Revealing critical mechanisms in determining sorghum resistance to drought and salt using mRNA, small RNA and degradome sequencing.

BACKGROUND: Plant growth and development are severely threatened by drought and salt stresses. Compared with structural genes, transcription factors (TFs) play more pivotal roles in plant growth and stress adaptation. However, the underlying mechanisms of sorghum adapting to drought and salt are insufficient, and systematic analysis of TFs in response to the above stresses is lacking. RESULTS: In this study, TFs were identified in sorghum and model plants (Arabidopsis thaliana and rice), and gene number and conserved domain were compared between sorghum and model plants. According to syntenic analysis, the expansion of sorghum and rice TFs may be due to whole-genome duplications. Between sorghum and model plants TFs, specific conserved domains were identified and they may be related to functional diversification of TFs. Forty-five key genes in sorghum, including four TFs, were likely responsible for drought adaption based on differently expression analysis. MiR5072 and its target gene (Sobic.001G449600) may refer to the determination of sorghum drought resistance according to small RNA and degradome analysis. Six genes were associated with drought adaptation of sorghum based on weighted gene co-expression network analysis (WGCNA). Similarly, the core genes in response to salt were also characterized using the above methods. Finally, 15 candidate genes, particularly two TFs (Sobic.004G300300, HD-ZIP; Sobic.003G244100, bZIP), involved in combined drought and salt resistance of sorghum were identified. CONCLUSIONS: In summary, the findings in this study help clarify the molecular mechanisms of sorghum responding to drought and salt. We identified candidate genes and provide important genetic resource for potential development of drought-tolerant and salt-tolerant sorghum plants.

Automated detection of myocardial infarction based on an improved state refinement module for LSTM/GRU.

Myocardial infarction (MI) is a common cardiovascular disease caused by the blockages of coronary arteries. The visual inspection of electrocardiogram (ECG) is the main diagnosis pattern, while it is taxing and time-consuming. Motivated from state refinement module for long short term memory (SRM-LSTM), we proposed two improved state refinement frameworks based on LSTM and gated recurrent unit (GRU) called ISRM-LSTM and ISRM-GRU. Both are capable of adaptively refining current states of sample points in ECG with a message passing mechanism than existing LSTM. To evaluate the validity, both are installed into convolutional network architecture and standard LSTM, GRU and Residual networks are employed as control groups across the Physikalisch-Technische Bundesanstalt database. Empirical results confirm noticeable performance improvements than control groups and several existing algorithms with an accuracy of 99.1%. To our knowledge, both modules are the first attempt to consider the interaction characteristics into deep network and improve interpretability exhibiting considerable potentials on lightweight devices thanks to only utilization of three channel ECGs.

Metabarcoding and Metabolome Analyses Reveal Mechanisms of Leymus chinensis Growth Promotion by Fairy Ring of Leucocalocybe mongolica.

Fairy rings are a unique ecological phenomenon caused by the growth of the fungal mycelium in the soil. Fairy rings formed by Leucocalocybe mongolica (LM) are generally distributed in the Mongolian Plateau, where they promote plant growth without fertilization and alleviate fertilizer use. We previously investigated the soil factors regulating growth promotion in a fairy ring ecosystem; however, the aspects of the plant (Leymus chinensis, LC) that promote growth have not been explored. Therefore, the present study investigated the endophyte diversity and metabolome of LC in an LM fairy ring ecosystem. We analyzed the leaf and root samples of LC from the DARK (FR) and OUT (CK) zones. The fairy rings significantly improved the fungal diversity of roots and leaves and the bacterial diversity of leaves in the FR zone. Ralstonia was the dominant bacteria detected in the LC leaves. In addition, Marasmius, another fairy ring fungal genus, was also detected with a high abundance in the roots of the FR zone. Furthermore, widely targeted metabolome analysis combined with KEGG annotation identified 1011 novel metabolites from the leaves and roots of LC and seven pathways significantly regulated by the fairy ring in the FR zone. The fairy ring ecosystem significantly downregulated the flavonoid metabolism in the leaves and roots of LC. The correlation analysis found Ralstonia is a potential regulatory factor of flavonoid biosynthesis in LC. In addition, salicylic acid and jasmonic acid were found upregulated in the leaves, probably related to Marasmius enrichment. Thus, the study details plant factors associated with enhanced growth in an LM fairy ring ecosystem. These findings lay a theoretical foundation for developing the fairy ring ecosystem in an agricultural system.

