RESUMEN
CD8(+) T cells play a critical role in limiting peripheral virus replication, yet how they locate virus-infected cells within tissues is unknown. Here, we have examined the environmental signals that CD8(+) T cells use to localize and eliminate virus-infected skin cells. Epicutaneous vaccinia virus (VV) infection, mimicking human smallpox vaccination, greatly increased expression of the CXCR3 chemokine receptor ligands CXCL9 and CXCL10 in VV-infected skin. Despite normal T cell numbers in the skin, Cxcr3(-/-) mice exhibited dramatically impaired CD8(+)-T-cell-dependent virus clearance. Intravital microscopy revealed that Cxcr3(-/-) T cells were markedly deficient in locating, engaging, and killing virus-infected cells. Further, transfer of wild-type CD8(+) T cells restored viral clearance in Cxcr3(-/-) animals. These findings demonstrate a function for CXCR3 in enhancing the ability of tissue-localized CD8(+) T cells to locate virus-infected cells and thereby exert anti-viral effector functions.
Asunto(s)
Queratinocitos/inmunología , Infecciones por Poxviridae/inmunología , Receptores CXCR3/inmunología , Piel/inmunología , Linfocitos T Citotóxicos/inmunología , Virus Vaccinia/inmunología , Traslado Adoptivo , Animales , Movimiento Celular , Quimiocina CXCL10/genética , Quimiocina CXCL10/inmunología , Quimiocina CXCL9/genética , Quimiocina CXCL9/inmunología , Femenino , Regulación de la Expresión Génica , Humanos , Queratinocitos/patología , Queratinocitos/virología , Ratones Transgénicos , Infecciones por Poxviridae/genética , Infecciones por Poxviridae/patología , Infecciones por Poxviridae/virología , Receptores CXCR3/deficiencia , Receptores CXCR3/genética , Transducción de Señal , Piel/patología , Piel/virología , Linfocitos T Citotóxicos/patología , Linfocitos T Citotóxicos/trasplante , Carga ViralRESUMEN
Infection with (SAg)-producing bacteria may precede or follow infection with or vaccination against influenza A viruses (IAVs). However, how SAgs alter the breadth of IAV-specific CD8+ T cell (TCD8) responses is unknown. Moreover, whether recall responses mediating heterosubtypic immunity to IAVs are manipulated by SAgs remains unexplored. We employed wild-type (WT) and mutant bacterial SAgs, SAg-sufficient/deficient Staphylococcus aureus strains, and WT, mouse-adapted and reassortant IAV strains in multiple in vivo settings to address the above questions. Contrary to the popular view that SAgs delete or anergize T cells, systemic administration of staphylococcal enterotoxin B (SEB) or Mycoplasma arthritidis mitogen before intraperitoneal IAV immunization enlarged the clonal size of 'select' IAV-specific TCD8 and reshuffled the hierarchical pattern of primary TCD8 responses. This was mechanistically linked to the TCR Vß makeup of the impacted clones rather than their immunodominance status. Importantly, SAg-expanded TCD8 retained their IFN-γ production and cognate cytolytic capacities. The enhancing effect of SEB on immunodominant TCD8 was also evident in primary responses to vaccination with heat-inactivated and live attenuated IAV strains administered intramuscularly and intranasally, respectively. Interestingly, in prime-boost immunization settings, the outcome of SEB administration depended strictly upon the time point at which this SAg was introduced. Accordingly, SEB injection before priming raised CD127highKLRG1low memory precursor frequencies and augmented the anamnestic responses of SEB-binding TCD8. By comparison, introducing SEB before boosting diminished recall responses to IAV-derived epitopes drastically and indiscriminately. This was accompanied by lower Ki67 and higher Fas, LAG-3 and PD-1 levels consistent with a pro-apoptotic and/or exhausted phenotype. Therefore, SAgs can have contrasting impacts on anti-IAV immunity depending on the naïve/memory status and the TCR composition of exposed TCD8. Finally, local administration of SEB or infection with SEB-producing S. aureus enhanced pulmonary TCD8 responses to IAV. Our findings have clear implications for superinfections and prophylactic vaccination.
