Synergistic pathogenicity of vertically transmitted chicken infectious anemia virus and avian leukosis virus subgroup J coinfection in chickens.

Avian leukemia virus subgroup J (ALV-J) and chicken infectious anemia virus (CIAV) can be vertically transmitted; however, the pathogenicity of vertically transmitted coinfection with these 2 pathogens has not been studied. In this study, we created a model of chick morbidity in which chicks carried either ALV-J, CIAV, or both viruses via embryo inoculation. Thereafter, we analyzed the effects of vertically transmitted coinfection with CIAV and ALV-J on the pathogenicity of ALV-J and performed a purification assay based on hatching, mortality viremia positivity, and detection of fecal ALV-p27 antigen rates, and body weight. The hatching rate of the ALV-J+CIAV group was 68.57%, lower than those of the single infection and control groups. The survival curve showed that the mortality rates of the CIAV and ALV-J coinfection groups were higher than those of the single infection and control groups. Body weight statistics showed that coinfection aggravated the 7-d growth inhibition effect. The results of ALV-p27 antigen detection in cell culture supernatants showed that the positivity rates of the ALV-J and ALV-J+CIAV groups were 100% at all ages and 0% in the control group. The results of ALV-p27 antigen detection by anal swabs showed that the positivity rates of the ALV-J group were 92.86, 90.90, 88.89, and 93.33% at all ages, and that the ALV-J p27 positivity detection rate of anal swabs was lower than that of plasma virus isolation. The immune organ index of the ALV-J+CIAV group was significantly or very significantly lower than those of the single infection and control groups. The immune organ viral load showed that coinfection with CIAV and ALV-J promoted the proliferation of ALV-J and CIAV in immune organs. Coinfection with ALV-J and CIAV reduced chicken embryo hatchability and increased chick mortality and growth inhibition relative to their respective single infections. Additionally, coinfection with ALV-J + CIAV was even more detrimental in inducing immune organ atrophy (e.g., the thymus, spleen, and bursa), and promoted individual virus replication during coinfection.

Avian leukosis virus usurps the cellular SERBP1 protein to enhance its transcription and promote productive infections in avian cells.

Avian leukosis virus subgroup K (ALV-K) is composed of newly emerging isolates, which cluster separately from the well-characterized subgroups A, B, C, D, E, and J in sequence analysis, and exhibits a specific host range and a unique pattern of superinfection interference. Avian leukosis virus subgroup K replicate more slowly in avian cells than other ALV strains, leading to escaped detection during ALV eradication, but the underlying mechanism are largely unknown. In our previous study, we have reported that JS11C1 and most of other suspected ALV-K strains possessed unique mutations in the U3 region. Here, we selected 5 mutations in some important transcriptional regulation elements to explore the possible factor contributing for the lower activity of LTR, including CA-TG mutation in the CAAT box, 21 nt deletion in the CAAT box, A-G and A-T mutations in the CArG boxes, 11 nt insertion in the PRE boxes, and C-T mutation in the TATA box. On the basis of infectious clone of JS11C1, we demonstrated that the 11 nt fragment in the PRE boxes was associated with the transcription activity of LTR, the enhancer ability of U3, and the replication capacity of the virus. Notably, we determined the differential U3-protein interaction profile of ALVs and found that the 11 nt fragment specifically binds to cellular SERPINE1 mRNA binding protein 1 (SERBP1) to increase the LTR activity and enhance virus replication. Collectively, these findings reveal that a 11 nt fragment in the U3 gene contributed to its binding ability to the cellular SERBP1 to enhance its transcription and the infectious virus productions in avian cells. This study highlighted the vital role of host factor in retrovirus replication and thus provides a new perspective to elucidate the interaction between retrovirus and its host and a molecular basis to develop efficient strategies against retroviruses.

Transcriptome-wide N6-methyladenosine modification and microRNA jointly regulate the infection of avian leukosis virus subgroup J in vitro.

