RESUMEN
Although macrophages are armed with potent antibacterial functions, Mycobacterium tuberculosis (Mtb) replicates inside these innate immune cells. Determinants of macrophage intrinsic bacterial control, and the Mtb strategies to overcome them, are poorly understood. To further study these processes, we used an affinity tag purification mass spectrometry (AP-MS) approach to identify 187 Mtb-human protein-protein interactions (PPIs) involving 34 secreted Mtb proteins. This interaction map revealed two factors involved in Mtb pathogenesis-the secreted Mtb protein, LpqN, and its binding partner, the human ubiquitin ligase CBL. We discovered that an lpqN Mtb mutant is attenuated in macrophages, but growth is restored when CBL is removed. Conversely, Cbl-/- macrophages are resistant to viral infection, indicating that CBL regulates cell-intrinsic polarization between antibacterial and antiviral immunity. Collectively, these findings illustrate the utility of this Mtb-human PPI map for developing a deeper understanding of the intricate interactions between Mtb and its host.
Asunto(s)
Proteínas Bacterianas/genética , VIH/genética , Interacciones Huésped-Patógeno , Mycobacterium tuberculosis/genética , Proteínas Proto-Oncogénicas c-cbl/genética , Factores de Virulencia/genética , Animales , Proteínas Bacterianas/inmunología , Línea Celular Tumoral , Chlamydia trachomatis/genética , Chlamydia trachomatis/inmunología , Regulación de la Expresión Génica , VIH/inmunología , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/inmunología , Humanos , Linfocitos/microbiología , Linfocitos/virología , Macrófagos/microbiología , Macrófagos/virología , Ratones , Mycobacterium tuberculosis/inmunología , Cultivo Primario de Células , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-cbl/deficiencia , Proteínas Proto-Oncogénicas c-cbl/inmunología , Células RAW 264.7 , Transducción de Señal , Factores de Virulencia/inmunologíaRESUMEN
The COVID-19 pandemic has revealed how an emerging pathogen can cause a sudden and dramatic increase in demand for viral testing. Testing pooled samples could meet this demand; however, the sensitivity of reverse transcription quantitative polymerase chain reaction (RT-qPCR), the gold standard, significantly decreases with an increasing number of samples pooled. Here, we introduce detection of intact virus by exogenous-nucleotide reaction (DIVER), a method that quantifies intact virus and is robust to sample dilution. As demonstrated using two models of severe acute respiratory syndrome coronavirus 2, DIVER first tags membraned particles with exogenous oligonucleotides, then captures the tagged particles on beads functionalized with a virus-specific capture agent (in this instance, angiotensin-converting enzyme 2), and finally quantifies the oligonucleotide tags using qPCR. Using spike-presenting liposomes and spike-pseudotyped lentivirus, we show that DIVER can detect 1 × 105 liposomes and 100 plaque-forming units of lentivirus and can successfully identify positive samples in pooling experiments. Overall, DIVER is well positioned for efficient sample pooling and clinical validation.
Asunto(s)
COVID-19 , Pandemias , COVID-19/diagnóstico , Humanos , Liposomas , Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2/genética , Sensibilidad y EspecificidadRESUMEN
Natural killer (NK) cells recognize and kill cancer cells and infected cells by engaging cell surface ligands that are induced preferentially or exclusively on these cells. These ligands are recognized by activating receptors on NK cells, such as NKG2D. In addition to activation by cell surface ligands, the acquisition of optimal effector activity by NK cells is driven in vivo by cytokines and other signals. This review addresses a developing theme in NK cell biology: that NK-activating ligands on cells, and the provision of cytokines and other signals that drive high effector function in NK cells, are driven by abnormalities that arise from transformation or the infected state. The pathways include genomic damage, which causes self DNA to be exposed in the cytosol of affected cells, where it activates the DNA sensor cGAS. The resulting signaling induces NKG2D ligands and also mobilizes NK cell activation. Other key pathways that regulate NKG2D ligands include PI-3 kinase activation, histone acetylation, and the integrated stress response. This review summarizes the roles of these pathways and their relevance in both viral infections and cancer.
