AT-rich Region Research Articles

Identification and characterization of transposable element AhMITE1 in the genomes of cultivated and two wild peanuts

BackgroundThe cultivated peanut (Arachis hypogaea L., AABB) is an allotetraploid hybrid between two diploid peanuts, A. duranensis (AA genome) and A. ipaensis (BB genome). Miniature inverted-repeat transposable elements (MITEs), some of which are known as active nonautonomous DNA transposons with high copy numbers, play important roles in genome evolution and diversification. AhMITE1, a member of the MITE family of transposons, but information on the peanut genomes is still limited. Here, we analyzed AhMITE1, AuMITE1 and ApMITE1 in the cultivated (A. hypogaea) and two wild peanut (A. duranensis and A. ipaensis) genomes.ResultsThe cultivated and the two wild peanut genomes harbored 142, 14 and 21 AhMITE1, AuMITE1 and ApMITE1 family members, respectively. These three family members exhibited highly conserved TIR sequences, and insertions preferentially occurred within 2 kb upstream and downstream of gene-coding and AT-rich regions. Phylogenetic and pairwise nucleotide diversity analysis showed that AhMITE1 and ApMITE1 family members have undergone one round of amplification bursts during the evolution of the peanut genome. PCR analyses were performed in 23 peanut varieties and demonstrated that AhMITE1 is an active transposon and that hybridization or chemical mutagenesis can promote the mobilization of AhMITE1.ConclusionsAhMITE1, AuMITE1 and ApMITE1 family members were identified based on local BLAST search with MAK between the cultivated and the two wild peanut genomes. The phylogenetic, nucleotide diversity and variation copy numbers of AhMITE1, AuMITE1 and ApMITE1 members provides opportunities for investigating their roles during peanut evolution. These findings will contribute to knowledge on diversity of AhMITE1, provide information about the potential impact on the gene expression and promote the development of DNA markers in peanut.

Diversity of the repetitive DNA fraction in Cestrum, the genus with the largest genomes within Solanaceae.

Cestrum species present large genomes (2C = ~ 24 pg), a high occurrence of B chromosomes and great diversity in heterochromatin bands. Despite this diversity, karyotypes maintain the chromosome number 2n = 16 (except when they present B chromosomes), and a relative similarity in chromosome morphology and symmetry. To deepen our knowledge of the Cestrum genome composition, low-coverage sequencing data of C. strigilatum and C. elegans were compared, including cytogenomic analyses of seven species. Bioinformatics analyses showed retrotransposons comprising more than 70% of the repetitive fraction, followed by DNA transposons (~ 17%), but FISH assays using retrotransposon probes revealed inconspicuous and scattered signals. The four satellite DNA families here analyzed represented approximately 2.48% of the C. strigilatum dataset, andthese sequences were used as probes in FISH assays. Hybridization signals were colocalized with all AT- and GC-rich sequences associated with heterochromatin, including AT-rich Cold-Sensitive Regions (CSRs). Although satellite probes hybridized in almostall tested species, a satDNA family named CsSat49 was highlighted because it predominates in centromeric regions. Data suggest that the satDNA fraction is conserved in the genus, although there is variation in the number of FISH signals between karyotypes. Except to the absence of FISH signals with probes CsSat1 and CsSat72 in two species, the other satellites occurred in species of different phylogenetic clades. Some satDNA sequences have been detected in the B chromosomes, indicating that they are rich in preexisting sequences in the chromosomes of the A complement. This comparative study provides an important advance in the knowledge on genome organization and heterochromatin composition in Cestrum, especially on the distribution of satellite fractions between species and their importance for the B chromosome composition.

Characterization of the complete mitochondrial genome sequence of Coilia brachygnathus (Clupeiformes, Engraulidae) from Huai River and its phylogenetic position

The complete mitogenome sequence of Coilia brachygnathus (Kreyenberg & Pappenheim, 1908) from Wabu Lake in Huai River Basin was annotated and characterized in this study. This mitochondrial genome is a circular DNA molecule of 16.896 bp in size with 57.52% AT content, including 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and an AT-rich region (control region) as other bony fishes. There are a total of 10 overlap locations and 15 intergenic spacer regions throughout the mitogenome of C. brachygnathus. All PCGs employed a standard ATG as a start codon, except cytochrome c oxidase 1 (cox1) with GTG. In addition, TAA or TAG was identified as the typical stop codon. A phylogenetic tree reconstructed with the maximum likelihood method depicted a clone relationship with eight species of genus Coilia and our previous study based on the amino acid sequences of 13 mitochondrial PCGs. The complete mitochondrial genome is a valuable resource in classifying and conserving C. brachygnathus.

