RESUMO
RAB GTPases (RABs) control intracellular membrane trafficking with high precision. In the present study, we carried out a short hairpin RNA (shRNA) screen focused on a library of 62 RABs during infection with porcine reproductive and respiratory syndrome virus 2 (PRRSV-2), a member of the family Arteriviridae. We found that 13 RABs negatively affect the yield of PRRSV-2 progeny virus, whereas 29 RABs have a positive impact on the yield of PRRSV-2 progeny virus. Further analysis revealed that PRRSV-2 infection transcriptionally regulated RAB18 through RIG-I/MAVS-mediated canonical NF-κB activation. Disrupting RAB18 expression led to the accumulation of lipid droplets (LDs), impaired LDs catabolism, and flawed viral replication and assembly. We also discovered that PRRSV-2 co-opts chaperone-mediated autophagy (CMA) for lipolysis via RAB18, as indicated by the enhanced associations between RAB18 and perlipin 2 (PLIN2), CMA-specific lysosomal associated membrane protein 2A (LAMP2A), and heat shock protein family A (Hsp70) member 8 (HSPA8/HSC70) during PRRSV-2 infection. Knockdown of HSPA8 and LAMP2A impacted on the yield of PRRSV-2 progeny virus, implying that the virus utilizes RAB18 to promote CMA-mediated lipolysis. Importantly, we determined that the C-terminal domain (CTD) of HSPA8 could bind to the switch II domain of RAB18, and the CTD of PLIN2 was capable of associating with HSPA8, suggesting that HSPA8 facilitates the interaction between RAB18 and PLIN2 in the CMA process. In summary, our findings elucidate how PRRSV-2 hijacks CMA-mediated lipid metabolism through innate immune activation to enhance the yield of progeny virus, offering novel insights for the development of anti-PRRSV-2 treatments.
Assuntos
Autofagia Mediada por Chaperonas , Vírus da Síndrome Respiratória e Reprodutiva Suína , Suínos , Animais , Lipólise , Regulação para Cima , Proteínas rab de Ligação ao GTP/genética , Proteínas de Membrana Lisossomal , RNA Interferente PequenoRESUMO
Pseudorabies virus (PRV) is recognized as the aetiological agent responsible for Aujeszky's disease, or pseudorabies, in swine populations. Rab6, a member of the small GTPase family, is implicated in various membrane trafficking processes, particularly exocytosis regulation. Its involvement in PRV infection, however, has not been documented previously. In our study, we observed a significant increase in the Rab6 mRNA and protein levels in both PK-15 porcine kidney epithelial cells and porcine alveolar macrophages, as well as in the lungs and spleens of mice infected with PRV. The overexpression of wild-type Rab6 and its GTP-bound mutant facilitated PRV proliferation, whereas the GDP-bound mutant form of Rab6 had no effect on viral propagation. These findings indicated that the GTPase activity of Rab6 was crucial for the successful spread of PRV. Further investigations revealed that the reduction in Rab6 levels through knockdown significantly hampered PRV proliferation and disrupted virus assembly and egress. At the molecular level, Rab6 was found to interact with the PRV glycoproteins gB and gE, both of which are essential for viral assembly and egress. Our results collectively suggest that PRV exploits Rab6 to expedite its assembly and egress and identify Rab6 as a promising novel target for therapeutic treatment for PRV infection.
Assuntos
Herpesvirus Suídeo 1 , Montagem de Vírus , Liberação de Vírus , Proteínas rab de Ligação ao GTP , Animais , Camundongos , Linhagem Celular , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/metabolismo , Pseudorraiva/virologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Suínos , Doenças dos Suínos/virologia , Montagem de Vírus/genética , Liberação de Vírus/genéticaRESUMO
Chromatin dynamics regulated by epigenetic modification is crucial in genome stability and gene expression. Various epigenetic mechanisms have been identified in the pathogenesis of human diseases. Here, we examined the effects of ten epigenetic agents on pseudorabies virus (PRV) infection by using GFP-reporter assays. Inhibitors of bromodomain protein 4 (BRD4), which receives much more attention in cancer than viral infection, was found to exhibit substantial anti-viral activity against PRV as well as a range of DNA and RNA viruses. We further demonstrated that BRD4 inhibition boosted a robust innate immune response. BRD4 inhibition also de-compacted chromatin structure and induced the DNA damage response, thereby triggering the activation of cGAS-mediated innate immunity and increasing host resistance to viral infection both in vitro and in vivo. Mechanistically, the inhibitory effect of BRD4 inhibition on viral infection was mainly attributed to the attenuation of viral attachment. Our findings reveal a unique mechanism through which BRD4 inhibition restrains viral infection and points to its potent therapeutic value for viral infectious diseases.