Co-exposure of chronic stress and alumina nanoparticles aggravates hippocampal microglia pyroptosis by activating cathepsin B/NLRP3 signaling pathway.

Combined exposure of chronic stress and alumina nanoparticles (AlNPs) aggravates hippocampal injury, but the pathogenesis is unevaluated. This study aimed to investigate the effect and mechanism of co-exposure to chronic stress and AlNPs on hippocampal microglia pyroptosis. In this study, chronic restraint stress (CRS) alone caused NLRP3-mediated hippocampal microglia pyroptosis, but AlNPs did not. Moreover, co-exposure to CRS and AlNPs exacerbated hippocampal microglia pyroptosis, resulting in more severe hippocampal damage and behavioral deficits in rats. Protein-protein interaction network predicted that cathepsin B was a potential regulatory protein of NLRP3. CRS up-regulated cathepsin B expression which had a more pronounced increase in co-exposure group. Whereas, caspase-1 inhibitor VX-765 alleviated hippocampal microglia pyroptosis and behavioral deficits in rats. Consistent with in vivo results, co-exposure of corticosterone and AlNPs aggravated NLRP3-mediated pyroptosis and cathepsin B expression in HAPI cells. Nevertheless, the pyroptosis of HAPI cells was inhibited by cathepsin B inhibitor CA-074Me and NLRP3 knockout, respectively. NLRP3 agonist nigericin failed to promote the pyroptosis of HAPI cells in the presence of cathepsin B inhibition. These results demonstrated that co-exposure to chronic stress and AlNPs could aggravate hippocampal microglia pyroptosis by activating cathepsin B/NLRP3 signaling pathway, resulting in hippocampal damage and behavioral deficits.

The Divergent Roles of the Rice bcl-2 Associated Athanogene (BAG) Genes in Plant Development and Environmental Responses.

Bcl-2-associated athanogene (BAG), a group of proteins evolutionarily conserved and functioned as co-chaperones in plants and animals, is involved in various cell activities and diverse physiological processes. However, the biological functions of this gene family in rice are largely unknown. In this study, we identified a total of six BAG members in rice. These genes were classified into two groups, OsBAG1, -2, -3, and -4 are in group I with a conserved ubiquitin-like structure and OsBAG5 and -6 are in group Ⅱ with a calmodulin-binding domain, in addition to a common BAG domain. The BAG genes exhibited diverse expression patterns, with OsBAG4 showing the highest expression level, followed by OsBAG1 and OsBAG3, and OsBAG6 preferentially expressed in the panicle, endosperm, and calli. The co-expression analysis and the hierarchical cluster analysis indicated that the OsBAG1 and OsBAG3 were co-expressed with primary cell wall-biosynthesizing genes, OsBAG4 was co-expressed with phytohormone and transcriptional factors, and OsBAG6 was co-expressed with disease and shock-associated genes. ß-glucuronidase (GUS) staining further indicated that OsBAG3 is mainly involved in primary young tissues under both primary and secondary growth. In addition, the expression of the BAG genes under brown planthopper (BPH) feeding, N, P, and K deficiency, heat, drought and plant hormones treatments was investigated. Our results clearly showed that OsBAGs are multifunctional molecules as inferred by their protein structures, subcellular localizations, and expression profiles. BAGs in group I are mainly involved in plant development, whereas BAGs in group II are reactive in gene regulations and stress responses. Our results provide a solid basis for the further elucidation of the biological functions of plant BAG genes.

Automated detection of premature ventricular contraction based on the improved gated recurrent unit network.