Asunto(s)
Infecciones/inmunología , Células Asesinas Naturales/inmunología , Neoplasias/inmunología , Animales , Citotoxicidad Inmunológica , Daño del ADN , Humanos , Vigilancia Inmunológica , Activación de Linfocitos , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Nucleotidiltransferasas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de SeñalRESUMEN
Host receptor usage by Kaposi's sarcoma-associated herpesvirus (KSHV) has been best studied using primary microvascular endothelial and fibroblast cells, although the virus infects a wide variety of cell types in culture and in natural infections. In these two infection models, KSHV adheres to the cell though heparan sulfate (HS) binding and then interacts with a complex of EphA2, xCT, and integrins α3ß1, αVß3, and αVß5 to catalyze viral entry. We dissected this receptor complex at the genetic level with CRISPR-Cas9 to precisely determine receptor usage in two epithelial cell lines. Surprisingly, we discovered an infection mechanism that requires HS and EphA2 but is independent of αV- and ß1-family integrin expression. Furthermore, infection appears to be independent of the EphA2 intracellular domain. We also demonstrated that while two other endogenous Eph receptors were dispensable for KSHV infection, transduced EphA4 and EphA5 significantly enhanced infection of cells lacking EphA2.IMPORTANCE Our data reveal an integrin-independent route of KSHV infection and suggest that multiple Eph receptors besides EphA2 can promote and regulate infection. Since integrins and Eph receptors are large protein families with diverse expression patterns across cells and tissues, we propose that KSHV may engage with several proteins from both families in different combinations to negotiate successful entry into diverse cell types.
Asunto(s)
Infecciones por Herpesviridae/virología , Herpesvirus Humano 8 , Integrina alfa3beta1/genética , Integrina alfaVbeta3/genética , Receptores de Vitronectina/genética , Internalización del Virus , Sistemas CRISPR-Cas , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Células Endoteliales/virología , Efrina-A2/genética , Fibroblastos/virología , Edición Génica , Regulación Viral de la Expresión Génica , Células HeLa , Herpesvirus Humano 8/fisiología , Humanos , Integrina alfa3beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Pinocitosis , Receptor EphA2 , Receptores de Vitronectina/metabolismo , Transducción de Señal/genéticaRESUMEN
To efficiently establish a persistent infection, Kaposi's sarcoma-associated herpesvirus (KSHV; also known as HHV8) dedicates a large amount of its coding potential to produce proteins that antagonize the immune system of its host. These viral immunomodulators interfere with both the innate and adaptive immune responses and most of them are homologous to cellular proteins, suggesting that they have been pirated from the host during viral evolution. In this Review, I present recent advances in the understanding of immune evasion by KSHV, with a particular focus on the virally encoded modulators of immune responses that are unique to this virus.
Asunto(s)
Herpesvirus Humano 8/inmunología , Modelos Inmunológicos , Proteínas Virales/inmunología , Animales , ADN Viral/genética , ADN Viral/inmunología , Herpesvirus Humano 8/genética , Humanos , Sarcoma de Kaposi/inmunología , Sarcoma de Kaposi/virología , Proteínas Virales/genéticaRESUMEN
Lytic gammaherpesvirus infection restricts host gene expression by promoting widespread degradation of cytoplasmic mRNA through the activity of the viral endonuclease SOX. Though generally assumed to be selective for cellular transcripts, the extent to which SOX impacts viral mRNA stability has remained unknown. We addressed this issue using the model murine gammaherpesvirus MHV68 and, unexpectedly, found that all stages of viral gene expression are controlled through mRNA degradation. Using both comprehensive RNA expression profiling and half-life studies we reveal that the levels of the majority of viral mRNAs but not noncoding RNAs are tempered by MHV68 SOX (muSOX) activity. The targeting of viral mRNA by muSOX is functionally significant, as it impacts intracellular viral protein abundance and progeny virion composition. In the absence of muSOX-imposed gene expression control the viral particles display increased cell surface binding and entry as well as enhanced immediate early gene expression. These phenotypes culminate in a viral replication defect in multiple cell types as well as in vivo, highlighting the importance of maintaining the appropriate balance of viral RNA during gammaherpesviral infection. This is the first example of a virus that fails to broadly discriminate between cellular and viral transcripts during host shutoff and instead uses the targeting of viral messages to fine-tune overall gene expression.
