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1.
Cell ; 157(6): 1460-1472, 2014 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-24906157

RESUMEN

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/análisis
2.
Proc Natl Acad Sci U S A ; 120(33): e2303155120, 2023 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-37561786

RESUMEN

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/metabolismo
3.
Proc Natl Acad Sci U S A ; 115(19): 4998-5003, 2018 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-29691324

RESUMEN

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/genética
4.
Proc Natl Acad Sci U S A ; 114(23): 6104-6109, 2017 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-28533400

RESUMEN

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/inmunología
5.
J Virol ; 90(8): 3929-43, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26842472

RESUMEN

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/genética
6.
PLoS Pathog ; 11(4): e1004811, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25875600

RESUMEN

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 Genética
7.
PLoS Pathog ; 10(5): e1004058, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24787765

RESUMEN

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/metabolismo
8.
Med Microbiol Immunol ; 204(3): 273-84, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25894764

RESUMEN

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 Sistemas
9.
J Gen Virol ; 95(Pt 4): 933-939, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24394698

RESUMEN

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.


Asunto(s)
Citomegalovirus/fisiología , Interacciones Huésped-Patógeno , Evasión Inmune , Receptor fas/antagonistas & inhibidores , Receptor fas/inmunología , Células Cultivadas , Fibroblastos/virología , Humanos
10.
J Virol ; 87(19): 10489-500, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23885075

RESUMEN

The human cytomegalovirus (HCMV) virion envelope contains a complex consisting of glycoproteins gH and gL plus proteins encoded by the UL128 locus (UL128L): pUL128, pUL130, and pUL131A. UL128L is necessary for efficient infection of myeloid, epithelial, and endothelial cells but limits replication in fibroblasts. Consequently, disrupting mutations in UL128L are rapidly selected when clinical isolates are cultured in fibroblasts. In contrast, bacterial artificial chromosome (BAC)-cloned strains TB40-BAC4, FIX, and TR do not contain overt disruptions in UL128L, yet no virus reconstituted from them has been reported to acquire mutations in UL128L in vitro. We performed BAC mutagenesis and reconstitution experiments to test the hypothesis that these strains contain subtle mutations in UL128L that were acquired during passage prior to BAC cloning. Compared to strain Merlin containing wild-type UL128L, all three strains produced higher yields of cell-free virus. Moreover, TB40-BAC4 and FIX spread cell to cell more rapidly than wild-type Merlin in fibroblasts but more slowly in epithelial cells. The differential growth properties of TB40-BAC4 and FIX (but not TR) were mapped to single-nucleotide substitutions in UL128L. The substitution in TB40-BAC4 reduced the splicing efficiency of UL128, and that in FIX resulted in an amino acid substitution in UL130. Introduction of these substitutions into Merlin dramatically increased yields of cell-free virus and increased cell-to-cell spread in fibroblasts but reduced the abundance of pUL128 in the virion and the efficiency of epithelial cell infection. These substitutions appear to represent mutations in UL128L that permit virus to be propagated in fibroblasts while retaining epithelial cell tropism.


Asunto(s)
Infecciones por Citomegalovirus/virología , Citomegalovirus/fisiología , Fibroblastos/virología , Glicoproteínas de Membrana/metabolismo , Epitelio Pigmentado de la Retina/virología , Proteínas del Envoltorio Viral/metabolismo , Western Blotting , Células Cultivadas , Cromosomas Artificiales Bacterianos/genética , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/virología , Feto , Fibroblastos/metabolismo , Prepucio/metabolismo , Prepucio/virología , Variación Genética , Humanos , Masculino , Glicoproteínas de Membrana/genética , Mutagénesis , Mutación/genética , Plásmidos/genética , Epitelio Pigmentado de la Retina/metabolismo , Tropismo , Proteínas del Envoltorio Viral/genética , Virión/fisiología , Internalización del Virus
11.
J Immunol ; 188(6): 2794-804, 2012 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-22345649

RESUMEN

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.