BACKGROUND AND OBJECTIVE: Premature ventricular contraction (PVC) is the common arrhythmia disease, affecting thousands of individuals worldwide. However, the traditional PVC detection is cumbersome by visually inspecting electrocardiogram (ECG) signals. METHODS: In this work, we specially propose an improved gated recurrent unit (IGRU) by setting a scale parameter into existing bidirectional GRU (BGRU) model for PVC signals recognition, which is used to alleviate the problem of information redundancy in BGRU. To verify the effectiveness, IGRU model will be embedded into a convolutional network frame and existing GRU and BGRU models are employed as control groups for a fair comparison. RESULTS: The results exhibit that the model attains better model performance than control groups and several state-of-the-art algorithms with the accuracy of 98.3% and 97.9% with the MIT-BIH arrhythmia database and China Physiological Signal Challenge 2018 database. Besides, motivated from the waveform characteristics of ECG signals in PVC, the proposed model can provide certain physiological interpretability for physicians and researchers. CONCLUSIONS: To our knowledge, this is the first attempt to re-design the existing GRU network for ECG signals classification, thus exhibiting great application potentials especially in lightweight equipment such as mobile phone and camera.

Transcriptome and MiRNAomics Analyses Identify Genes Associated with Cytoplasmic Male Sterility in Cotton (Gossypium hirsutum L.).

Cytoplasmic male sterility (CMS) is important for large-scale hybrid seed production. Rearrangements in the mitochondrial DNA (mtDNA) for the cotton (Gossypium hirsutum L.) CMS line J4A were responsible for pollen abortion. However, the expression patterns of nuclear genes associated with pollen abortion and the molecular basis of CMS for J4A are unknown, and were the objectives of this study by comparing J4A with the J4B maintainer line. Cytological evaluation of J4A anthers showed that microspore abortion occurs during meiosis preventing pollen development. Changes in enzyme activity of mitochondrial respiratory chain complex IV and mitochondrial respiratory chain complex V and the content of ribosomal protein and ATP during anther abortion were observed for J4A suggesting insufficient synthesis of ATP hindered pollen production. Additionally, levels of sucrose, starch, soluble sugar, and fructose were significantly altered in J4A during the meiosis stage, suggesting reduced sugar metabolism contributed to sterility. Transcriptome and miRNAomics analyses identified 4461 differentially expressed mRNAs (DEGs) and 26 differentially expressed microRNAs (DEMIs). Pathway enrichment analysis indicated that the DEMIs were associated with starch and sugar metabolism. Six deduced target gene regulatory pairs that may participate in CMS were identified, ghi-MIR7484-10/mitogen-activated protein kinase kinase 6 (MAPKK6), ghi-undef-156/agamous-like MADS-box protein AGL19 (AGL19), ghi-MIR171-1-22/SNF1-related protein kinase regulatory subunit gamma-1 and protein trichome birefringence-like 38, and ghi-MIR156-(8/36)/WRKY transcription factor 28 (WRKY28). Overall, a putative CMS mechanism involving mitochondrial dysfunction, the ghi-MIR7484-10/MAPKK6 network, and reduced glucose metabolism was suggested, and ghi-MIR7484-10/MAPKK6 may be related to abnormal microspore meiosis and induction of excessive sucrose accumulation in anthers.

Uncovering genomic regions controlling plant architectural traits in hexaploid wheat using different GWAS models.

