RESUMEN
Latent membrane protein 1 (LMP1) is an EBV-encoded transforming protein that strongly mimics the B cell-activating properties of a normal cellular membrane protein, CD40. LMP1 and CD40 both associate with the cytoplasmic adapter proteins called TNFR-associated factors (TRAFs). TRAFs 1, 2, and 3 bind to a region of LMP1 that is essential for EBV to transform B lymphocytes, carboxyl-terminal activating region (CTAR) 1. However, studies of transiently overexpressed LMP1 molecules, primarily in epithelial cells, indicated that a second region, CTAR2, is largely responsible for LMP1-mediated activation of NF-kappaB and c-Jun N-terminal kinase. To better understand LMP1 signaling in B lymphocytes, we performed a structure-function analysis of the LMP1 C-terminal cytoplasmic domain stably expressed in B cell lines. Our results demonstrate that LMP1-stimulated Ig production, surface molecule up-regulation, and NF-kappaB and c-Jun N-terminal kinase activation require both CTAR1 and CTAR2, and that these two regions may interact to mediate LMP1 signaling. Furthermore, we find that the function of CTAR1, but not CTAR2, correlates with TRAF binding and present evidence that as yet unidentified cytoplasmic proteins may associate with LMP1 to mediate some of its signaling activities.
Asunto(s)
Linfocitos B/inmunología , Proteínas/fisiología , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/farmacología , Animales , Antígenos CD/metabolismo , Inmunoglobulina M/metabolismo , Interleucina-6/biosíntesis , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Mutación , FN-kappa B/metabolismo , Estructura Terciaria de Proteína , Transducción de Señal , Relación Estructura-Actividad , Factor 1 Asociado a Receptor de TNF , Factor 2 Asociado a Receptor de TNF , Factor 3 Asociado a Receptor de TNF , Células Tumorales Cultivadas , Regulación hacia Arriba , Proteínas de la Matriz Viral/genéticaRESUMEN
Members of the imidazoquinoline molecule family, including imiquimod and resiquimod (R-848), have potent antiviral and antitumor activities. Imiquimod cream (5%) (Aldara) is currently indicated for treatment of external genital and perianal warts. Previous characterization of these compounds has focused upon their ability to activate monocytes and dendritic cells, but recent studies have shown that resiquimod also stimulates B lymphocytes to proliferate and express an activated phenotype. This suggests that resiquimod could potentially serve as an effective vaccine adjuvant in stimulating a humoral immune response. This study shows that resiquimod mimics effects of the T-dependent CD40 signal in both mouse and human B cell lines. Resiquimod, like CD40, stimulates antibody secretion, cytokine production, protection from apoptosis, and CD80 upregulation. In addition, it shows synergy with signals delivered by the B cell antigen receptor and heightens CD40-mediated B cell activation, demonstrating that resiquimod can enhance antigen-specific responses in B lymphocytes.
Asunto(s)
Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Antígenos CD40/inmunología , Imidazoles/farmacología , Factores Inmunológicos/farmacología , Activación de Linfocitos/efectos de los fármacos , Adyuvantes Inmunológicos/farmacología , Animales , Anticuerpos Monoclonales , Apoptosis/efectos de los fármacos , Linfocitos B/citología , Linfocitos B/metabolismo , Antígeno B7-1/metabolismo , División Celular/efectos de los fármacos , Línea Celular , Islas de CpG/inmunología , Sinergismo Farmacológico , Humanos , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/inmunología , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Latent membrane protein 1 (LMP1) is required for EBV-induced immortalization of human B cells, and expression of the protein in the absence of other viral proteins leads to an activated phenotype in B cells. It has been well documented that LMP1 causes B cells to up-regulate adhesion molecules, such as LFA-1 and ICAM-1, and coactivation molecules, such as B7-1 and CD23, as well as to activate NF-kappaB. Ligation of the endogenous B cell CD40 molecule also induces these and other activated phenotypic changes. Here, we report that expression of LMP1 also activates B cells to secrete Ig and IL-6 and rescues them from B cell receptor-mediated growth arrest analogous to CD40 signaling. Furthermore, an HLA-A2LMP1 chimeric construct demonstrates that the oligomerization of the carboxyl-terminal 200 amino acids of LMP1 is sufficient for B cell signaling. Finally, we demonstrate that LMP1 and CD40 signaling pathways interact cooperatively in inducing B cell effector functions.