N6-methyladenosine (m6A) methylation in transcripts has been suggested to influence tumorigenesis in liver tumors caused by the avian leukosis virus subgroup J (ALV-J). However, m6A modifications during ALV-J infection in vitro remain unclear. Herein, we performed m6A and RNA sequencing in ALV-J-infected chicken fibroblasts (DF-1). A total of 51 differentially expressed genes containing differentially methylated peaks were identified, which were markedly enriched in microRNAs (miRNAs) in cancer cells as well as apoptosis, mitophagy and autophagy, RNA degradation, and Hippo and MAPK signaling pathways. Correlation analysis indicated that YTHDC1 (m6A-reader gene) plays a key role in m6A modulation during ALV-J infection. The env gene of ALV-J harbored the strongest peak, and untranslated regions and long terminal repeats also contained peaks of different degrees. To the best of our knowledge, this is the first thorough analysis of m6A patterns in ALV-J-infected DF-1 cells. Combined with miRNA profiles, this study provides a useful basis for future research into the key pathways of ALV-J infection associated with m6A alteration.

Metabolomics analysis of CEF cells infected with avian leukosis virus subgroup J based on UHPLC-QE-MS.

Avian leukosis virus subgroup J (ALV-J) is a retrovirus that can cause immunosuppression and tumors in chicken. However, relative pathogenesis is still not clear. At present, metabolomics has shown great potential in the screening of tumor metabolic markers, prognostic evaluation, and drug target design. In this study, we utilize an untargeted metabolomics approach based on ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) to analyze the metabolic changes in chicken embryo fibroblast (CEF) cells infected by ALV-J. We found that ALV-J infection significantly altered a wealth of metabolites compared with control group. Additionally, most of the differentially expressed metabolites belonged to lipid metabolism, purine nucleotide metabolism and amino acid metabolism. Among them, the proportion of lipid metabolites account for the highest proportion (around 31%). Results suggest that these changes may be conductive to the formation of virion, thereby promoting the replication of ALV-J. These data provided metabolic evidence and potential biomarkers for the cellular metabolic changes induced by ALV-J, and provided important insight for further understanding the replication needs and pathogenesis of ALV-J.

PMAIP1 promotes J subgroup avian leukosis virus replication by regulating mitochondrial function.

Avian leukosis virus Subgroup J (ALV-J) exhibits high morbidity and pathogenicity, affecting approximately 20% of poultry farms. It induces neoplastic diseases and immunosuppression. Phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), a proapoptotic mitochondrial protein in the B-cell lymphoma-2 (Bcl-2) family, plays a role in apoptosis in cancer cells. However, the connection between the PMAIP1 gene and ALV-J pathogenicity remains unexplored. This study investigates the potential impact of the PMAIP1 gene on ALV-J replication and its regulatory mechanisms. Initially, we examined PMAIP1 expression using quantitative real-time PCR (qRT-PCR) in vitro and in vivo. Furthermore, we manipulated PMAIP1 expression in chicken fibroblast cells (DF-1) and assessed its effects on ALV-J infection through qRT-PCR, immunofluorescence assay (IFA), and western blotting (WB). Our findings reveal a significant down-regulation of PMAIP1 in the spleen, lung, and kidney, coupled with an up-regulation in the bursa and liver of ALV-J infected chickens compared to uninfected ones. Additionally, DF-1 cells infected with ALV-J displayed a notable up-regulation of PMAIP1 at 6, 12, 24, 48, 74, and 108 h. Over-expression of PMAIP1 enhanced ALV-J replication, interferon expression, and proinflammatory factors. Conversely, interference led to contrasting results. Furthermore, we observed that PMAIP1 promotes virus replication by modulating mitochondrial function. In conclusion, the PMAIP1 gene facilitates virus replication by regulating mitochondrial function, thereby enriching our understanding of mitochondria-related genes and their involvement in ALV-J infection, offering valuable insights for avian leukosis disease resistance strategies.

3'UTR of ALV-J can affect viral replication through promoting transcription and mRNA nuclear export.

IMPORTANCE: 3'UTRs can affect gene transcription and post-transcriptional regulation in multiple ways, further influencing the function of proteins in a unique manner. Recently, ALV-J has been mutating and evolving rapidly, especially the 3'UTR of viral genome. Meanwhile, clinical symptoms caused by ALV-J have changed significantly. In this study, we found that the ALV-J strains containing △-r-TM-type 3'UTR are the most abundant. By constructing ALV-J infectious clones and subgenomic vectors containing different 3'UTRs, we prove that 3'UTRs directly affect viral tissue preference and can promote virus replication as an enhancer. ALV-J strain containing 3'UTR of △-r-TM proliferated fastest in primary cells. All five forms of 3'UTRs can assist intron-containing viral mRNA nuclear export, with similar efficiency. ALV-J mRNA half-life is not influenced by different 3'UTRs. Our results dissect the roles of 3'UTR on regulating viral replication and pathogenicity, providing novel insights into potential anti-viral strategies.