Asunto(s)
Memoria Inmunológica/inmunología , Virus de la Influenza A/inmunología , Superantígenos/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Epítopos/inmunología , Femenino , Humanos , Memoria Inmunológica/fisiología , Virus de la Influenza A/metabolismo , Gripe Humana/inmunología , Gripe Humana/metabolismo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Staphylococcus aureus/inmunología , Superantígenos/fisiología , Sobreinfección/inmunología , VacunaciónRESUMEN
The importance of antiviral CD8+ T cell recognition of alternative reading frame (ARF)-derived peptides is uncertain. In this study, we describe an epitope (NS1-ARF21-8) present in a predicted 14-residue peptide encoded by the +1 register of NS1 mRNA in the influenza A virus (IAV). NS1-ARF21-8 elicits a robust, highly functional CD8+ T cell response in IAV-infected BALB/c mice. NS1-ARF21-8 is presented from unspliced NS mRNA, likely from downstream initiation on a Met residue that comprises the P1 position of NS1-ARF21-8 Derived from a 14-residue peptide with no apparent biological function and negligible impacts on IAV infection, infectivity, and pathogenicity, NS1-ARF21-8 provides a clear demonstration of how immunosurveillance exploits natural errors in protein translation to provide antiviral immunity. We further show that IAV infection enhances a model cellular ARF translation, which potentially has important implications for virus-induced autoimmunity.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/metabolismo , Virus de la Influenza A/fisiología , Gripe Humana/inmunología , Infecciones por Orthomyxoviridae/inmunología , Proteínas no Estructurales Virales/metabolismo , Empalme Alternativo , Animales , Modelos Animales de Enfermedad , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Vigilancia Inmunológica , Ratones , Ratones Endogámicos BALB C , Sistemas de Lectura Abierta/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunologíaRESUMEN
It is uncertain how antiviral lymphocytes are activated in draining lymph nodes, the site where adaptive immune responses are initiated. Here, using intravital microscopy we show that after infection of mice with vaccinia virus (a large DNA virus) or vesicular stomatitis virus (a small RNA virus), virions drained to the lymph node and infected cells residing just beneath the subcapsular sinus. Naive CD8+ T cells rapidly migrated to infected cells in the peripheral interfollicular region and then formed tight interactions with dendritic cells, leading to complete T cell activation. Thus, antigen presentation at the lymph node periphery, not at lymphocyte exit sites in deeper lymph node venules, as dogma dictates, has a dominant function in antiviral CD8+ T cell activation.
Asunto(s)
Presentación de Antígeno , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Ganglios Linfáticos/inmunología , Activación de Linfocitos , Animales , Células Presentadoras de Antígenos/inmunología , Movimiento Celular , Células Dendríticas/inmunología , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Ratones , Ratones Transgénicos , Virus Vaccinia/inmunología , Vesiculovirus/inmunologíaRESUMEN
Superantigens (SAgs) released by common Gram-positive bacterial pathogens have been reported to delete, anergize, or activate mouse T cells. However, little is known about their effects on preexisting memory CD8+ T cell (TCD8) pools. Furthermore, whether SAgs manipulate human memory TCD8 responses to cognate antigens is unknown. We used a human peripheral blood mononuclear cell culture system and a nontransgenic mouse model in which the impact of stimulation by two fundamentally distinct SAgs, staphylococcal enterotoxin B and Mycoplasma arthritidis mitogen, on influenza virus- and/or cytomegalovirus-specific memory TCD8 could be monitored. Bacterial SAgs surprisingly expanded antiviral memory TCD8 generated naturally through infection or artificially through vaccination. Mechanistically, this was a T cell-intrinsic and T cell receptor ß-chain variable-dependent phenomenon. Importantly, SAg-expanded TCD8 displayed an effector memory phenotype and were capable of producing interferon-γ and destroying target cells ex vivo or in vivo. These findings have clear implications for antimicrobial defense and rational vaccine design.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Superantígenos/inmunología , Adulto , Animales , Antígenos Bacterianos/inmunología , Enterotoxinas/inmunología , Femenino , Humanos , Memoria Inmunológica/inmunología , Vacunas contra la Influenza/inmunología , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Orthomyxoviridae/inmunología , Adulto JovenRESUMEN