Wheat is a major food crop worldwide. The plant architecture is a complex trait mostly influenced by plant height, tiller number, and leaf morphology. Plant height plays a crucial role in lodging and thus affects yield and grain quality. In this study, a wheat population was genotyped by using Illumina iSelect 90K single nucleotide polymorphism (SNP) assay and finally 22,905 high-quality SNPs were used to perform a genome-wide association study (GWAS) for plant architectural traits employing four multi-locus GWAS (ML-GWAS) and three single-locus GWAS (SL-GWAS) models. As a result, 174 and 97 significant SNPs controlling plant architectural traits were detected by ML-GWAS and SL-GWAS methods, respectively. Among these SNP makers, 43 SNPs were consistently detected, including seven across multiple environments and 36 across multiple methods. Interestingly, five SNPs (Kukri_c34553_89, RAC875_c8121_1490, wsnp_Ex_rep_c66315_64480362, Ku_c5191_340, and tplb0049a09_1302) consistently detected across multiple environments and methods, played a role in modulating both plant height and flag leaf length. Furthermore, candidate SNPs (BS00068592_51, Kukri_c4750_452 and BS00022127_51) constantly repeated in different years and methods associated with flag leaf width and number of tillers. We also detected several SNPs (Jagger_c6772_80, RAC875_c8121_1490, BS00089954_51, Excalibur_01167_1207, and Ku_c5191_340) having common associations with more than one trait across multiple environments. By further appraising these GWAS methods, the pLARmEB and FarmCPU models outperformed in SNP detection compared to the other ML-GWAS and SL-GWAS methods, respectively. Totally, 152 candidate genes were found to be likely involved in plant growth and development. These finding will be helpful for better understanding of the genetic mechanism of architectural traits in wheat.

Appraising the Genetic Architecture of Kernel Traits in Hexaploid Wheat Using GWAS.

Kernel morphology is one of the major yield traits of wheat, the genetic architecture of which is always important in crop breeding. In this study, we performed a genome-wide association study (GWAS) to appraise the genetic architecture of the kernel traits of 319 wheat accessions using 22,905 single nucleotide polymorphism (SNP) markers from a wheat 90K SNP array. As a result, 111 and 104 significant SNPs for Kernel traits were detected using four multi-locus GWAS models (mrMLM, FASTmrMLM, FASTmrEMMA, and pLARmEB) and three single-locus models (FarmCPU, MLM, and MLMM), respectively. Among the 111 SNPs detected by the multi-locus models, 24 SNPs were simultaneously detected across multiple models, including seven for kernel length, six for kernel width, six for kernels per spike, and five for thousand kernel weight. Interestingly, the five most stable SNPs (RAC875_29540_391, Kukri_07961_503, tplb0034e07_1581, BS00074341_51, and BobWhite_049_3064) were simultaneously detected by at least three multi-locus models. Integrating these newly developed multi-locus GWAS models to unravel the genetic architecture of kernel traits, the mrMLM approach detected the maximum number of SNPs. Furthermore, a total of 41 putative candidate genes were predicted to likely be involved in the genetic architecture underlining kernel traits. These findings can facilitate a better understanding of the complex genetic mechanisms of kernel traits and may lead to the genetic improvement of grain yield in wheat.

Multi-Locus Genome-Wide Association Studies for 14 Main Agronomic Traits in Barley.

The agronomic traits, including morphological and yield component traits, are important in barley breeding programs. In order to reveal the genetic foundation of agronomic traits of interest, in this study 122 doubled haploid lines from a cross between cultivars "Huaai 11" (six-rowed and dwarf) and "Huadamai 6" (two-rowed) were genotyped by 9680 SNPs and phenotyped 14 agronomic traits in 3 years, and the two datasets were used to conduct multi-locus genome-wide association studies. As a result, 913 quantitative trait nucleotides (QTNs) were identified by five multi-locus GWAS methods to be associated with the above 14 traits and their best linear unbiased predictions. Among these QTNs and their adjacent genes, 39 QTNs (or QTN clusters) were repeatedly detected in various environments and methods, and 10 candidate genes were identified from gene annotation. Nineteen QTNs and two genes (sdw1/denso and Vrs1) were previously reported, and eight candidate genes need to be further validated. The Vrs1 gene, controlling the number of rows in the spike, was found to be associated with spikelet number of main spike, spikelet number per plant, grain number per plant, grain number per spike, and 1,000 grain weight in multiple environments and by multi-locus GWAS methods. Therefore, the above results evidenced the feasibility and reliability of genome-wide association studies in doubled haploid population, and the QTNs and their candidate genes detected in this study are useful for marker-assisted selection breeding, gene cloning, and functional identification in barley.

