RESUMO
A systematic quantitative analysis of temporal changes in host and viral proteins throughout the course of a productive infection could provide dynamic insights into virus-host interaction. We developed a proteomic technique called "quantitative temporal viromics" (QTV), which employs multiplexed tandem-mass-tag-based mass spectrometry. Human cytomegalovirus (HCMV) is not only an important pathogen but a paradigm of viral immune evasion. QTV detailed how HCMV orchestrates the expression of >8,000 cellular proteins, including 1,200 cell-surface proteins to manipulate signaling pathways and counterintrinsic, innate, and adaptive immune defenses. QTV predicted natural killer and T cell ligands, as well as 29 viral proteins present at the cell surface, potential therapeutic targets. Temporal profiles of >80% of HCMV canonical genes and 14 noncanonical HCMV open reading frames were defined. QTV is a powerful method that can yield important insights into viral infection and is applicable to any virus with a robust in vitro model.
Assuntos
Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Interações Hospedeiro-Patógeno , Proteômica , Virologia/métodos , Humanos , Evasão da Resposta Imune , Células Matadoras Naturais/imunologia , Transdução de Sinais , Linfócitos T/imunologia , Proteínas Virais/análiseRESUMO
Human cytomegalovirus (HCMV) is a paradigm of pathogen immune evasion and sustains lifelong persistent infection in the face of exceptionally powerful host immune responses through the concerted action of multiple immune-evasins. These reduce NK cell activation by inhibiting ligands for activating receptors, expressing ligands for inhibitory receptors, or inhibiting synapse formation. However, these functions only inhibit direct interactions with the infected cell. To determine whether the virus also expresses soluble factors that could modulate NK function at a distance, we systematically screened all 170 HCMV canonical protein-coding genes. This revealed that UL4 encodes a secreted and heavily glycosylated protein (gpUL4) that is expressed with late-phase kinetics and is capable of inhibiting NK cell degranulation. Analyses of gpUL4 binding partners by mass spectrometry identified an interaction with TRAIL. gpUL4 bound TRAIL with picomolar affinity and prevented TRAIL from binding its receptor, thus acting as a TRAIL decoy receptor. TRAIL is found in both soluble and membrane-bound forms, with expression of the membrane-bound form strongly up-regulated on NK cells in response to interferon. gpUL4 inhibited apoptosis induced by soluble TRAIL, while also binding to the NK cell surface in a TRAIL-dependent manner, where it blocked NK cell degranulation and cytokine secretion. gpUL4 therefore acts as an immune-evasin by inhibiting both soluble and membrane-bound TRAIL and is a viral-encoded TRAIL decoy receptor. Interestingly, gpUL4 could also suppress NK responses to heterologous viruses, suggesting that it may act as a systemic virally encoded immunosuppressive agent.
Assuntos
Citomegalovirus , Células Matadoras Naturais , Humanos , Citomegalovirus/fisiologia , Evasão da Resposta Imune , Glicoproteínas/metabolismo , ApoptoseRESUMO
Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defenses. In exploring the finding that HCMV infection up-regulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory antiviral cytokine TNFα, we found that the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype 'sheddase', a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in its UL/b' region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with an HCMV double-deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins (P < 0.05) in an ADAM17-dependent fashion. These included reported substrates of ADAM17 with established immunological functions such as TNFR2 and jagged1, but also numerous unreported host and viral targets, such as nectin1, UL8, and UL144. Regulation of TNFα-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation.