The interactions between programmed death-1 (PD-1) and its ligands hamper tumor-specific CD8+ T cell (TCD8) responses, and PD-1-based "checkpoint inhibitors" have shown promise in certain cancers, thus revitalizing interest in immunotherapy. PD-1-targeted therapies reverse TCD8 exhaustion/anergy. However, whether they alter the epitope breadth of TCD8 responses remains unclear. This is an important question because subdominant TCD8 are more likely than immunodominant clones to escape tolerance mechanisms and may contribute to protective anticancer immunity. We have addressed this question in an in vivo model of TCD8 responses to well-defined epitopes of a clinically relevant oncoprotein, large T Ag. We found that unlike other coinhibitory molecules (CTLA-4, LAG-3, TIM-3), PD-1 was highly expressed by subdominant TCD8, which correlated with their propensity to favorably respond to PD-1/PD-1 ligand-1 (PD-L1)-blocking Abs. PD-1 blockade increased the size of subdominant TCD8 clones at the peak of their primary response, and it also sustained their presence, thus giving rise to an enlarged memory pool. The expanded population was fully functional as judged by IFN-γ production and MHC class I-restricted cytotoxicity. The selective increase in subdominant TCD8 clonal size was due to their enhanced survival, not proliferation. Further mechanistic studies utilizing peptide-pulsed dendritic cells, recombinant vaccinia viruses encoding full-length T Ag or epitope mingenes, and tumor cells expressing T Ag variants revealed that anti-PD-1 invigorates subdominant TCD8 responses by relieving their lysis-dependent suppression by immunodominant TCD8 To our knowledge, our work constitutes the first report that interfering with PD-1 signaling potentiates epitope spreading in tumor-specific responses, a finding with clear implications for cancer immunotherapy and vaccination.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Epítopos/inmunología , Inmunidad Celular , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Transducción de Señal/inmunología , Animales , Linfocitos T CD8-positivos/patología , Muerte Celular/genética , Muerte Celular/inmunología , Línea Celular Tumoral , Epítopos/genética , Femenino , Interferón gamma/genética , Interferón gamma/inmunología , Ratones , Proteínas de Neoplasias/genética , Neoplasias Experimentales/genética , Receptor de Muerte Celular Programada 1/genética , Transducción de Señal/genéticaRESUMEN
Skin infection with the poxvirus vaccinia (VV) elicits a powerful, inflammatory cellular response that clears virus infection in a coordinated, spatially organized manner. Given the high concentration of pro-inflammatory effectors at areas of viral infection, it is unclear how tissue pathology is limited while virus-infected cells are being eliminated. To better understand the spatial dynamics of the anti-inflammatory response to a cutaneous viral infection, we first screened cytokine mRNA expression levels after epicutaneous (ec.) VV infection and found a large increase the anti-inflammatory cytokine IL-10. Ex vivo analyses revealed that T cells in the skin were the primary IL-10-producing cells. To understand the distribution of IL-10-producing T cells in vivo, we performed multiphoton intravital microscopy (MPM) of VV-infected mice, assessing the location and dynamic behavior of IL-10 producing cells. Although virus-specific T cells were distributed throughout areas of the inflamed skin lacking overt virus-infection, IL-10+ cells closely associated with large keratinocytic foci of virus replication where they exhibited similar motility patterns to bulk antigen-specific CD8+ T cells. Paradoxically, neutralizing secreted IL-10 in vivo with an anti-IL-10 antibody increased viral lesion size and viral replication. Additional analyses demonstrated that IL-10 antibody administration decreased recruitment of CCR2+ inflammatory monocytes, which were important for reducing viral burden in the infected skin. Based upon these findings, we conclude that spatially concentrated IL-10 production limits cutaneous viral replication and dissemination, likely through modulation of the innate immune repertoire at the site of viral growth.