ALV-miRNA-p19-01 Promotes Viral Replication via Targeting Dual Specificity Phosphatase 6.

MicroRNAs (miRNAs) are a group of regulatory noncoding RNAs, serving as major regulators with a sequence-specific manner in multifarious biological processes. Although a series of viral families have been proved to encode miRNAs, few reports were available regarding the function of ALV-J-encoded miRNA. Here, we reported a novel miRNA (designated ALV-miRNA-p19-01) in ALV-J-infected DF-1 cells. We found that ALV-miRNA-p19-01 is encoded by the genome of the ALV-J SCAU1903 strain (located at nucleotides site 779 to 801) in a classic miRNA biogenesis manner. The transfection of DF-1 cells with ALV-miRNA-p19-01 enhanced ALV-J replication, while the blockage of ALV-miRNA-p19-01 suppressed ALV-J replication. Furthermore, our data showed that ALV-miRNA-p19-01 promotes ALV-J replication by directly targeting the cellular gene dual specificity phosphatase 6 through regulating ERK2 activity.

TMT-based proteomic analysis reveals integrins involved in the synergistic infection of reticuloendotheliosis virus and avian leukosis virus subgroup J.

BACKGROUND: Co-infection with the avian leukosis virus subgroup J (ALV-J) and the reticuloendotheliosis virus (REV) increases mutual viral replication, causing a more serious pathogenic effect by accelerating the progression of neoplasia and extending the tumor spectrum. However, the molecular mechanism underlying the synergistic replication of ALV-J and REV remains unclear. RESULTS: Here, we performed this study to compare the differentially expressed proteins among CEF cells infected with ALV-J, REV or both at the optimal synergistic infection time using TMT-based quantitative proteomics. We identified a total of 719 (292 upregulated and 427 downregulated) and 64 (35 upregulated and 29 downregulated) proteins by comparing co-infecting both viruses with monoinfecting ALV-J and REV, respectively. GO annotation and KEGG pathway analysis showed the differentially expressed proteins participated in virus-vector interaction, biological adhesion and immune response pathways in the synergistic actions of ALV-J and REV at the protein levels. Among the differentially expressed proteins, a large number of integrins were inhibited or increased in the co-infection group. Further, eight integrins, including ITGα1, ITGα3, ITGα5, ITGα6, ITGα8, ITGα9, ITGα11 and ITGß3, were validated in CEF cells by qRT-PCR or western blot. CONCLUSIONS: These findings proved that integrins may be key regulators in the mechanism of synergistic infection of REV and ALV-J, which will provide more insight into the pathogenesis of synergism of REV and ALV-J at protein level.

Novel Function of Avian p53 in Binding to ALV-J LTR Contributes to Its Antiviral Roles.

Accumulating evidence suggests that p53 is involved in viral infection. However, it remains elusive whether avian p53 orchestrates avian leukosis virus (ALV) replication. We showed that p53 recruits the histone deacetylase 1 and 2 (HDAC1/2) complex to the ALV promoter to shut off ALV's promoter activity and viral replication. HDAC1/2 binding to the ALV promoter was abolished in the absence of p53. Moreover, we collected samples in ALV-infected chickens and found that the acetylation status of ALV-bound H3 and H4 histones correlated with ALV viremia. HDAC inhibitors (HDACi) potently increase ALV replication, but HDACi-promoted viral replication is dramatically reduced in cells with p53 depletion. These data demonstrate that p53 is critical for inhibition ALV replication and suggest that future studies of ALV replication need to account for the potential effects of p53 activity. IMPORTANCE Rous sarcoma virus (RSV)/ALV was the first retrovirus to be discovered, which was really the first hint that cancer, or a tumor, could be transmitted by a virus. The specific mechanisms that regulate ALV replication during infection remain poorly understood. Here, we show that avian p53 and HDAC complex inhibit ALV promoter activity and replication, and p53 inhibits ALV replication through binding to the ALV promoter. We demonstrated that the acetylation status of ALV-bound H3 and H4 histones correlates with ALV viremia level using clinical samples collected from commercial poultry. These findings identify both p53-mediated inhibition on ALV replication and a potential role for virus-induced tumorigenesis.

Knock-Out of Retrovirus Receptor Gene Tva in the Chicken Confers Resistance to Avian Leukosis Virus Subgroups A and K and Affects Cobalamin (Vitamin B12)-Dependent Level of Methylmalonic Acid.

