RESUMEN
The complex interplay between caspase-8 and receptor-interacting protein (RIP) kinase RIP 3 (RIPK3) driving extrinsic apoptosis and necroptosis is not fully understood. Murine cytomegalovirus triggers both apoptosis and necroptosis in infected cells; however, encoded inhibitors of caspase-8 activity (M36) and RIP3 signaling (M45) suppress these antiviral responses. Here, we report that this virus activates caspase-8 in macrophages to trigger apoptosis that gives rise to secondary necroptosis. Infection with double-mutant ΔM36/M45mutRHIM virus reveals a signaling pattern in which caspase-8 activates caspase-3 to drive apoptosis with subsequent RIP3-dependent activation of mixed lineage kinase domain-like (MLKL) leading to necroptosis. This combined cell death signaling is highly inflammatory, greater than either apoptosis induced by ΔM36 or necroptosis induced by M45mutRHIM virus. IL-6 production by macrophages is dramatically increased during double-mutant virus infection and correlates with faster antiviral responses in the host. Collaboratively, M36 and M45 target caspase-8 and RIP3 pathways together to suppress this proinflammatory cell death. This study reveals the effect of antiviral programmed cell death pathways on inflammation, shows that caspase-8 activation may go hand-in-hand with necroptosis in macrophages, and revises current understanding of independent and collaborative functions of M36 and M45 in blocking apoptotic and necroptotic cell death responses.
Asunto(s)
Apoptosis , Infecciones por Herpesviridae/veterinaria , Muromegalovirus/metabolismo , Ribonucleótido Reductasas/metabolismo , Enfermedades de los Roedores/fisiopatología , Proteínas Virales/metabolismo , Animales , Caspasa 8/genética , Caspasa 8/inmunología , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/fisiopatología , Infecciones por Herpesviridae/virología , Interacciones Huésped-Patógeno , Ratones , Muromegalovirus/clasificación , Muromegalovirus/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Ribonucleótido Reductasas/genética , Enfermedades de los Roedores/genética , Enfermedades de los Roedores/inmunología , Enfermedades de los Roedores/virología , Proteínas Virales/genéticaRESUMEN
Cytomegaloviruses utilize overlapping and embedded reading frames as a way to efficiently package and express all genes necessary to carry out a complex lifecycle. Murine cytomegalovirus encodes a mitochondrial-localized inhibitor of Bak oligomerization (vIBO) from m41.1, a reading frame that is embedded within the m41 gene. The m41.1-encoded mitochondrial protein and m41-encoded Golgi-localized protein have both been implicated in cell death suppression; however, their contribution to viral infection within the host has not been investigated. Here, we report that mitochondrial-localized m41.1 (vIBO) is required for optimal viral replication in macrophages and has a modest impact on dissemination in infected mice. In contrast, Golgi-localized m41 protein is dispensable during acute infection and dissemination as well as for latency. All together, these data indicate that the primary evolutionary focus of this locus is to maintain mitochondrial function through inhibition of Bak-mediated death pathways in support of viral pathogenesis.
Asunto(s)
Regulación Viral de la Expresión Génica , Infecciones por Herpesviridae/patología , Muromegalovirus/genética , Muromegalovirus/fisiología , Proteínas Virales/metabolismo , Factores de Virulencia/metabolismo , Secuencia de Aminoácidos , Estructuras Animales/virología , Animales , Secuencia de Bases , Modelos Animales de Enfermedad , Células Epiteliales/virología , Fibroblastos/virología , Eliminación de Gen , Infecciones por Herpesviridae/virología , Macrófagos/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Muromegalovirus/patogenicidad , Carga Viral , Proteínas Virales/genética , Factores de Virulencia/genéticaRESUMEN
Human herpesvirus 8 (HHV-8), or Kaposi's sarcoma (KS)-associated herpesvirus, has been linked to all forms of KS. The results of most current serological assays for the detection of HHV-8-specific antibodies have low levels of concordance among themselves. To establish a sensitive and specific testing strategy that can be used to screen for HHV-8-specific antibodies, three HHV-8 proteins, ORF65, ORF73, and K8.1A, were expressed by using baculoviral vectors in insect cells and incorporated into a monoclonal antibody-enhanced immunofluorescence assay (mIFA) termed the Sf9 three-antigen mIFA. The results obtained by this mIFA were compared to those obtained by a standard mIFA with an HHV-8-infected B-cell line (BC3 mIFA). Test sera were obtained from patients diagnosed with KS, human immunodeficiency virus type 1-infected patients at high risk for HHV-8 infection, and healthy controls from a local blood bank. The combined use of both assays had a sensitivity of 94% and a specificity of 96%. The performance of these two assays when they were used together indicates that they may be useful for the reliable detection of HHV-8-specific immunoglobulin G antibodies in a population.