Assuntos
Citomegalovirus , Fator de Necrose Tumoral alfa , Humanos , Citomegalovirus/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Proteoma/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Proteômica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Citocinas/metabolismo , Membrana Celular/metabolismo , Metaloproteases/metabolismo , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Virais/metabolismoRESUMO
CD58 is an adhesion molecule that is known to play a critical role in costimulation of effector cells and is intrinsic to immune synapse structure. Herein, we describe a virally encoded gene that inhibits CD58 surface expression. Human cytomegalovirus (HCMV) UL148 was necessary and sufficient to promote intracellular retention of CD58 during HCMV infection. Blocking studies with antagonistic anti-CD58 mAb and an HCMV UL148 deletion mutant (HCMV∆UL148) with restored CD58 expression demonstrated that the CD2/CD58 axis was essential for the recognition of HCMV-infected targets by CD8+ HCMV-specific cytotoxic T lymphocytes (CTLs). Further, challenge of peripheral blood mononuclear cells ex vivo with HCMV∆UL148 increased both CTL and natural killer (NK) cell degranulation against HCMV-infected cells, including NK-driven antibody-dependent cellular cytotoxicity, showing that UL148 is a modulator of the function of multiple effector cell subsets. Our data stress the effect of HCMV immune evasion functions on shaping the immune response, highlighting the capacity for their potential use in modulating immunity during the development of anti-HCMV vaccines and HCMV-based vaccine vectors.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Evasão da Resposta Imune , Imunidade Celular , Células Matadoras Naturais/imunologia , Proteínas Virais de Fusão/imunologia , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Transformada , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/patologia , Humanos , Células Matadoras Naturais/patologia , Proteínas Virais de Fusão/genéticaRESUMO
Molluscum contagiosum virus (MCV) is a common cause of benign skin lesions in young children and currently the only endemic human poxvirus. Following the infection of primary keratinocytes in the epidermis, MCV induces the proliferation of infected cells and this results in the production of wart-like growths. Full productive infection is observed only after the infected cells differentiate. During this prolonged replication cycle the virus must avoid elimination by the host immune system. We therefore sought to investigate the function of the two major histocompatibility complex class-I-related genes encoded by the MCV genes mc033 and mc080. Following insertion into a replication-deficient adenovirus vector, codon-optimized versions of mc033 and mc080 were expressed as endoglycosidase-sensitive glycoproteins that localized primarily in the endoplasmic reticulum. MC080, but not MC033, downregulated cell-surface expression of endogenous classical human leucocyte antigen (HLA) class I and non-classical HLA-E by a transporter associated with antigen processing (TAP)-independent mechanism. MC080 exhibited a capacity to inhibit or activate NK cells in autologous assays in a donor-specific manner. MC080 consistently inhibited antigen-specific T cells being activated by peptide-pulsed targets. We therefore propose that MC080 acts to promote evasion of HLA-I-restricted cytotoxic T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Regulação para Baixo/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Evasão da Resposta Imune/imunologia , Células Matadoras Naturais/imunologia , Vírus do Molusco Contagioso/imunologia , Apresentação de Antígeno/imunologia , Linhagem Celular , Retículo Endoplasmático/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Queratinócitos/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas Virais/imunologiaRESUMO
Human cytomegalovirus (HCMV) strains that have been passaged in vitro rapidly acquire mutations that impact viral growth. These laboratory-adapted strains of HCMV generally exhibit restricted tropism, produce high levels of cell-free virus, and develop susceptibility to natural killer cells. To permit experimentation with a virus that retained a clinically relevant phenotype, we reconstructed a wild-type (WT) HCMV genome using bacterial artificial chromosome technology. Like clinical virus, this genome proved to be unstable in cell culture; however, propagation of intact virus was achieved by placing the RL13 and UL128 genes under conditional expression. In this study, we show that WT-HCMV produces extremely low titers of cell-free virus but can efficiently infect fibroblasts, epithelial, monocyte-derived dendritic, and Langerhans cells via direct cell-cell transmission. This process of cell-cell transfer required the UL128 locus, but not the RL13 gene, and was significantly less vulnerable to the disruptive effects of IFN, cellular restriction factors, and neutralizing antibodies compared with cell-free entry. Resistance to neutralizing antibodies was dependent on high-level expression of the pentameric gH/gL/gpUL128-131A complex, a feature of WT but not passaged strains of HCMV.