The chicken Tva cell surface protein, a member of the low-density lipoprotein receptor family, has been identified as an entry receptor for avian leukosis virus of classic subgroup A and newly emerging subgroup K. Because both viruses represent an important concern for the poultry industry, we introduced a frame-shifting deletion into the chicken tva locus with the aim of knocking-out Tva expression and creating a virus-resistant chicken line. The tva knock-out was prepared by CRISPR/Cas9 gene editing in chicken primordial germ cells and orthotopic transplantation of edited cells into the testes of sterilized recipient roosters. The resulting tva -/- chickens tested fully resistant to avian leukosis virus subgroups A and K, both in in vitro and in vivo assays, in contrast to their susceptible tva +/+ and tva +/- siblings. We also found a specific disorder of the cobalamin/vitamin B12 metabolism in the tva knock-out chickens, which is in accordance with the recently recognized physiological function of Tva as a receptor for cobalamin in complex with transcobalamin transporter. Last but not least, we bring a new example of the de novo resistance created by CRISPR/Cas9 editing of pathogen dependence genes in farm animals and, furthermore, a new example of gene editing in chicken.

ACSL1 Inhibits ALV-J Replication by IFN-â Signaling and PI3K/Akt Pathway.

J subgroup avian leukosis virus (ALV-J) infection causes serious immunosuppression problems, leading to hematopoietic malignancy tumors in chicken. It has been demonstrated that interferon-stimulated genes (ISGs) could limit ALV-J replication; nevertheless, the underlying mechanisms remain obscure. Here, we demonstrate that Long-chain Acyl-CoA synthetase 1 (ACSL1) is an interferon (IFN)-stimulated gene that specifically restricts the replication of ALV-J due to the higher IFN-I production. More importantly, ACSL1 induces primary monocyte-derived macrophages (MDMs) to pro-inflammatory phenotypic states during ALV-J infection, and ACSL1 mediates apoptosis through the PI3K/Akt signaling pathway in ALV-J-infected primary monocyte-derived macrophages (MDMs). Overall, these results provide evidence that ACSL1 contributes to the antiviral response against ALV-J.

Mutual regulation between chicken telomerase reverse transcriptase and the Wnt/ß-catenin signalling pathway inhibits apoptosis and promotes the replication of ALV-J in LMH cells.

This study aimed to explore the mutual regulation between chicken telomerase reverse transcriptase (chTERT) and the Wnt/ß-catenin signalling pathway and its effects on cell growth and avian leukosis virus subgroup J (ALV-J) replication in LMH cells. First, LMH cells stably overexpressing the chTERT gene (LMH-chTERT cells) and corresponding control cells (LMH-NC cells) were successfully constructed with a lentiviral vector expression system. The results showed that chTERT upregulated the expression of ß-catenin, Cyclin D1, TCF4 and c-Myc. chTERT expression level and telomerase activity were increased when cells were treated with LiCl. When the cells were treated with ICG001 or IWP-2, the activity of the Wnt/ß-catenin signalling pathway was significantly inhibited, and chTERT expression and telomerase activity were also inhibited. However, when the ß-catenin gene was knocked down by small interfering RNA (siRNA), the changes in chTERT expression and telomerase activity were consistent with those in cells treated with ICG001 or IWP-2. These results indicated that chTERT and the Wnt/ß-catenin signalling pathway can be mutually regulated. Subsequently, we found that chTERT not only shortened the cell cycle to promote proliferation but also inhibited apoptosis by downregulating the expression of Caspase 3, Caspase 9 and BAX; upregulating BCL-2 and BCL-X expression; and promoting autophagy. Moreover, chTERT significantly enhanced the migration ability of LMH cells, upregulated the protein and mRNA expression of ALV-J and increased the virus titre. ALV-J replication promoted chTERT expression and telomerase activity.

The tyrosine phosphatase SHP-2 dephosphorylated by ALV-J via its Env efficiently promotes ALV-J replication.