Asunto(s)
Regulación Viral de la Expresión Génica/fisiología , Estabilidad del ARN , ARN Mensajero/metabolismo , Rhadinovirus/fisiología , Virión/metabolismo , Replicación Viral/fisiología , Animales , Chlorocebus aethiops , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/metabolismo , Ratones , Células 3T3 NIH , ARN Mensajero/genética , Células Vero , Virión/genéticaRESUMEN
Activation-induced cytidine deaminase (AID) is specifically induced in germinal center B cells to carry out somatic hypermutation and class-switch recombination, two processes responsible for antibody diversification. Because of its mutagenic potential, AID expression and activity are tightly regulated to minimize unwanted DNA damage. Surprisingly, AID expression has been observed ectopically during pathogenic infections. However, the function of AID outside of the germinal centers remains largely uncharacterized. In this study, we demonstrate that infection of human primary naïve B cells with Kaposi's sarcoma-associated herpesvirus (KSHV) rapidly induces AID expression in a cell intrinsic manner. We find that infected cells are marked for elimination by Natural Killer cells through upregulation of NKG2D ligands via the DNA damage pathway, a pathway triggered by AID. Moreover, without having a measurable effect on KSHV latency, AID impinges directly on the viral fitness by inhibiting lytic reactivation and reducing infectivity of KSHV virions. Importantly, we uncover two KSHV-encoded microRNAs that directly regulate AID abundance, further reinforcing the role for AID in the antiviral response. Together our findings reveal additional functions for AID in innate immune defense against KSHV with implications for a broader involvement in innate immunity to other pathogens.
Asunto(s)
Linfocitos B/inmunología , Citidina Desaminasa/inmunología , Regulación Enzimológica de la Expresión Génica/inmunología , Herpesvirus Humano 8/fisiología , Inmunidad Innata/fisiología , Latencia del Virus/inmunología , Linfocitos B/enzimología , Células Cultivadas , Citidina Desaminasa/biosíntesis , Femenino , Centro Germinal/enzimología , Centro Germinal/inmunología , Humanos , Células Asesinas Naturales/enzimología , Células Asesinas Naturales/inmunología , Masculino , Subfamilia K de Receptores Similares a Lectina de Células NK/biosíntesis , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunologíaRESUMEN
The fate of developing T cells is determined by the strength of T cell receptor (TCR) signal they receive in the thymus. This process is finely regulated through the tuning of positive and negative regulators in thymocytes. The Family with sequence similarity 49 member B (Fam49b) protein is a newly discovered negative regulator of TCR signaling that has been shown to suppress Rac-1 activity in vitro in cultured T cell lines. However, the contribution of Fam49b to the thymic development of T cells is unknown. To investigate this important issue, we generated a novel mouse line deficient in Fam49b (Fam49b-KO). We observed that Fam49b-KO double positive (DP) thymocytes underwent excessive negative selection, whereas the positive selection stage was unaffected. Fam49b deficiency impaired the survival of single positive thymocytes and peripheral T cells. This altered development process resulted in significant reductions in CD4 and CD8 single-positive thymocytes as well as peripheral T cells. Interestingly, a large proportion of the TCRγδ+ and CD8αα+TCRαß+ gut intraepithelial T lymphocytes were absent in Fam49b-KO mice. Our results demonstrate that Fam49b dampens thymocytes TCR signaling in order to escape negative selection during development, uncovering the function of Fam49b as a critical regulator of the selection process to ensure normal thymocyte development and peripheral T cells survival.
Asunto(s)
Receptores de Antígenos de Linfocitos T , Transducción de Señal , Timocitos , Animales , Ratones , Supervivencia Celular , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Timocitos/metabolismo , Timocitos/citologíaRESUMEN
Natural killer (NK) cells are lymphocytes that play a major role in the elimination of virally-infected cells and tumor cells. NK cells recognize and target abnormal cells through activation of stimulatory receptors such as NKG2D. NKG2D ligands are self-proteins, which are absent or expressed at low levels on healthy cells but are induced upon cellular stress, transformation, or viral infection. The exact molecular mechanisms driving expression of these ligands remain poorly understood. Here we show that murine cytomegalovirus (MCMV) infection activates the phosphatidylinositol-3-kinase (PI3K) pathway and that this activation is required for the induction of the RAE-1 family of mouse NKG2D ligands. Among the multiple PI3K catalytic subunits, inhibition of the p110α catalytic subunit blocks this induction. Similarly, inhibition of p110α PI3K reduces cell surface expression of RAE-1 on transformed cells. Many viruses manipulate the PI3K pathway, and tumors frequently mutate the p110α oncogene. Thus, our findings suggest that dysregulation of the PI3K pathway is an important signal to induce expression of RAE-1, and this may represent a commonality among various types of cellular stresses that result in the induction of NKG2D ligands.