Assuntos
Técnicas de Cultura de Células/métodos , Citomegalovirus/genética , Citomegalovirus/metabolismo , Anticorpos Neutralizantes , Linhagem Celular , Células Cultivadas , Cromossomos Artificiais Bacterianos/metabolismo , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/metabolismo , Fibroblastos/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Mutação , Fenótipo , Tropismo/imunologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Replicação Viral/imunologiaRESUMO
The genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination, and gene loss. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analyzed, in the process enabling the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating variation; (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination; (iii) mutants with nonfunctional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae; and (iv) intrahost variation in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology, and pathogenesis of HCMV.
Assuntos
Sequência de Bases , Infecções por Citomegalovirus/virologia , Citomegalovirus/genética , Genoma Viral , Recombinação Genética , DNA Viral/genética , Bases de Dados de Ácidos Nucleicos , Conjuntos de Dados como Assunto , Evolução Molecular , Genes Virais , Variação Genética , Genoma Viral/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Análise de Sequência de DNA , Sequenciamento Completo do GenomaRESUMO
UNLABELLED: Clinical human cytomegalovirus (HCMV) strains invariably mutate when propagatedin vitro Mutations in gene RL13 are selected in all cell types, whereas in fibroblasts mutants in the UL128 locus (UL128L; genes UL128, UL130, and UL131A) are also selected. In addition, sporadic mutations are selected elsewhere in the genome in all cell types. We sought to investigate conditions under which HCMV can be propagated without incurring genetic defects. Bacterial artificial chromosomes (BACs) provide a stable, genetically defined source of viral genome. Viruses were generated from BACs containing the genomes of strains TR, TB40, FIX, and Merlin, as well as from Merlin-BAC recombinants containing variant nucleotides in UL128L from TB40-BAC4 or FIX-BAC. Propagation of viruses derived from TR-BAC, TB40-BAC4, and FIX-BAC in either fibroblast or epithelial cells was associated with the generation of defects around the prokaryotic vector, which is retained in the unique short (US) region of viruses. This was not observed for Merlin-BAC, from which the vector is excised in derived viruses; however, propagation in epithelial cells was consistently associated with mutations in the unique longb' (UL/b') region, all impacting on gene UL141. Viruses derived from Merlin-BAC in fibroblasts had mutations in UL128L, but mutations occurred less frequently with recombinants containing UL128L nucleotides from TB40-BAC4 or FIX-BAC. Viruses derived from a Merlin-BAC derivative in which RL13 and UL128L were either mutated or repressed were remarkably stable in fibroblasts. Thus, HCMV containing a wild-type gene complement can be generatedin vitroby deriving virus from a self-excising BAC in fibroblasts and repressing RL13 and UL128L. IMPORTANCE: Researchers should aim to study viruses that accurately represent the causative agents of disease. This is problematic for HCMV because clinical strains mutate rapidly when propagatedin vitro, becoming less cell associated, altered in tropism, more susceptible to natural killer cells, and less pathogenic. Following isolation from clinical material, HCMV genomes can be stabilized by cloning into bacterial artificial chromosomes (BACs), and then virus is regenerated by DNA transfection. However, mutations can occur not only during isolation prior to BAC cloning but also when virus is regenerated. We have identified conditions under which BAC-derived viruses containing an intact, wild-type genome can be propagatedin vitrowith minimal risk of mutants being selected, enabling studies of viruses expressing the gene complement of a clinical strain. However, even under these optimized conditions, sporadic mutations can occur, highlighting the advisability of sequencing the HCMV stocks used in experiments.
