RESUMO
Vaccinia virus (VACV) encodes an innate immune evasion protein, E3, which contains an N-terminal Z-nucleic acid binding (Zα) domain that is critical for pathogenicity in mice. Here we demonstrate that the N terminus of E3 is necessary to inhibit an IFN-primed virus-induced necroptosis. VACV deleted of the Zα domain of E3 (VACV-E3LΔ83N) induced rapid RIPK3-dependent cell death in IFN-treated L929 cells. Cell death was inhibited by the RIPK3 inhibitor, GSK872, and infection with this mutant virus led to phosphorylation and aggregation of MLKL, the executioner of necroptosis. In 293T cells, induction of necroptosis depended on expression of RIPK3 as well as the host-encoded Zα domain-containing DNA sensor, DAI. VACV-E3LΔ83N is attenuated in vivo, and pathogenicity was restored in either RIPK3- or DAI-deficient mice. These data demonstrate that the N terminus of the VACV E3 protein prevents DAI-mediated induction of necroptosis.
Assuntos
DNA Forma Z/metabolismo , Glicoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Vaccinia virus/metabolismo , Proteínas Virais/metabolismo , Animais , Caspases/metabolismo , Morte Celular , Linhagem Celular , Sobrevivência Celular , DNA Forma Z/química , Glicoproteínas/genética , Humanos , Imunidade Inata , Interferon Tipo I/química , Interferon Tipo I/farmacologia , Camundongos , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Vaccinia virus/imunologia , Vaccinia virus/patogenicidade , Proteínas Virais/química , VirulênciaRESUMO
UNLABELLED: The vaccinia virus (VACV) E3 protein has been shown to be important for blocking activation of the cellular innate immune system and allowing viral replication to occur unhindered. Mutation or deletion of E3L severely affects viral host range and pathogenesis. While the monkeypox virus (MPXV) genome encodes a homologue of the VACV E3 protein, encoded by the F3L gene, the MPXV gene is predicted to encode a protein with a truncation of 37 N-terminal amino acids. VACV with a genome encoding a similarly truncated E3L protein (VACV-E3LΔ37N) has been shown to be attenuated in mouse models, and infection with VACV-E3LΔ37N has been shown to lead to activation of the host antiviral protein kinase R pathway. In this report, we present data demonstrating that, despite containing a truncated E3 homologue, MPXV phenotypically resembles a wild-type (wt) VACV rather than VACV-E3LΔ37N. Thus, MPXV appears to contain a gene or genes that can suppress the phenotypes associated with an N-terminal truncation in E3. The suppression maps to sequences outside F3L, suggesting that the suppression is extragenic in nature. Thus, MPXV appears to have evolved mechanisms to minimize the effects of partial inactivation of its E3 homologue. IMPORTANCE: Poxviruses have evolved to have many mechanisms to evade host antiviral innate immunity; these mechanisms may allow these viruses to cause disease. Within the family of poxviruses, variola virus (which causes smallpox) is the most pathogenic, while monkeypox virus is intermediate in pathogenicity between vaccinia virus and variola virus. Understanding the mechanisms of monkeypox virus innate immune evasion will help us to understand the evolution of poxvirus innate immune evasion capabilities, providing a better understanding of how poxviruses cause disease.
Assuntos
Evasão da Resposta Imune , Imunidade Inata , Interferon Tipo I/imunologia , Monkeypox virus/genética , Proteínas de Ligação a RNA/genética , Vaccinia virus/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Evolução Biológica , Linhagem Celular , Chlorocebus aethiops , Cricetulus , Células Epiteliais/imunologia , Células Epiteliais/virologia , Expressão Gênica , Células HeLa , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Humanos , Interferon Tipo I/genética , Dados de Sequência Molecular , Monkeypox virus/imunologia , Monkeypox virus/patogenicidade , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/imunologia , Coelhos , Alinhamento de Sequência , Transdução de Sinais , Vaccinia virus/imunologia , Vaccinia virus/patogenicidade , Células Vero , Proteínas Virais/química , Proteínas Virais/imunologia , Replicação ViralRESUMO
Necroptosis has been implicated as a critical cell death pathway in cancers, Alzheimer's and other neurodegenerative diseases, and virus-infected cells. Necroptosis occurs when mixed-lineage kinase domain-like protein (MLKL) punctures the cytoplasmic membrane allowing a rapid influx of water leading to a loss of cellular integrity. As its role in human disease becomes apparent, methods identifying necroptosis will need to be further developed and optimized. Here we describe identification of necroptosis through quantifying cell death with pathway inhibitors and using western blots to identify end points of MLKL activation and protein-protein interactions leading to it.
Assuntos
Fibroblastos/virologia , Immunoblotting/métodos , Necroptose/genética , Proteínas Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Acrilamidas/farmacologia , Animais , Benzotiazóis/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/química , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Corantes Fluorescentes/química , Regulação da Expressão Gênica , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Necroptose/efeitos dos fármacos , Oligopeptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Multimerização Proteica/efeitos dos fármacos , Quinolinas/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Succinimidas/química , Sulfonamidas/farmacologia , Vaccinia virus/crescimento & desenvolvimentoRESUMO
Necroptosis mediated by Z-nucleic-acid-binding protein (ZBP)1 (also called DAI or DLM1) contributes to innate host defense against viruses by triggering cell death to eliminate infected cells. During infection, vaccinia virus (VACV) protein E3 prevents death signaling by competing for Z-form RNA through an N-terminal Zα domain. In the absence of this E3 domain, Z-form RNA accumulates during the early phase of VACV infection, triggering ZBP1 to recruit receptor interacting protein kinase (RIPK)3 and execute necroptosis. The C-terminal E3 double-strand RNA-binding domain must be retained to observe accumulation of Z-form RNA and induction of necroptosis. Substitutions of Zα from either ZBP1 or the RNA-editing enzyme double-stranded RNA adenosine deaminase (ADAR)1 yields fully functional E3 capable of suppressing virus-induced necroptosis. Overall, our evidence reveals the importance of Z-form RNA generated during VACV infection as a pathogen-associated molecular pattern (PAMP) unleashing ZBP1/RIPK3/MLKL-dependent necroptosis unless suppressed by viral E3.
Assuntos
Necroptose/fisiologia , Proteínas de Ligação a RNA/metabolismo , Vaccinia virus/fisiologia , Proteínas Virais/metabolismo , Adenosina Desaminase/metabolismo , Morte Celular , Humanos , Necroptose/genética , Proteínas Quinases/metabolismo , RNA de Cadeia Dupla , Proteínas de Ligação a RNA/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Vaccinia virus/genéticaRESUMO
Monkeypox virus (MPXV) infection fails to activate the host anti-viral protein, PKR, despite lacking a full-length homologue of the vaccinia virus (VACV) PKR inhibitor, E3. Since PKR can be activated by dsRNA produced during a viral infection, we have analyzed the accumulation of dsRNA in MPXV-infected cells. MPXV infection led to less accumulation of dsRNA than VACV infection. Because in VACV infections accumulation of abnormally low amounts of dsRNA is associated with mutations that lead to resistance to the anti-poxvirus drug isatin beta-thiosemicarbazone (IBT), we investigated the effects of treatment of MPXV-infected cells with IBT. MPXV infection was eight-fold more resistant to IBT than wild-type vaccinia virus (wtVACV). These results demonstrate that MPXV infection leads to the accumulation of less dsRNA than wtVACV, which in turn likely leads to a decreased capacity for activation of the dsRNA-dependent host enzyme, PKR.