RESUMEN
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.
Asunto(s)
Citomegalovirus , Células Asesinas Naturales , Humanos , Citomegalovirus/fisiología , Evasión Inmune , Glicoproteínas/metabolismo , ApoptosisRESUMEN
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.
Asunto(s)
Citomegalovirus , Factor de Necrosis Tumoral alfa , Humanos , Citomegalovirus/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Proteoma/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Proteómica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Citocinas/metabolismo , Membrana Celular/metabolismo , Metaloproteasas/metabolismo , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Virales/metabolismoRESUMEN
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.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Regulación hacia Abajo/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Evasión Inmune/inmunología , Células Asesinas Naturales/inmunología , Virus del Molusco Contagioso/inmunología , Presentación de Antígeno/inmunología , Línea Celular , Retículo Endoplásmico/inmunología , Interacciones Huésped-Patógeno/inmunología , Humanos , Queratinocitos/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas Virales/inmunologíaRESUMEN
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.
Asunto(s)
Secuencia de Bases , Infecciones por Citomegalovirus/virología , Citomegalovirus/genética , Genoma Viral , Recombinación Genética , ADN Viral/genética , Bases de Datos de Ácidos Nucleicos , Conjuntos de Datos como Asunto , Evolución Molecular , Genes Virales , Variación Genética , Genoma Viral/genética , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , Análisis de Secuencia de ADN , Secuenciación Completa del GenomaRESUMEN
Human cytomegalovirus (HCMV) is an important pathogen with multiple immune evasion strategies, including virally facilitated degradation of host antiviral restriction factors. Here, we describe a multiplexed approach to discover proteins with innate immune function on the basis of active degradation by the proteasome or lysosome during early-phase HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, with high confidence we identified 35 proteins enriched in antiviral restriction factors. A final screen employed a comprehensive panel of viral mutants to predict viral genes that target >250 human proteins. This approach revealed that helicase-like transcription factor (HLTF), a DNA helicase important in DNA repair, potently inhibits early viral gene expression but is rapidly degraded during infection. The functionally unknown HCMV protein UL145 facilitates HLTF degradation by recruiting the Cullin4 E3 ligase complex. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses.
Asunto(s)
Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Proteínas/química , Proteínas Virales/química , Citomegalovirus/genética , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/virología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Humanos , Evasión Inmune , Estabilidad Proteica , Proteínas/genética , Proteínas/inmunología , Proteómica , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
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.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Evasión Inmune , Inmunidad Celular , Células Asesinas Naturales/inmunología , Proteínas Virales de Fusión/inmunología , Linfocitos T CD8-positivos/patología , Línea Celular Transformada , Citomegalovirus/genética , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/patología , Humanos , Células Asesinas Naturales/patología , Proteínas Virales de Fusión/genéticaRESUMEN
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.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Citomegalovirus/genética , Citomegalovirus/metabolismo , Anticuerpos Neutralizantes , Línea Celular , Células Cultivadas , Cromosomas Artificiales Bacterianos/metabolismo , Citomegalovirus/patogenicidad , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/metabolismo , Fibroblastos/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Mutación , Fenotipo , Tropismo/inmunología , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Replicación Viral/inmunologíaRESUMEN
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.
Asunto(s)
Infecciones por Citomegalovirus/virología , Vacunas contra Citomegalovirus/inmunología , Citomegalovirus/genética , Citomegalovirus/inmunología , Recombinación Genética , Anticuerpos Antivirales/inmunología , Citomegalovirus/clasificación , Citomegalovirus/aislamiento & purificación , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/prevención & control , Vacunas contra Citomegalovirus/administración & dosificación , Vacunas contra Citomegalovirus/genética , Genoma Viral , Genómica , Humanos , InmunizaciónRESUMEN
The human cytomegalovirus (HCMV) US12 family consists of ten sequentially arranged genes (US12-21) with poorly characterized function. We now identify novel natural killer (NK) cell evasion functions for four members: US12, US14, US18 and US20. Using a systematic multiplexed proteomics approach to quantify ~1300 cell surface and ~7200 whole cell proteins, we demonstrate that the US12 family selectively targets plasma membrane proteins and plays key roles in regulating NK ligands, adhesion molecules and cytokine receptors. US18 and US20 work in concert to suppress cell surface expression of the critical NKp30 ligand B7-H6 thus inhibiting NK cell activation. The US12 family is therefore identified as a major new hub of immune regulation.