Complete mitochondrial genomes of Papilio nephelus chaon and Papilio epycides (Lepidoptera: Papilionidae: Papilioninae) and phylogenetic analysis

The complete mitochondrial genome (mitogenome) sequences of Papilio nephelus chaon and Papilio epycides were sequenced by Illumina and analyzed in this study. They are 15,287 bp and 15,012 bp in size, respectively, and contains 13 protein-coding genes (PCGs), 22 tRNA genes (tRNAs), 2 rRNA genes (rRNAs), and 1 AT-rich control region (CR). The phylogenetic relationships of 56 species in the Papilionidae were inferred based on concatenated nucleotide sequences by using Maximum Likelihood with the selected best-fit model GTR + F+R6. The phylogenetic analysis showed that P. nephelus chaon and P. epycides were located in the genus Papilio. This study provides a basis for further study on mitogenome and phylogenetics of the Papilionidae.

Precise annotation of Drosophila mitochondrial genomes leads to insights into AT-rich regions

In the present study, we performed precise annotation of Drosophila melanogaster, D. simulans, D. grimshawi, Bactrocera oleae mitochondrial (mt) genomes using pan RNA-seq analysis. Using PacBio cDNA-seq data from D. simulans, we precisely annotated the Transcription Initiation Sites (TISs) of the mt Heavy and Light strands in Drosophila mt genomes and reported that the polyA(+) and polyA(-) motifs in the CRs are associated with TISs. The discovery of the conserved polyA(+) and polyA(-) motifs provides insights into many polyA and polyT sequences in CRs of insect mt genomes, leading to reveal the mt transcription and its regulation in invertebrates. Notably, we propose that: (1) polyA/polyT motifs in CRs function as signals to initiate mtDNA transcription; (2) the duplication, recombination or mutation of these polyA/polyT sequences formed the AT-rich regions during evolution; and (3) since CRs of many invertebrate species still contain many polyA/polyT sequences, there is a high probability that several TISs and TTSs exist in invertebrate mt genomes.

Invading viral DNA triggers dsRNA synthesis by RNA polymerase II to activate antiviral RNA interference in Drosophila.

dsRNA sensing triggers antiviral responses against RNA and DNA viruses in diverse eukaryotes. In Drosophila, Invertebrate iridescent virus 6 (IIV-6), a large DNA virus, triggers production of small interfering RNAs (siRNAs) by the dsRNA sensor Dicer-2. Here, we show that host RNA polymerase II (RNAPII) bidirectionally transcribes specific AT-rich regions of the IIV-6 DNA genome to generate dsRNA. Both replicative and naked IIV-6 genomes trigger production of dsRNA in Drosophila cells, implying direct sensing of invading DNA. Loquacious-PD, a Dicer-2 co-factor essential for the biogenesis of endogenous siRNAs, is dispensable for processing of IIV-6-derived dsRNAs, which suggests that they are distinct. Consistent with this finding, inhibition of the RNAPII co-factor P-TEFb affects the synthesis of endogenous, but not virus-derived, dsRNA. Altogether, our results suggest that a non-canonical RNAPII complex recognizes invading viral DNA to synthesize virus-derived dsRNA, which activates the antiviral siRNA pathway in Drosophila.

Complete mitochondrial genome analysis and molecular phylogenetic implications of Kennelia xylinana (Lepidoptera: Tortricidae).

Kennelia is a small genus in Tortricidae that is distributed in the Oriental and Palaearctic regions, and its taxonomic position within the subfamily Olethreutinae is controversial. For a comprehensive understanding of the genus, we sequenced the mitogenome of Kennelia xylinana, the type species of Kennelia, and Ancylis unculana, a species of Enarmoniini; analyzed the mitogenome characteristics of K. xylinana; and explored its phylogenetic position. Similar to other members of Lepidoptera, the mitogenome of K. xylinana is 15,762-bp long and consists of 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes, and a noncoding control region. In particular, we found a structure (TATAATTAATAA)11 in the middle of the AT-rich region. Based on the Bayesian inference and maximum likelihood analyses of the 13 PCGs of 40 tortricid species, representing 8 tribes of 2 subfamilies, K. xylinana was clustered with two members of Enarmoniini, A. unculana and Loboschiza koenigiana, and formed highly supported monophyly. The results indicate that Kennelia should be placed in the tribe Enarmoniini.