Avian leukosis virus subgroup J (ALV-J) generally induces hemangioma, myeloid leukosis, and immunosuppression in chickens, causing significant poultry industry economic losses worldwide. The unusual env gene of ALV-J, with low homology to other subgroups of ALVs, is associated with its unique pathogenesis. However, the exact molecular basis for the pathogenesis and oncogenesis of ALV-J is still not fully understood. In this study, ALV-J infection and the overexpression of Env could efficiently downregulate the phosphorylation of SHP-2 (pSHP-2) in vitro and in vivo. The membrane-spanning domain (MSD) in Env Gp37 was the functional domain responsible for pSHP-2 downregulation. Moreover, the overexpression of SHP-2 could effectively promote the replication of ALV-J, whereas knockout or allosteric inhibition of SHP-2 could inhibit ALV-J replication. In addition, the knockout of endogenous chicken SHP-2 could significantly increase the proliferation ability of DF-1 cells. All these data demonstrate that SHP-2 dephosphorylated by ALV-J Env could efficiently promote ALV-J replication, highlighting the important role of SHP-2 in the pathogenesis of ALV-J and providing a new target for developing antiviral drugs against ALV-J.

Avian leukosis virus subgroup J infection alters viral composition in the chicken gut.

Chicken is one of the economically important poultry species. Avian leucosis virus subgroup J (ALV-J) has emerged as a serious cause of mortality and suboptimal performance of domestic chickens. Changes in virome may contribute to pathogenesis. Thus, it is important to investigate the effects of ALV-J infection on the composition of the virome in chicken. In the study metagenomic sequencing was used to characterize the virome of feces collected from the AVL-J infected chickens and the controls. Our results indicated that the chicken gut virome contained a diverse range of viruses that can be found in mammal, reptile, fish, and frogs. Furthermore, at the order, family and genus levels, AVL-J infection significantly altered the chicken gut virome composition. The predominant order was Herpesvirales, accounting for more than 96% of the chicken gut virome. Furthermore, the relative abundance of Caudovirales in the controls was higher than that in the AVL-J-infected chickens. At the family level, the relative abundance of Herpesviridae, Myoviridae, Alloherpesviridae, and Genomoviridae was significantly altered in the AVL-J-infected chickens compared with that in the controls. Additionally, the relative abundance of 15 genera showed a significant difference between the AVL-J-infected chickens and controls. These results will increase our understanding of the viral diversity and changes in the virome of chicken gut, with implications in chicken health.

Cytokine inducible SH2-containing protein potentiate J subgroup avian leukosis virus replication and suppress antiviral responses in DF-1 chicken fibroblast cells.

Cytokine-inducible Srchomology2 (SH2)-containing protein (CIS) belongs to the suppressors of cytokine signaling (SOCS) protein family function as a negative feedback loop inhibiting cytokine signal transduction. J subgroup avian leukosis virus (ALV-J), a commonly-seen avian virus with a feature of immunosuppression, poses an unmeasurable threat to the poultry industry across the world. However, commercial medicines or vaccines are still no available for this virus. This study aims to evaluate the potential effect of chicken CIS in antiviral response and its role on ALV-J replication. The results showed that ALV-J strain SCAU-HN06 infection induced CIS expression in DF-1 cells, which was derived from chicken embryo free of endogenous avian sarcoma-leukosis virus (ASLV) like sequences. By overexpressing CIS, the expression of chicken type I interferon (IFN-I) and interferon-stimulated genes (ISGs; PKR, ZAP, CH25H, CCL4, IFIT5, and ISG12) were both suppressed. Meanwhile, data showed that CIS overexpression also increased viral yield. Interestingly, knockdown of CIS enhanced induction of IFN-I and ISGs and inhibited viral replication. Collectively, we proved that modulation of CIS expression not only affected SCAU-HN06 replication in vitro but also altered the expression of IFN-I and ISGs that act as an essential part of antiviral innate immune system. Our data provide a potential target for developing antiviral agents for ALV-J.

Chicken interferon regulatory factor 7 (IRF7) can control ALV-J virus infection by triggering type I interferon production through affecting genes related with innate immune signaling pathway.