Asunto(s)
Interleucina-10/metabolismo , Virus Vaccinia/inmunología , Vaccinia/inmunología , Animales , Citocinas/genética , Citocinas/metabolismo , Femenino , Interleucina-10/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Monocitos/inmunología , Piel/inmunología , Organismos Libres de Patógenos Específicos , Linfocitos T/inmunología , Vaccinia/virología , Replicación ViralRESUMEN
Influenza A virus gene segment 7 encodes two proteins: the M1 protein translated from unspliced mRNA and the M2 protein produced by mRNA splicing and largely encoded by the M1 +1 reading frame. To better understand the generation of defective ribosomal products relevant to MHC class I Ag presentation, we engineered influenza A virus gene segment 7 to encode the model H-2 K(b) class I peptide ligand SIINFEKL at the M2 protein C terminus. Remarkably, after treating virus-infected cells with the RNA splicing inhibitor spliceostatin A to prevent M2 mRNA generation, K(b)-SIINFEKL complexes were still presented on the cell surface at levels ≤60% of untreated cells. Three key findings indicate that SIINFEKL is produced by cytoplasmic translation of unspliced M1 mRNA initiating at CUG codons within the +1 reading frame: 1) synonymous mutation of CUG codons in the M2-reading frame reduced K(b)-SIINFEKL generation; 2) K(b)-SIINFEKL generation was not affected by drug-mediated inhibition of AUG-initiated M1 synthesis; and 3) K(b)-SIINFEKL was generated in vitro and in vivo from mRNA synthesized in the cytoplasm by vaccinia virus, and hence cannot be spliced. These findings define a viral defective ribosomal product generated by cytoplasmic noncanonical translation and demonstrate the participation of CUG-codon-based translation initiation in pathogen immunosurveillance.
Asunto(s)
Virus Defectuosos/genética , Virus de la Influenza A/genética , Péptidos/genética , Ribosomas/metabolismo , Proteínas de la Matriz Viral/genética , Animales , Presentación de Antígeno/efectos de los fármacos , Línea Celular , Virus Defectuosos/química , Virus Defectuosos/efectos de los fármacos , Virus Defectuosos/metabolismo , Genes MHC Clase I , Células HeLa , Humanos , Virus de la Influenza A/química , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/metabolismo , Ratones , Ratones Endogámicos C57BL , Péptidos/química , Biosíntesis de Proteínas , Piranos/farmacología , Empalme del ARN/efectos de los fármacos , Compuestos de Espiro/farmacologíaRESUMEN
The nature of crosspriming immunogens for CD8(+) T cell responses is highly controversial. By using a panel of T cell receptor-like antibodies specific for viral peptides bound to mouse D(b) major histocompatibility complex class I molecules, we show that an exceptional peptide (PA(224-233)) expressed as a viral minigene product formed a sizeable cytosolic pool continuously presented for hours after protein synthesis was inhibited. PA(224-233) pool formation required active cytosolic heat-shock protein 90 but not ER g96 and uniquely enabled crosspriming by this peptide. These findings demonstrate that exceptional class I binding oligopeptides that escape proteolytic degradation are potent crosspriming agents. Thus, the feeble immunogenicity of natural proteasome products in crosspriming can be attributed to their evanescence in donor cells and not an absolute inability of cytosolic oligopeptides to be transferred to and presented by professional antigen-presenting cells.
Asunto(s)
Antígenos Virales/inmunología , Linfocitos T CD8-positivos/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Virus de la Influenza A/inmunología , Péptidos/inmunología , Animales , Anticuerpos/inmunología , Antígenos Virales/metabolismo , Linfocitos T CD8-positivos/inmunología , Línea Celular , Femenino , Proteínas HSP90 de Choque Térmico/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Ratones , Péptidos/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
The influenza A virus (IAV) genome is divided into eight distinct RNA segments believed to be copackaged into virions with nearly perfect efficiency. Here, we describe a mutation in IAV nucleoprotein (NP) that enhances replication and transmission in guinea pigs while selectively reducing neuraminidase (NA) gene segment packaging into virions. We show that incomplete IAV particles lacking gene segments contribute to the propagation of the viral population through multiplicity reactivation under conditions of widespread coinfection, which we demonstrate commonly occurs in the upper respiratory tract of guinea pigs. NP also dramatically altered the functional balance of the viral glycoproteins on particles by selectively decreasing NA expression. Our findings reveal novel functions for NP in selective control of IAV gene packaging and balancing glycoprotein expression and suggest a role for incomplete gene packaging during host adaptation and transmission.
