CRISPR-Cas9-mediated chicken prmt5 gene knockout and its critical role in interferon regulation.

Protein arginine methyltransferase 5 (PRMT5), a type II arginine methyltransferase, controls arginine dimethylation of a variety of substrates. While many papers have reported the function of mammalian PRMT5, it remains unclear how PRMT5 functions in chicken cells. In this study, we found that chicken (ch) PRMT5 is widely expressed in a variety of chicken tissues and is distributed in both the cytoplasm and the nucleus. Ectopic expression of chPRMT5 significantly suppresses chIFN-ß activation induced by chMDA5. In addition, a prmt5 gene-deficient DF-1 cell line was constructed using CRISPR/Cas9. In comparison with the wild-type cells, the prmt5-/- DF-1 cells displays normal morphology and maintain proliferative capacity. Luciferase reporter assay and overexpression showed that prmt5-/- DF-1 cells had increased IFN-ß production. With identified chicken PRMT5 and CRISPR/Cas9 knockout performed in DF-1 cells, we uncovered a functional link of chPRMT5 in suppression of IFN-ß production and interferon-stimulated gene expression.

PRMT5 Facilitates Infectious Bursal Disease Virus Replication through Arginine Methylation of VP1.

The infectious bursal diseases virus (IBDV) polymerase, VP1 protein, is responsible for transcription, initial translation and viral genomic replication. Knowledge about the new kind of post-translational modification of VP1 supports identification of novel drugs against the virus. Because the arginine residue is known to be methylated by protein arginine methyltransferase (PRMT) enzyme, we investigated whether IBDV VP1 is a substrate for known PRMTs. In this study, we show that VP1 is specifically associated with and methylated by PRMT5 at the arginine 426 (R426) residue. IBDV infection causes the accumulation of PRMT5 in the cytoplasm, which colocalizes with VP1 as a punctate structure. In addition, ectopic expression of PRMT5 significantly enhances the viral replication. In the presence of PMRT5, enzyme inhibitor and knockout of PRMT5 remarkably decreased viral replication. The polymerase activity of VP1 was severely damaged when R426 mutated to alanine, resulting in impaired viral replication. Our study reports a novel form of post-translational modification of VP1, which supports its polymerase function to facilitate the viral replication. IMPORTANCE Post-translational modification of infectious bursal disease virus (IBDV) VP1 is important for the regulation of its polymerase activity. Investigation of the significance of specific modification of VP1 can lead to better understanding of viral replication and can probably also help in identifying novel targets for antiviral compounds. Our work demonstrates the molecular mechanism of VP1 methylation mediated by PRMT5, which is critical for viral polymerase activity, as well as viral replication. Our study expands a novel insight into the function of arginine methylation of VP1, which might be useful for limiting the replication of IBDV.

Phosphorylation of VP1 Mediated by CDK1-Cyclin B1 Facilitates Infectious Bursal Disease Virus Replication.

Infectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus belonging to the genus Avibirnavirus in the family Birnaviridae. It can cause serious failure of vaccination in young poultry birds with impaired immune systems. Post-translational modifications of the VP1 protein are essential for viral RNA transcription, genome replication, and viral multiplication. Little information is available so far regarding the exact mechanism of phosphorylation of IBDV VP1 and its significance in the viral life cycle. Here, we provide several lines of evidence that the cyclin-dependent kinase 1 (CDK1)-cyclin B1 complex phosphorylates VP1, which facilitates viral replication. We show that the CDK1-cyclin B1 specifically interacts with VP1 and phosphorylates VP1 on the serine 7 residue, located in the N-terminal 7SPAQ10 region, which follows the optimal phosphorylation motif of CDK1, p-S/T-P. Additionally, IBDV infection drives the cytoplasmic accumulation of CDK1-cyclin B1, which co-localizes with VP1, supporting the kinase activity of CDK1-cyclin B1. Treatment with CDK1 inhibitor RO3306 and knockdown of CDK1-cyclin B1 severely disrupts the polymerase activity of VP1, resulting in diminished viral replication. Moreover, the replication of S7A mutant recombinant IBDV was significantly decreased compared to that of wild-type (WT) IBDV. Thus, CDK1-cyclin B1 is a crucial enzyme which phosphorylates IBDV VP1 on serine 7, which is necessary both for the polymerase activity of VP1 and for viral replication. IMPORTANCE Infectious bursal disease virus still poses a great economic threat to the global poultry farming industry. Detailed information on the steps of viral genome replication is essential for the development of antiviral therapeutics. Phosphorylation is a common post-translational modification in several viral proteins. There is a lack of information regarding the significance of VP1 phosphorylation and its role in modulating the viral life cycle. In this study, we found that CDK1-cyclin B1 accumulates in the cytoplasm and phosphorylates VP1 on serine 7. The presence of a CDK1 inhibitor and the silencing of CDK1-cyclin B1 decrease IBDV replication. The mutation of VP1 serine 7 to alanine reduces VP1 polymerase activity, disrupting the viral life cycle, which suggests that this residue serves an essential function. Our study offers novel insights into the regulatory mechanism of VP1 phosphorylation.