The Carniolan Honeybee from Slovenia-A Complete and Annotated Mitochondrial Genome with Comparisons to Closely Related Apis mellifera Subspecies.

Simple SummaryThe western honeybee, Apis mellifera, is a globally distributed bee species with many recognised subspecies, one of which is Apis mellifera carnica, the Carniolan honeybee. Apis m. carnica is native to southern Central Europe and parts of the Balkans, with the locus classicus in Slovenia. It is also widely popular with beekeepers in parts of Central and Northern Europe and other parts of the world, including the USA, Canada, and even New Zealand. In Slovenia, A. m. carnica is protected, with measures to conserve the subspecies’ autochthonous domestic population in place. Such efforts depend heavily upon genomic and phylogenetic information. In this study, we sequenced and annotated the mitochondrial genome of a specimen from Slovenia and compared the obtained data with a previously published sample of the A. m. carnica from Austria and the closely related Italian honeybee A. m. ligustica. We found several features unique to the new mitochondrial genome. We also phylogenetically analyzed the relationship between our sequence and the selected available A. mellifera mitochondrial sequences. The acquired position of the sequenced A. m. carnica from Slovenia on the phylogenetic tree brings new evidence for close relationships among C and O lineages and reflects their recent historical matrilinear ancestry.The complete mitochondrial genome of the Carniolan honeybee (Apis mellifera carnica) from Slovenia, a homeland of this subspecies, was acquired in two contigs from WGS data and annotated. The newly obtained mitochondrial genome is a circular closed loop of 16,447 bp. It comprises 37 genes (13 protein coding genes, 22 tRNA genes, and 2 rRNA genes) and an AT-rich control region. The order of the tRNA genes resembles the order characteristic of A. mellifera. The mitogenomic sequence of A. m. carnica from Slovenia contains 44 uniquely coded sites in comparison to the closely related subspecies A. m. ligustica and to A. m. carnica from Austria. Furthermore, 24 differences were recognised in comparison between A. m. carnica and A. m. ligustica subspecies. Among them, there are three SNPs that affect translation in the nd2, nd4, and cox2 genes, respectively. The phylogenetic placement of A. m. carnica from Slovenia within C lineage deviates from the expected position and changes the perspective on relationship between C and O lineages. The results of this study represent a valuable addition to the information available in the phylogenomic studies of A. mellifera—a pollinator species of worldwide importance. Such genomic information is essential for this local subspecies’ conservation and preservation as well as its breeding and selection.

New data on the mitochondrial genome of Nematocera (lower Diptera): features, structures and phylogenetic implications

Abstract Nematocera (lower Diptera) is a taxonomically diverse group with ~52 000 species in 40 families, including many important agricultural and sanitary pests. The number and composition of nematoceran infraorders have long been subject to debate. The use of mitochondrial genomes for resolving these questions shows considerable promise. Herein, we sequenced and annotated the complete or nearly complete mitochondrial genomes of eight nematoceran species from seven families, representing the first mitochondrial genomes of two infraorders and six families. They range in size from 14 447 to 16 255 bp and all contain 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and an AT-rich region. Like other nematocerans, the arrangement of genes of their mitochondrial genomes is identical to the presumed ancestral arrangement. A phylogenetic analysis was conducted by using mitochondrial genomes of 116 representatives from Diptera, Mecoptera and Siphonaptera. Our phylogenetic results support both new and traditional arrangements. The small, highly specialized family Deuterophlebiidae appears to be sister to all remaining Diptera, while Nymphomyiidae and Blephariceridae are nested within Culicomorpha and Psychodomorpha, respectively; hence, the recognition of ‘Blephariceromorpha’ as a valid monophyletic infraorder is discouraged. Anisopodidae is supported as the sister group of Brachycera.

