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1.
Mol Cell Proteomics ; 13(8): 2132-46, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24969177

RESUMEN

Herpesviral capsids are assembled in the host cell nucleus before being translocated into the cytoplasm for further maturation. The crossing of the nuclear envelope represents a major event that requires the formation of the nuclear egress complex (NEC). Previous studies demonstrated that human cytomegalovirus (HCMV) proteins pUL50 and pUL53, as well as their homologs in all members of Herpesviridae, interact with each other at the nuclear envelope and form the heterodimeric core of the NEC. In order to characterize further the viral and cellular protein content of the multimeric NEC, the native complex was isolated from HCMV-infected human primary fibroblasts at various time points and analyzed using quantitative proteomics. Previously postulated components of the HCMV-specific NEC, as well as novel potential NEC-associated proteins such as emerin, were identified. In this regard, interaction and colocalization between emerin and pUL50 were confirmed by coimmunoprecipitation and confocal microscopy analyses, respectively. A functional validation of viral and cellular NEC constituents was achieved through siRNA-mediated knockdown experiments. The important role of emerin in NEC functionality was demonstrated by a reduction of viral replication when emerin expression was down-regulated. Moreover, under such conditions, reduced production of viral proteins and deregulation of viral late cytoplasmic maturation were observed. Combined, these data prove the functional importance of emerin as an NEC component, associated with pUL50, pUL53, pUL97, p32/gC1qR, and further regulatory proteins. Summarized, our findings provide the first proteomics-based characterization and functional validation of the HCMV-specific multimeric NEC.


Asunto(s)
Citomegalovirus/fisiología , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteómica/métodos , Proteínas Virales/metabolismo , Animales , Fibroblastos/virología , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Ratones
2.
J Exp Med ; 203(8): 1843-50, 2006 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-16831899

RESUMEN

Members of the alpha- and beta-subfamily of herpesviridae encode glycoproteins that specifically bind to the Fc part of immunoglobulin (Ig)G. Plasma membrane resident herpesviral Fc receptors seem to prevent virus-specific IgG from activating antibody-dependent effector functions. We show that the mouse cytomegalovirus (MCMV) molecule fcr-1 promotes a rapid down-regulation of NKG2D ligands murine UL16-binding protein like transcript (MULT)-1 and H60 from the cell surface. Deletion of the m138/fcr-1 gene from the MCMV genome attenuates viral replication to natural killer (NK) cell response in an NKG2D-dependent manner in vivo. A distinct N-terminal module within the fcr-1 ectodomain in conjunction with the fcr-1 transmembrane domain was required to dispose MULT-1 to degradation in lysosomes. In contrast, down-modulation of H60 required the complete fcr-1 ectodomain, implying independent modes of fcr-1 interaction with the NKG2D ligands. The results establish a novel viral strategy for down-modulating NK cell responses and highlight the impressive diversity of Fc receptor functions.


Asunto(s)
Proteínas Portadoras/metabolismo , Regulación hacia Abajo , Antígenos de Histocompatibilidad Clase I/metabolismo , Glicoproteínas de Membrana/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Muromegalovirus/metabolismo , Receptores Fc/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas Virales/metabolismo , Animales , Proteínas Portadoras/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Inmunoglobulinas/inmunología , Ligandos , Lisosomas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Antígenos de Histocompatibilidad Menor/inmunología , Muromegalovirus/fisiología , Células 3T3 NIH , Subfamilia K de Receptores Similares a Lectina de Células NK , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Receptores Inmunológicos/inmunología , Receptores de Células Asesinas Naturales , Replicación Viral/fisiología
3.
J Exp Med ; 201(2): 211-20, 2005 Jan 17.
Artículo en Inglés | MEDLINE | ID: mdl-15642742

RESUMEN

The NK cell-activating receptor NKG2D interacts with three different cellular ligands, all of which are regulated by mouse cytomegalovirus (MCMV). We set out to define the viral gene product regulating murine UL16-binding protein-like transcript (MULT)-1, a newly described NKG2D ligand. We show that MCMV infection strongly induces MULT-1 gene expression, but surface expression of this glycoprotein is nevertheless completely abolished by the virus. Screening a panel of MCMV deletion mutants defined the gene m145 as the viral regulator of MULT-1. The MCMV m145-encoded glycoprotein turned out to be necessary and sufficient to regulate MULT-1 by preventing plasma membrane residence of MULT-1. The importance of MULT-1 in NK cell regulation in vivo was confirmed by the attenuating effect of the m145 deletion that was lifted after NK cell depletion. Our findings underline the significance of escaping MULT-1/NKG2D signaling for viral survival and maintenance.