Asunto(s)
Virus de la Influenza A/fisiología , Gripe Humana/transmisión , Neuraminidasa/genética , Nucleoproteínas/fisiología , Proteínas Virales/fisiología , Genes Virales , Humanos , Virus de la Influenza A/genética , Gripe Humana/virología , Ensamble de Virus , Replicación ViralRESUMEN
Green fluorescent protein (GFP) and other fluorescent proteins are essential tools for biological research. When fused to peptides or proteins as a reporter, GFP enables localization and quantitation of gene products in otherwise unmanipulated live cells or organisms. We previously reported that a sizable fraction of nascent GFP is post-translationally converted into a 20-kDa Triton X-100-insoluble proteasome substrate (Qian, S. B., Princiotta, M. F., Bennink, J. R., and Yewdell, J. W. (2006) J. Biol. Chem. 281, 392-400; Dolan, B. P., Li, L., Veltri, C. A., Ireland, C. M., Bennink, J. R., and Yewdell, J. W. (2011) J. Immunol. 186, 2065-2072). Here, we show that a similarly sized fragment is generated by all GFP and red fluorescent protein family members we examined. We demonstrate that fragmentation is a by-product of GFP chromophore rearrangement. A non-rearranging GFP mutant fails to fragment and generates diminished levels of K(b)-SIINFEKL complexes when SIINFEKL is genetically fused to either the C- or N-terminal domains of GFP fusion proteins. Instructively, another fragmenting GFP mutant that cannot create the functional chromophore but still generates fragments also demonstrates diminished K(b)-SIINFEKL generation. However, the mutant and wild-type fragments differ fundamentally in that wild-type fragments are rapidly liberated from the intact molecule and degraded quickly, accounting for increased K(b)-SIINFEKL generation. In the fragmenting mutant, the fragments are generated slowly and remain associated, likely in a native conformation based on their original structural description (Barondeau, D. P., Kassmann, C. J., Tainer, J. A., and Getzoff, E. D. (2006) J. Am. Chem. Soc. 128, 4685-4693). The wild-type GFP fragments represent the first biochemically defined natural defective ribosomal products to contribute peptides for immunosurveillance, enabling quantitation of peptide generation efficiency from this source of defective ribosomal products. More broadly, given the wide use of fluorescent proteins, their ubiquitous and abundant fragmentation must be considered when interpreting experiments using these extremely useful probes.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Proteínas Luminiscentes/química , Fragmentos de Péptidos/inmunología , Presentación de Antígeno , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/inmunología , Monitorización Inmunológica , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Proteína Fluorescente RojaRESUMEN
Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several N-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as N-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4°C and well at 37°C. A de novo designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of N-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple N-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that N-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from N-linked glycans.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/inmunología , Afinidad de Anticuerpos , Variación Antigénica/genética , Variación Antigénica/inmunología , Línea Celular , Perros , Glicosilación , Aparato de Golgi/inmunología , Humanos , Vacunas contra la Influenza/inmunología , Células de Riñón Canino Madin Darby , Ratones , Estructura Terciaria de Proteína , Proteínas Recombinantes/inmunología , VacunaciónRESUMEN
Translational fidelity, essential for protein and cell function, requires accurate transfer RNA (tRNA) aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of one error per 10,000 to 100,000 couplings. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower. Here we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to tenfold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code.
Asunto(s)
Inmunidad Innata , Metionina/metabolismo , Estrés Oxidativo/fisiología , Aminoacilación de ARN de Transferencia/fisiología , Adenoviridae/fisiología , Animales , Código Genético , Células HeLa , Humanos , Ligandos , Metionina/genética , Ratones , Modelos Genéticos , NADPH Oxidasas/metabolismo , Orthomyxoviridae/fisiología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Metionina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Especificidad por Sustrato , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismo , Aminoacilación de ARN de Transferencia/efectos de los fármacosRESUMEN
Transfer RNAs (tRNAs) are central to protein synthesis and impact translational speed and fidelity by their abundance. Here we examine the extent to which viruses manipulate tRNA populations to favor translation of their own genes. We study two very different viruses: influenza A virus (IAV), a medium-sized (13 kB genome) RNA virus; and vaccinia virus (VV), a large (200 kB genome) DNA virus. We show that the total cellular tRNA population remains unchanged following viral infection, whereas the polysome-associated tRNA population changes dramatically in a virus-specific manner. The changes in polysome-associated tRNA levels reflect the codon usage of viral genes, suggesting the existence of local tRNA pools optimized for viral translation.