Assuntos
Cromossomos Artificiais Bacterianos , Citomegalovirus/crescimento & desenvolvimento , Cultura de Vírus/métodos , Linhagem Celular , Citomegalovirus/genética , Células Epiteliais , Fibroblastos , Genes Virais , Genoma Viral , Instabilidade Genômica , Humanos , Técnicas In Vitro , Glicoproteínas de Membrana/genética , Proteínas do Envelope Viral/genéticaRESUMO
Human cytomegalovirus (HCMV) US2, US3, US6 and US11 act in concert to prevent immune recognition of virally infected cells by CD8+ T-lymphocytes through downregulation of MHC class I molecules (MHC-I). Here we show that US2 function goes far beyond MHC-I degradation. A systematic proteomic study using Plasma Membrane Profiling revealed US2 was unique in downregulating additional cellular targets, including: five distinct integrin α-chains, CD112, the interleukin-12 receptor, PTPRJ and thrombomodulin. US2 recruited the cellular E3 ligase TRC8 to direct the proteasomal degradation of all its targets, reminiscent of its degradation of MHC-I. Whereas integrin α-chains were selectively degraded, their integrin ß1 binding partner accumulated in the ER. Consequently integrin signaling, cell adhesion and migration were strongly suppressed. US2 was necessary and sufficient for degradation of the majority of its substrates, but remarkably, the HCMV NK cell evasion function UL141 requisitioned US2 to enhance downregulation of the NK cell ligand CD112. UL141 retained CD112 in the ER from where US2 promoted its TRC8-dependent retrotranslocation and degradation. These findings redefine US2 as a multifunctional degradation hub which, through recruitment of the cellular E3 ligase TRC8, modulates diverse immune pathways involved in antigen presentation, NK cell activation, migration and coagulation; and highlight US2's impact on HCMV pathogenesis.
Assuntos
Evasão da Resposta Imune/imunologia , Glicoproteínas de Membrana/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Citomegalovirus/imunologia , Citometria de Fluxo , Humanos , Immunoblotting , Imunoprecipitação , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Proteômica/métodos , RNA Interferente Pequeno , Transdução GenéticaRESUMO
CD200 receptor (CD200R) negatively regulates peripheral and mucosal innate immune responses. Viruses, including herpesviruses, have acquired functional CD200 orthologs, implying that viral exploitation of this pathway is evolutionary advantageous. However, the role that CD200R signaling plays during herpesvirus infection in vivo requires clarification. Utilizing the murine cytomegalovirus (MCMV) model, we demonstrate that CD200R facilitates virus persistence within mucosal tissue. Specifically, MCMV infection of CD200R-deficient mice (CD200R(-/-)) elicited heightened mucosal virus-specific CD4 T cell responses that restricted virus persistence in the salivary glands. CD200R did not directly inhibit lymphocyte effector function. Instead, CD200R(-/-) mice exhibited enhanced APC accumulation that in the mucosa was a consequence of elevated cellular proliferation. Although MCMV does not encode an obvious CD200 homolog, productive replication in macrophages induced expression of cellular CD200. CD200 from hematopoietic and non-hematopoietic cells contributed independently to suppression of antiviral control in vivo. These results highlight the CD200-CD200R pathway as an important regulator of antiviral immunity during cytomegalovirus infection that is exploited by MCMV to establish chronicity within mucosal tissue.