Chicken PRMT1 promotes infectious bursal disease virus replication via suppressing IFN-ß production.

The protein arginine methyltransferase (PRMT) family, such as PRMT1, regulates the arginine methylation of various substrates. Many studies have examined the role of PRMT1 in mammals, however, it is still unknown how PRMT1 works in chickens. To investigate the effect of chicken PRMT1 (chPRMT1) on regulating IFN-ß production and IBDV replication, chPRMT1 knock out DF-1 cells were constructed in this study. First, we found that chPRMT1 was widely expressed in a variety of chicken tissues and that it was distributed in the cytoplasm and nucleus of DF-1 cells. Additionally, IFN-ß activation was inhibited by chPRMT1 at the step of chMAVS. In addition, chPRMT1 knock out DF-1 cells were constructed using CRISPR-Cas9 technique. The morphology and viability of chPRMT1 knock out DF-1 cells were similar with the wild-type cells. In addition, the IFN-ß as well as interferon stimulate genes activation induced by chMAVS in PRMT1 knock out DF-1 cells were significantly higher than that in WT cells. Furthermore, ectopic expression of chPRMT1 significantly supports IBDV replication. We also found that the ability of IBDV replication in PRMT1 knock out DF-1 cells was remarkably lower than that of in WT cells, suggesting that PRMT1 negatively regulate IBDV replication via suppressing IFN-ß production. In conclusion, the PRMT1 knock out DF-1 cells were constructed, which was further used to demonstrate an inhibitory role of chPRMT1 in IFN-ß production, and a contributor of chPRMT1 in IBDV replication.

Characterization and functional analysis of chicken dsRNA binding protein hnRNPU.

In mammals, heterogeneous ribonucleoprotein U (hnRNPU), also named as nuclear matrix protein-nuclear scaffold attachment factor (SAFA), was originally identified as a DNA/RNA interactor protein. It has been reported that human hnRNPU facilitates IFN-ß generation after vesicular stomatitis virus (VSV) infection. Nevertheless, the role of chicken hnRNPU (chhnRNPU) in IFN-ß regulation as well as in infectious bursal diseases virus (IBDV) replication is still unclear. Here, we found that chhnRNPU inhibits IFN-ß production via interacting with MDA5 and MAVS, and facilitates IBDV replication via associating with genomic dsRNA of IBDV. Firstly, chicken hnRNPU (chhnRNPU) was widely expressed in different tissues of chickens and was distributed in the nucleus of DF-1 cells. Overexpression of chhnRNPU significantly suppresses IFN-ß promoter activities induced by MDA5 and MAVS. Additionally, immunoprecipitated by dsRNA antibodies, which followed LC-MS analysis demonstrate that chhnRNPU is a partner of viral genomic dsRNA. chhnRNPU is translocated from nucleus to cytosol to co-localize with replication complex of IBDV after IBDV infection. Over-expression of chhnRNPU significantly promotes IBDV replication, which was determined by western blotting, qRT-PCR and TCID50 assay. Furthermore, knock down chhnRNPU by siRNA remarkably facilitates IFN-ß production, and inhibits IBDV proliferation. These data collectively reveal that chhnRNPU positively regulates IBDV replication via negatively regulating IFN-ß response.