Asunto(s)
Proteínas Portadoras/metabolismo , Citomegalovirus/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/fisiología , Proteínas Virales/genética , Animales , Anticuerpos Monoclonales/inmunología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Citomegalovirus/metabolismo , Regulación hacia Abajo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Activación de Linfocitos/genética , Proteínas de la Membrana , Ratones , Mutación , Subfamilia K de Receptores Similares a Lectina de Células NK , Receptores Inmunológicos/metabolismo , Receptores de Células Asesinas Naturales , Análisis de Secuencia de Proteína , Proteínas Virales/metabolismo
4.
J Virol ; 83(16): 8198-207, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19494006

RESUMEN

The NKG2D receptor is one of the most potent activating natural killer cell receptors involved in antiviral responses. The mouse NKG2D ligands MULT-1, RAE-1, and H60 are regulated by murine cytomegalovirus (MCMV) proteins m145, m152, and m155, respectively. In addition, the m138 protein interferes with the expression of both MULT-1 and H60. We show here that one of five RAE-1 isoforms, RAE-1delta, is resistant to downregulation by MCMV and that this escape has functional importance in vivo. Although m152 retained newly synthesized RAE-1delta and RAE-1gamma in the endoplasmic reticulum, no viral regulator was able to affect the mature RAE-1delta form which remains expressed on the surfaces of infected cells. This differential susceptibility to downregulation by MCMV is not a consequence of faster maturation of RAE-1delta compared to RAE-1gamma but rather an intrinsic property of the mature surface-resident protein. This difference can be attributed to the absence of a PLWY motif from RAE-1delta. Altogether, these findings provide evidence for a novel mechanism of host escape from viral immunoevasion of NKG2D-dependent control.


Asunto(s)
Regulación hacia Abajo , Infecciones por Herpesviridae/genética , Proteínas de la Membrana/genética , Muromegalovirus/inmunología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Expresión Génica , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Células Asesinas Naturales/inmunología , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Muromegalovirus/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Homología de Secuencia de Aminoácido , Proteínas Virales/genética , Proteínas Virales/inmunología
5.
J Immunother Cancer ; 8(1)2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32503945

RESUMEN

BACKGROUND: The use of checkpoint inhibitors has revolutionized cancer therapy. Unfortunately, these therapies often cause immune-related adverse effects, largely due to a lack of tumor specificity. METHODS: We stained human natural killer cells using fusion proteins composed of the extracellular portion of various tumor markers fused to the Fc portion of human IgG1, and identified Nectin4 as a novel TIGIT ligand. Next, we generated a novel Nectin4 blocking antibody and demonstrated its efficacy as a checkpoint inhibitor in killing assays and in vivo. RESULTS: We identify Nectin4 to be a novel ligand of TIGIT. We showed that, as opposed to all other known TIGIT ligands, which bind also additional receptors, Nectin4 interacts only with TIGIT. We show that the TIGIT-Nectin4 interaction inhibits natural killer cell activity, a critical part of the innate immune response. Finally, we developed blocking Nectin4 antibodies and demonstrated that they enhance tumor killing in vitro and in vivo. CONCLUSION: We discovered that Nectin4 is a novel ligand for TIGIT and demonstrated that specific antibodies against it enhance tumor cell killing in vitro and in vivo. Since Nectin4 is expressed almost exclusively on tumor cells, our Nectin4-blocking antibodies represent a combination of cancer specificity and immune checkpoint activity, which may prove more effective and safe for cancer immunotherapy.


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Inmunoterapia/métodos , Receptores Inmunológicos/metabolismo , Animales , Femenino , Humanos , Ligandos , Ratones
6.
J Phys Chem B ; 112(16): 4876-83, 2008 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-18386861

RESUMEN

To understand the origin of high enantioselectivity of Burkholderia cepacia lipase (BCL) toward secondary alcohol, (R,S)-1-phenoxy-2-hydroxybutane (1), and its ester (E1), we determined the crystal structure of BCL complexed with phosphonate analogue of S-E1 and accomplished a series of MM, MC, and QM/MM studies. We have found that the inhibitor in the S configuration binds into the BCL active site in the same manner as the R isomer, with an important difference: while in case of the R-inhibitor the H-bond between its alcohol oxygen and catalytic His286 can be formed, in the case of the S-inhibitor this is not possible. Molecular modeling for both E1 enantiomers revealed orientations in which all hydrogen bonds characteristic of productive binding are formed. To check the possibility of chemical transformation, four different orientations of the substrate (two for each enantiomer) were chosen, and a series of ab initio QM/MM calculations were accomplished. Starting from the covalent complex, we modeled the ester (E1) hydrolysis and the alcohol (1) esterification. The calculations revealed that ester release is possible starting with all four covalent complexes. Alcohol release from the BCL-E1 complex in which the S-substrate is bound in the same manner as the S-inhibitor in the crystal structure however is not possible. These results show that the crystallographically determined binding modes should be taken with caution when modeling chemical reactions.