Detection of QTLs for seedling characteristics in barley (Hordeum vulgare L.) grown under hydroponic culture condition.

BACKGROUND: Seedling characteristics play significant roles in the growth and development of barley (Hordeum vulgare L.), including stable stand establishment, water and nutrients uptake, biotic resistance and abiotic stresses, and can influence yield and quality. However, the genetic mechanisms underlying seedling characteristics in barley are largely unknown and little research has been done. In the present work, 21 seedling-related characteristics are assessed in a barley double haploid (DH) population, grown under hydroponic conditions. Of them, leaf age (LAG), shoot height (SH), maximum root length (MRL), main root number (MRN) and seedling fresh weight (SFW) were investigated at the 13th, 20th, 27th, and 34th day after germination. The objectives were to identify quantitative trait loci (QTLs) underlying these seedling characteristics using a high-density linkage map and to reveal the QTL expression pattern by comparing the QTLs among four different seedling growth stages. RESULTS: A total of 70 QTLs were distributed over all chromosomes except 4H, and, individually, accounted for 5.01%-77.78% of phenotypic variation. Out of the 70 detected QTLs, 23 showed a major effect on 14 seedling-related characteristics. Ten co-localized chromosomal regions on 2H (five regions), 3H (two regions) and 7H (three regions) involved 39 QTLs (55.71%), each simultaneously influenced more than one trait. Meanwhile, 9 co-localized genomic regions involving 22 QTLs for five seedling characteristics (LAG, SH, MRL, MRN and SFW) at the 13th, 20th, 27th and 34th day-old seedling were common for two or more growth stages of seedling. QTL in the vicinity of Vrs1 locus on chromosome 2H with the favorable alleles from Huadamai 6 was found to have the largest main effects on multiple seedling-related traits. CONCLUSIONS: Six QTL cluster regions associated with 16 seedling-related characteristics were observed on chromosome 2H, 3H and 7H. The majority of the 29 regions identified for five seedling characteristics were selectively expressed at different developmental stages. The genetic effects of 9 consecutive expression regions displayed different developmental influences at different developmental stages. These findings enhanced our understanding of a genetic basis underlying seedling characteristics in barley. Some QTLs detected here could be used for marker-assisted selection (MAS) in barley breeding.

SNP-based high density genetic map and mapping of btwd1 dwarfing gene in barley.

A high-density linkage map is a valuable tool for functional genomics and breeding. A newly developed sequence-based marker technology, restriction site associated DNA (RAD) sequencing, has been proven to be powerful for the rapid discovery and genotyping of genome-wide single nucleotide polymorphism (SNP) markers and for the high-density genetic map construction. The objective of this research was to construct a high-density genetic map of barley using RAD sequencing. 1894 high-quality SNP markers were developed and mapped onto all seven chromosomes together with 68 SSR markers. These 1962 markers constituted a total genetic length of 1375.8 cM and an average of 0.7 cM between adjacent loci. The number of markers within each linkage group ranged from 209 to 396. The new recessive dwarfing gene btwd1 in Huaai 11 was mapped onto the high density linkage maps. The result showed that the btwd1 is positioned between SNP marks 7HL_6335336 and 7_249275418 with a genetic distance of 0.9 cM and 0.7 cM on chromosome 7H, respectively. The SNP-based high-density genetic map developed and the dwarfing gene btwd1 mapped in this study provide critical information for position cloning of the btwd1 gene and molecular breeding of barley.

QTL underlying some agronomic traits in barley detected by SNP markers.