Serum investigation of antibodies against porcine circovirus 4 Rep and Cap protein in Jiangxi Province, China.

In 2019, a novel porcine circovirus 4 (PCV4) was first identified in Hunan Province, China. The circular PCV4 DNA was detected in both diseased and healthy pigs. Recently, PCV4 prevalence surveys have been analyzed in many provinces in both China and South Korea with low positive rates. However, no serological data has been conducted to investigate the prevalence of PCV4 in pigs from Jiangxi Province. To address this issue, an indirect anti-PCV4 antibody enzyme-linked immunosorbent assay (ELISA) based on Cap and Rep protein as a coating antigen was established and applied to study the serum epidemiology of PCV4 in Jiangxi Province. Purified PCV4-His-tagged Cap and Rep were used as the coating antigen to develop an ELISA detection kit. There was no cross-reaction of the Cap/Rep-based ELISA with antisera against PCV2, TGEV and PRRSV, indicating a high specificity of this ELISA assay. The intra-assay coefficient variations (CVs) of Cap-based were 1.239%-9.796%, Rep-based 1.288%-5.011%, and inter-assay CVs of 1.167%-4.694% and 1.621%-8.979%, respectively, indicating a good repeatability. Finally, a total number of 507 serum samples were collected from Jiangxi Province to test for antibody prevalence of PCV4, and 17 (3.35%) and 36 (7.10%) of the samples were Cap and Rep antibody positive, respectively. In summary, our established ELISA kit could be used to detect PCV4 antibodies in serum with good repeatability and high specificity. In addition, field samples detection results showed that the antibody of PCV4 was poorly distributed in intensive pig farms in Jiangxi Province, China.

Frequency detection of porcine circovirus-like viruses in pigs with porcine respiratory disease.

The emergence of the circular replication-associated protein (Rep) encoding single-stranded (CRESS) DNA viruses in different hosts has been associated with serious diseases, such as porcine diarrhea. The prevalence and pathogenicity of porcine circovirus-like virus (Po-Circo-like virus (PCLV)), a member of CRESS DNA virus, has not been fully illustrated. In order to understand the frequency of PCLV in pigs with respiratory disease, 519 healthy tissues (268 lungs, 201 lymph nodes and 50 hearts) and 380 tissues (212 lungs, 124 lymph nodes and 44 hearts) diagnosed with respiratory disease were collected for analyzing the prevalence of the PCLV infection using the Tag-Man qPCR assay. In addition, the complete genome of 43 PCLV strains were then sequenced, which were subsequently to analyze their characteristics. We found that 31.7 % (285/899) samples were tested positive for PCL virus. It is interesting to note that just 9.6 % (50/519) of the healthy samples were tested positive for PCLV, 61.8 % (235/380) of the diseased samples were PCLV positive. Analysis of the full genome of 43 PCLV strains showed that the genome of 42 PCLV strains included two distinct stem-loop structures, but the genome of PCLV FJ5-2020 strain contained no stem-loop structures. A phylogenetic tree analysis based on the Rep protein, PCLV could be classified into four genotypes: PCLVa, PCLVb, PCLVc, and PCLVd. In conclusion, this is the first report that the high frequency of PCLV in association in respiratory diseased pigs. PCLV strains were divided into four new genotypes of PCLV.

SUMOylation of optineurin is critical for inhibiting interferon ß production.

