RESUMEN
Downregulation of MHC class I molecules is a strategy developed by some viruses to escape cellular immune responses. Myxoma virus (MV), a poxvirus causing rabbit myxomatosis, encodes MV-LAP that is known to increase MHC-I endocytosis and degradation through a C(4)HC(3) motif critical for an E3 ubiquitin ligase activity. Here, we performed a functional mapping of MV-LAP and showed that not only the C(4)HC(3) motif is necessary for a marked downregulation of MHC-I but also a conserved region in the C-terminal part of the protein. We also showed that the putative transmembrane domains are responsible for a specific subcellular localization of the protein: they retain MV-LAP in the ER in transfected cells and in the endolysosomal compartments in infected cells. We observed that a specific MV infection context is necessary for a fully efficient downregulation of MHC-I. Our data suggest that the functionality of viral LAP factors, inherited by herpes- and poxviruses from mammalian cells, is more complex than anticipated.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Proteínas de la Membrana/fisiología , Myxoma virus/fisiología , Proteínas Virales/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Secuencia Conservada , ADN Viral/genética , Regulación hacia Abajo , Genes Virales , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Myxoma virus/genética , Mixomatosis Infecciosa/genética , Mixomatosis Infecciosa/inmunología , Mapeo Peptídico , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Transfección , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Shope fibroma virus (SFV) is one of the few poxviruses that induce cutaneous tumours, whereas myxoma virus, a closely related leporipoxvirus, does not. However, both have a virally encoded homologue of the epidermal growth factor (namely SFGF and MGF, respectively) that is considered to be crucial for poxvirus tumorigenesis. In this study, the role of viral growth factors in the context of infection with SFV, a tumorigenic leporipoxvirus, was investigated. An SFV mutant was engineered with the sfgf gene deleted and replaced with mgf. Macroscopic, histological and cytological examinations led to the conclusion that growth factors are indeed important for the development and maintenance of fibromas, provided that they are expressed in the proper viral context. However, they are not exchangeable and MGF cannot substitute for SFGF in the genesis of fibromas. It is likely that factors other than viral epidermal growth factor homologues influence the development of tumours.
Asunto(s)
Transformación Celular Neoplásica , Virus del Fibroma del Conejo/fisiología , Factor de Crecimiento Transformador alfa/fisiología , Animales , Sustancias de Crecimiento/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Infecciones por Poxviridae/patología , Conejos , Neoplasias Cutáneas/etiología , Infecciones Tumorales por Virus/patologíaRESUMEN
NF-kappaB is one of the most important elements that coordinate stress-induced, immune, and inflammatory responses. Myxoma virus, a member of the Poxviridae family responsible for rabbit myxomatosis, codes for several factors that help its survival in the host. In this study, we focused on the product of the M150R gene. We show that the protein has nine ankyrin repeats (ANKs), with the eighth having a close similarity with the nuclear localization signal-containing ANK of I-kappaBalpha, which regulates NF-kappaB activity by sequestering it in the cytosol. Because the viral protein is targeted to the nucleus, it was named MNF, for myxoma nuclear factor. This localization was lost when the eighth ANK was removed. In tumor necrosis factor alpha-treated cells, MNF and NF-kappaB colocalized as dotted spots in the nucleus. In vivo experiments with a knockout virus showed that MNF is a critical virulence factor, with its deletion generating an almost apathogenic virus. Detailed histological examinations revealed an increase in the inflammatory process in the absence of MNF, consistent with the interference of MNF with the NF-kappaB-induced proinflammatory pathway. Because MNF has homologs in other poxviruses, such as vaccinia, cowpox, and variola viruses, this protein is probably part of a key mechanism that contributes to the immunogenic and pathogenic properties of these viruses.