Asunto(s)
Citomegalovirus/inmunología , Citomegalovirus/patogenicidad , Interacciones Huésped-Patógeno , Factores Inmunológicos/antagonistas & inhibidores , Células Asesinas Naturales/inmunología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas Virales/metabolismo , Humanos , Evasión Inmune , ProteómicaRESUMEN
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.
Asunto(s)
Cromosomas Artificiales Bacterianos , Citomegalovirus/crecimiento & desarrollo , Cultivo de Virus/métodos , Línea Celular , Citomegalovirus/genética , Células Epiteliales , Fibroblastos , Genes Virales , Genoma Viral , Inestabilidad Genómica , Humanos , Técnicas In Vitro , Glicoproteínas de Membrana/genética , Proteínas del Envoltorio Viral/genéticaRESUMEN
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.
Asunto(s)
Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Citomegalovirus/fisiología , Animales , Citomegalovirus/clasificación , Evolución Molecular , Regulación Viral de la Expresión Génica , Genes Virales , Variación Genética , Genoma Viral , Humanos , Mutación , Selección Genética , Biología de SistemasRESUMEN
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.
Asunto(s)
Evasión Inmune/inmunología , Glicoproteínas de Membrana/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Cromatografía Líquida de Alta Presión , Citomegalovirus/inmunología , Citometría de Flujo , Humanos , Immunoblotting , Inmunoprecipitación , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Espectrometría de Masas , Proteínas de la Membrana/metabolismo , Proteómica/métodos , ARN Interferente Pequeño , Transducción GenéticaRESUMEN
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.
Asunto(s)
Antígenos CD/inmunología , Infecciones por Citomegalovirus/inmunología , Macrófagos/inmunología , Membrana Mucosa/inmunología , Membrana Mucosa/virología , Animales , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Macrófagos/metabolismo , Macrófagos/virología , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
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.
Asunto(s)
Citomegalovirus/inmunología , Citomegalovirus/fisiología , Interferón Tipo I/inmunología , Replicación Viral , Células Cultivadas , Humanos , Interferón Tipo I/deficiencia , Ensayo de Placa ViralRESUMEN
Immune evasion genes help human cytomegalovirus (HCMV) establish lifelong persistence. Without immune pressure, laboratory-adapted HCMV strains have undergone genetic alterations. Among these, the deletion of the UL/b' domain is associated with loss of virulence. In a screen of UL/b', we identified pUL135 as a protein responsible for the characteristic cytopathic effect of clinical HCMV strains that also protected from natural killer (NK) and T cell attack. pUL135 interacted directly with abl interactor 1 (ABI1) and ABI2 to recruit the WAVE2 regulatory complex to the plasma membrane, remodel the actin cytoskeleton and dramatically reduce the efficiency of immune synapse (IS) formation. An intimate association between F-actin filaments in target cells and the IS was dispelled by pUL135 expression. Thus, F-actin in target cells plays a critical role in synaptogenesis, and this can be exploited by pathogens to protect against cytotoxic immune effector cells. An independent interaction between pUL135 and talin disrupted cell contacts with the extracellular matrix.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Citomegalovirus/inmunología , Proteínas Virales/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Proteínas del Citoesqueleto/metabolismo , Interacciones Huésped-Patógeno , Humanos , Sinapsis Inmunológicas/virología , Inmunomodulación , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/virología , Talina/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Human Papillomavirus (HPV) infection is the primary cause of cervical neoplasia. HPV DNA is integrated into the human genome in the majority of cervical cancers. The nature of integration may differ with integration incorporating a single copy of HPV or occurring in concatenated form. Our understanding of HPV tumorigenesis is largely based on studies using characterised cell lines with defined integration sites; these cell lines provide an invaluable standard for validation of diagnostic assays. Cell lines also further understanding of integration mechanisms in clinical samples. The objective of this study was to explore integration assays and to investigate integration events in cell lines where HPV is integrated in concatenated form. Restriction site PCR and detection of integrated papillomavirus sequences were performed on DNA from SiHa and CaSki. A novel integration site on Xq27.3 and HPV genome rearrangements were detected in CaSki DNA. However, where integration was previously detected by FISH in CaSki, and reported to be integrated in concatenated form, integration was not detected by DIPS or RS-PCR. The data presented illustrate that HPV copy number can hinder integration detection; this needs consideration when interpreting results from tests applied to clinical samples.