Targeting the vital kinase protein TBK1, optineurin (OPTN) performs as a suppressor involved in controlling RNA virus-induced interferon ß (IFN-ß) production. It has been determined that OPTN is altered by phosphorylation and ubiquitination, which are crucial for the generation of IFN-ß and mitophagy. In this study, endogenous OPTN was first discovered in human cells at a molecular mass of about 115 kD. The SUMOylation band was verified to be the higher molecular mass band of the OPTN. Additionally, the OPTN SUMOylation band was seen in cells from several species, including mouse, rabbit, bovine, porcine, etc., but not in cells from avian animals (chicken and duck). This finding suggests that OPTN SUMOylation is well conserved in mammals but not in avian animals. Additionally, it was determined that some lysine residues in the human OPTN had SUMOylation sites that followed the consensus motif. LPS, VSV infection, starvation, and RNA virus infection are a few of the stimuli that encourage endogens OPTN SUMOylation. OPTN SUMOylation is essential for OPTN biological activity, as evidenced by the stark differences in the cellular distribution of mutant OPTN SUMOylation sites from wild type (WT) OPTN. Additionally, we discovered that non-SUMOylated OPTN lost its ability to block both IFN-ß production. Our findings offer a preliminary understanding of how OPTN SUMOylation regulates IFN-ß production.

Detection of Porcine Circovirus 1/2/3 and Genetic Analysis of Porcine Circovirus 2 in Wild Boar from Jiangxi Province of China.

A number of disorders that harm pig production are linked to porcine circoviruses, including PCV2. PCV2 infection is a substantial contributor to porcine-circovirus-associated illnesses (PCAS) and the post-weaning multi-systemic wasting syndrome (PMWS), which have a significant negative economic impact on pig production. Additionally, PCV infection has been labeled as a global concern to cattle and wildlife. This study's objectives were to examine the prevalence of PCV1/2/3 in Jiangxi Province, China, and to clarify the epidemiological significance of wild boar in PCV epidemiology. The 2020 hunting seasons resulted in the collection of 138 wild boar samples for PCV1/2/3 detection, which was followed by the genetic clarification of PCV2 strains. According to our data, 21.7% (30/138) of the population had PCV1 positivity, 22.5% (31/138) had PCV2 positivity, and 5.8% (8/138) had PCV3 positivity. Additionally, 10 out of 138 wild boar samples had PCV1 and PCV2 co-infections, while 5 out of 138 wild boar samples had PCV2 and PC3 co-infections. Nineteen full-length PCV2 genomes measuring 1767 nt were recovered from various animal tissues using conventional PCR. Eighteen out of nineteen PCV2 strains were identified as PCV2b by phylogenetic tree analysis, which was completed by the reference strain HLJ2015 obtained from domestic pigs in 2015. Additionally, one genotype of PCV2d JX11-2020 (MW889021) shared a sub-branch with the referenced strain TJ (AY181946), which was isolated in domestic pigs in 2002. This finding raises the possibility that domestic pigs could contract PCV2 strains from wild boar, posing a serious threat to the Jiangxi province of China's pig production industry.

First complete genomic sequence analysis of porcine circovirus type 4 (PCV4) in wild boars.

Porcine circovirus type 4 (PCV4), a unique circovirus with a different classification from other existing circovirus, was discovered in domestic pigs in several provinces of China. In this study, in order to investigate the epidemiology and genetic diversity of PCV4 in wild boars (Sus scrofa), a total number of 138 wild boar samples were collected from five different areas in Jiangxi Province of China, between January 2020 and December 2020. Taqman based real-time PCR were used to test PCV4 as well as PCV1, PCV2, and PCV3. Among 138 samples, 30 samples (21.7%) were positive for PCV1, 31 samples (22.5%) were positive for PCV2, 8 samples (5.8%) were positive for PCV3 and 27 samples (19.6%) were positive for PCV4, respectively. Some of the samples were co-infected with multiple PCVs. In this study, we successfully sequenced the complete genome of two PCV4 strains, which shared 98.5-99.8% of their genomic nucleotide similarity with the other five PCV4 strains discovered in domestic pigs. Phylogenetic analysis showed that the two PCV4 strains derived from wild boars were located in a closed relative branch with other PCV4 strains derived from domestic pigs, but were distinguished from other circovirus. These results of this study not only expand our understanding of the prevalence of PCVs, especially PCV4, in wild boars in Jiangxi province of China, but also showed the molecular epidemiology of PCV4. Nevertheless, the impact of wild boars infected with PCV4 on intensive farmed pigs industry remains to be further explored.

Chicken TAX1BP1 suppresses type I interferon production via degrading chicken MAVS and facilitates infectious bursal diseases virus replication.

