RESUMO
Pseudorabies virus (PRV), an α-herpesvirus, induces immunosuppression and can lead to severe neurological diseases. N-methyl-D-aspartate receptor (NMDAR), an important excitatory nerve receptor in the central nervous system, is linked to various nervous system pathologies. The link between NMDAR and PRV-induced neurological diseases has not been studied. In vivo studies revealed that PRV infection triggers a reduction in hippocampal NMDAR expression, mediated by inflammatory processes. Extensive hippocampal neuronal degeneration was found in mice on the 6th day by hematoxylin-eosin staining, which was strongly correlated with increased NMDAR protein expression. In vitro studies utilizing the CCK-8 assay demonstrated that treatment with an NMDAR antagonist significantly heightened the cytotoxic effects of PRV on T lymphocytes. Notably, NMDAR inhibition did not affect the replication ability of PRV. However, it facilitated the accumulation of pro-inflammatory cytokines in PRV-infected T cells and enhanced the transcription of the CD25 gene through the secretion of interleukin-2 (IL-2), consequently exacerbating immunosuppression. In this study, we found that NMDAR has functional activity in T lymphocytes and is crucial for the inflammatory and immune responses triggered by PRV infection. These discoveries highlight the significant role of NMDAR in PRV-induced neurological disease pathogenesis.
Assuntos
Herpesvirus Suídeo 1 , Pseudorraiva , Receptores de N-Metil-D-Aspartato , Animais , Camundongos , Herpesvirus Suídeo 1/imunologia , Receptores de N-Metil-D-Aspartato/imunologia , Receptores de N-Metil-D-Aspartato/metabolismo , Pseudorraiva/virologia , Pseudorraiva/imunologia , Linfócitos T/imunologia , Linfócitos T/virologia , Hipocampo/virologia , Hipocampo/imunologia , Citocinas/metabolismo , Citocinas/imunologia , Citocinas/genética , Terapia de Imunossupressão , Tolerância Imunológica , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Interleucina-2/imunologia , Interleucina-2/genéticaRESUMO
Pseudorabies virus (PRV) is one of the herpes viruses that can infect a wide range of animals including pigs, cattle, sheep, mice, and wild animals. PRV is a neurotropic alphaherpesvirus capable of infecting a variety of mammals. There is a rising interest in the targeted application of probiotic bacteria to prevent viral diseases, including PRV. In this study, the surface expression of enhanced green fluorescent protein (EGFP) on recombinant Lactiplantibacillus plantarum NC8 (rNC8) through the LP3065 LPxTG motif of Lactobacillus plantarum WCFS1 was generated. The surface expression was observed through confocal microscopy. Dendritic cell targeting peptides (DCpep) were also fused with LPxTG that help to bind with mouse DCs. The PRV-gD was cloned in LP3065 LPxTG, resulting in the generation of rNC8-LP3065-gD. Inactivated rNC8-LP3065-gD was administered intravenously in mice on days 1 and 7 at a dose of 200 µL (109 CFU/mouse) for monitoring immunogenicity. Subsequently, a challenge dose of PRV TJ (104 TCID50) was administered intramuscularly at 14 days post-immunization. The survival rate of the immunized mice reached 80% (4/5) with no significant signs of illness. A significant rise in anti-gD antibodies was detected in the immunized mice by ELISA. Quantitative PCR (qPCR) results showed decreased viral loading in different body tissues. Flow cytometry of lymphocytes derived from mice spleen indicated an increase in CD3+CD4+ T cells, but CD3+CD8+ T cells were not detected. Moreover, it offers a model to delineate immune correlates with rNC8-induced immunity against swine viral diseases.
Assuntos
Herpesvirus Suídeo 1 , Pseudorraiva , Animais , Herpesvirus Suídeo 1/imunologia , Herpesvirus Suídeo 1/genética , Camundongos , Pseudorraiva/prevenção & controle , Pseudorraiva/imunologia , Pseudorraiva/virologia , Feminino , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Lactobacillus plantarum/genética , Lactobacillus plantarum/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/genética , Camundongos Endogâmicos BALB C , Vacinas contra Pseudorraiva/imunologia , Suínos , Proteínas de Fluorescência Verde/genética , Técnicas de Visualização da Superfície CelularRESUMO
Serine/threonine kinase receptor-associated protein (STRAP) serves as a scaffold protein and is engaged in a variety of cellular activities, although its importance in antiviral innate immunity is unknown. We discovered that STRAP works as an interferon (IFN)-inducible positive regulator, facilitating type I IFN signaling during pseudorabies virus infection. Mechanistically, STRAP interacts with TBK1 to activate type I IFN signaling. Both the CT and WD40 7 - 6 domains contribute to the function of STRAP. Furthermore, TBK1 competes with PRV-UL50 for binding to STRAP, and STRAP impedes the degradation of TBK1 mediated by PRV-UL50, thereby increasing the interaction between STRAP and TBK1. Overall, these findings reveal a previously unrecognized role for STRAP in innate antiviral immune responses during PRV infection. STRAP could be a potential therapeutic target for viral infectious diseases.