Assuntos
Antígenos CD/imunologia , Infecções por Citomegalovirus/imunologia , Macrófagos/imunologia , Mucosa/imunologia , Mucosa/virologia , Animais , Citomegalovirus/imunologia , Infecções por Citomegalovirus/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Imunofluorescência , Macrófagos/metabolismo , Macrófagos/virologia , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Human cytomegalovirus (HCMV) is an important opportunistic pathogen in immunocompromised patients and a major cause of congenital birth defects when acquired in utero. In the 1990s, four chimeric viruses were constructed by replacing genome segments of the high passage Towne strain with segments of the low passage Toledo strain, with the goal of obtaining live attenuated vaccine candidates that remained safe but were more immunogenic than the overly attenuated Towne vaccine. The chimeras were found to be safe when administered to HCMV-seronegative human volunteers, but to differ significantly in their ability to induce seroconversion. This suggests that chimera-specific genetic differences impacted the ability to replicate or persist in vivo and the consequent ability to induce an antibody response. To identify specific genomic breakpoints between Towne and Toledo sequences and establish whether spontaneous mutations or rearrangements had occurred during construction of the chimeras, complete genome sequences were determined. No major deletions or rearrangements were observed, although a number of unanticipated mutations were identified. However, no clear association emerged between the genetic content of the chimeras and the reported levels of vaccine-induced HCMV-specific humoral or cellular immune responses, suggesting that multiple genetic determinants are likely to impact immunogenicity. In addition to revealing the genome organization of the four vaccine candidates, this study provided an opportunity to probe the genetics of HCMV attenuation in humans. The results may be valuable in the future design of safe live or replication-defective vaccines that optimize immunogenicity and efficacy.
Assuntos
Infecções por Citomegalovirus/virologia , Vacinas contra Citomegalovirus/imunologia , Citomegalovirus/genética , Citomegalovirus/imunologia , Recombinação Genética , Anticorpos Antivirais/imunologia , Citomegalovirus/classificação , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/prevenção & controle , Vacinas contra Citomegalovirus/administração & dosagem , Vacinas contra Citomegalovirus/genética , Genoma Viral , Genômica , Humanos , ImunizaçãoRESUMO
The effect of abrogating the interferon (IFN) response on human cytomegalovirus (HCMV) replication was investigated using primary human cells engineered to block either the production of or the response to type I IFNs. In IFN-deficient cells, HCMV produced larger plaques and spread and replicated more rapidly than in parental cells. These cells demonstrate the vital role of IFNs in controlling HCMV replication and provide useful tools to investigate the IFN response to HCMV.
Assuntos
Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Interferon Tipo I/imunologia , Replicação Viral , Células Cultivadas , Humanos , Interferon Tipo I/deficiência , Ensaio de Placa ViralRESUMO
NKG2D plays a major role in controlling immune responses through the regulation of natural killer (NK) cells, αß and γδ T-cell function. This activating receptor recognizes eight distinct ligands (the MHC Class I polypeptide-related sequences (MIC) A andB, and UL16-binding proteins (ULBP)1-6) induced by cellular stress to promote recognition cells perturbed by malignant transformation or microbial infection. Studies into human cytomegalovirus (HCMV) have aided both the identification and characterization of NKG2D ligands (NKG2DLs). HCMV immediate early (IE) gene up regulates NKGDLs, and we now describe the differential activation of ULBP2 and MICA/B by IE1 and IE2 respectively. Despite activation by IE functions, HCMV effectively suppressed cell surface expression of NKGDLs through both the early and late phases of infection. The immune evasion functions UL16, UL142, and microRNA(miR)-UL112 are known to target NKG2DLs. While infection with a UL16 deletion mutant caused the expected increase in MICB and ULBP2 cell surface expression, deletion of UL142 did not have a similar impact on its target, MICA. We therefore performed a systematic screen of the viral genome to search of addition functions that targeted MICA. US18 and US20 were identified as novel NK cell evasion functions capable of acting independently to promote MICA degradation by lysosomal degradation. The most dramatic effect on MICA expression was achieved when US18 and US20 acted in concert. US18 and US20 are the first members of the US12 gene family to have been assigned a function. The US12 family has 10 members encoded sequentially through US12-US21; a genetic arrangement, which is suggestive of an 'accordion' expansion of an ancestral gene in response to a selective pressure. This expansion must have be an ancient event as the whole family is conserved across simian cytomegaloviruses from old world monkeys. The evolutionary benefit bestowed by the combinatorial effect of US18 and US20 on MICA may have contributed to sustaining the US12 gene family.