Mammalian TAX1BP1 (TAX1 binding protein 1), originally identified as a partner of the HTLV-1 viral oncoprotein, functions in regulation of cellular cytokine production. TAX1BP1 plays an important signal transduction regulator, specifically modulating innate immune signaling pathways including NF-B and IRF3. The function of TAX1BP1, which regulates the innate immune response in mammals, has been well studied in previous reports, but the role of chicken TAX1BP1 (chTAX1) in IFN regulation and infectious bursal disease virus (IBDV) replication is still unclear. In this report, chTAX1 was successfully cloned and sub-inserted into a eukaryotic expression vector. The critical regions of chTAX1, such as LC3 binding motif, ubiquitin binding motif, are highly conserved compared to other organisms. We also found that chTAX1 inhibits IFN expression by promoting degradation of chicken MAVS (chMAVS). In addition, the distribution of chTAX1 altered and translocated to co-localize with both VP1 and VP3 after IBDV infection. Overexpression of chTAX1 promotes IBDV replication and knockdown of chTAX1 by RNA interference suppresses IBDV replication. In summary, our data initially indicate that chTAX1 is a suppressor of IFN expression as well as a promoter of IBDV replication.

An Improved, Dual-Direction, Promoter-Driven, Reverse Genetics System for the Infectious Bursal Disease Virus (IBDV).

The infectious bursal disease virus (IBDV), one member of the Birnaviridae family, causes immunosuppression in young chickens by damaging the mature B cells of the bursa of Fabricius (BF), the central immune system of young chickens. The genome of IBDV is a bisegmented, double-strand RNA (dsRNA). Reverse genetics systems for IBDV allow the generation of genetically manipulated infectious virus via transfected plasmid DNA, encoding the two genomic viral RNA segments as well as major viral proteins. For this purpose, the minus-sense of both segment A and segment B are inserted into vectors between the polymerase I promoter and the corresponding terminator I. These plasmids facilitate the transcription of the viral minus-sense genome but copy the plus-sense genome as well viral protein translation depends on the activity of VP1 and VP3, when transfected into 293T cells. To further improve rescue efficiency, dual-direction promoters were generated based on the polymerase II promoter in the reverse direction in the backbone of the pCDNA3.0 vector. Therefore, the polymerase I promoter transcribes the viral minus-sense genome in the forward direction and the polymerase II promoter transcribes viral mRNA, translated into viral proteins that produce infectious IBDV. We also found that the rescue efficiency of transfecting two plasmids is significantly higher than that of transfecting four plasmids. In addition, this dual-direction promoter rescue system was used to generate R186A mutant IBDV since Arg186 is the arginine monomer-methylation site identified by LC-MS. Our data furtherly showed that the Arg186 monomer methylation mutant was due to a reduction in VP1 polymerase activity as well as virus replication, suggesting that the Arg186 methylation site is essential for IBDV replication.

Development and Primary Application of an Indirect ELISA Based on Rep Protein to Analyze Antibodies against Porcine Cocirvirus-like Virus (PCLV).

Porcine circovirus-like virus (PCLV) is a member of circovirus that contains a single-strand DNA genome, which may be one of the pathogens that causes diarrheal symptoms in pigs. The Rep protein encoded by the genome of PCLV may be responsible for viral genome replication. The development of serological detection methods for PCLV is of great necessity for clinical diagnosis, as well as epidemiological investigations. Therefore, this study attempted to build an indirect enzyme-linked immunosorbent assay (ELISA) to examine antibodies against PCLV based on the His-tagged recombinant Rep protein. Full-length PCLV Rep protein was induced and expressed in E. coli and was purified as an antigen to establish an ELISA detection kit. The purified Rep protein was used to inject into mice to produce specific antibodies. There was no cross-reaction of Rep-based ELISA with antisera against other porcine viruses. The intra-assay and inter-assay coefficient variations (CVs) were 0.644-8.211% and 0.859-7.246%, respectively, indicating good repeatability. The non-cross-reaction with TGEV, PRRSV and PCV2 testing showed high sensitivity and high specificity for this ELISA assay. A total of 1593 serum samples collected from different pig farms in Jiangxi Province were tested for anti-PCLV Rep antibodies, and 284 (17.83%) of the 1593 samples were Rep antibody positive. Altogether, the indirect ELISA detection tool developed in this study could be applied to examine serum of PCLV antibodies with good repeatability, high sensitivity and high specificity. In addition, field sample detection results suggested that the PCLV antibody has a low prevalence in pig populations in Jiangxi Province of China.