Assuntos
Herpesvirus Suídeo 1 , Imunidade Inata , Interferon Tipo I , Proteínas Serina-Treonina Quinases , Animais , Linhagem Celular , Herpesvirus Suídeo 1/imunologia , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Pseudorraiva/imunologia , Pseudorraiva/virologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
Background: The cGAS-STING axis-mediated type I interferon pathway is a crucial strategy for host defense against DNA virus infection. Numerous evasion strategies developed by the pseudorabies virus (PRV) counteract host antiviral immunity. To what extent PRV-encoded proteins evade the cGAS-STING signaling pathway is unknown. Methods: Using US2 stably expressing cell lines and US2-deficient PRV model, we revealed that the PRV tegument protein US2 reduces STING protein stability and downregulates STING-mediated antiviral signaling. Results: To promote K48-linked ubiquitination and STING degradation, US2 interacts with the LBD structural domain of STING and recruits the E3 ligase TRIM21. TRIM21 deficiency consistently strengthens the host antiviral immune response brought on by PRV infection. Additionally, US2-deficient PRV is less harmful in mice. Conclusions: Our study implies that PRV US2 inhibits IFN signaling by a new mechanism that selectively targets STING while successfully evading the host antiviral response. As a result, the present study reveals a novel strategy by which PRV evades host defense and offers explanations for why the Bartha-K61 classical vaccine strain failed to offer effective defense against PRV variant strains in China, indicating that US2 may be a key target for developing gene-deficient PRV vaccines.
Assuntos
Herpesvirus Suídeo 1 , Imunidade Inata , Pseudorraiva , Transdução de Sinais , Proteínas do Envelope Viral , Animais , Humanos , Camundongos , Células HEK293 , Herpesvirus Suídeo 1/imunologia , Herpesvirus Suídeo 1/fisiologia , Interações Hospedeiro-Patógeno/imunologia , Evasão da Resposta Imune , Proteínas de Membrana/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/genética , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Pseudorraiva/imunologia , Pseudorraiva/virologia , Ribonucleoproteínas/imunologia , Ribonucleoproteínas/metabolismo , Ubiquitinação , Proteínas do Envelope Viral/metabolismoRESUMO
Introduction: Variant pseudorabies virus (PRV) is a newly emerged zoonotic pathogen that can cause human blindness. PRV can take advantage of its large genome and multiple non-essential genes to construct recombinant attenuated vaccines carrying foreign genes. However, a major problem is that the foreign genes in recombinant PRV are only integrated into the genome for independent expression, rather than assembled on the surface of virion. Methods: We reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) Cap gene into the extracellular domain of the PRV gE gene using the Cre-loxP recombinant system combined with the CRISPR-Cas9 gene editing system. This recombinant PRV (PRV-Cap), with the envelope-embedded Cap protein, exhibits a similar replication ability to its parental virus. Results: An immunogenicity assay revealed that PRV-Cap immunized mice have 100% resistance to lethal PRV and PCV2 attacks. Neutralization antibody and ELISPOT detections indicated that PRV-Cap can enhance neutralizing antibodies to PRV and produce IFN-γ secreting T cells specific for both PRV and PCV2. Immunological mechanistic investigation revealed that initial immunization with PRV-Cap stimulates significantly early activation and expansion of CD69+ T cells, promoting the activation of CD4 Tfh cell dependent germinal B cells and producing effectively specific effector memory T and B cells. Booster immunization with PRV-Cap recalled the activation of PRV-specific IFN-γ+IL-2+CD4+ T cells and IFN-γ+TNF-α+CD8+ T cells, as well as PCV2-specific IFN-γ+TNF-α+CD8+ T cells. Conclusion: Collectively, our data suggested an immunological mechanism in that the recombinant PRV with envelope-assembled PCV2 Cap protein can serve as an excellent vaccine candidate for combined immunity against PRV and PCV2, and provided a cost-effective method for the production of PRV- PCV2 vaccine.
Assuntos
Infecções por Circoviridae , Circovirus , Herpesvirus Suídeo 1 , Animais , Circovirus/imunologia , Circovirus/genética , Camundongos , Herpesvirus Suídeo 1/imunologia , Herpesvirus Suídeo 1/genética , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/prevenção & controle , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Vacinas Virais/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/genética , Suínos , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/genética , Vacinas Sintéticas/imunologia , Pseudorraiva/imunologia , Pseudorraiva/prevenção & controle , Feminino , Camundongos Endogâmicos BALB CRESUMO
Stimulator of IFN genes (STING) is a critical component of the innate immune system, playing an essential role in defending against DNA virus infections. However, the mechanisms governing basal STING regulation remain poorly understood. In this study, we demonstrate that the basal level of STING is critically maintained by hypoxia-inducible factor 1 (HIF-1)α through transcription. Under normal conditions, HIF-1α binds constitutively to the promoter region of STING, actively promoting its transcription. Knocking down HIF-1α results in a decrease in STING expression in multiple cell lines and zebrafish, which in turn reduces cellular responses to synthetic dsDNAs, including cell signaling and IFN production. Moreover, this decrease in STING levels leads to an increase in cellular susceptibility to DNA viruses HSV-1 and pseudorabies virus. These findings unveil a (to our knowledge) novel role of HIF-1α in maintaining basal STING levels and provide valuable insights into STING-mediated antiviral activities and associated diseases.