In order to breed new birds with strong disease resistance, it is necessary to first understand the mechanism of avian antiviral response. Interferon regulatory factor 7 (IRF7) is not only a member of type I interferons (IFNs) regulatory factor (IRFs) family, but also a major regulator of the IFN response in mammals. However, whether IRF7 is involved in the host innate immune response remains unclear in poultry, due to the absence of IRF3. Here, we first observed by HE stains that with the increase of the time of ALV-J challenge, the thymus was obviously loose and swollen, the arrangement of liver cell was disordered, and the bursa of fabricius formed vacuolated. Real-time PCR detection showed that the expression level of IRF7 gene and related immune genes in ALV-J group was significantly higher than that in control group (P < 0.05). To further study the role of chicken IRF7 during avian leukosis virus subgroup J (ALV-J) infection, we constructed an induced IRF7 overexpression and interfered chicken embryo fibroblasts (CEFs) cell and performed in vitro infection using low pathogenic ALV-J and virus analog poly(I:C). In ALV-J and poly(I:C) stimulated CEFs cells, the expression level of STAT1, IFN-α, IFN-ß, TLR3 and TLR7 were increased after IRF7 overexpressed, while the results were just the opposite after IRF7 interfered, which indicating that IRF7 may be associated with Toll-like receptor signaling pathway and JAK-STAT signaling pathway. These findings suggest that chicken IRF7 is an important regulator of IFN and is involved in chicken anti-ALV-J innate immunity.

Screening active fractions from Pinus massoniana pollen for inhibiting ALV-J replication and their structure activity relationship investigation.

The objective was to identify the active fractions of polysaccharide against replication of ALV-J and elucidate their structure activity relationship. The optimal extraction conditions were extracting temperature 90℃, pH 9 and the ratio of liquid to solid 30:1. Under these conditions, extraction yield of total polysaccharide was 6.5 % ± 0.19 %. Total polysaccharide was then purified by DEAE-52 cellulose and Sephadex G-200 gel. Three fractions, PPP-1, PPP-2, and PPP-3, were identified with molecular weight of 463.70, 99.41, and 26.97 kDa, respectively. Three polysaccharide fractions were all composed of 10 monosaccharides in different proportions. Compared with PPP-1, which was mainly composed of glucose, PPP-2 and PPP-3 contained a higher proportion of galactose, glucuronic acid and galacturonic acid. The Congo red assay indicated that the PPP-2 may have a triple helical structure, while PPP-1 and PPP-3 were absent. In vitro assay showed that there was no significant cytotoxicity among the polysaccharide fractions under the concentration of 800 µg mL-1 (P > 0.05). The antiviral test showed that PPP-2 had the strongest activity, indicating PPP-2 was the major antiviral component. The structure-activity relationship showed that the antiviral activities of polysaccharide fractions were affected by their monosaccharide composition, molecular weight, and triple helical structure, which was a result of a combination of multiple molecular structural factors. These results showed that the PPP-2 could be exploited as a valued product for replacing synthetic antiviral drugs, and provided support for future applications of polysaccharide from Pinus massoniana pollen as a useful source for antiviral agent.

Regulatory effects of chicken TRIM25 on the replication of ALV-A and the MDA5-mediated type I interferon response.

This study focuses on the immunoregulatory effects of chicken TRIM25 on the replication of subgroup A of avian leukosis virus (ALV-A) and the MDA5-mediated type I interferon response. The ALV-A-SDAU09C1 strain was inoculated into DF1 cells and 1-day-old SPF chickens, and the expression of TRIM25 was detected at different time points after inoculation. A recombinant overexpression plasmid containing the chicken TRIM25 gene (TRIM25-GFP) was constructed and transfected into DF1 cells to analyse the effects of the overexpression of chicken TRIM25 on the replication of ALV-A and the expression of MDA5, MAVS and IFN-ß. A small interfering RNA targeting chicken TRIM25 (TRIM25-siRNA) was prepared and transfected into DF1 cells to assess the effects of the knockdown of chicken TRIM25 on the replication of ALV-A and the expression of MDA5, MAVS and IFN-ß. The results showed that chicken TRIM25 was significantly upregulated at all time points both in ALV-A-infected cells and in ALV-A-infected chickens. Overexpression of chicken TRIM25 in DF1 cells dramatically decreased the antigenic titres of ALV-A in the cell supernatant and upregulated the relative expression of MDA5, MAVS and IFN-ß induced by ALV-A or by poly(I:C); in contrast, knockdown of chicken TRIM25 significantly increased the antigenic titres of ALV-A and downregulated the relative expression of MDA5, MAVS and IFN-ß. It can be concluded that chicken TRIM25 can inhibit the replication of ALV-A and upregulate the MDA5 receptor-mediated type I interferon response in chickens. This study can help improve the understanding of the antiviral activities of chicken TRIM25 and enrich the knowledge of antiviral responses in chickens.

The Bipartite Sequence Motif in the N and C Termini of gp85 of Subgroup J Avian Leukosis Virus Plays a Crucial Role in Receptor Binding and Viral Entry.