Selenium protects against cadmium-induced cardiac injury by attenuating programmed cell death via PI3K/AKT/PTEN signaling.

Cadmium (Cd) is an environmental pollutant that has an enormous influence on agricultural production, but selenium (Se) can alleviate its toxicity. The present study aimed to illustrate the effects of Se on Cd-induced heart injury. All 40 rabbits were randomly divided into four groups: control group, Se [0.5 mg kg-1 ·body weight (BW)] group, Cd (1 mg kg-1 ·BW) group, and Se + Cd group. After 30 days of feeding, morphological changes, the levels of oxidative stress and myocardial enzyme, the content of cardiac troponin T, programmed cell death (pyroptosis, autophagy and apoptosis), and PI3K/AKT/PTEN transduction capacity were observed. The results showed that Cd destroyed the physiological balance of trace elements and caused myocardial damage, increased the cardiac oxidative damage and led to programmed cell death. Coadministration of Se prominently ameliorated histological lesions and improved cardiac function of hearts in Cd-induced rabbits. Furthermore, Se exerted detoxification and oxidation resistance, maintained trace element homeostasis, and alleviated the changes of mRNA and protein levels of pyroptosis-, autophagy- and apoptosis-controlling factors and PI3K/AKT/PTEN signal molecules caused by Cd. In conclusion, Se might protect against Cd-induced pyroptosis, autophagy and apoptosis by interfering with PI3K/AKT/PTEN signaling in heart.

The synergistic effect of traditional Chinese medicine prescription and rumen-protected γ-aminobutyric acid on beef cattle under heat stress.

Traditional Chinese medicine (TCM) prescription or rumen-protected GABA (RP-GABA) can effectively relieve the heat stress (HS) in cattle, but the joint effects of TCM and RP-GABA on HS in beef cattle are not fully clarified. To investigate the effects of TCM or/and RP-GABA on growth performance, antioxidant capacity, serum parameters and heat shock proteins (HSPs) expression in beef cattle under HS ambient. A total of 40 Jinjiang yellow cattle were randomly divided into four groups: (a) control group (basal diet, BD), (b) TCM group (BD+TCM), (c) GABA group (BD+RP-GABA) and (d) TCM+GABA group (BD+TCM plus RP-GABA). Results indicated that the average daily feed intake (ADFI) was significantly elevated in the TCM+GABA group (p < 0.05), whereas, average daily gain (ADG) was elevated (p < 0.05) in the group of TCM (38.5%), GABA (35.4%) and TCM+GABA (41.5%) compared with the control group. Meanwhile, TCM+GABA exhibited prominently more positive effects in terms of SOD, BUN, T-CHO, TG, HDL-C and HSP70 (p < 0.05 or p < 0.01) than the control and other treatment groups. Therefore, TCM or GABA can effectively moderate the HS response in beef cattle by ameliorating antioxidant capacity, serum parameters and HSPs expression, meanwhile, the combination of them exerts a synergistic effect on HS alleviation.

Chicken optineurin suppresses MDA5-mediated interferon ß production.