BACKGROUND: Increasing the yield of barley (Hordeum vulgare L.) is a main breeding goal in developing barley cultivars. A high density genetic linkage map containing 1894 SNP and 68 SSR markers covering 1375.8 cM was constructed and used for mapping quantitative traits. A late-generation double haploid population (DH) derived from the Huaai 11 × Huadamai 6 cross was used to identify QTLs and QTL × environment interactions for ten traits affecting grain yield including length of main spike (MSL), spikelet number on main spike (SMS), spikelet number per plant (SLP), grain number per plant (GP), grain weight per plant (GWP), grain number per spike (GS), thousand grain weight (TGW), grain weight per spike (GWS), spike density (SPD) and spike number per plant (SP). RESULTS: In single environment analysis using composite interval mapping (CIM), a total of 221 QTLs underlying the ten traits were detected in five consecutive years (2009-2013). The QTLs detected in each year were 50, 48, 41, 41 and 41 for the year 2009 to 2013. The QTLs associated with these traits were generally clustered on chromosome 2H, 4H and 7H. In multi-environment analysis, a total of 111 significant QTLs including 18 for MSL, 16 for SMS, 15 for SPD, 5 for SP, 4 for SLP, 14 for TGW, 5 for GP, 11 for GS, 8 for GWP, and 15 for GWS were detected in the five years. Most QTLs showed significant QTL × environment interactions (QEI), nine QTLs (qIMSL3-1, qIMSL4-1, qIMSL4-2, qIMSL6-1, qISMS7-1, qISPD2-7, qISPD7-1, qITGW3-1 and qIGWS4-3) were detected with minimal QEI effects and stable in different years. Among 111 QTLs,71 (63.40 %) QTLs were detected in both single and multiple environments. CONCLUSIONS: Three main QTL cluster regions associated with the 10 agronomic traits on chromosome 2H, 4H and 7H were detected. The QTLs for SMS, SLP, GP and GWP were located in the region near Vrs1 on chromosome 2H. The QTLs underlying SMS, SPD and SLP were clustered on chromosome 4H. On the terminal of chromosome 7H, there was a QTL cluster associated with TGW, SPD, GWP and GWS. The information will be useful for marker-assisted selection (MAS) in barley breeding.

Effects of Porphyromonas gingivalis extracellular vesicles on human periodontal ligament fibroblasts.

The purpose of this study was to investigate the influencing mechanism of Porphyromonas gingivalis extracellular vesicles on human periodontal ligament fibroblasts to better understand the pathogenesis of periodontitis, the major cause of adult tooth loss. Human periodontal ligament fibroblasts were cultured and randomly assigned to a control group and an extracellular vesicles (ECV) group. The ECV group was exposed to isolated Porphyromonas gingivalis extracellular vesicles; the control group was not exposed. Western blotting was used to detect expression of matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1), and RT-PCR was used to detect mRNA expression of alkaline phosphatase (ALP). When human periodontal ligament fibroblasts were processed by Porphyromonas gingivalis extracellular vesicles (ECV), protein expression levels of both MMP-1 and TIMP-1 were significantly higher than that of the control group (P<0.05). In contrast, ALP mRNA expression in human periodontal ligament fibroblasts processed by ECV was significantly lower than that of the control group (P<0.05). Porphyromonas gingivalis extracellular vesicles can up-regulate expression of MMP-1 and TIMP-1 protein and ALP mRNA of human periodontal fibroblasts.

Functional and genetic studies of the substrate specificity of coronavirus infectious bronchitis virus 3C-like proteinase.