Assuntos
Herpesvirus Humano 1 , Subunidade alfa do Fator 1 Induzível por Hipóxia , Imunidade Inata , Proteínas de Membrana , Peixe-Zebra , Animais , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peixe-Zebra/imunologia , Herpesvirus Humano 1/imunologia , Herpesvirus Suídeo 1/imunologia , Imunidade Celular , Regulação da Expressão Gênica/imunologia , Transdução de Sinais/imunologia , Transcrição Gênica/imunologia , Regiões Promotoras Genéticas , Células HEK293 , Linhagem Celular , Herpes Simples/imunologia , Pseudorraiva/imunologiaRESUMO
Although vaccination with inactivated vaccines is a popular preventive method against pseudorabies virus (PRV) infection, inactivated vaccines have poor protection efficiency because of their weak immunogenicity. The development of an effective adjuvant is urgently needed to improve the efficacy of inactivated PRV vaccines. In this study, a promising nanocomposite adjuvant named as MIL@A-SW01-C was developed by combining polyacrylic acid-coated metal-organic framework MIL-53(Al) (MIL@A) and squalene (oil)-in-water emulsion (SW01) and then mixing it with a carbomer solution. One part of the MIL@A was loaded onto the oil/water interface of SW01 emulsion via hydrophobic interaction and coordination, while another part was dispersed in the continuous water phase using carbomer. MIL@A-SW01-C showed good biocompatibility, high PRV (antigen)-loading capability, and sustained antigen release. Furthermore, the MIL@A-SW01-C adjuvanted PRV vaccine induced high specific serum antibody titers, increased splenocyte proliferation and cytokine secretion, and a more balanced Th1/Th2 immune response compared with commercial adjuvants, such as alum and biphasic 201. In the mouse challenge experiment, two- and one-shot vaccinations resulted in survival rates of 73.3 % and 86.7 %, respectively. After one-shot vaccination, the host animal pigs were also challenged with wild PRV. A protection rate of 100 % was achieved, which was much higher than that observed with commercial adjuvants. This study not only establishes the superiority of MIL@A-SW01-C composite nanoadjuvant for inactivated PRV vaccine in mice and pigs but also presents an effective method for developing promising nanoadjuvants. STATEMENT OF SIGNIFICANCE: We have developed a nanocomposite of MIL-53(Al) and oil-in-water emulsion (MIL@A-SW01-C) as a promising adjuvant for the inactivated PRV vaccines. MIL@A-SW01-C has good biocompatibility, high PRV (antigen) loading capability, and prolonged antigen release. The developed nanoadjuvant induced much higher specific IgG antibody titers, increased splenocyte proliferation and cytokine secretion, and a more balanced Th1/Th2 immune response than commercial adjuvants alum and biphasic 201. In mouse challenge experiments, survival rates of 73.3 % and 86.7 % were achieved from two-shot and one-shot vaccinations, respectively. At the same time, a protection rate of 100 % was achieved with the host animal pigs challenged with wild PRV.
Assuntos
Adjuvantes Imunológicos , Emulsões , Animais , Adjuvantes Imunológicos/farmacologia , Emulsões/química , Camundongos , Suínos , Herpesvirus Suídeo 1/imunologia , Vacinas contra Pseudorraiva/imunologia , Camundongos Endogâmicos BALB C , Óleos/química , Feminino , Água/química , Vacinas de Produtos Inativados/imunologia , Pseudorraiva/prevenção & controle , Pseudorraiva/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Citocinas/metabolismoRESUMO
Pseudorabies virus (PRV), an alphaherpesvirus, is a neglected zoonotic pathogen. Dectin-1 sensing of ß-glucan (BG) induces trained immunity, which can possibly form a new strategy for the prevention of viral infection. However, alphaherpesvirus including PRV have received little to no investigation in the context of trained immunity. Here, we found that BG pretreatment improved the survival rate, weight loss outcomes, alleviated histological injury and decreased PRV copy number of tissues in PRV-infected mice. Type I interferons (IFNs) including IFN-α/ß levels in serum were significantly increased by BG. However, these effects were abrogated in the presence of Dectin-1 antagonist. Dectin-1-mediated effect of BG was also confirmed in porcine and murine macrophages. These results suggested that BG have effects on type I IFNs with antiviral property involved in Dectin-1. In piglets, oral or injected immunization with BG and PRV vaccine could significantly elevated the level of PRV-specific IgG and type I IFNs. And it also increased the antibody levels of porcine reproductive and respiratory syndrome virus vaccine and classical swine fever vaccine that were later immunized, indicating a broad-spectrum effect on improving vaccine immunity. On the premise that the cost was greatly reducing, the immunological effect of oral was better than injection administration. Our findings highlighted that BG induced type I IFNs related antiviral effect against PRV involved in Dectin-1 and potential application value as a feed additive to help control the spread of PRV and future emerging viruses.