Asunto(s)
Virus de la Influenza A/genética , Biosíntesis de Proteínas , ARN de Transferencia/metabolismo , Virus Vaccinia/genética , Codón , Genes Virales , Células HeLa , Humanos , Interferones/farmacología , Polirribosomas/química , ARN de Transferencia/análisis , Replicación ViralRESUMEN
To better understand the generation of MHC class I-associated peptides, we used a model antigenic protein whose proteasome-mediated degradation is rapidly and reversibly controlled by Shield-1, a cell-permeant drug. When expressed from a stably transfected gene, the efficiency of antigen presentation is ~2%, that is, one cell-surface MHC class I-peptide complex is generated for every 50 folded source proteins degraded upon Shield-1 withdrawal. By contrast, when the same protein is expressed by vaccinia virus, its antigen presentation efficiency is reduced ~10-fold to values similar to those reported for other vaccinia virus-encoded model antigens. Virus infection per se does not modify the efficiency of antigen processing. Rather, the efficiency difference between cellular and virus-encoded antigens is based on whether the antigen is synthesized from transgene- vs. virus-encoded mRNA. Thus, class I antigen-processing machinery can distinguish folded proteins based on the precise details of their synthesis to modulate antigen presentation efficiency.
Asunto(s)
Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I/metabolismo , Animales , Presentación de Antígeno/genética , Secuencia de Bases , Línea Celular , Células HeLa , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Ratones , Ovalbúmina/genética , Ovalbúmina/inmunología , Ovalbúmina/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
How the ribosome-bound nascent chain folds to assume its functional tertiary structure remains a central puzzle in biology. In contrast to refolding of a denatured protein, cotranslational folding is complicated by the vectorial nature of nascent chains, the frequent ribosome pausing, and the cellular crowdedness. Here, we present a strategy called folding-associated cotranslational sequencing that enables monitoring of the folding competency of nascent chains during elongation at codon resolution. By using an engineered multidomain fusion protein, we demonstrate an efficient cotranslational folding immediately after the emergence of the full domain sequence. We also apply folding-associated cotranslational sequencing to track cotranslational folding of hemagglutinin in influenza A virus-infected cells. In contrast to sequential formation of distinct epitopes, the receptor binding domain of hemagglutinin follows a global folding route by displaying two epitopes simultaneously when the full sequence is available. Our results provide direct evidence of domain-wise global folding that occurs cotranslationally in mammalian cells.
Asunto(s)
Biosíntesis de Proteínas/fisiología , Pliegue de Proteína , Células HEK293 , Glicoproteínas Hemaglutininas del Virus de la Influenza/biosíntesis , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Sensitivity is essential in CD8+ T-cell killing of virus-infected cells and tumor cells. Although the affinity of the T-cell receptor (TCR) for antigen is relatively low, the avidity of T cell-antigen-presenting cell interactions is greatly enhanced by increasing the valence of the interaction. It is known that TCRs cluster into protein islands after engaging their cognate antigen (peptides bound to MHC molecules). Here, we show that mouse K(b) class I molecules segregate into preformed, long-lasting (hours) clusters on the antigen-presenting cell surface based on their bound viral peptide. Peptide-specific K(b) clustering occurs when source antigens are expressed by vaccinia or vesicular stomatitis virus, either as proteasome-liberated precursors or free intracellular peptides. By contrast, K(b)-peptide complexes generated by incubating cells with synthetic peptides are extensively intermingled on the cell surface. Peptide-specific complex sorting is first detected in the Golgi complex, and compromised by removing the K(b) cytoplasmic tail. Peptide-specific clustering is associated with increased T-cell sensitivity: on a per-complex basis, endogenous SIINFEKL activates T cells more efficiently than synthetic SIINFEKL, and wild-type K(b) presents endogenous SIINFEKL more efficiently than tailless K(b). We propose that endogenous processing generates peptide-specific clusters of class I molecules to maximize the sensitivity and speed of T-cell immunosurveillance.