Coronavirus (CoV) 3C-like proteinase (3CLpro), located in nonstructural protein 5 (nsp5), processes the replicase polyproteins 1a and 1ab (pp1a and pp1ab) at 11 specific sites to produce 12 mature nonstructural proteins (nsp5 to nsp16). Structural and biochemical studies suggest that a conserved Gln residue at the P1 position is absolutely required for efficient cleavage. Here, we investigate the effects of amino acid substitution at the P1 position of 3CLpro cleavage sites of infectious bronchitis virus (IBV) on the cleavage efficiency and viral replication by in vitro cleavage assays and reverse genetic approaches. Our results demonstrated that a P1-Asn substitution at the nsp4-5/Q2779, nsp5-6/Q3086, nsp7-8/Q3462, nsp8-9/Q3672, and nsp9-10/Q3783 sites, a P1-Glu substitution at the nsp8-9/Q3672 site, and a P1-His substitution at the nsp15-16/Q6327 site were tolerated and allowed recovery of infectious mutant viruses, albeit with variable degrees of growth defects. In contrast, a P1-Asn substitution at the nsp6-7/Q3379, nsp12-13/Q4868, nsp13-14/Q5468, and nsp14-15/Q5989 sites, as well as a P1-Pro substitution at the nsp15-16/Q6327 site, abolished 3CLpro-mediated cleavage at the corresponding position and blocked the recovery of infectious viruses. Analysis of the effects of these lethal mutations on RNA synthesis suggested that processing intermediates, such as the nsp6-7, nsp12-13, nsp13-14, nsp14-15, and nsp15-16 precursors, may function in negative-stranded genomic RNA replication, whereas mature proteins may be required for subgenomic RNA (sgRNA) transcription. More interestingly, a mutant 3CLpro with either a P166S or P166L mutation was selected when an IBV infectious cDNA clone carrying the Q6327N mutation at the nsp15-16 site was introduced into cells. Either of the two mutations was proved to enhance significantly the 3CLpro-mediated cleavage efficiency at the nsp15-16 site with a P1-Asn substitution and compensate for the detrimental effects on recovery of infectious virus.

Interaction of the coronavirus infectious bronchitis virus membrane protein with beta-actin and its implication in virion assembly and budding.

Coronavirus M protein is an essential component of virion and plays pivotal roles in virion assembly, budding and maturation. The M protein is integrated into the viral envelope with three transmembrane domains flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. In this study, we showed co-purification of the M protein from coronavirus infectious bronchitis virus (IBV) with actin. To understand the cellular factors that may be involved in virion assembly, budding and maturation processes, IBV M was used as the bait in a yeast two-hybrid screen, resulting in the identification of beta-actin as a potentially interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation and immunofluorescence microscopy in mammalian cells, and mutation of amino acids A159 and K160 on the M protein abolished the interaction. Introduction of the A159-K160 mutation into an infectious IBV clone system blocks the infectivity of the clone, although viral RNA replication and subgenomic mRNA transcription were actively detected. Disruption of actin filaments with cell-permeable agent cytochalasin D at early stages of the infection cycle led to the detection of viral protein synthesis in infected cells but not release of virus particles to the cultured media. However, the same treatment at late stages of the infection cycle did not affect the release of virus particles to the media, suggesting that disruption of the actin filaments might block virion assembly and budding, but not release of the virus particles. This study reveals an essential function of actin in the replication cycle of coronavirus.

Proteolytic processing of polyproteins 1a and 1ab between non-structural proteins 10 and 11/12 of Coronavirus infectious bronchitis virus is dispensable for viral replication in cultured cells.

Coronavirus 3C-like proteinase (3CLpro) plays important roles in viral life cycle through extensive processing of the polyproteins 1a and 1ab into 12 mature, non-structural proteins (nsp5-nsp16). Structural and biochemical studies have revealed that all confirmed 3CLpro cleavage sites have a conserved Gln residue at the P1 position, which is thought to be absolutely required for efficient cleavage. Recent studies on murine hepatitis virus (MHV) showed that processing of the 1a polyprotein at the position between nsp10-nsp11 is essential for viral replication. In this report, we investigated the requirement of processing at the equivalent position for replication of avian coronavirus infectious bronchitis virus (IBV), using an infectious cloning system. The results showed that mutation of the P1 Gln to Pro or deletion of the Gln residue in the nsp10-nsp11/12 site completely abolished the 3CLpro-mediated processing, but allowed production of infectious recombinant viruses with variable degrees of growth defect, suggesting that cleavage at the nsp10-nsp11/12 site of IBV is dispensable for viral replication in cultured cells. This study would pave a way for potential vaccine development by generation of attenuated IBV from field isolates through manipulation of the nsp10-nsp11/12 cleavage site. Similar approaches would be also applicable to other human and animal coronaviruses.