Assuntos
Citomegalovirus , Antígenos de Histocompatibilidade Classe I/metabolismo , Evasão da Resposta Imune , Células Matadoras Naturais/imunologia , Lisossomos/metabolismo , Proteólise , Proteínas Virais/fisiologia , Adulto , Proteínas de Bactérias/metabolismo , Células Cultivadas , Citomegalovirus/imunologia , Citomegalovirus/patogenicidade , Inibidores Enzimáticos/farmacologia , Humanos , Evasão da Resposta Imune/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Leupeptinas/farmacologia , Proteínas Luminescentes/metabolismo , Lisossomos/efeitos dos fármacos , Macrolídeos/farmacologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/fisiologia , Proteólise/efeitos dos fármacos , Proteínas Recombinantes/metabolismoRESUMO
In celebrating the 60th anniversary of the first isolation of human cytomegalovirus (HCMV), we reflect on the merits and limitations of the viral strains currently being used to develop urgently needed treatments. HCMV research has been dependent for decades on the high-passage strains AD169 and Towne, heavily exploiting their capacity to replicate efficiently in fibroblasts. However, the genetic integrity of these strains is so severely compromised that great caution needs to be exercised when considering their past and future use. It is now evident that wild-type HCMV strains are not readily propagated in vitro. HCMV mutants are rapidly selected during isolation in fibroblasts, reproducibly affecting gene RL13, the UL128 locus (which includes genes UL128, UL130 and UL131A) and often the U(L)/b' region. As a result, the virus becomes less cell associated, altered in tropism and less pathogenic. This problem is not restricted to high-passage strains, as even low-passage strains can harbour biologically significant mutations. Cloning and manipulation of the HCMV genome as a bacterial artificial chromosome (BAC) offers a means of working with stable, genetically defined strains. To this end, the low-passage strain Merlin genome was cloned as a BAC and sequentially repaired to match the viral sequence in the original clinical sample from which Merlin was derived. Restoration of UL128L to wild type was detrimental to growth in fibroblasts, whereas restoration of RL13 impaired growth in all cell types tested. Stable propagation of phenotypically wild-type virus could be achieved only by placing both regions under conditional expression. In addition to the development of these tools, the Merlin transcriptome and proteome have been characterized in unparalleled detail. Although Merlin may be representative of the clinical agent, high-throughput whole-genome deep sequencing studies have highlighted the remarkable high level of interstrain variation present in circulating virus. There is a need to develop systems capable of addressing the significance of this diversity, free from the confounding effects of genetic changes associated with in vitro adaptation. The generation of a set of BAC clones, each containing the genome of a different HCMV strain repaired to match the sequence in the clinical sample, would provide a pathway to address the biological and clinical effects of natural variation in wild-type HCMV.
Assuntos
Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Animais , Citomegalovirus/classificação , Evolução Molecular , Regulação Viral da Expressão Gênica , Genes Virais , Variação Genética , Genoma Viral , Humanos , Mutação , Seleção Genética , Biologia de SistemasRESUMO
Human cytomegalovirus (HCMV) is known to evade extrinsic pro-apoptotic pathways not only by downregulating cell surface expression of the death receptors TNFR1, TRAIL receptor 1 (TNFRSF10A) and TRAIL receptor 2 (TNFRSF10B), but also by impeding downstream signalling events. Fas (CD95/APO-1/TNFRSF6) also plays a prominent role in apoptotic clearance of virus-infected cells, so its fate in HCMV-infected cells needs to be addressed. Here, we show that cell surface expression of Fas was suppressed in HCMV-infected fibroblasts from 24 h onwards through the late phase of productive infection, and was dependent on de novo virus-encoded gene expression but not virus DNA replication. Significant levels of the fully glycosylated (endoglycosidase-H-resistant) Fas were retained within HCMV-infected cells throughout the infection within intracellular membranous structures. HCMV infection provided cells with a high level of protection against Fas-mediated apoptosis. Downregulation of Fas was observed with HCMV strains AD169, FIX, Merlin and TB40.