Assuntos
Herpesvirus Suídeo 1 , Interferon Tipo I , Lectinas Tipo C , Pseudorraiva , beta-Glucanas , Animais , beta-Glucanas/farmacologia , beta-Glucanas/administração & dosagem , Camundongos , Suínos , Lectinas Tipo C/imunologia , Pseudorraiva/imunologia , Pseudorraiva/prevenção & controle , Interferon Tipo I/imunologia , Herpesvirus Suídeo 1/imunologia , Herpesvirus Suídeo 1/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Antivirais/farmacologia , Vacinas Virais/imunologia , FemininoRESUMO
Herpesviruses antagonize host antiviral responses through a myriad of molecular strategies culminating in the death of the host cells. Pseudorabies virus (PRV) is a significant veterinary pathogen in pigs, causing neurological sequalae that ultimately lead to the animal's demise. PRV is known to trigger apoptotic cell death during the late stages of infection. The virion host shutdown protein (VHS) encoded by UL41 plays a crucial role in the PRV infection process. In this study, we demonstrate that UL41 inhibits PRV-induced activation of inflammatory cytokine and negatively regulates the cGAS-STING-mediated antiviral activity by targeting IRF3, thereby inhibiting the translocation and phosphorylation of IRF3. Notably, mutating the conserved amino acid sites (E192, D194, and D195) in the RNase domain of UL41 or knocking down UL41 inhibits the immune evasion of PRV, suggesting that UL41 may play a crucial role in PRV's evasion of the host immune response during infection. These results enhance our understanding of how PRV structural proteins assist the virus in evading the host immune response.
Assuntos
Herpesvirus Suídeo 1 , Evasão da Resposta Imune , Fator Regulador 3 de Interferon , NF-kappa B , Herpesvirus Suídeo 1/imunologia , Herpesvirus Suídeo 1/genética , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Animais , Suínos , NF-kappa B/metabolismo , NF-kappa B/genética , NF-kappa B/imunologia , Humanos , Interferons/imunologia , Interferons/metabolismo , Interferons/genética , Pseudorraiva/virologia , Pseudorraiva/imunologia , Linhagem Celular , Interações Hospedeiro-Patógeno/imunologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/imunologia , Células HEK293 , Fosforilação , Transporte ProteicoRESUMO
In recent years, pseudorabies virus (PRV) variants have resulted in an epidemic in swine herds and huge economic losses in China. Therefore, it is essential to develop an efficacious vaccine against the spread of PRV variants. Here, the triple-gene-deletion virus and the triple-gene-deletion plus gC virus were constructed by homologous recombination (HR). And then, their growth capacity, proliferation ability, and immune efficacy were evaluated. The results showed that the growth kinetics of the recombinant viruses were similar to those of the parental strain PRV-AH. Compared with the triple-gene-deletion virus group, the more dominant level of neutralizing antibody (NA) can be induced in the triple-gene-deletion plus gC virus group with the same 106.0 TCID50 dose after 4 and 6 weeks post-initial immunization (PII) (p < 0.0001). In addition, the antibody titers in mice immunized with the triple-gene-deletion plus gC virus were significantly higher than those immunized with triple-gene deletion virus with the same 105.0 TCID50 dose after 6 weeks PII (p < 0.001). More importantly, in the triple-gene-deletion plus gC virus group with 105.0 TCID50, the level of NA was close to that in the triple-gene deletion virus group with 106.0 TCID50 at 6 weeks PII. Meanwhile, the cytokines IL-4 and IFN-γ in sera were tested by enzyme-linked immunosorbent assay (ELISA) in each group. The highest level of IL-4 or IFN-γ was also elicited in the triple-gene deletion plus gC virus group at a dose of 106.0 TCID50. After challenge with PRV-AH, the survival rates of the triple-gene deletion plus gC virus immunized groups were higher than those of other groups. In immunized groups with 105.0 TCID50, the survival rate shows a significant difference between the triple-gene deletion plus gC virus group (75%, 6/8) and the triple-gene deletion virus group (12.5%, 1/8). In general, the immune efficacy of the PRV TK/gI/gE-deleted virus can be increased with additional gC insertion in mice, which has potential for developing an attenuated vaccine candidate for PRV control.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Deleção de Genes , Herpesvirus Suídeo 1 , Vacinas contra Pseudorraiva , Pseudorraiva , Animais , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Camundongos , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Pseudorraiva/prevenção & controle , Pseudorraiva/imunologia , Pseudorraiva/virologia , Vacinas contra Pseudorraiva/imunologia , Vacinas contra Pseudorraiva/genética , Vacinas contra Pseudorraiva/administração & dosagem , Camundongos Endogâmicos BALB C , Suínos , Feminino , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Recombinação Homóloga , Citocinas/metabolismo , ChinaRESUMO