Assuntos
Proteínas de Ciclo Celular/imunologia , Dano ao DNA/imunologia , Vírus de DNA/imunologia , Imunidade Inata , Proteínas Nucleares/imunologia , Vírus de RNA/imunologia , Fatores de Transcrição/imunologia , Células A549 , Animais , Chlorocebus aethiops , Infecções por Vírus de DNA/imunologia , Cães , Feminino , Células HEK293 , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Células RAW 264.7 , Infecções por Vírus de RNA/imunologia , Suínos , Células VeroRESUMO
Autophagy maintains cellular homeostasis by degrading organelles, proteins, and lipids in lysosomes. Autophagy is involved in the innate and adaptive immune responses to a variety of pathogens. Some viruses can hijack host autophagy to enhance their replication. However, the role of autophagy in porcine reproductive and respiratory syndrome virus (PRRSV) infection is unclear. Here, we show that N-Myc downstream-regulated gene 1 (NDRG1) deficiency induced autophagy, which facilitated PRRSV replication by regulating lipid metabolism. NDRG1 mRNA is expressed ubiquitously in most porcine tissues and most strongly in white adipose tissue. PRRSV infection downregulated the expression of NDRG1 mRNA and protein, while NDRG1 deficiency contributed to PRRSV RNA replication and progeny virus assembly. NDRG1 deficiency reduced the number of intracellular lipid droplets (LDs), but the expression levels of key genes in lipogenesis and lipolysis were not altered. Our results also show that NDRG1 deficiency promoted autophagy and increased the subsequent yields of hydrolyzed free fatty acids (FFAs). The reduced LD numbers, increased FFA levels, and enhanced PRRSV replication were abrogated in the presence of an autophagy inhibitor. Overall, our findings suggest that NDRG1 plays a negative role in PRRSV replication by suppressing autophagy and LD degradation.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped single-positive-stranded RNA virus, causes acute respiratory distress in piglets and reproductive failure in sows. It has led to tremendous economic losses in the swine industry worldwide since it was first documented in the late 1980s. Vaccination is currently the major strategy used to control the disease. However, conventional vaccines and other strategies do not provide satisfactory or sustainable prevention. Therefore, safe and effective strategies to control PRRSV are urgently required. The significance of our research is that we demonstrate a previously unreported relationship between PRRSV, NDRG1, and lipophagy in the context of viral infection. Furthermore, our data point to a new role for NDRG1 in autophagy and lipid metabolism. Thus, NDRG1 and lipophagy will have significant implications for understanding PRRSV pathogenesis for developing new therapeutics.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulação para Baixo , Ácidos Graxos não Esterificados/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Animais , Autofagia , Células HEK293 , Humanos , Masculino , Filogenia , Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Suínos , Replicação ViralRESUMO
OBJECTIVE: The purpose of the article is to evaluate the changes in lipid metabolism in bovine mammary-gland epithelial MAC-T cells after PKM2 knockdown. RESULTS: MAC-T cells stably expressing low levels of PKM2 were established with lentivirus-mediated small hairpin RNA. Although the knockdown of PKM2 had no effect on MAC-T cell growth, the reduced expression of PKM2 attenuated the mRNA and protein expression of key enzymes involved in sterol synthesis through the SREBP pathway. CONCLUSIONS: The downregulation of PKM2 significantly influenced lipid synthesis in bovine mammary-gland epithelial MAC-T cells. These findings extend our understanding of the crosstalk between glycolysis and lipid metabolism in bovine mammary-gland epithelial cells.
Assuntos
Proteínas de Transporte/genética , Metabolismo dos Lipídeos/genética , Glândulas Mamárias Animais/metabolismo , Proteínas de Membrana/genética , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Hormônios Tireóideos/genética , Animais , Proteínas de Transporte/metabolismo , Bovinos , Células Epiteliais/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Glicólise/genética , Lipídeos/biossíntese , Proteínas de Membrana/metabolismo , RNA Mensageiro/genética , Transdução de Sinais , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Linfócitos T/metabolismo , Hormônios Tireóideos/metabolismo , Proteínas de Ligação a Hormônio da TireoideRESUMO
Feline bocavirus (FBoV) is a globally distributed linear, single-stranded DNA virus infect cats, currently classified into three distinct genotypes. Although FBoV can lead to systemic infections, its complete pathogenic potential remains unclear. In this study, 289 blood samples were collected from healthy cats in Harbin, revealing an overall FBoV prevalence of 12.1%. Notably, genotypes 1 and 3 of FBoV were found co-circulating among the cat population in Harbin. Additionally, recombination events were detected, particularly in the newly discovered NG/104 and DL/102 strains. Furthermore, negative selection sites were predominantly observed across the protein coding genes of FBoV. These findings suggest a co-circulation of genetically diverse FBoV strains among cats in Harbin, indicate that purifying selection is the primary driving force shaping the genomic evolution of FBoV, and also underscore the importance of comprehensive surveillance efforts to enhance our understanding of the epidemiology and evolutionary characteristics of FBoV.