Xenogeneic nucleoid-associated EnrR thwarts H-NS silencing of bacterial virulence with unique DNA binding

Type III and type VI secretion systems (T3/T6SS) are encoded in horizontally acquired genomic islands (GIs) that play crucial roles in evolution and virulence in bacterial pathogens. T3/T6SS expression is subjected to tight control by the host xenogeneic silencer H-NS, but how this mechanism is counteracted remains to be illuminated. Here, we report that xenogeneic nucleoid-associated protein EnrR encoded in a GI is essential for virulence in pathogenic bacteria Edwardsiella and Salmonella. We showed that EnrR plays critical roles in T3/T6SS expression in these bacteria. Various biochemical and genetic analyses demonstrated that EnrR binds and derepresses the promoter of esrB, the critical regulator of T3/T6SS, to promote their expression by competing with H-NS. Additionally, EnrR targets AT-rich regions, globally modulates the expression of ∼363 genes and is involved in various cellular processes. Crystal structures of EnrR in complex with a specific AT-rich palindromic DNA revealed a new DNA-binding mode that involves conserved HTH-mediated interactions with the major groove and contacts of its N-terminal extension to the minor groove in the symmetry-related duplex. Collectively, these data demonstrate that EnrR is a virulence activator that can antagonize H-NS, highlighting a unique mechanism by which bacterial xenogeneic regulators recognize and regulate foreign DNA.

The complete mitochondrial genome of major malaria vector Anopheles anthropophagus (Diptera: Culicidae) in China

Anopheles anthropophagus (Xu and Feng 1975) is the major vector of malaria in Eastern and Southern China. The species An. anthropophagus is considered a synonym of An. lesteri (Baisas & Hu, 1936), although they differ in several key biological characteristics. Here, we report the complete mitochondrial genome of An. anthropophagus for the first time. The mitogenome of An. anthropophagus is a typical circular, double-stranded molecule with a total length of 15,413 base pairs, and contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and an AT-rich control region. A phylogenetic analysis of the complete mitogenomes of 16 species of Anopheles (Culicidae) revealed that An. anthropophagus is closely related to An. sinensis (Wiedemann 1828), in the family Culicidae. The An. anthropophagus mitogenome provides new data for further taxonomic and phylogenetic studies of the genus Anopheles.

Molecular detection of a novel Ancylostoma sp. by whole mtDNA sequence from pangolin Manis javanica

BackgroundAncylostoma species are hematophagous parasites that cause chronic hemorrhage in various animals and humans. Pangolins, also known as scaly anteaters, are mammals that live in soil environments where they are readily exposed to soil-borne parasitic nematodes. However, only a limited number of helminth species have been identified in this animal host so far.MethodsAncylostoma sp. was isolated from a wild pangolin, and the complete mitochondrial (mt) genome of Ancylostoma sp. was obtained by Illumina sequencing of total genomic DNA.ResultsThe circular complete mt genome that was assembled had a total length of 13,757 bp and comprised 12 protein-coding genes (PCGs), 22 transfer ribosomal RNAs, two ribosomal RNAs (rRNAs), two non-coding regions and one AT-rich region, but lacked the gene coding for ATPase subunit 8 (atp8). The overall AT content of the mt genome of Ancylostoma sp. was 76%, which is similar to that of other nematodes. The PCGs used two start codons (ATT and TTG) and three stop codons (TAA, TAG, and T). The nucleotide identity of the 12 PCGs ranged from 83.1% to 89.7% and had the highest sequence identity with Ancylostoma caninum among species in the Ancylostomatidae family. Also, the pangolin-derived Ancylostoma sp. lacked repeat sequences in the non-coding regions and in the unique sequence of the short non-coding regions, which differentiated it from other Ancylostoma species. In addition, phylogenetic analyses of 18S rRNA and mtDNA sequences revealed that the Ancylostoma sp. was positioned in a separate branch in the subfamily Ancylostomatinae along with other Ancylostoma species.ConclusionsThe Ancylostoma sp. isolated from a pangolin in this study was identified as a possible new Ancylostoma species. The identification of this Ancylostoma sp. from pangolin enriches our knowledge of the species in the Ancylostomatidae family and provides information that will lead to a better understanding of the taxonomy, diagnostics, and biology of hookworms.Graphical

The complete mitochondrial genome of basiprionota bisignata (Boheman, 1862) (Coleoptera: Chrysomelidae)

The complete mitochondrial genome of Basiprionota bisignata (Boheman, 1862) (a species of leaf beetles) was successfully sequenced, annotated, and analyzed in this study. This mitochondrial genome is a circular DNA molecule of 16,069 bp in size with 78.5% AT content, including 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and an AT-rich region (control region). The gene order is consistent with the putative ancestral arrangement of insects. All PCGs are initiated by ATN (A/T/C/G) condons and terminated with TAA/G or their incomplete form single T-. All tRNAs can be folded into common clover leaf secondary structures, except for trnS1. The phylogenetic tree was reconstructed using maximum likelihood analysis, and the topology recovered the monophyly of Cassidinae and the sister relationship between Basiprionota and the clade (Thlaspida + Aspidomorph).