Assuntos
Citomegalovirus/fisiologia , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Receptor fas/antagonistas & inibidores , Receptor fas/imunologia , Células Cultivadas , Fibroblastos/virologia , HumanosRESUMO
Human CMV (HCMV)-encoded NK cell-evasion functions include an MHC class I homolog (UL18) with high affinity for the leukocyte inhibitory receptor-1 (CD85j, ILT2, or LILRB1) and a signal peptide (SP(UL40)) that acts by upregulating cell surface expression of HLA-E. Detailed characterization of SP(UL40) revealed that the N-terminal 14 aa residues bestowed TAP-independent upregulation of HLA-E, whereas C region sequences delayed processing of SP(UL40) by a signal peptide peptidase-type intramembrane protease. Most significantly, the consensus HLA-E-binding epitope within SP(UL40) was shown to promote cell surface expression of both HLA-E and gpUL18. UL40 was found to possess two transcription start sites, with utilization of the downstream site resulting in translation being initiated within the HLA-E-binding epitope (P2). Remarkably, this truncated SP(UL40) was functional and retained the capacity to upregulate gpUL18 but not HLA-E. Thus, our findings identify an elegant mechanism by which an HCMV signal peptide differentially regulates two distinct NK cell-evasion pathways. Moreover, we describe a natural SP(UL40) mutant that provides a clear example of an HCMV clinical virus with a defect in an NK cell-evasion function and exemplifies issues that confront the virus when adapting to immunogenetic diversity in the host.
Assuntos
Proteínas do Capsídeo/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Evasão da Resposta Imune/imunologia , Células Matadoras Naturais/imunologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Northern Blotting , Western Blotting , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Membrana Celular/imunologia , Membrana Celular/metabolismo , Separação Celular , Citomegalovirus/genética , Citomegalovirus/imunologia , Citomegalovirus/metabolismo , Infecções por Citomegalovirus , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Células Matadoras Naturais/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais/genética , Proteínas Virais/imunologia , Antígenos HLA-ERESUMO
Deep sequencing was used to bring high resolution to the human cytomegalovirus (HCMV) transcriptome at the stage when infectious virion production is under way, and major findings were confirmed by extensive experimentation using conventional techniques. The majority (65.1%) of polyadenylated viral RNA transcription is committed to producing four noncoding transcripts (RNA2.7, RNA1.2, RNA4.9, and RNA5.0) that do not substantially overlap designated protein-coding regions. Additional noncoding RNAs that are transcribed antisense to protein-coding regions map throughout the genome and account for 8.7% of transcription from these regions. RNA splicing is more common than recognized previously, which was evidenced by the identification of 229 potential donor and 132 acceptor sites, and it affects 58 protein-coding genes. The great majority (94) of 96 splice junctions most abundantly represented in the deep-sequencing data was confirmed by RT-PCR or RACE or supported by involvement in alternative splicing. Alternative splicing is frequent and particularly evident in four genes (RL8A, UL74A, UL124, and UL150A) that are transcribed by splicing from any one of many upstream exons. The analysis also resulted in the annotation of four previously unrecognized protein-coding regions (RL8A, RL9A, UL150A, and US33A), and expression of the UL150A protein was shown in the context of HCMV infection. The overall conclusion, that HCMV transcription is complex and multifaceted, has implications for the potential sophistication of virus functionality during infection. The study also illustrates the key contribution that deep sequencing can make to the genomics of nuclear DNA viruses.