Adjuvants including aluminum adjuvant (Alum) and oil-water emulsion have been widely used in inactivated pseudorabies virus (PRV) vaccines to improve their performance, however, they are not sufficient to protect from PRV infection because of the weak immune response and poor Th1-type immune response. Divalent manganese ion (Mn2+) has been reported to increase the cellular immune response significantly. In this work, a xanthan gum and carbomer-dispersed Mn2+-loaded tannic acid-polyethylene glycol (TPMnXC) nanoparticle colloid is developed and used as an adjuvant to improve the performance of the inactivated PRV vaccine. The good in vitro and in vivo biocompatibility of the developed TPMnXC colloid has been confirmed by the cell viability assay, erythrocyte hemolysis, blood routine analysis, and histological analysis of mouse organs and injection site. The TPMnXC-adjuvanted inactivated PRV vaccine (TPMnXC@PRV) significantly promotes higher and more balanced immune responses indicating with an increased specific total IgG antibody and IgG2a/IgG1 ratio, efficient splenocytes proliferation, and elevated Th1- and Th2-type cytokine secretion than those of control groups. Wild PRV challenge experiment is performed using mice as a model animal, achieving a protection rate of up to 86.67 %, which is much higher than those observed from the commercial Alum. This work not only demonstrates the high potentiality of TPMnXC in practical applications but also provides a new way to develop the Mn2+-loaded nanoadjuvant for veterinary vaccines.
Assuntos
Adjuvantes Imunológicos , Herpesvirus Suídeo 1 , Imunidade Celular , Imunidade Humoral , Manganês , Nanopartículas , Polissacarídeos Bacterianos , Taninos , Animais , Camundongos , Adjuvantes Imunológicos/farmacologia , Nanopartículas/química , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Taninos/química , Taninos/farmacologia , Manganês/química , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/farmacologia , Polissacarídeos Bacterianos/imunologia , Herpesvirus Suídeo 1/imunologia , Vacinas contra Pseudorraiva/imunologia , Vacinas de Produtos Inativados/imunologia , Pseudorraiva/prevenção & controle , Pseudorraiva/imunologia , Feminino , Citocinas/metabolismo , Camundongos Endogâmicos BALB C , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , PolifenóisRESUMO
Natural immunity is the first defense line of the host immune system, which plays a significant role in combating foreign pathogenic microorganisms. The IFN-ß (interferon-beta) signaling pathway, being a typical example of innate immunity, plays a vital function. This study aimed to elucidate the function of pseudorabies virus (PRV) UL38 protein (unique long region 38) in suppressing the activation of the IFN-ß signaling pathway. The findings from our study indicate that the PRV UL38 protein effectively hampers the activation of IFN-ß by poly (dA: dT) (poly(deoxyadenylic-deoxythymidylic)) and 2'3'-cGAMP (2'-3'-cyclic GMP-AMP). Furthermore, UL38 exhibits spatial co-localization with STING (stimulator of interferon genes) and effectively hinders STING dimerization. Subsequently, STING was downgraded to suppress the production of IFN-ß and ISGs (interferon stimulated genes). Immunoprecipitation analysis revealed that the interaction between UL38 and STING, which subsequently initiated the degradation of STING via selective autophagy mediated by TOLLIP (toll interacting protein). To summarize, this research elucidates the function of UL38 in counteracting the cGAS (cGAMP synthase)-STING-induced IFN-ß pathway. The PRV UL38 protein may attenuate the activation of IFN-ß as a means of regulating the virus's persistence in the host.