AT-hook peptides bind the major and minor groove of AT-rich DNA duplexes.

The mammalian high mobility group protein AT-hook 2 (HMGA2) houses three motifs that preferentially bind short stretches of AT-rich DNA regions. These DNA binding motifs, known as ‘AT-hooks’, are traditionally characterized as being unstructured. Upon binding to AT-rich DNA, they form ordered assemblies. It is this disordered-to-ordered transition that has implicated HMGA2 as a protein actively involved in many biological processes, with abnormal HMGA expression linked to a variety of health problems including diabetes, obesity, and oncogenesis. In the current work, the solution binding dynamics of the three ‘AT-hook’ peptides (ATHPs) with AT-rich DNA hairpin substrates were studied using DNA UV melting studies, fluorescence spectroscopy, native ion mobility spectrometry-mass spectrometry (IMS-MS), solution isothermal titration calorimetry (ITC) and molecular modeling. Results showed that the ATHPs bind to the DNA to form a single, 1:1 and 2:1, ‘key-locked’ conformational ensemble. The molecular models showed that 1:1 and 2:1 complex formation is driven by the capacity of the ATHPs to bind to the minor and major grooves of the AT-rich DNA oligomers. Complementary solution ITC results confirmed that the 2:1 stoichiometry of ATHP: DNA is originated under native conditions in solution.

High mobility group A3 enhances transcription of the DNA demethylase gene SlDML2 to promote tomato fruit ripening.

DNA methylation plays an important role in regulating tomato (Solanum lycopersicum) fruit ripening. Although SlDML2, a DNA demethylase (DML) gene, is critically involved in tomato fruit ripening, little is known about genes that regulate its expression. Using yeast one-hybrid screening, we identified a High Mobility Group A protein, named SlHMGA3, and demonstrated its binding activity to the AT-rich region of the SlDML2 promoter. We produced slhmga3 tomato mutants using CRISPR/Cas9 and observed that slhmga3 fruit reached the breaker stage much later than fruit from the wild-type. We further demonstrated that at the initiation stage of fruit ripening, the increased expression of SlDML2 and ethylene biosynthetic and signaling genes was significantly delayed in slhmga3 fruit, along with delays in ethylene production and demethylation and activation of ripening-associated transcription factor genes. Our results demonstrate that SlHMGA3 plays a role in enhancing SlDML2 expression, and its effects on tomato fruit ripening are largely through DNA demethylation of ripening-associated transcription factor genes.

Chromatin remodeling factors OsYAF9 and OsSWC4 interact to promote internode elongation in rice.

Deposition of H2A.Z and H4 acetylation by SWI2/SNF2-Related 1 Chromatin Remodeling (SWR1) and Nucleosome Acetyltransferase of H4 (NuA4) complexes in specific regulatory regions modulates transcription and development. However, little is known about these complexes in Oryza sativa (rice) development. Here, we reported that OsYAF9 and OsSWC4, two subunits of SWR1 and NuA4 complexes, are involved in rice vegetative and reproductive development. Loss of OsYAF9 resulted in reduced height, fewer tillers, fewer pollen grains, and defects in embryogenesis and seed filling. OsYAF9 directly interacted with OsSWC4 in vitro and in vivo. Loss of OsSWC4 function exhibited defects in pollen germination and failure to generate seeds, whereas knockdown of OsSWC4 resulted in reduced height and fewer tillers. The reduced height caused by OsYAF9 mutation and OsSWC4 knockdown was due to shorter internodes and defects in cell elongation, and this phenotype was rescued with gibberellin (GA) treatment, suggesting that both OsYAF9 and OsSWC4 are involved in the GA biosynthesis pathway. OsSWC4 was directly bound to the AT-rich region of GA biosynthesis genes, which in turn accomplished H2A.Z deposition and H4 acetylation at the GA biosynthesis genes with OsYAF9. Together, our study provides insights into the mechanisms involving OsSWC4 and OsYAF9 forming a protein complex to promote rice internode elongation with H2A.Z deposition and H4 acetylation.