Asunto(s)
Núcleo Celular/metabolismo , Inflamación/metabolismo , Inflamación/virología , Myxoma virus/metabolismo , Myxoma virus/patogenicidad , FN-kappa B/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Genes Virales/genética , Inflamación/complicaciones , Inflamación/patología , Datos de Secuencia Molecular , Myxoma virus/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Infecciones por Poxviridae/complicaciones , Infecciones por Poxviridae/metabolismo , Infecciones por Poxviridae/patología , Infecciones por Poxviridae/virología , ARN Viral/biosíntesis , ARN Viral/genética , Conejos , Eliminación de Secuencia , Carga Viral , Proteínas Virales/química , Proteínas Virales/genética , Virulencia/genéticaRESUMEN
Down-modulation of major histocompatibility class I (MHC-I) molecules is a viral strategy for survival in the host. Myxoma virus, a member of the Poxviridae family responsible for rabbit myxomatosis, can down-modulate the expression of MHC-I molecules, but the viral factor(s) has not been described. We cloned and characterized a gene coding for an endoplasmic reticulum (ER)-resident protein containing an atypical zinc finger and two transmembrane domains, which we called myxoma virus leukemia-associated protein (MV-LAP). MV-LAP down-regulated surface MHC-I and Fas-CD95 molecules upon transfection; the mechanism probably involves an exacerbation of endocytosis and was lost when the ER retention signal was removed. In addition, the lytic activity of MHC-I-restricted antigen-specific cytolytic T lymphocytes (CTL) against myxoma virus-infected antigen-presenting target cells was significantly reduced, revealing a strong correlation between MHC-I down-regulation by MV-LAP and CTL killing in vitro. In vivo experiments with a knockout virus showed that MV-LAP is a virulence factor, potentially involved in the immunosuppression characteristic of myxomatosis. Data bank analysis revealed that MV-LAP has homologs in herpesviruses and other poxviruses. We propose the name "scrapins" to define a new group of ER-resident surface cellular receptor abductor proteins. The down-regulation of cell surface molecules by scrapins probably helps protect infected cells during viral infections.
Asunto(s)
Regulación hacia Abajo/fisiología , Antígenos de Histocompatibilidad Clase I/metabolismo , Proteínas de la Membrana/metabolismo , Myxoma virus/patogenicidad , Mixomatosis Infecciosa/inmunología , Proteínas Virales/metabolismo , Receptor fas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Myxoma virus/inmunología , Myxoma virus/fisiología , Mixomatosis Infecciosa/fisiopatología , Mixomatosis Infecciosa/virología , Conejos , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Virales/genética , Receptores Virales/metabolismo , Análisis de Secuencia de ADN , Linfocitos T Citotóxicos/inmunología , Proteínas Virales/genética , VirulenciaRESUMEN
Myxoma virus (MV), a member of the family Poxviridae, is the causative agent of myxomatosis, a fatal disease of the European rabbit. The MV genome is a linear, double-stranded DNA molecule that encodes several factors important for evasion of the host immune system. Sequencing the right-end region of the MV genome identified an 801 bp open reading frame (ORF) encoding a polypeptide that belongs to the serpin superfamily. To date, two MV-encoded serpins have been characterized: SERP-1 binds to several targets and is an anti-inflammatory molecule, whereas Serp2 is essential for virus virulence and has both anti-inflammatory and anti-apoptotic effects. Thus, Serp3 is the third MV-encoded serpin. DNA sequence analysis of Serp3 indicated a similarity to poxvirus late promoters, which was confirmed by mRNA expression analysis. Serp3 has an atypical serpin motif and has significant sequence deletions as compared to most cellular and viral serpins. However, molecular modelling studies suggested that Serp3 can retain the overall serpin fold. Insertional inactivation of the serp3 ORF led to a significant attenuation of virulence in vivo (as measured by the increase in survival of infected rabbits) and limited dissemination of the virus to secondary sites of infection. In rabbits infected with a Serp3 deletion mutant (MV-Serp3(-)), the main histopathological feature is the absence of secondary myxomas. Both wild-type MV and MV-Serp3(-) replicate at comparable levels in vivo. Serp3 may represent a significant virulence factor of MV and probably acts in synergy with other viral proteins.