Asunto(s)
Infecciones por Citomegalovirus/fisiopatología , Células Asesinas Naturales/metabolismo , Subfamilia K de Receptores Similares a Lectina de Células NK/biosíntesis , Proteínas Asociadas a Matriz Nuclear/biosíntesis , Proteínas de Transporte Nucleocitoplasmático/biosíntesis , Fosfatidilinositol 3-Quinasa/fisiología , Receptores de Células Asesinas Naturales/fisiología , Animales , Dominio Catalítico/fisiología , Línea Celular Tumoral , Transformación Celular Viral , Fosfatidilinositol 3-Quinasa Clase I , Fibroblastos/virología , Células Asesinas Naturales/inmunología , Ligandos , Ratones , Muromegalovirus/inmunología , Fosfatidilinositol 3-Quinasas/fisiologíaRESUMEN
During a lytic gammaherpesvirus infection, host gene expression is severely restricted by the global degradation and altered 3' end processing of mRNA. This host shutoff phenotype is orchestrated by the viral SOX protein, yet its functional significance to the viral lifecycle has not been elucidated, in part due to the multifunctional nature of SOX. Using an unbiased mutagenesis screen of the murine gammaherpesvirus 68 (MHV68) SOX homolog, we isolated a single amino acid point mutant that is selectively defective in host shutoff activity. Incorporation of this mutation into MHV68 yielded a virus with significantly reduced capacity for mRNA turnover. Unexpectedly, the MHV68 mutant showed little defect during the acute replication phase in the mouse lung. Instead, the virus exhibited attenuation at later stages of in vivo infections suggestive of defects in both trafficking and latency establishment. Specifically, mice intranasally infected with the host shutoff mutant accumulated to lower levels at 10 days post infection in the lymph nodes, failed to develop splenomegaly, and exhibited reduced viral DNA levels and a lower frequency of latently infected splenocytes. Decreased latency establishment was also observed upon infection via the intraperitoneal route. These results highlight for the first time the importance of global mRNA degradation during a gammaherpesvirus infection and link an exclusively lytic phenomenon with downstream latency establishment.
Asunto(s)
Infecciones por Herpesviridae/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Factores de Transcripción SOX/metabolismo , Proteínas Virales/metabolismo , Latencia del Virus/fisiología , Animales , Células COS , Chlorocebus aethiops , Femenino , Células HEK293 , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/patología , Humanos , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Ratones , Células 3T3 NIH , ARN Mensajero/genética , Rhadinovirus/genética , Factores de Transcripción SOX/genética , Células Vero , Proteínas Virales/genéticaRESUMEN
Multiple receptors within the innate immune system have evolved to recognize nucleic acids as signatures of viral infection. It is believed that this specificity is essential for viral detection, as viruses often lack other invariant features that can serve as suitable targets for innate receptors. One such innate receptor, TLR9, has been implicated in the detection of many dsDNA viruses. In this study, we investigate the detection of murine gammaherpesvirus 68 (MHV68) by TLR9. We find that the genomic DNA of the murine CMV, a very potent inducer of innate responses. Genome-wide analysis of the number of stimulatory versus nonstimulatory CpG motifs present in the genome of each virus reveals that the MHV68 genome contains only a fraction of the number of immunostimulatory motifs present in murine CMV. Notably, MHV68 appears to have selectively suppressed the number of stimulatory motifs through cytosine to thymine conversion. These data suggest that certain viruses may have evolved and modified their genomic content to avoid recognition by nucleic acid-sensing receptors of the innate immune system.