The mitochondrial genome of Grapsus albolineatus (Decapoda: Brachyura: Grapsidae) and phylogenetic associations in Brachyura

Complete mitochondrial genomes (mitogenomes) can provide useful information for phylogenetic relationships, gene rearrangement, and evolutionary traits. In this study, we determined the complete mitochondrial DNA sequence of the herbivorous crab Grapsus albolineatus. It is a typical metazoan mitochondrial genome. The total size is 15,583 bp, contains the entire set of 37 genes, and has an AT-rich region. Then, 23 of the 37 genes were encoded by the heavy (+) strand while 14 are encoded by the light (−) strand. Compared with the pan-crustacean ground pattern, two tRNA genes (tRNA-His and tRNA-Gln) were rearranged and the tandem duplication/random loss model was used to explain the observed gene rearrangements. The phylogenetic results showed that all Grapsidae crabs clustered together as a group. Furthermore, the monophyly of each family was well supported, with the exception of Menippidae. In general, the results obtained in this study will contribute to the better understanding of gene rearrangements in Grapsidae crab mitogenomes and provide new insights into the phylogeny of Brachyura.

T-DNA integration and its effect on gene expression in dual Bt gene transgenic Populus ×euramericana cv. Neva

The integration of Bacillus thuringiensis (Bt) genes is often accompanied by unintended effects along with the improved resistance to targeted pests. The insertion information of transfer DNA (T-DNA) and the expression information of upstream and downstream genes are of great significance for related research on unexpected effects and molecular-level mechanisms. In this study, six dual Bt transgenic Populus × euramericana cv. Neva (poplar 107) lines were used as research objects. We determined growth and physiological indices, and characterized the T-DNA integration using next-generation sequencing technology. The transgenic and non-transgenic lines showed no significant difference in growth index. However, while insect resistance was enhanced, effects related to the integration sites of the Bt gene occurred. A total of 15 insertion sites were detected among the six transgenic lines, and T-DNA preferentially inserted into AT-rich regions of the poplar genome. RNA sequencing and weighted gene co-expression network analysis were used to explore the effects of T-DNA insertion on the gene expression of poplar 107. As a result, the metabolic pathways most affected by the Bt gene were starch and sucrose metabolism and pentose and gluconate conversion. The expression of exogenous Bt had a greater impact than the insertion position or copy number on poplar 107 gene expression, and the Cry1Ac toxin protein also played a major role. This study provides theoretical support for the cultivation and management of poplar 107 new varieties, and provides reference for Poplar insect resistance breeding and its molecular response to Bt gene.

The complete mitochondrial genome of Clepsis pallidana (Lepidoptera: Tortricidae) with phylogenetic implications

The leaf roller, Clepsis pallidana (Fabricius, 1776) (Lepidoptera: Tortricidae), is an important pest of many crops in China. The circular genome is 15,679 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a non-coding AT-rich region. The base composition of the whole mitogenome is 41.2% A, 40.1% T, 11.1% C, and 7.7% G, which shows a strong AT bias. Phylogenetic analysis based on 13 PCGs showed that C. pallidana was closely related to Epiphyas postvittana and clustered within Archipini clade, which was consistent with the traditional classification.

African swine fever virus I267L acts as an important virulence factor by inhibiting RNA polymerase III-RIG-I-mediated innate immunity.

ASFV is a large DNA virus that is highly pathogenic in domestic pigs. How this virus is sensed by the innate immune system as well as why it is so virulent remains enigmatic. In this study, we show that the ASFV genome contains AT-rich regions that are recognized by the DNA-directed RNA polymerase III (Pol-III), leading to viral RNA sensor RIG-I-mediated innate immune responses. We further show that ASFV protein I267L inhibits RNA Pol-III-RIG-I-mediated innate antiviral responses. I267L interacts with the E3 ubiquitin ligase Riplet, disrupts Riplet-RIG-I interaction and impairs Riplet-mediated K63-polyubiquitination and activation of RIG-I. I267L-deficient ASFV induces higher levels of interferon-β, and displays compromised replication both in primary macrophages and pigs compared with wild-type ASFV. Furthermore, I267L-deficiency attenuates the virulence and pathogenesis of ASFV in pigs. These findings suggest that ASFV I267L is an important virulence factor by impairing innate immune responses mediated by the RNA Pol-III-RIG-I axis.