Asunto(s)
Linfocitos B/fisiología , Antígenos CD40/fisiología , Proteínas Oncogénicas Virales/fisiología , Proteínas de la Matriz Viral/fisiología , Animales , Molécula 1 de Adhesión Intercelular/biosíntesis , Interleucina-6/metabolismo , Antígeno-1 Asociado a Función de Linfocito/biosíntesis , Ratones , FN-kappa B/metabolismo , Receptores de Antígenos de Linfocitos B/fisiología , Células Tumorales CultivadasRESUMEN
All retroviruses (except the spumaretroviruses) contain a nucleocapsid (NC) protein that encodes one or two copies of the Zn2+-finger sequence -Cys-X2-Cys-X4-His-X4-Cys-. This region has been shown to be essential for recognition and packaging of the genomic RNA during virion particle assembly. Additionally, this region has been shown to be involved in early infection events in a wide spectrum of retroviruses, including mammalian type C [e.g., murine leukemia virus (MuLV)], human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus, and other retroviruses. Mutations in the two Zn2+-fingers of the NC protein of simian immunodeficiency virus strain Mne [SIV(Mne)] have been generated. The resulting virions contained the normal complement of processed viral proteins with densities indistinguishable from wild-type SIV(Mne). All of the mutants had electron micrograph morphologies similar to those of immature particles observed in wild-type preparations. RNA packaging was less affected by mutations in the NC protein of SIV(Mne) than has been observed for similar mutants in the MuLV and HIV-1 systems. Nevertheless, in vitro replication of SIV(Mne) NC mutants was impaired to levels comparable to those observed for MuLV and HIV-1 NC mutants; replication defective NC mutants are typically 10(5)- to 10(6)-fold less infectious than similar levels of wild-type virus. One mutant, DeltaCys33-Cys36, was also found to be noninfectious in vivo when mutant virus was administered intravenously to a pig-tailed macaque. NC mutations can therefore be used to generate replication defective virions for candidate vaccines in the SIV macaque model for primate lentiviral diseases.
Asunto(s)
Mutación , Proteínas de la Nucleocápside/genética , Virus de la Inmunodeficiencia de los Simios/genética , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Línea Celular Transformada , Cisteína , Humanos , Ratones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/análisis , Virus de la Inmunodeficiencia de los Simios/fisiología , Virus de la Inmunodeficiencia de los Simios/ultraestructura , Virión , Replicación ViralRESUMEN
The p6(Gag) protein of human immunodeficiency virus type 1 (HIV-1) is produced as the carboxyl-terminal sequence within the Gag polyprotein. The amino acid composition of this protein is high in hydrophilic and polar residues except for a patch of relatively hydrophobic amino acids found in the carboxyl-terminal 16 amino acids. Internal cleavage of p6(Gag) between Y36 and P37, apparently by the HIV-1 protease, removes this hydrophobic tail region from approximately 30% of the mature p6(Gag) proteins in HIV-1MN. To investigate the importance of this cleavage and the hydrophobic nature of this portion of p6(Gag), site-directed mutations were made at the minor protease cleavage site and within the hydrophobic tail. The results showed that all of the single-amino-acid-replacement mutants exhibited either reduced or undetectable cleavage at the site yet almost all were nearly as infectious as wild-type virus, demonstrating that processing at this site is not important for viral replication. However, one exception, Y36F, was 300-fold as infectious the wild type. In contrast to the single-substitution mutants, a virus with two substitutions in this region of p6(Gag), Y36S-L41P, could not infect susceptible cells. Protein analysis showed that while the processing of the Gag precursor was normal, the double mutant did not incorporate Env into virus particles. This mutant could be complemented with surface glycoproteins from vesicular stomatitis virus and murine leukemia virus, showing that the inability to incorporate Env was the lethal defect for the Y36S-L41P virus. However, this mutant was not rescued by an HIV-1 Env with a truncated gp41(TM) cytoplasmic domain, showing that it is phenotypically different from the previously described MA mutants that do not incorporate their full-length Env proteins. Cotransfection experiments with Y36S-L41P and wild-type proviral DNAs revealed that the mutant Gag dominantly blocked the incorporation of Env by wild-type Gag. These results show that the Y36S-L41P p6(Gag) mutation dramatically blocks the incorporation of HIV-1 Env, presumably acting late in assembly and early during budding.
Asunto(s)
Productos del Gen env/fisiología , Productos del Gen gag/fisiología , VIH-1/fisiología , Ensamble de Virus , Secuencia de Aminoácidos , Productos del Gen env/química , Productos del Gen gag/química , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Relación Estructura-Actividad , Replicación ViralRESUMEN
We have identified three types of cytoskeletal proteins inside human immunodeficiency virus type 1 (HIV-1) virions by analyzing subtilisin-digested particles. HIV-1 virions were digested with protease, and the treated particles were isolated by sucrose density centrifugation. This method removes both exterior viral proteins and proteins associated with microvesicles that contaminate virion preparations. Since the proteins inside the virion are protected from digestion by the viral lipid envelope, they can be isolated and analyzed after treatment. Experiments presented here demonstrated that this procedure removed more than 95% of the protein associated with microvesicles. Proteins in digested HIV-1(MN) particles from infected H9 and CEM(ss) cell lines were analyzed by high-pressure liquid chromatography, protein sequencing, and immunoblotting. The data revealed that three types of cytoskeletal proteins are present in virions at different concentrations relative to the molar level of Gag: actin (approximately 10 to 15%), ezrin and moesin (approximately 2%), and cofilin (approximately 2 to 10%). Our analysis of proteins within virus particles detected proteolytic fragments of alpha-smooth muscle actin and moesin that were cleaved at sites which might be recognized by HIV-1 protease. These cleavage products are not present in microvesicles from uninfected cells. Therefore, these processed proteins are most probably produced by HIV-1 protease digestion. The presence of these fragments, as well as the incorporation of a few specific cytoskeletal proteins into virions, suggests an active interaction between cytoskeletal and viral proteins.