Assuntos
Autofagia , Herpesvirus Suídeo 1 , Interferon beta , Proteínas de Membrana , Nucleotidiltransferases , Transdução de Sinais , Animais , Humanos , Linhagem Celular , Células HEK293 , Herpesvirus Suídeo 1/fisiologia , Herpesvirus Suídeo 1/imunologia , Interações Hospedeiro-Patógeno , Imunidade Inata , Interferon beta/metabolismo , Interferon beta/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Pseudorraiva/virologia , Pseudorraiva/metabolismo , Pseudorraiva/imunologia , Proteínas Virais/metabolismo , Proteínas Virais/genética , Suínos , MesocricetusRESUMO
Alphaherpesvirus pseudorabies virus (PRV) causes severe economic losses to the global pig industry and has garnered increasing attention due to its broad host range including humans. PRV has developed a variety of strategies to antagonize host antiviral innate immunity. However, the underlying mechanisms have not been fully elucidated. In our previous work, we demonstrated that non-muscle myosin heavy chain IIA (NMHC-IIA), a multifunctional cytoskeleton protein, attenuates innate immune responses triggered by RNA viruses. In the current study, we reported a previously unrecognized role of NMHC-IIA in counteracting PRV-induced cyclic GMP-AMP synthase (cGAS)-dependent type I interferon (IFN-I) production. Mechanistically, PRV infection led to an elevation of NMHC-IIA, strengthening the interaction between poly (ADP-ribose) polymerase 1 (PARP1) and cGAS. This interaction impeded cGAS recognition of PRV DNA and hindered downstream signaling activation. Conversely, inhibition of NMHC-IIA by Blebbistatin triggered innate immune responses and enhanced resistance to PRV proliferation both in vitro and in vivo. Taken together, our findings unveil that PRV utilizes NMHC-IIA to antagonize host antiviral immune responses via impairing DNA sensing by cGAS. This in-depth understanding of PRV immunosuppression not only provides insights for potential PRV treatment strategies but also highlights NMHC-IIA as a versatile immunosuppressive regulator usurped by both DNA and RNA viruses. Consequently, NMHC-IIA holds promise as a target for the development of broad-spectrum antiviral drugs.IMPORTANCECyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) axis plays a vital role in counteracting alphaherpesvirus infections. Alphaherpesviruses exploit various strategies for antagonizing cGAS-STING-mediated antiviral immune responses. However, limited examples of pseudorabies virus (PRV)-caused immunosuppression have been documented. Our findings reveal a novel role of non-muscle myosin heavy chain IIA (NMHC-IIA) in suppressing PRV-triggered innate immune responses to facilitate viral propagation both in vitro and in vivo. In detail, NMHC-IIA recruits poly (ADP-ribose) polymerase 1 (PARP1) to augment its interaction with cGAS, which impairs cGAS recognition of PRV DNA. Building on our previous demonstration of NMHC-IIA's immunosuppressive role during RNA virus infections, these findings indicate that NMHC-IIA acts as a broad-spectrum suppressor of host antiviral innate immunity in response to both DNA and RNA viruses. Therefore, NMHC-IIA will be a promising target for the development of comprehensive antiviral strategies.
Assuntos
Herpesvirus Suídeo 1 , Imunidade Inata , Miosina não Muscular Tipo IIA , Pseudorraiva , Animais , Humanos , Camundongos , Linhagem Celular , DNA Viral/imunologia , Células HEK293 , Herpesvirus Suídeo 1/imunologia , Interferon Tipo I/metabolismo , Interferon Tipo I/imunologia , Cadeias Pesadas de Miosina/metabolismo , Cadeias Pesadas de Miosina/imunologia , Miosina não Muscular Tipo IIA/metabolismo , Nucleotidiltransferases/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Pseudorraiva/imunologia , Pseudorraiva/virologia , Transdução de Sinais , SuínosRESUMO
Pseudorabies virus (PRV) is a highly contagious viral disease, which leads to severe financial losses in the breeding industry worldwide. Presently, PRV is mainly controlled using live attenuated and inactivated vaccines. However, these vaccines have an innate tendency to lose their structural conformation upon exposure to environmental and chemical stressors and cannot provide full protection against the emerging prevalent PRV variants. In this work, first, we synthesized aminated ZIF-7/8 nanoparticles (NPs), and then chemical bond-coated alginate dialdehyde (ADA, a type of dioxide alginate saccharide) on their surface via Schiff base reaction to obtain ZIF-7/8-ADA NPs. The as-fabricated ZIF-7/8-ADA NPs exhibited high stability, monodispersity and a high loading ratio of antigen. Furthermore, the ZIF-7/8-ADA NPs showed good biocompatibility in vitro and in vivo. Using ZIF-7/8-ADA NPs as an adjuvant and inactivated PRV as a model antigen, we constructed a PR vaccine through a simple mixture. The immunity studies indicated that ZIF-7/8-ADA induced an enhancement in the Th1/Th2 immune response, which was superior to that of the commercial ISA201, alum adjuvant and ZIF-7/8. Due to the pH-sensitive release of the antigen in lysosomes, the as-prepared PR vaccine subsequently accelerated the antigen presentation and improved the immune responses in vitro and in vivo. The results of PRV challenge using mice as the model demonstrated that ZIF-7/8-ADA achieved the same preventive effect as the commercial ISA201 and was much better than the alum adjuvant, and thus can serve as a promising delivery system and adjuvant to enhance humoral and cellular responses against PRV infection.