Chicken MDA5 (chMDA5), the essential accepted pattern recognition receptors for detecting cytoplasmic viral RNA in chicken, initiates interferon ß (IFN-ß) generation. However, there is an incomplete elucidation of regulating chMDA5-mediated IFN-ß production. NEMO-related protein, optineurin, was identified as inhibitors of virus triggered IFN-ß induction in human or mice. In this study, full length of chicken optineurin (chOPTN) was cloned from chicken embryo fibroblast, and its role in inhibiting IFN-ß signaling pathway was further explored. Full-length chOPTN encodes 547 amino acids residues and contains unique LC3 interaction region and ubiquitin binding domain. Chicken optineurin mRNA and protein are widely expressed in different tissues, especially the heart, kidney, and bursal fabricius (BF). Overexpressed chOPTN not only inhibits poly I:C or homos-induced human IFN-ß promoter activation in 293T cells but also suppresses poly I:C, infectious bursal disease virus (IBDV) genome double-strand RNA (dsRNA), and chMDA5-induced chicken IFN-ß (chIFN-ß) promoter activation. In addition, we first revealed that chOPTN negatively regulates chIFN-ß production via inhibiting ubiquitination of chicken TBK1, which is dependent on the ubiquitin-binding domain of chOPTN. Moreover, chIFN-ß stimulus, poly I:C, and IBDV genome dsRNA improve chOPTN expression. Endogenous chOPTN expression is also upregulated by IBDV infection in 293T, DF-1 cells, as well as in BF. Therefore, our results suggested that chOPTN plays an inhibition role of chMDA5-mediated chIFN-ß signaling pathway in chicken cells.

Inhibition of autophagy enhances cadmium-induced apoptosis in duck renal tubular epithelial cells.

Increasing evidence indicates autophagy and apoptosis are involved in the toxicity mechanism of heavy metals. Our previous studies showed that cadmium (Cd) could induce autophagy and apoptosis in duck kidneys in vivo, nevertheless, the interaction between them has yet to be elucidated. Herein, the cells were either treated with 3CdSO4·8H2O (0, 1.25, 2.5, 5.0 µM Cd) or/and 3-methyladenine (3-MA) (2.5 µM) for 12 h and the indictors related autophagy and apoptosis were detected to assess the correlation between autophagy and apoptosis induced by Cd in duck renal tubular epithelial cells. The results demonstrated that Cd exposure notably elevated intracellular and extracellular Cd contents, the number of autophagosomes and LC3 puncta, up-regulated LC3A, LC3B, Beclin-1, Atg5 mRNA levels, and Beclin-1 and LC3II/LC3I protein levels, down-regulated mTOR, p62 and Dynein mRNA levels and p62 protein level. Additionally, autophagy inhibitor 3-MA decreased Beclin-1, LC3II/LC3I protein levels and increased p62 protein level. Moreover, co-treatment with Cd and 3-MA could notably elevate Caspase-3, Cyt C, Bax, and Bak-1 mRNA levels, Caspase-3 and cleaved Caspase-3 protein levels, and cell apoptotic rate as well as cell damage, decreased mitochondrial membrane potential (MMP), Bcl-2 mRNA level and the ratio of Bcl-2 to Bax compared to treatment with Cd alone. Overall, these results indicate Cd exposure can induce autophagy in duck renal tubular epithelial cells, and inhibition of autophagy might aggravate Cd-induced apoptosis through mitochondria-mediated pathway.

SUMO1 Modification Facilitates Avibirnavirus Replication by Stabilizing Polymerase VP1.

SUMOylation is a posttranslational modification that has crucial roles in diverse cellular biological pathways and in various viral life cycles. In this study, we found that the VP1 protein, the RNA-dependent RNA polymerase of avibirnavirus infectious bursal disease virus (IBDV), regulates virus replication by SUMOylation during infection. Our data demonstrated that the polymerase VP1 is efficiently modified by small ubiquitin-like modifier 1 (SUMO1) in avibirnavirus-infected cell lines. Mutation analysis showed that residues 404I and 406I within SUMO interaction motif 3 of VP1 constitute the critical site for SUMO1 modification. Protein stability assays showed that SUMO1 modification enhanced significantly the stability of polymerase VP1 by inhibiting K48-linked ubiquitination. A reverse genetic approach showed that only IBDV with I404C/T and I406C/F mutations of VP1 could be rescued successfully with decreased replication ability. Our data demonstrated that SUMO1 modification is essential to sustain the stability of polymerase VP1 during IBDV replication and provides a potential target for designing antiviral drugs targeting IBDV.IMPORTANCE SUMOylation is an extensively discussed posttranslational modification in diverse cellular biological pathways. However, there is limited understanding about SUMOylation of viral proteins of IBDV during infection. In the present study, we revealed a SUMO1 modification of VP1 protein, the RNA-dependent RNA polymerase of avibirnavirus infectious bursal disease virus (IBDV). The required site of VP1 SUMOylation comprised residues 404I and 406I of SUMO interaction motif 3, which was essential for maintaining its stability by inhibiting K48-linked ubiquitination. We also showed that IBDV with SUMOylation-deficient VP1 had decreased replication ability. These data demonstrated that the SUMOylation of IBDV VP1 played an important role in maintaining IBDV replication.