Asunto(s)
Alcoholes/química , Alcoholes/metabolismo , Burkholderia cepacia/enzimología , Lipasa/química , Lipasa/metabolismo , Sitios de Unión , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Catálisis , Cristalografía por Rayos X , Esterificación , Enlace de Hidrógeno , Modelos Moleculares , Estructura Molecular , Fosfatos/química , Fosfatos/metabolismo , Estructura Terciaria de Proteína , Difracción de Rayos X
7.
Viruses ; 7(8): 4582-601, 2015 Aug 11.
Artículo en Inglés | MEDLINE | ID: mdl-26270673

RESUMEN

Replication of human cytomegalovirus (HCMV) is characterized by a tight virus-host cell interaction. Cyclin-dependent protein kinases (CDKs) are functionally integrated into viral gene expression and protein modification. The HCMV-encoded protein kinase pUL97 acts as a CDK ortholog showing structural and functional similarities. Recently, we reported an interaction between pUL97 kinase with a subset of host cyclins, in particular with cyclin T1. Here, we describe an interaction of pUL97 at an even higher affinity with cyclin B1. As a striking feature, the interaction between pUL97 and cyclin B1 proved to be strictly dependent on pUL97 activity, as interaction could be abrogated by treatment with pUL97 inhibitors or by inserting mutations into the conserved kinase domain or the nonconserved C-terminus of pUL97, both producing loss of activity. Thus, we postulate that the mechanism of pUL97-cyclin B1 interaction is determined by an active pUL97 kinase domain.


Asunto(s)
Ciclina B1/metabolismo , Citomegalovirus/fisiología , Interacciones Huésped-Patógeno , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Mapeo de Interacción de Proteínas , Replicación Viral , Sitios de Unión , Dominio Catalítico , Células Cultivadas , Análisis Mutacional de ADN , Humanos , Unión Proteica
8.
Med Microbiol Immunol ; 197(2): 159-66, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18259774

RESUMEN

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes morbidity risk in immunologically suppressed and immunodeficient patients including congenital infections. Approaches to curb the consequences of HCMV infections are restricted by a lack of complete understanding of viral pathogenesis. The infection of mice with murine cytomegalovirus (MCMV) as a model of HCMV infection has been particularly useful in elucidating the role of innate and adaptive immune response mechanisms. A large number of cytomegalovirus genes modulate the innate and the adaptive host immune response. The products of several MCMV genes are involved in subverting the natural killer (NK) cell response by down-modulating cellular ligands for the NKG2D receptor expressed on NK cells and CD8(+) T cells. Mutant viruses lacking these immunoevasion genes are attenuated with respect to virus growth in vivo. Given the importance of the NKG2D receptor in controlling both NK- and T cell-mediated immunity, it is of tremendous importance to understand the molecular mechanisms and consequences of viral regulation of the NKG2D ligands.


Asunto(s)
Infecciones por Citomegalovirus/inmunología , Muromegalovirus/inmunología , Receptores Inmunológicos/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Células Asesinas Naturales/inmunología , Ligandos , Ratones , Subfamilia K de Receptores Similares a Lectina de Células NK , Receptores de Células Asesinas Naturales
9.
J Virol ; 79(5): 2920-30, 2005 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15709011

RESUMEN

Both human and mouse cytomegaloviruses (CMVs) encode proteins that inhibit the activation of NK cells by down-regulating cellular ligands for the activating NK cell receptor NKG2D. Up to now, three ligands for the NKG2D receptor, named RAE-1, H60, and MULT-1, have been identified in mice. The resistance of mouse strains to murine CMV (MCMV) infection is determined by their ability to generate an effective NK cell response. The MCMV gene m152, a member of the m145 gene family, down-regulates the expression of RAE-1 in order to avoid NK cell control in vivo. Here we report that the m155 gene, another member of the m145 gene family, encodes a protein that interferes with the expression of H60 on the surfaces of infected cells. Deletion of the m155 gene leads to an only partial restoration of H60 expression on the cell surface, suggesting the involvement of another, so far unknown, viral inhibitor. In spite of this, an m155 deletion mutant virus shows NK cell-dependent attenuation in vivo. The acquisition of endo-beta-N-acetylglucosaminidase H resistance and the preserved half-life of H60 in MCMV-infected cells indicate that the m155-mediated effect must take place in a compartment after H60 exits from the ERGIC-cis-Golgi compartment.


Asunto(s)
Glicoproteínas de Membrana/inmunología , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Muromegalovirus/inmunología , Muromegalovirus/patogenicidad , Receptores Inmunológicos/metabolismo , Proteínas Virales/inmunología , Animales , Secuencia de Bases , ADN Viral/genética , Regulación hacia Abajo , Eliminación de Gen , Genes Virales , Humanos , Células Asesinas Naturales/inmunología , Ligandos , Glicoproteínas de Membrana/genética , Ratones , Muromegalovirus/genética , Mutagénesis Sitio-Dirigida , Mutación , Subfamilia K de Receptores Similares a Lectina de Células NK , Receptores de Células Asesinas Naturales , Proteínas Virales/genética
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