Assuntos
Adjuvantes Imunológicos , Alginatos , Estruturas Metalorgânicas , Nanopartículas , Animais , Alginatos/química , Alginatos/farmacologia , Camundongos , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Estruturas Metalorgânicas/química , Estruturas Metalorgânicas/farmacologia , Pseudorraiva/prevenção & controle , Pseudorraiva/imunologia , Herpesvirus Suídeo 1/imunologia , Vacinas contra Pseudorraiva/imunologia , Vacinas contra Pseudorraiva/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Portadores de Fármacos/química , Vacinação/métodos , Camundongos Endogâmicos BALB C , FemininoRESUMO
Pseudorabies virus (PRV) is a member of the genus Varicellovirus, family Herpesviridae and causes Aujeszky's disease to lead to huge economic losses in the global pig industry. The Non-POU domain-containing octamer-binding protein (NONO), as a Drosophila behavior/human splicing (DBHS) protein, plays a key role in multiple biological functions in cells, including transcriptional regulation, RNA splicing, DNA repair and so on. However, whether swine NONO (sNONO) inhibits PRV infection is less understood. In this study, we showed that sNONO was a crucial host factor for antagonizing PRV infection and positive regulated transcription levels of ISGs. After PRV infection, sNONO enhanced the activation of IFN-ß promoter and IFN-ß expression. Furthermore, knockout of sNONO in PAM-KNU cells impaired activation of type I IFN pathway and increased PRV propagation. Taken together, we have first elucidated the anti-PRV function and mechanism of sNONO, which may provide a new strategy for preventing DNA virus infection.
Assuntos
Proteínas de Ligação a DNA , Pseudorraiva , Proteínas de Ligação a RNA , Doenças dos Suínos , Animais , Proteínas de Ligação a DNA/genética , Herpesvirus Suídeo 1 , Interferon beta/imunologia , Pseudorraiva/imunologia , Proteínas de Ligação a RNA/genética , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Fatores de TranscriçãoRESUMO
Pseudorabies virus (PRV) is a porcine alphaherpesvirus that belongs to the Herpesviridae family. We showed earlier that infection of porcine epithelial cells with PRV triggers activation of the nuclear factor κB (NF-κB) pathway, a pivotal signaling axis in the early immune response. However, PRV-induced NF-κB activation does not lead to NF-κB-dependent gene expression. Here, using electrophoretic mobility shift assays (EMSAs), we show that PRV does not disrupt the ability of NF-κB to interact with its κB target sites. Assessing basal cellular transcriptional activity in PRV-infected cells by quantitation of prespliced transcripts of constitutively expressed genes uncovered a broad suppression of cellular transcription by PRV, which also affects the inducible expression of NF-κB target genes. Host cell transcription inhibition was rescued when viral genome replication was blocked using phosphonoacetic acid (PAA). Remarkably, we found that host gene expression shutoff in PRV-infected cells correlated with a substantial retention of the NF-κB subunit p65, the TATA box binding protein, and RNA polymerase II-essential factors required for (NF-κB-dependent) gene transcription-in expanding PRV replication centers in the nucleus and thereby away from the host chromatin. This study reveals a potent mechanism used by the alphaherpesvirus PRV to steer the protein production capacity of infected cells to viral proteins by preventing expression of host genes, including inducible genes involved in mounting antiviral responses. IMPORTANCE Herpesviruses are highly successful pathogens that cause lifelong persistent infections of their host. Modulation of the intracellular environment of infected cells is imperative for the success of virus infections. We reported earlier that a DNA damage response in epithelial cells infected with the alphaherpesvirus pseudorabies virus (PRV) results in activation of the hallmark proinflammatory NF-κB signaling axis but, remarkably, that this activation does not lead to NF-κB-induced (proinflammatory) gene expression. Here, we report that PRV-mediated inhibition of host gene expression stretches beyond NF-κB-dependent gene expression and in fact reflects a broad inhibition of host gene transcription, which correlates with a substantial recruitment of essential host transcription factors in viral replication compartments in the nucleus, away from the host chromatin. These data uncover a potent alphaherpesvirus mechanism to interfere with production of host proteins, including proteins involved in antiviral responses.
Assuntos
Herpesvirus Suídeo 1 , Pseudorraiva , Doenças dos Suínos , Transcrição Gênica , Animais , Herpesvirus Suídeo 1/fisiologia , Interações entre Hospedeiro e Microrganismos , NF-kappa B/genética , NF-kappa B/metabolismo , Pseudorraiva/imunologia , Pseudorraiva/fisiopatologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/fisiopatologiaRESUMO
The alphaherpesvirus pseudorabies virus (PRV) is the etiologic agent of swine Aujeszky's disease, which can cause huge economic losses to the pig industry. PRV can overcome a type I interferon (IFN)-induced antiviral state in host cells through its encoded EP0 protein. However, the exact role of EP0 in this process is poorly defined. Here, we report that EP0 transcriptionally represses IFN regulatory factor 9 (IRF9), a critical component in the IFN signaling pathway, thereby reducing the cellular levels of IRF9 and inhibiting IFN-induced gene transcription. This activity of EP0 is mediated by its C-terminal region independently of the RING domain. Moreover, compared with EP0 wild-type PRV, EP0-deficient PRV loses the ability to efficiently decrease cellular IRF9, while reintroducing the C-terminal region of EP0 back into the EP0-deficient virus restores the activity. Together, these results suggest that EP0 can transcriptionally modulate IRF9-mediated antiviral pathways through its C-terminal region, contributing to PRV innate immune evasion. IMPORTANCE Alphaherpesviruses can establish lifelong infections and cause many diseases in humans and animals. Pseudorabies virus (PRV) is a swine alphaherpesvirus that threatens pig production. Using PRV as a model, we found that alphaherpesvirus can utilize its encoded early protein EP0 to inhibit the IFN-induced upregulation of antiviral proteins by reducing the basal expression levels of IRF9 through repressing its transcription. Our findings reveal a mechanism employed by alphaherpesvirus to evade the immune response and indicate that EP0 is an important viral protein in pathogenesis and a potential target for antiviral drug development.
