RESUMEN
Monkeypox virus (MPXV) causes a smallpox-like disease in humans. Clinical and epidemiological studies provide evidence of pathogenicity differences between two geographically distinct monkeypox virus clades: the West African and Congo Basin. Genomic analysis of strains from both clades identified a â¼10 kbp deletion in the less virulent West African isolates sequenced to date. One absent open reading frame encodes the monkeypox virus homologue of the complement control protein (CCP). This modulatory protein prevents the initiation of both the classical and alternative pathways of complement activation. In monkeypox virus, CCP, also known as MOPICE, is a â¼24 kDa secretory protein with sequence homology to this superfamily of proteins. Here we investigate CCP expression and its role in monkeypox virulence and pathogenesis. CCP was incorporated into the West African strain and removed from the Congo Basin strain by homologous recombination. CCP expression phenotypes were confirmed for both wild type and recombinant monkeypox viruses and CCP activity was confirmed using a C4b binding assay. To characterize the disease, prairie dogs were intranasally infected and disease progression was monitored for 30 days. Removal of CCP from the Congo Basin strain reduced monkeypox disease morbidity and mortality, but did not significantly decrease viral load. The inclusion of CCP in the West African strain produced changes in disease manifestation, but had no apparent effect on disease-associated mortality. This study identifies CCP as an important immuno-modulatory protein in monkeypox pathogenesis but not solely responsible for the increased virulence seen within the Congo Basin clade of monkeypox virus.
Asunto(s)
Activación de Complemento , Monkeypox virus/inmunología , Monkeypox virus/patogenicidad , Mpox/inmunología , Mpox/virología , Proteínas Virales/inmunología , Animales , Secuencia de Bases , Proteína de Unión al Complemento C4b/inmunología , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Masculino , Datos de Secuencia Molecular , Monkeypox virus/genética , Sistemas de Lectura Abierta/genética , Recombinación Genética , Sciuridae , Carga Viral , Proteínas Virales/genéticaRESUMEN
Type 1 interferons (T1-IFNs) play a major role in antiviral defense, but when or how they protect during infections that spread through the lympho-hematogenous route is not known. Orthopoxviruses, including those that produce smallpox and mousepox, spread lympho-hematogenously. They also encode a decoy receptor for T1-IFN, the T1-IFN binding protein (T1-IFNbp), which is essential for virulence. We demonstrate that during mousepox, T1-IFNs protect the liver locally rather than systemically, and that the T1-IFNbp attaches to uninfected cells surrounding infected foci in the liver and the spleen to impair their ability to receive T1-IFN signaling, thus facilitating virus spread. Remarkably, this process can be reversed and mousepox cured late in infection by treating with antibodies that block the biological function of the T1-IFNbp. Thus, our findings provide insights on how T1-IFNs function and are evaded during a viral infection in vivo, and unveil a novel mechanism for antibody-mediated antiviral therapy.
Asunto(s)
Anticuerpos Antivirales/farmacología , Virus de la Ectromelia/metabolismo , Ectromelia Infecciosa/inmunología , Receptor de Interferón alfa y beta/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Factores de Virulencia/antagonistas & inhibidores , Animales , Anticuerpos Antivirales/inmunología , Línea Celular , Cricetinae , Virus de la Ectromelia/inmunología , Virus de la Ectromelia/patogenicidad , Ectromelia Infecciosa/tratamiento farmacológico , Ectromelia Infecciosa/metabolismo , Femenino , Hígado/inmunología , Hígado/metabolismo , Hígado/virología , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Receptor de Interferón alfa y beta/inmunología , Receptor de Interferón alfa y beta/metabolismo , Bazo/inmunología , Bazo/metabolismo , Bazo/virología , Virus de la Viruela/inmunología , Virus de la Viruela/metabolismo , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismo , Acoplamiento Viral/efectos de los fármacosRESUMEN
The vaccinia virus (VACV) complement control protein (VCP) is an immunomodulatory protein that is both secreted from and expressed on the surface of infected cells. Surface expression of VCP occurs though an interaction with the viral transmembrane protein A56 and is dependent on a free N-terminal cysteine of VCP. Although A56 and VCP have been shown to interact in infected cells, the mechanism remains unclear. To investigate if A56 is sufficient for surface expression, we transiently expressed VCP and A56 in eukaryotic cell lines and found that they interact on the cell surface in the absence of other viral proteins. Since A56 contains three extracellular cysteines, we hypothesized that one of the cysteines may be unpaired and could therefore form a disulfide bridge with VCP. To test this, we generated a series of A56 mutants in which each cysteine was mutated to a serine, and we found that mutation of cysteine 162 abrogated VCP cell surface expression. We also tested the ability of other poxvirus complement control proteins to bind to VACV A56. While the smallpox homolog of VCP is able to bind VACV A56, the ectromelia virus (ECTV) VCP homolog is only able to bind the ECTV homolog of A56, indicating that these proteins may have coevolved. Surface expression of poxvirus complement control proteins may have important implications in viral pathogenesis, as a virus that does not express cell surface VCP is attenuated in vivo. This suggests that surface expression of VCP may contribute to poxvirus pathogenesis.
Asunto(s)
Membrana Celular/virología , Poxviridae/patogenicidad , Proteínas Virales/metabolismo , Línea Celular , Cisteína/genética , Cisteína/metabolismo , Disulfuros , Humanos , Mutagénesis Sitio-DirigidaRESUMEN
Multiple monkeypox virus (MPXV) animal models have been discussed in previous studies, but no small animal models, nor most non-human primate models, demonstrated the protracted asymptomatic incubation phase seen in systemic human orthopoxvirus illness. Herein, we characterize a black-tailed prairie dog (PD) (Cynomys ludovicianus) model of infection, via intranasal and intradermal exposures, with the two MPXV clades. Daily observations of the animals were made (food consumption, general symptoms, disease presentation), while weights and virus evaluations (ocular, nasal, oropharyngeal, faeces, blood) were obtained/made every third day. Generalized rash became apparent 9-12 days post-infection for all animals. Individual animals demonstrated a range of symptoms consistent with human monkeypox disease. Measurable viraemias and excretas were similar for both clade-representative strains and persisted until at least day 21. Greater morbidity was observed in Congo Basin strain-challenged animals and mortality was observed only in the Congo Basin strain-challenged animals. The PD model is valuable for the study of strain-dependent differences in MPXV. Additionally, the model closely mimics human systemic orthopoxvirus disease and may serve as a valuable non-human surrogate for investigations of antivirals and next generation orthopoxvirus vaccines.