Asunto(s)
Genoma Viral/inmunología , Muromegalovirus/inmunología , Rhadinovirus/inmunología , Receptor Toll-Like 9/antagonistas & inhibidores , Receptor Toll-Like 9/genética , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Animales , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/virología , Células HEK293 , Humanos , Inmunidad Innata/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Muromegalovirus/genética , Rhadinovirus/genética , Receptor Toll-Like 9/metabolismoRESUMEN
Cell surface heparan sulfate (HS) is an important coreceptor for many cytokines, chemokines, and growth factors. In this study, we report that splenic murine B cells express very little HS and that upon infection with either gammaherpesvirus (murine gammaherpesvirus 68) or betaherpesvirus (murine cytomegalovirus), HS is rapidly upregulated at the surface of B cells. HS upregulation was not observed in mice deficient for the type I IFN (IFN-I) receptor. Additionally, treatment of wild-type mice with the IFN-I inducer polyinosine polycytidylic acid triggered HS expression at the B cell surface. Similarly, incubation of purified splenic B cells with IFN-I, TLR ligands, or BCR stimulators ex vivo resulted in a drastic increase in HS surface expression. We found that IFN-I induced an increase in the surface expression of HS-modified syndecan 4 as well as that of an unidentified heparan sulfate proteoglycan. Finally, IFN-I treatment increased B cell responsiveness to APRIL, a cytokine involved in B cell survival and T cell-independent B cell responses. Enzymatic removal of HS from IFN-I-treated B cells inhibited APRIL. Altogether, our results indicate that upon herpesvirus infection in mice, HS is rapidly upregulated at the surface of B cells due to the action of IFN-I, potentially increasing B cell responsiveness to cytokines. Induction of HS expression at the B cell surface by stimulators of the innate immune response likely plays a key role in the development of a robust immune response.
Asunto(s)
Linfocitos B/metabolismo , Heparitina Sulfato/biosíntesis , Infecciones por Herpesviridae/inmunología , Interferón Tipo I/metabolismo , Animales , Linfocitos B/inmunología , Western Blotting , Separación Celular , Citometría de Flujo , Gammaherpesvirinae/inmunología , Heparitina Sulfato/inmunología , Inmunoprecipitación , Interferón Tipo I/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Muromegalovirus/inmunología , Bazo/citología , Bazo/inmunología , Regulación hacia ArribaRESUMEN
MHC-E restricted CD8 T cells show promise in vaccine settings, but their development and specificity remain poorly understood. Here we focus on a CD8 T cell population reactive to a self-peptide (FL9) bound to mouse MHC-E (Qa-1b) that is presented in response to loss of the MHC I processing enzyme ERAAP, termed QFL T cells. We find that mature QFL thymocytes are predominantly CD8αß+CD4-, show signs of agonist selection, and give rise to both CD8αα and CD8αß intraepithelial lymphocytes (IEL), as well as memory phenotype CD8αß T cells. QFL T cells require the MHC I subunit ß-2 microglobulin (ß2m), but do not require Qa1b or classical MHC I for positive selection. However, QFL thymocytes do require Qa1b for agonist selection and full functionality. Our data highlight the relaxed requirements for positive selection of an MHC-E restricted T cell population and suggest a CD8αß+CD4- pathway for development of CD8αα IELs.
Asunto(s)
Linfocitos T CD8-positivos , Receptores de Antígenos de Linfocitos T alfa-beta , Animales , Ratones , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Timocitos/metabolismo , Genes MHC Clase IIRESUMEN
The endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP) plays a crucial role in shaping the peptide-major histocompatibility complex (MHC) class I repertoire and maintaining immune surveillance. While murine cytomegalovirus (MCMV) has multiple strategies for manipulating the antigen processing pathway to evade immune responses, the host has also developed ways to counter viral immune evasion. In this study, we find that MCMV modulates ERAAP and induces an interferon γ (IFN-γ)-producing CD8+ T cell effector response that targets uninfected ERAAP-deficient cells. We observe that ERAAP downregulation during infection leads to the presentation of the self-peptide FL9 on non-classical Qa-1b, thereby eliciting Qa-1b-restricted QFL T cells to proliferate in the liver and spleen of infected mice. QFL T cells upregulate effector markers upon MCMV infection and are sufficient to reduce viral load after transfer to immunodeficient mice. Our study highlights the consequences of ERAAP dysfunction during viral infection and provides potential targets for anti-viral therapies.