Ubiquitination Is Essential for Avibirnavirus Replication by Supporting VP1 Polymerase Activity.

Ubiquitination is critical for several cellular physical processes. However, ubiquitin modification in virus replication is poorly understood. Therefore, the present study aimed to determine the presence and effect of ubiquitination on polymerase activity of viral protein 1 (VP1) of avibirnavirus. We report that the replication of avibirnavirus is regulated by ubiquitination of its VP1 protein, the RNA-dependent RNA polymerase of infectious bursal disease virus (IBDV). In vivo detection revealed the ubiquitination of VP1 protein in IBDV-infected target organs and different cells but not in purified IBDV particles. Further analysis of ubiquitination confirms that VP1 is modified by K63-linked ubiquitin chain. Point mutation screening showed that the ubiquitination site of VP1 was at the K751 residue in the C terminus. The K751 ubiquitination is independent of VP1's interaction with VP3 and eukaryotic initiation factor 4A II. Polymerase activity assays indicated that the K751 ubiquitination at the C terminus of VP1 enhanced its polymerase activity. The K751-to-R mutation of VP1 protein did not block the rescue of IBDV but decreased the replication ability of IBDV. Our data demonstrate that the ubiquitination of VP1 is crucial to regulate its polymerase activity and IBDV replication.IMPORTANCE Avibirnavirus protein VP1, the RNA-dependent RNA polymerase, is responsible for IBDV genome replication, gene expression, and assembly. However, little is known about its chemical modification relating to its polymerase activity. In this study, we revealed the molecular mechanism of ubiquitin modification of VP1 via a K63-linked ubiquitin chain during infection. Lysine (K) residue 751 at the C terminus of VP1 is the target site for ubiquitin, and its ubiquitination is independent of VP1's interaction with VP3 and eukaryotic initiation factor 4A II. The K751 ubiquitination promotes the polymerase activity of VP1 and unubiquitinated VP1 mutant IBDV significantly impairs virus replication. We conclude that VP1 is the ubiquitin-modified protein and reveal the mechanism by which VP1 promotes avibirnavirus replication.

Binding of the pathogen receptor HSP90AA1 to avibirnavirus VP2 induces autophagy by inactivating the AKT-MTOR pathway.

Autophagy is an essential component of host innate and adaptive immunity. Viruses have developed diverse strategies for evading or utilizing autophagy for survival. The response of the autophagy pathways to virus invasion is poorly documented. Here, we report on the induction of autophagy initiated by the pathogen receptor HSP90AA1 (heat shock protein 90 kDa α [cytosolic], class A member 1) via the AKT-MTOR (mechanistic target of rapamycin)-dependent pathway. Transmission electron microscopy and confocal microscopy revealed that intracellular autolysosomes packaged avibirnavirus particles. Autophagy detection showed that early avibirnavirus infection not only increased the amount of light chain 3 (LC3)-II, but also upregulated AKT-MTOR dephosphorylation. HSP90AA1-AKT-MTOR knockdown by RNA interference resulted in inhibition of autophagy during avibirnavirus infection. Virus titer assays further verified that autophagy inhibition, but not induction, enhanced avibirnavirus replication. Subsequently, we found that HSP90AA1 binding to the viral protein VP2 resulted in induction of autophagy and AKT-MTOR pathway inactivation. Collectively, our findings suggest that the cell surface protein HSP90AA1, an avibirnavirus-binding receptor, induces autophagy through the HSP90AA1-AKT-MTOR pathway in early infection. We reveal that upon viral recognition, a direct connection between HSP90AA1 and the AKT-MTOR pathway trigger autophagy, a critical step for controlling infection.