Asunto(s)
Antígenos Virales/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Péptidos/química , Animales , Presentación de Antígeno/inmunología , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Línea Celular , Citoplasma/metabolismo , Aparato de Golgi/metabolismo , Ratones , Complejo de la Endopetidasa Proteasomal/metabolismo , Microglobulina beta-2/metabolismoRESUMEN
No antiviral vaccine is perfect. For some important pathogens, there are no effective vaccines. Many current vaccines are based on the working principles of Jenner and Pasteur, that is, empiric administration of attenuated or inactivated forms of the pathogen. Tapping the full potential of vaccination requires a thorough understanding of the mechanism of immune activation by pathogens and their individual components. Though the rate of discovery continues to accelerate, the complexity of the immune system is daunting, particularly when integrated into the overall physiology of the host. Here, we review the application of multiphoton microscopy to examine host-pathogen interactions, focusing on our recent efforts to understand mouse CD8(+) T-cell responses to viruses at the level of cellular interactions in lymph nodes draining the infection site. We also discuss our recent efforts to understand the influence of the sympathetic nervous system on antiviral immunity, with the ultimate goal of appreciating the traditional elements of immunity as just one facet of the total organismal response to infection and immunization.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Ganglios Linfáticos/inmunología , Microscopía de Fluorescencia por Excitación Multifotónica , Vacunas Virales , Animales , Linfocitos B/inmunología , Células Dendríticas/inmunología , Diseño de Fármacos , Interacciones Huésped-Patógeno , Humanos , Activación de Linfocitos , Ratones , Sistema Nervioso Simpático/fisiología , Virosis/inmunología , Virosis/prevención & controlRESUMEN
Influenza A virus (IAV) infects a remarkably wide variety of avian and mammalian hosts. Evolution finely hones IAV genes to optimally infect and be transmitted in a particular host species. Sporadically, IAV manages to jump between species, introducing novel antigenic strains into the new host population that wreak havoc until herd immunity develops. IAV adaptation to new hosts typically involves reassortment of IAV gene segments from coinfecting virus strains adapted to different hosts in conjunction with multiple adaptive mutations in the various IAV genes. To better understand host adaptation between mammalian species in real time, we passaged mouse-adapted A/PR8/34 (PR8) in guinea pigs. Guinea pigs, unlike mice, support spontaneous and robust IAV transmission. For some IAV strains, including PR8, adaptation is required for a virus to attain transmissibility, providing an opportunity to understand the evolution of transmissibility in guinea pigs. Multiple guinea pig-adapted PR8 mutants generated by serial nasal wash passaging in independent lines replicated more efficiently and were transmitted by cocaging. All transmissible variants possessed one of two nonsynonymous mutations in M1, either alone or in combination with mutations in PB2, HA, NP, or NA. Rapid reassortment between independently selected variants combined beneficial mutations in NP and M1 to form the fittest virus capable of being transmitted. These findings provide further insight into genetic determinants in NP and M1 involved in PR8 IAV adaptation to be transmitted in a new host and clearly show the benefit of a segmented genome in rapidly generating optimal combinations of mutations in IAV evolution.
Asunto(s)
Adaptación Biológica , Virus de la Influenza A/genética , Mutación Missense , Proteínas de Unión al ARN/genética , Recombinación Genética , Proteínas del Núcleo Viral/genética , Proteínas de la Matriz Viral/genética , Animales , Modelos Animales de Enfermedad , Femenino , Cobayas , Ratones , Cavidad Nasal/virología , Proteínas de la Nucleocápside , Infecciones por Orthomyxoviridae/transmisión , Infecciones por Orthomyxoviridae/virología , Pase SeriadoRESUMEN
Influenza A virus (IAV) remains an important human pathogen largely because of antigenic drift, the rapid emergence of antibody escape mutants that precludes durable vaccination. The most potent neutralizing antibodies interact with cognate epitopes in the globular "head" domain of hemagglutinin (HA), a homotrimeric glycoprotein. The H1 HA possesses five distinct regions defined by a large number of mouse monoclonal antibodies (MAbs), i.e., Ca1, Ca2, Cb, Sa, and Sb. Ca1-Ca2 sites require HA trimerization to attain full antigenicity, consistent with their locations on opposite sides of the trimer interface. Here, we show that full antigenicity of Cb and Sa sites also requires HA trimerization, as revealed by immunofluorescence microscopy of IAV-infected cells and biochemically by pulse-chase radiolabeling experiments. Surprisingly, epitope antigenicity acquired by HA trimerization persists following acid triggering of the globular domains dissociation and even after proteolytic release of monomeric heads from acid-treated HA. Thus, the requirement for HA trimerization by trimer-specific MAbs mapping to the Ca, Cb, and Sa sites is not dependent upon the bridging of adjacent monomers in the native HA trimer. Rather, complete antigenicity of HA (and, by inference, immunogenicity) requires a final folding step that accompanies its trimerization. Once this conformational change occurs, HA trimers themselves would not necessarily be required to induce a highly diverse neutralizing response to epitopes in the globular domain.