viral endonuclease is required for virus growth in macrophages but not in Vero cells. This finding supports the existence of a viral reparative system to maintain virus viability in the infected macrophage.     

In silico studies of the African swine fever virus DNA polymerase X support an induced-fit mechanism

The African swine fever virus DNA polymerase X (pol X), a member of the X family of DNA polymerases, is thought to be involved in base excision repair. Kinetics data indicate that pol X catalyzes DNA polymerization with low fidelity, suggesting a role in viral mutagenesis. Though pol X lacks the fingers domain that binds the DNA in other members of the X family, it binds DNA tightly. To help interpret details of this interaction, molecular dynamics simulations of free pol X at different salt concentrations and of pol X bound to gapped DNA, in the presence and in the absence of the incoming nucleotide, are performed. Anchors for the simulations are two NMR structures of pol X without DNA and a model of one NMR structure plus DNA and incoming nucleotide. Our results show that, in its free form, pol X can exist in two stable conformations that interconvert to one another depending on the salt concentration. When gapped double stranded DNA is introduced near the active site, pol X prefers an open conformation, regardless of the salt concentration. Finally, under physiological conditions, in the presence of both gapped DNA and correct incoming nucleotide, and two divalent ions, the thumb subdomain of pol X undergoes a large conformational change, closing upon the DNA. These results predict for pol X a substrate-induced conformational change triggered by the presence of DNA and the correct incoming nucleotide in the active site, as in DNA polymerase beta. The simulations also suggest specific experiments (e.g., for mutants Phe-102Ala, Val-120Gly, and Lys-85Val that may reveal crucial DNA binding and active-site organization roles) to further elucidate the fidelity mechanism of pol X.     

Differential expansion of highly repeated DNA sequences in the swine subgenomes

Differences in highly repeated DNA sequences among three swine breeds genomes were detected by means of whole‐comparative genomic hybridization (W‐CGH). The results showed that Duroc, Iberian and Landrace/Large White breeds share similar DNA sequences in their centromeric regions, but the number of copies of the highly repeated DNA sequences building the blocks of heterochromatin in the metacentric chromosomes is differentially expanded among them. That is not the case in the acrocentric subgenome where the chromosomes share similar sequence composition and number of copies among the three breeds in the centromeric regions. The highly repeated DNA sequences in the chromosome Y also displayed differences among the breeds studied. The reported results are discussed in the light of the possible evolutionary tendencies of these particular DNA sequences.     

Interactions of the DNA polymerase X of African swine fever virus with double-stranded DNA. Functional structure of the complex

Interactions of the polymerase X of African swine fever virus with the double-stranded DNA (dsDNA) have been studied with fluorescent dsDNA oligomers, using quantitative fluorescence titrations, analytical ultracentrifugation, and fluorescence energy transfer techniques. Studies with unmodified dsDNAs were performed, using competition titration method. ASV pol X binds the dsDNA with a site-size of n=10(+/-2) base-pairs, which is significantly shorter than the total site-size of 16(+/-2) nucleotides of the enzyme-ssDNA complex. The small site size indicates that the enzyme binds the dsDNA exclusively using the proper DNA-binding subsite. Fluorescence energy transfer studies between the tryptophan residue W92 and the acceptor, located at the 5' or 3' end of the dsDNA, suggest strongly that the proper DNA-binding subsite is located on the non-catalytic C-terminal domain. Moreover, intrinsic interactions with the dsDNA 10-mer or 20-mer are accompanied by the same net number of ions released, independent of the length of the DNA, indicating the same length of the DNA engaged in the complex. The dsDNA intrinsic affinity is about two orders of magnitude higher than the ssDNA affinity, indicating that the proper DNA-binding subsite is, in fact, the specific dsDNA-binding site. Surprisingly, ASFV pol X binds the dsDNA with significant positive cooperativity, which results from protein-protein interactions. Cooperative interactions are accompanied by the net ion release, with anions participating in the ion-exchange process. The significance of these results for ASFV pol X activity in the recognition of damaged DNA is discussed.     

Morphological study on the entry of African swine fever virus into cells

The early interactions between African swine fever virus (ASFV) and monkey kidney cells in culture, and the effect of chloroquine were studied by electron microscopy. Our results indicate that ASFV uptake occurs by endocytosis: after attachment to the cell surface, the virions were seen in coated pits and were internalized by endocytosis in endosomes and finally in lysosomes. Virions in coated vesicles were never seen. All these steps were completed in about 15 min. Direct penetration of viruses through the plasma membrane was never observed. In order to elucidate the participation of an acidic intracellular compartment in the penetration of ASFV, we studied the effect of chloroquine, a lysosomotropic drug known to increase the pH of acidic intracellular vacuoles and to inhibit ASFV infection. In the presence of this drug there were no apparent alterations on binding, endocytosis and intracellular distribution of the viral particles. The main effect of chloroquine was to retain the virions in lysosomes. When the drug was removed from the medium, the viral particles disappeared and images of binding of viral membranes with the membranes of the intracellular vacuoles were obtained, suggesting that the inhibited step is the uncoating of the virus. Viral fusion with the plasma membrane was obtained when the medium was acidified to pH 5-6. These results suggest that ASFV enters the cells by adsorptive endocytosis and that the uncoating process takes place intracellularly in a way similar to that described for Semliki Forest virus and other enveloped viruses.     

Identification of an African swine fever virus gene with similarity to a myeloid differentiation primary response gene and a neurovirulence-associated gene of herpes simplex virus.

Here we describe an open reading frame (LMW23-NL) in the African swine fever virus genome that possesses striking similarity to a murine myeloid differentiation primary response gene (MyD116) and the neurovirulence-associated gene (ICP34.5) of herpes simplex virus. In all three proteins, a centrally located acidic region precedes a highly conserved, hydrophilic 56-amino-acid domain located at the carboxy terminus. LMW23-NL predicts a highly basic protein of 184 amino acids with an estimated molecular mass of 21.3 kDa. The similarity of LMW23-NL to genes involved in myeloid cell differentiation and viral host range suggests a role for it in African swine fever virus host range.     

African swine fever virus assembles a single membrane derived from rupture of the endoplasmic reticulum

Collective evidence argues that two members of the nucleocytoplasmic large DNA viruses (NCLDVs) acquire their membrane from open membrane intermediates, postulated to be derived from membrane rupture. We now study membrane acquisition of the NCLDV African swine fever virus. By electron tomography (ET), the virion assembles a single bilayer, derived from open membrane precursors that collect as ribbons in the cytoplasm. Biochemically, lumenal endoplasmic reticulum (ER) proteins are released into the cytosol, arguing that the open intermediates are ruptured ER membranes. ET shows that viral capsid assembles on the convex side of the open viral membrane to shape it into an icosahedron. The viral capsid is composed of tiny spikes with a diameter of ～5 nm, connected to the membrane by a 6 nm wide structure displaying thin striations, as observed by several complementary electron microscopy imaging methods. Immature particles display an opening that closes after uptake of the viral genome and core proteins, followed by the formation of the mature virion. Together with our previous data, this study shows a common principle of NCLDVs to build a single internal envelope from open membrane intermediates. Our data now provide biochemical evidence that these open intermediates result from rupture of a cellular membrane, the ER.