Asunto(s)
Presentación de Antígeno , Muromegalovirus , Animales , Ratones , Aminopeptidasas/metabolismo , Linfocitos T CD8-positivos , Retículo Endoplásmico/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Leucil Aminopeptidasa/metabolismo , Péptidos/metabolismoRESUMEN
The production of cytokines such as type I interferon (IFN) is an essential component of innate immunity. Insufficient amounts of cytokines lead to host sensitivity to infection, whereas abundant cytokine production can lead to inflammation. A tight regulation of cytokine production is, thus, essential for homeostasis of the immune system. IFN-α production during RNA virus infection is mediated by the master transcription factor IRF7, which is activated upon ubiquitination by TRAF6 and phosphorylation by IKKε and TBK1 kinases. We found that Fas-associated death domain (FADD), first described as an apoptotic protein, is involved in regulating IFN-α production through a novel interaction with TRIM21. TRIM21 is a member of a large family of proteins that can impart ubiquitin modification onto its cellular targets. The interaction between FADD and TRIM21 enhances TRIM21 ubiquitin ligase activity, and together they cooperatively repress IFN-α activation in Sendai virus-infected cells. FADD and TRIM21 can directly ubiquitinate IRF7, affect its phosphorylation status, and interfere with the ubiquitin ligase activity of TRAF6. Conversely, a reduction of FADD and TRIM21 levels leads to higher IFN-α induction, IRF7 phosphorylation, and lower titers of RNA virus of infected cells. We conclude that FADD and TRIM21 together negatively regulate the late IFN-α pathway in response to viral infection.
Asunto(s)
Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Interferón-alfa/biosíntesis , Infecciones por Respirovirus/metabolismo , Ribonucleoproteínas/metabolismo , Virus Sendai/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Línea Celular , Perros , Proteína de Dominio de Muerte Asociada a Fas/genética , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Gripe Humana/genética , Gripe Humana/metabolismo , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Interferón-alfa/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Infecciones por Respirovirus/genética , Ribonucleoproteínas/genética , Virus Sendai/genética , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genéticaRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two RING finger E3 ubiquitin ligases (MIR1 and MIR2) that mediate ubiquitination and degradation of cellular proteins important for the establishment of an efficient antiviral immune response. MIR1 and MIR2 share 30% sequence identity; however, their substrate preferences are varied. MIR1 has been shown to primarily downregulate major histocompatibility complex class I (MHC-I), whereas MIR2 can downregulate a wide range of cell surface proteins. Many of the MIR substrates are thought to be present in lipid raft microdomains, a subregion of the plasma membrane known to be important for a wide range of signal transduction events. Palmitoylation is a posttranslational modification that increases recruitment of transmembrane proteins to lipid rafts. In this study, we investigated the importance of palmitoylation for MIR function. We present evidence that MIR2-mediated downregulation of MHC-I and platelet endothelial cell adhesion molecule 1 (PECAM-1) but not other substrates is inhibited in the presence of the drug 2-bromohexadecanoic acid (2-Br), a chemical inhibitor of palmitoylation. Biochemical analysis indicates that MIR2 is directly palmitoylated on cysteine 146. Mutation of this cysteine to a phenylalanine prevents MIR2 palmitoylation and blocks the ability of MIR2 to downregulate MHC-I and PECAM-I but not B7.2 and intercellular adhesion molecule 1 (ICAM-I), consistent with the phenotype observed after 2-Br treatment. Unpalmitoylated MIR2 does not interact with MHC-I and is thus unable to ubiquitinate and downregulate MHC-I from the cell surface. Furthermore, we observed that MIR2 is palmitoylated in vivo during lytic infection. Palmitoylation may act to regulate MIR2 function and localization during viral infection by allowing MIR2 to properly interact with and downregulate multiple substrates known to play an important role in the host immune response.
Asunto(s)
Infecciones por Herpesviridae/virología , Herpesvirus Humano 8/enzimología , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Regulación hacia Abajo , Células HEK293 , Células HeLa , Infecciones por Herpesviridae/metabolismo , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Lipoilación , Datos de Secuencia Molecular , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
NKG2D is a stimulatory receptor expressed by NK cells and some T cell subsets. Expression of the self-encoded ligands for NKG2D is presumably tightly regulated to prevent autoimmune disorders while allowing detection of infected cells and developing tumors. The NKG2D ligand Mult1 is regulated at multiple levels, with a final layer of regulation controlling protein stability. In this article, we report that Mult1 cell-surface expression was prevented by two closely related E3 ubiquitin ligases membrane-associated RING-CH (MARCH)4 and MARCH9, members of an E3 family that regulates other immunologically active proteins. Lysines within the cytoplasmic domain of Mult1 were essential for this repression by MARCH4 or MARCH9. Downregulation of Mult1 by MARCH9 was reversed by heat-shock treatment, which resulted in the dissociation of the two proteins and increased the amount of Mult1 at the cell surface. These results identify Mult1 as a target for the MARCH family of E3 ligases and show that induction of Mult1 in response to heat shock is due to regulated association with its E3 ligases.