Assuntos
Citomegalovirus/genética , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Transcriptoma , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Citomegalovirus/metabolismo , Éxons/genética , Genes Virais/genética , Genoma Viral/genética , Humanos , Immunoblotting , Masculino , Dados de Sequência Molecular , Poli A/genética , Splicing de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA/métodos , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
IL-10 is an immunomodulatory cytokine that acts to antagonize T cell responses elicited during acute and chronic infections. Thus, the IL-10R signaling pathway provides a potential therapeutic target in strategies aimed at combating infectious diseases. In this study, we set out to investigate whether IL-10 expression had an effect on NK cells. Murine CMV infection provides the best characterized in vivo system to evaluate the NK cell response, with NK cells being critical in the early control of acute infection. Blockade of IL-10R during acute murine CMV infection markedly reduced the accumulation of cytotoxic NK cells in the spleen and lung, a phenotype associated with a transient elevation of virus DNA load. Impaired NK cell responsiveness after IL-10R blockade was attributed to elevated levels of apoptosis observed in NK cells exhibiting an activated phenotype. Therefore, we conclude that IL-10 contributes to antiviral innate immunity during acute infection by restricting activation-induced death in NK cells.
Assuntos
Apoptose/imunologia , Infecções por Herpesviridae/imunologia , Imunidade Inata/imunologia , Interleucina-10/imunologia , Células Matadoras Naturais/imunologia , Animais , Separação Celular , Citometria de Fluxo , Interleucina-10/metabolismo , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Muromegalovirus/imunologia , Carga Viral/imunologiaRESUMO
TNF-like protein 1A (TL1A), a TNF superfamily cytokine that binds to death receptor 3 (DR3), is highly expressed in macrophage foam cell-rich regions of atherosclerotic plaques, although its role in foam cell formation has yet to be elucidated. We investigated whether TL1A can directly stimulate macrophage foam cell formation in both THP-1 and primary human monocyte-derived macrophages with the underlying mechanisms involved. We demonstrated that TL1A promotes foam cell formation in human macrophages in vitro by increasing both acetylated and oxidized low-density lipoprotein uptake, by enhancing intracellular total and esterified cholesterol levels and reducing cholesterol efflux. This imbalance in cholesterol homeostasis is orchestrated by TL1A-mediated changes in the mRNA and protein expression of several genes implicated in the uptake and efflux of cholesterol, such as scavenger receptor A and ATP-binding cassette transporter A1. Furthermore, through the use of virally delivered DR3 short-hairpin RNA and bone marrow-derived macrophages from DR3 knockout mice, we demonstrate that DR3 can regulate foam cell formation and contributes significantly to the action of TL1A in this process in vitro. We show, for the first time, a novel proatherogenic role for both TL1A and DR3 that implicates this pathway as a target for the therapeutic intervention of atherosclerosis.
Assuntos
Diferenciação Celular/imunologia , Células Espumosas/citologia , Células Espumosas/imunologia , Membro 25 de Receptores de Fatores de Necrose Tumoral/fisiologia , Transdução de Sinais/imunologia , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/fisiologia , Animais , Aterosclerose/imunologia , Aterosclerose/patologia , Transporte Biológico/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Feminino , Células Espumosas/patologia , Humanos , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Lipoproteínas LDL/metabolismo , Camundongos , Camundongos Knockout , Membro 25 de Receptores de Fatores de Necrose Tumoral/deficiência , Regulação para Cima/imunologiaRESUMO
Human cytomegalovirus (HCMV) UL141 induces protection against natural killer cell-mediated cytolysis by downregulating cell surface expression of CD155 (nectin-like molecule 5; poliovirus receptor), a ligand for the activating receptor DNAM-1 (CD226). However, DNAM-1 is also recognized to bind a second ligand, CD112 (nectin-2). We now show that HCMV targets CD112 for proteasome-mediated degradation by 48 h post-infection, thus removing both activating ligands for DNAM-1 from the cell surface during productive infection. Significantly, cell surface expression of both CD112 and CD155 was restored when UL141 was deleted from the HCMV genome. While gpUL141 alone is sufficient to mediate retention of CD155 in the endoplasmic reticulum, UL141 requires assistance from additional HCMV-encoded functions to suppress expression of CD112.