Asunto(s)
Proteínas de la Cápside/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Evasión Inmune/inmunología , Células Asesinas Naturales/inmunología , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Northern Blotting , Western Blotting , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Membrana Celular/inmunología , Membrana Celular/metabolismo , Separación Celular , Citomegalovirus/genética , Citomegalovirus/inmunología , Citomegalovirus/metabolismo , Infecciones por Citomegalovirus , Citometría de Flujo , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Células Asesinas Naturales/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Virales/genética , Proteínas Virales/inmunología , Antígenos HLA-E
12.
J Gen Virol ; 91(Pt 8): 2034-2039, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20410314

RESUMEN

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.


Asunto(s)
Citomegalovirus/inmunología , Citomegalovirus/patogenicidad , Tolerancia Inmunológica , Subunidad beta del Receptor de Interleucina-2/antagonistas & inhibidores , Células Asesinas Naturales/inmunología , Proteínas Virales/fisiología , Factores de Virulencia/fisiología , Células Cultivadas , Eliminación de Gen , Humanos , Receptores Virales/antagonistas & inhibidores , Proteínas Virales/genética , Proteínas Virales/inmunología , Factores de Virulencia/inmunología
13.
J Gen Virol ; 91(Pt 6): 1535-46, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20479471

RESUMEN

Mutations that occurred during adaptation of human cytomegalovirus to cell culture were monitored by isolating four strains from clinical samples, passaging them in various cell types and sequencing ten complete virus genomes from the final passages. Mutational dynamics were assessed by targeted sequencing of intermediate passages and the original clinical samples. Gene RL13 and the UL128 locus (UL128L, consisting of genes UL128, UL130 and UL131A) mutated in all strains. Mutations in RL13 occurred in fibroblast, epithelial and endothelial cells, whereas those in UL128L were limited to fibroblasts and detected later than those in RL13. In addition, a region containing genes UL145, UL144, UL142, UL141 and UL140 mutated in three strains. All strains exhibited numerous mutations in other regions of the genome, with a preponderance in parts of the inverted repeats. An investigation was carried out on the kinetic growth yields of viruses derived from selected passages that were predominantly non-mutated in RL13 and UL128L (RL13+UL128L+), or that were largely mutated in RL13 (RL13-UL128L+) or both RL13 and UL128L (RL13-UL128L-). RL13-UL128L- viruses produced greater yields of infectious progeny than RL13-UL128L+ viruses, and RL13-UL128L+ viruses produced greater yields than RL13+UL128L+ viruses. These results suggest strongly that RL13 and UL128L exert at least partially independent suppressive effects on growth in fibroblasts. As all isolates proved genetically unstable in all cell types tested, caution is advised in choosing and monitoring strains for experimental studies of vulnerable functions, particularly those involved in cell tropism, immune evasion or growth temperance.


Asunto(s)
Adaptación Biológica , Citomegalovirus/crecimiento & desarrollo , Citomegalovirus/genética , Mutación , Línea Celular , Citomegalovirus/aislamiento & purificación , Infecciones por Citomegalovirus/virología , Análisis Mutacional de ADN , ADN Viral/química , ADN Viral/genética , Células Endoteliales/virología , Células Epiteliales/virología , Fibroblastos/virología , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Pase Seriado , Proteínas Virales/genética
14.
J Virol ; 82(9): 4585-94, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18287244

RESUMEN

The adenovirus (Ad) early transcription unit 3 (E3) encodes multiple immunosubversive functions that are presumed to facilitate the establishment and persistence of infection. Indeed, the capacity of E3/19K to inhibit transport of HLA class I (HLA-I) to the cell surface, thereby preventing peptide presentation to CD8(+) T cells, has long been recognized as a paradigm for viral immune evasion. However, HLA-I downregulation has the potential to render Ad-infected cells vulnerable to natural killer (NK) cell recognition. Furthermore, expression of the immediate-early Ad gene E1A is associated with efficient induction of ligands for the key NK cell-activating receptor NKG2D. Here we show that while infection with wild-type Ad enhances synthesis of the NKG2D ligands, major histocompatibility complex class I chain-related proteins A and B (MICA and MICB), their expression on the cell surface is actively suppressed. Both MICA and MICB are retained within the endoplasmic reticulum as immature endoglycosidase H-sensitive forms. By analyzing a range of cell lines and viruses carrying mutated versions of the E3 gene region, E3/19K was identified as the gene responsible for this activity. The structural requirements within E3/19K necessary to sequester MICA/B and HLA-I are similar. In functional assays, deletion of E3/19K rendered Ad-infected cells more sensitive to NK cell recognition. We report the first NK evasion function in the Adenoviridae and describe a novel function for E3/19K. Thus, E3/19K has a dual function: inhibition of T-cell recognition and NK cell activation.