Asunto(s)
Proteínas Portadoras/inmunología , Regulación de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Proteínas de la Membrana/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Animales , Western Blotting , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Separación Celular , Células Cultivadas , Citometría de Flujo , Proteínas Ligadas a GPI , Expresión Génica , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/genética , Células Asesinas Activadas por Linfocinas/inmunología , Células Asesinas Activadas por Linfocinas/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Endoplasmic reticulum (ER) aminopeptidase associated with antigen processing (ERAAP) trims peptide precursors in the ER for presentation by major histocompatibility (MHC)-I molecules to surveying CD8+ T-cells. This function allows ERAAP to regulate the nature and quality of the peptide repertoire and, accordingly, the resulting immune responses. We recently showed that infection with murine cytomegalovirus leads to a dramatic loss of ERAAP levels in infected cells. In mice, this loss is associated with the activation of QFL T-cells, a subset of T-cells that monitor ERAAP integrity and eliminate cells experiencing ERAAP dysfunction. In this study, we aimed to identify host factors that regulate ERAAP expression level and determine whether these could be manipulated during viral infections. We performed a CRISPR knockout screen and identified ERp44 as a factor promoting ERAAP retention in the ER. ERp44's interaction with ERAAP is dependent on the pH gradient between the ER and Golgi. We hypothesized that viruses that disrupt the pH of the secretory pathway interfere with ERAAP retention. Here, we demonstrate that expression of the Envelope (E) protein from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) leads to Golgi pH neutralization and consequently decrease of ERAAP intracellular levels. Furthermore, SARS-CoV-2-induced ERAAP loss correlates with its release into the extracellular environment. ERAAP's reliance on ERp44 and a functioning ER/Golgi pH gradient for proper localization and function led us to propose that ERAAP serves as a sensor of disturbances in the secretory pathway during infection and disease.
RESUMEN
Modulation of T-cell receptor expression and signaling is essential to the survival of many viruses. The U24 protein expressed by human herpesvirus 6A, a ubiquitous human pathogen, has been previously shown to downregulate the T-cell receptor. Here, we show that U24 also mediates cell surface downregulation of a canonical early endosomal recycling receptor, the transferrin receptor, indicating that this viral protein acts by blocking early endosomal recycling. We present evidence that U24 is a C-tail-anchored protein that is dependent for its function on TRC40/Asna-1, a component of a posttranslational membrane insertion pathway. Finally, we find that U24 proteins from other roseoloviruses have a similar genetic organization and a conserved function that is dependent on a proline-rich motif. Inhibition of a basic cellular process by U24 has interesting implications not only for the pathogenicity of roseoloviruses but also for our understanding of the biology of endosomal transport.
Asunto(s)
Endocitosis/fisiología , Herpesvirus Humano 6/metabolismo , Herpesvirus Humano 7/metabolismo , Proteínas Virales/fisiología , Secuencia de Aminoácidos , Western Blotting , Línea Celular , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida , Humanos , Datos de Secuencia Molecular , Receptores de Transferrina/metabolismo , Homología de Secuencia de Aminoácido , Proteínas Virales/químicaRESUMEN
The persistence of the COVID-19 pandemic demands a dramatic increase in testing efficiency. Testing pooled samples for SARS-CoV-2 could meet this need; however, the sensitivity of RT-qPCR, the gold standard, significantly decreases with an increasing number of samples pooled. Here, we introduce DIVER, a method that quantifies intact virus and is robust to sample dilution. DIVER first tags viral particles with exogeneous oligonucleotides, then captures the tagged particles on ACE2-functionalized beads, and finally quantifies the oligonucleotide tags using qPCR. Using spike-presenting liposomes and Spike-pseudotyped lentivirus as SARS-CoV-2 models, we show that DIVER can detect 1×10 5 liposomes and 100 pfu lentivirus and can successfully identify positive samples in pooling experiments. Overall, DIVER is well-positioned for efficient sample pooling and expanded community surveillance.