Assuntos
Herpesvirus Suídeo 1 , Interferon Tipo I , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Pseudorraiva , Doenças dos Suínos , Animais , Antivirais/farmacologia , Regulação da Expressão Gênica/imunologia , Herpesvirus Suídeo 1/imunologia , Herpesvirus Suídeo 1/metabolismo , Interações entre Hospedeiro e Microrganismos/imunologia , Interferon Tipo I/metabolismo , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Pseudorraiva/imunologia , Pseudorraiva/virologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/metabolismoRESUMO
Pseudorabies virus (PRV) has evolved various immune evasion mechanisms that target host antiviral immune responses. However, it is unclear whether and how PRV encoded proteins modulate the cGAS-STING axis for immune evasion. Here, we show that PRV tegument protein UL13 inhibits STING-mediated antiviral signaling via regulation of STING stability. Mechanistically, UL13 interacts with the CDN domain of STING and recruits the E3 ligase RING-finger protein 5 (RNF5) to promote K27-/K29-linked ubiquitination and degradation of STING. Consequently, deficiency of RNF5 enhances host antiviral immune responses triggered by PRV infection. In addition, mutant PRV lacking UL13 impaired in antagonism of STING-mediated production of type I IFNs and shows attenuated pathogenicity in mice. Our findings suggest that PRV UL13 functions as an antagonist of IFN signaling via a novel mechanism by targeting STING to persistently evade host antiviral responses.
Assuntos
Herpesvirus Suídeo 1 , Proteínas de Membrana , Proteínas Quinases , Pseudorraiva , Ubiquitina-Proteína Ligases , Animais , Herpesvirus Suídeo 1/imunologia , Imunidade Inata , Proteínas de Membrana/imunologia , Camundongos , Proteínas Quinases/imunologia , Pseudorraiva/imunologia , Ubiquitina-Proteína Ligases/imunologia , Proteínas Virais/imunologiaRESUMO
Porcine circovirus-associated diseases (PCVADs) and pseudorabies (PR) are highly contagious and economically significant diseases of swine in China. Porcine circovirus type 3 (PCV3) is an emerging swine pathogen of PCVAD. Currently, no PCV3 vaccine is commercially available, and the epidemic caused by it is still spreading worldwide. In this study, we used the PRV variant strain HNX as the parental virus to construct recombinant PRV with TK/gE gene deletion and capsid (Cap) protein co-expression, named HNX-ΔTK/ΔgE-ORF2. The results revealed that PCV3 Cap protein can be detected in HNX-ΔTK/ΔgE-ORF2-infected PK-15 cells by both western blotting and immunofluorescence assays. Vaccination with HNX-ΔTK/ΔgE-ORF2 did not cause pruritus, ruffled fur, systemic infection, or inflammation (without high expression of interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) in plasma). Furthermore, HNX-ΔTK/ΔgE-ORF2 immunization induced an anti-Cap specific antibody, activated a PRV-specific cellular immune response, and provided 100 % protection to mice against the challenge of the virulent HNX strain. Thus, HNX-ΔTK/ΔgE-ORF2 appears to be a promising vaccine candidate against PRV and PCV3 for the control of the PRV variant and PCV3.
Assuntos
Proteínas do Capsídeo , Circovirus , Herpesvirus Suídeo 1 , Pseudorraiva , Vacinas Virais , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Circovirus/genética , Circovirus/imunologia , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/imunologia , Camundongos , Pseudorraiva/imunologia , Pseudorraiva/virologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Vacinas Virais/imunologiaRESUMO
Plasmacytoid dendritic cells (pDC) are important innate immune cells during the onset of viral infections as they are specialized in the production of massive amounts of antiviral type I interferon (IFN). Alphaherpesviruses such as herpes simplex virus (HSV) or pseudorabies virus (PRV) are double stranded DNA viruses and potent stimulators of pDC. Detailed information on how PRV activates porcine pDC is lacking. Using PRV and porcine primary pDC, we report here that PRV virions, so-called heavy (H-)particles, trigger IFNα production by pDC, whereas light (L-) particles that lack viral DNA and capsid do not. Activation of pDC requires endosomal acidification and, importantly, depends on the PRV gD envelope glycoprotein and O-glycosylations. Intriguingly, both for PRV and HSV-1, we found that L-particles suppress H-particle-mediated activation of pDC, a process which again depends on viral gD. This is the first report describing that gD plays a critical role in alphaherpesvirus-induced pDC activation and that L-particles directly interfere with alphaherpesvirus-induced IFNα production by pDC.