Asunto(s)
Proteínas E3 de Adenovirus/inmunología , Adenovirus Humanos/inmunología , Compartimento Celular , Antígenos de Histocompatibilidad Clase I/inmunología , Células Asesinas Naturales/inmunología , Receptores Inmunológicos/inmunología , Adenovirus Humanos/química , Expresión Génica , Inmunidad , Células Asesinas Naturales/virología , Ligandos , Receptores de Células Asesinas Naturales , Linfocitos T/inmunología
15.
Biotechniques ; 45(6): 659-62, 664-8, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19238796

RESUMEN

With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector of PCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector Vectors were optimized for high-throughput applications by making them "self-excising" through incorporating the I-SceI homing endonuclease into the vector removing the need to linearize vectors prior to transfection into packaging cells. AdZ vectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.


Asunto(s)
Adenoviridae/genética , Genes/fisiología , Ingeniería Genética/métodos , Vectores Genéticos , Adenoviridae/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Línea Celular , Cromosomas Artificiales Bacterianos/genética , Cromosomas Artificiales Bacterianos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Genes Sintéticos , Vectores Genéticos/metabolismo , Humanos , Proteínas Inmediatas-Precoces/genética , Mutagénesis Insercional , Análisis de Secuencia de ADN , Transactivadores/genética
16.
J Clin Virol ; 41(3): 206-12, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18069056

RESUMEN

Human cytomegalovirus (HCMV) causes lifelong, persistent infections and its survival is under intense, continuous selective pressure from the immune system. A key aspect of HCMV's capacity for survival lies in immune avoidance. In this context, cells undergoing productive infection exhibit remarkable resistance to natural killer (NK) cell-mediated cytolysis in vitro. To date, six genes encoding proteins (UL16, UL18, UL40, UL83, UL141 and UL142) and one encoding a microRNA (miR-UL112) have been identified as capable of suppressing NK cell recognition. Even though HCMV infection efficiently activates expression of ligands for the NK cell activating receptor NKG2D, at least three functions (UL16, UL142 and miR-UL112) act in concert to suppress presentation of these ligands on the cell surface. Although HCMV downregulates expression of endogenous MHC-I, it encodes an MHC-I homologue (UL18) and also upregulates the expression of cellular HLA-E through the action of UL40. The disruption of normal intercellular connections exposes ligands for NK cell activating receptors on the cell surface, notably CD155. HCMV overcomes this vulnerability by encoding a function (UL141) that acts post-translationally to suppress cell surface expression of CD155. The mechanisms by which HCMV systematically evades (or, more properly, modulates) NK cell recognition constitutes an area of growing understanding that is enhancing our appreciation of the basic mechanisms of NK cell function in humans.


Asunto(s)
Citomegalovirus/patogenicidad , Células Asesinas Naturales/inmunología , Proteínas Ligadas a GPI , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo
17.
Cell Host Microbe ; 24(3): 447-460.e11, 2018 09 12.
Artículo en Inglés | MEDLINE | ID: mdl-30122656

RESUMEN

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/inmunología
18.
Elife ; 62017 02 10.
Artículo en Inglés | MEDLINE | ID: mdl-28186488

RESUMEN

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 , Proteómica
19.
PLoS One ; 9(2): e89228, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24586613

RESUMEN

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.


Asunto(s)
Ciclo Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , ARN Polimerasa II/metabolismo , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , ADN Viral , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Purinas/farmacología , ARN Polimerasa II/genética , Roscovitina
20.
Cell Host Microbe ; 16(2): 201-214, 2014 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-25121749

RESUMEN

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/metabolismo
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