Assuntos
Bocavirus , Doenças do Gato , Variação Genética , Genótipo , Infecções por Parvoviridae , Filogenia , Gatos , Animais , China/epidemiologia , Doenças do Gato/virologia , Doenças do Gato/epidemiologia , Infecções por Parvoviridae/veterinária , Infecções por Parvoviridae/virologia , Infecções por Parvoviridae/epidemiologia , Bocavirus/genética , Bocavirus/classificação , Bocavirus/isolamento & purificação , Prevalência , Recombinação Genética , Genoma Viral , Evolução MolecularRESUMO
In the era of increased antibiotic resistance and ever-stricter control on antibiotic use, it is urgent to develop green, safe, and non-residue alternatives to antibiotics applied to the poultry industry. To this end, we supplied the potential Lactobacillus plantarum (L. plantarum) fermented Astragalus in the diet of laying hens, with a final addition of 3. Its effects have been assessed on laying performance, egg quality, antioxidant and immunological status, and intestinal microbiota, and are compared to the control group, to the Astragalus group containing 3 unfermented Astragalus, and to the L. plantarum group containing 2% L. plantarum [5 × 108 colony-forming unit (CFU) per milliliter (mL)]. During the second half of the experimental period (15 to 28 days), the egg production rate was considerably higher in the fermented Astragalus group than that in the other groups, with the fermented Astragalus group having the lowest feed conversion ratio. No significant difference (P > 0.05) was noted among treatments on egg quality. Fermented Astragalus-treated hens exhibited significantly increased catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in serum, and reduced malondialdehyde (MDA) in serum. Furthermore, fermented Astragalus supplementation resulted in a significant increase in ileal microbiota abundance relative to control. In conclusion, feeding laying hens with L. plantarum fermented Astragalus has beneficial effects on production, antioxidant potential, immunity, and ileal microbiota. L. plantarum fermented Astragalus is expected to be a novel feed additive used in poultry production.
RESUMO
Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a predominant DNA sensor inducing the activation of the innate immune responses that produce proinflammatory cytokines and type I interferons, which has been well-investigated in mammals. However, chicken cGAS (chcGAS), which participates in avian innate immunity, has not been well-investigated. Here, we cloned the complete open reading frame sequence of chcGAS. Multiple sequence alignment and phylogenetic analysis revealed that chcGAS was homologous to mammalian cGAS. The chcGAS mRNA was highly expressed in the bone marrow and ileum. The subcellular localization of chcGAS was mainly in the cytoplasm, and partial co-localization was observed in the endoplasmic reticulum. Through overexpression and RNA interference, we demonstrated that chcGAS responded to exogenous dsDNA, HS-DNA, and poly(dA:dT), and to self dsDNA from the DNA damage response, thereby triggering the activation of STING/TBK1/IRF7-mediated innate immunity in both chicken embryonic fibroblasts and chicken liver cancer cells. Furthermore, downregulation of chcGAS enhanced the infection of fowl adenovirus serotype 4 in LMH cells. Our results demonstrated that chcGAS was an important cytosolic DNA sensor activating innate immune responses and may shed light on a strategy for preventing infectious diseases in the poultry industry.
Assuntos
Adenoviridae/imunologia , Galinhas/imunologia , Galinhas/virologia , Citosol/metabolismo , DNA/metabolismo , Imunidade Inata , Nucleotídeos Cíclicos/metabolismo , Sorogrupo , Sequência de Aminoácidos , Animais , Linhagem Celular , Dano ao DNA , Etoposídeo/farmacologia , Perfilação da Expressão Gênica , Fator Regulador 7 de Interferon/metabolismo , Interferon beta/metabolismo , Interleucina-1beta/metabolismo , Nucleotídeos Cíclicos/química , Filogenia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
Myostatin (Mstn) is postulated to be a key determinant of muscle loss and cachexia in cancer. However, no experimental evidence supports a role for Mstn in cancer, particularly in regulating the survival and growth of cancer cells. In this study, we showed that the expression of Mstn was significantly increased in different tumor tissues and human cancer cells. Mstn knockdown inhibited the proliferation of cancer cells. A knockout (KO) of Mstn created by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 (CRISPR/Cas9) induced mitochondria-dependent apoptosis in HeLa cells. Furthermore, KO of Mstn reduced the lipid content. Molecular analyses demonstrated that the expression levels of fatty acid oxidation-related genes were upregulated and then increased rate of fatty acid oxidation. Mstn deficiency-induced apoptosis took place along with generation of reactive oxygen species (ROS) and elevated fatty acid oxidation, which may play a role in triggering mitochondrial membrane depolarization, the release of cytochrome c (Cyt-c), and caspase activation. Importantly, apoptosis induced by Mstn KO was partially rescued by antioxidants and etomoxir, thereby suggesting that the increased level of ROS was functionally involved in mediating apoptosis. Overall, our findings demonstrate a novel function of Mstn in regulating mitochondrial metabolism and apoptosis within cancer cells. Hence, inhibiting the production and function of Mstn may be an effective therapeutic intervention during cancer progression and muscle loss in cachexia.