Asunto(s)
Papillomaviridae/fisiología , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Integración Viral , Línea Celular Tumoral , Humanos , Papillomaviridae/genéticaRESUMEN
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.
Asunto(s)
Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Citomegalovirus/fisiología , Interacciones Huésped-Patógeno , Proteómica , Virología/métodos , Humanos , Evasión Inmune , Células Asesinas Naturales/inmunología , Transducción de Señal , Linfocitos T/inmunología , Proteínas Virales/análisisRESUMEN
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.
Asunto(s)
Citomegalovirus , Antígenos de Histocompatibilidad Clase I/metabolismo , Evasión Inmune , Células Asesinas Naturales/inmunología , Lisosomas/metabolismo , Proteolisis , Proteínas Virales/fisiología , Adulto , Proteínas Bacterianas/metabolismo , Células Cultivadas , Citomegalovirus/inmunología , Citomegalovirus/patogenicidad , Inhibidores Enzimáticos/farmacología , Humanos , Evasión Inmune/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Leupeptinas/farmacología , Proteínas Luminiscentes/metabolismo , Lisosomas/efectos de los fármacos , Macrólidos/farmacología , Subfamilia K de Receptores Similares a Lectina de Células NK/fisiología , Proteolisis/efectos de los fármacos , Proteínas Recombinantes/metabolismoRESUMEN
During primary infection, murine cytomegalovirus (MCMV) spreads systemically, resulting in virus replication and pathology in multiple organs. This disseminated infection is ultimately controlled, but the underlying immune defense mechanisms are unclear. Investigating the role of the cytokine IL-22 in MCMV infection, we discovered an unanticipated function for neutrophils as potent antiviral effector cells that restrict viral replication and associated pathogenesis in peripheral organs. NK-, NKT-, and T cell-secreted IL-22 orchestrated antiviral neutrophil-mediated responses via induction in stromal nonhematopoietic tissue of the neutrophil-recruiting chemokine CXCL1. The antiviral effector properties of infiltrating neutrophils were directly linked to the expression of TNF-related apoptosis-inducing ligand (TRAIL). Our data identify a role for neutrophils in antiviral defense, and establish a functional link between IL-22 and the control of antiviral neutrophil responses that prevents pathogenic herpesvirus infection in peripheral organs.
Asunto(s)
Infecciones por Herpesviridae/inmunología , Interleucinas/inmunología , Muromegalovirus/inmunología , Neutrófilos/inmunología , Ligando Inductor de Apoptosis Relacionado con TNF/biosíntesis , Animales , Antivirales , Quimiocina CXCL1/inmunología , Infecciones por Herpesviridae/patología , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Muromegalovirus/patogenicidad , Células T Asesinas Naturales/inmunología , Replicación Viral/inmunología , Interleucina-22RESUMEN
Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and RNA polymerase II mediated transcription. Several pharmacological CDK inhibitors are currently in clinical trials as potential cancer therapeutics and some of them also exhibit antiviral effects. Olomoucine II and roscovitine, purine-based inhibitors of CDKs, were described as effective antiviral agents that inhibit replication of a broad range of wild type human viruses. Olomoucine II and roscovitine show high selectivity for CDK7 and CDK9, with important functions in the regulation of RNA polymerase II transcription. RNA polymerase II is necessary for viral transcription and following replication in cells. We analyzed the effect of inhibition of CDKs by olomoucine II on gene expression from viral promoters and compared its effect to widely-used roscovitine. We found that both roscovitine and olomoucine II blocked the phosphorylation of RNA polymerase II C-terminal domain. However the repression of genes regulated by viral promoters was strongly dependent on gene localization. Both roscovitine and olomoucine II inhibited expression only when the viral promoter was not integrated into chromosomal DNA. In contrast, treatment of cells with genome-integrated viral promoters increased their expression even though there was decreased phosphorylation of the C-terminal domain of RNA polymerase II. To define the mechanism responsible for decreased gene expression after pharmacological CDK inhibitor treatment, the level of mRNA transcription from extrachromosomal DNA was determined. Interestingly, our results showed that inhibition of RNA polymerase II C-terminal domain phosphorylation increased the number of transcribed mRNAs. However, some of these mRNAs were truncated and lacked polyadenylation, which resulted in decreased translation. These results suggest that phosphorylation of RNA polymerase II C-terminal domain is critical for linking transcription and posttrancriptional processing of mRNA expressed from extrachromosomal DNA.