Subgroup J avian leukemia virus (ALV-J), belonging to the genus Alpharetrovirus, enters cells through its envelope surface unit (gp85) via specifically recognizing the cellular receptor chicken Na+/H+ exchanger type I (chNHE1), the 28 to 39 N-terminal residues of which were characterized as the minimal receptor functional domain in our previous studies. In this study, to further clarify the precise organization and properties of the interaction between ALV-J gp85 and chNHE1, we identified the chNHE1-binding domain of ALV-J gp85 using a series of gp85 mutants with segment substitutions and evaluating their effects on chNHE1 binding in protein-cell binding assays. Our results showed that hemagglutinin (HA) substitutions of amino acids (aa) 38 to 131 (N terminus of gp85) and aa 159 to 283 (C terminus of gp85) significantly inhibited the interaction between gp85 and chNHE1/chNHE1 loop 1. In addition, these HA-substituted chimeric gp85 proteins could not effectively block the entry of ALV-J into chNHE1-expressing cells. Furthermore, analysis of various N-linked glycosylation sites and cysteine mutants in gp85 revealed that glycosylation sites (N6 and N11) and cysteines (C3 and C9) were directly involved in receptor-gp85 binding and important for the entry of ALV-J into cells. Taken together, our findings indicated that the bipartite sequence motif, spanning aa 38 to 131 and aa 159 to 283, of ALV-J gp85 was essential for binding to chNHE1, with its two N-linked glycosylation sites and two cysteines being important for its receptor-binding function and subsequent viral infection steps.IMPORTANCE Infection of a cell by retroviruses requires the attachment and fusion of the host and viral membranes. The specific adsorption of envelope (Env) surface proteins to cell receptors is a key step in triggering infections and has been the target of antiviral drug screening. ALV-J is an economically important avian pathogen that belongs to the genus Alpharetrovirus and has a wider host range than other ALV subgroups. Our results showed that the amino acids 38 to 131 of the N terminus and 159 to 283 of the C terminus of ALV-J gp85 controlled the efficiency of gp85 binding to chNHE1 and were critical for viral infection. In addition, the glycosylation sites (N6 and N11) and cysteines (C3 and C9) of gp85 played a crucial role in the receptor binding and viral entry. These findings might help elucidate the mechanism of the entry of ALV-J into host cells and provide antiviral targets for the control of ALV-J.

Avian leukosis virus subgroup J and reticuloendotheliosis virus coinfection induced TRIM62 regulation of the actin cytoskeleton.

BACKGROUND: Coinfection with avian leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) is common in chickens, and the molecular mechanism of the synergistic pathogenic effects of the coinfection is not clear. Exosomes have been identified as new players in the pathogenesis of retroviruses. The different functions of exosomes depend on their cargo components. OBJECTIVES: The aim of this study was to investigate the function of co-regulation differentially expressed proteins in exosomes on coinfection of ALV-J and REV. METHODS: Here, viral replication in CEF cells infected with ALV-J, REV or both was detected by immunofluorescence microscopy. Then, we analyzed the exosomes isolated from supernatants of chicken embryo fibroblast (CEF) cells single infected and coinfected with ALV-J and REV by mass spectrometry. KEGG pathway enrichment analyzed the co-regulation differentially expressed proteins in exosomes. Next, we silenced and overexpressed tripartite motif containing 62 (TRIM62) to evaluate the effects of TRIM62 on viral replication and the expression levels of NCK-association proteins 1 (NCKAP1) and actin-related 2/3 complex subunit 5 (ARPC5) determined by quantitative reverse transcription polymerase chain reaction. RESULTS: The results showed that coinfection of ALV-J and REV promoted the replication of each other. Thirty proteins, including TRIM62, NCK-association proteins 1 (NCKAP1, also known as Nap125), and Arp2/3-5, ARPC5, were identified. NCKAP1 and ARPC5 were involved in the actin cytoskeleton pathway. TRIM62 negatively regulated viral replication and that the inhibition of REV was more significant than that on ALV-J in CEF cells coinfected with TRIM62. In addition, TRIM62 decreased the expression of NCKAP1 and increased the expression of ARPC5 in coinfected CEF cells. CONCLUSIONS: Collectively, our results indicated that coinfection with ALV-J and REV competitively promoted each other's replication, the actin cytoskeleton played an important role in the coinfection mechanism, and TRIM62 regulated the actin cytoskeleton.