RESUMO
Natural killer (NK) cells are innate lymphocytes that provide critical host defense against pathogens and cancer. Originally heralded for their early and rapid effector activity, NK cells have been recognized over the last decade for their ability to undergo adaptive immune processes, including antigen-driven clonal expansion and generation of long-lived memory. This review presents an overview of how NK cells lithely partake in both innate and adaptive responses and how this versatility is manifest in human NK cell-mediated immunity.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Animais , Humanos , Imunidade Celular , Células Matadoras NaturaisRESUMO
Human cytomegalovirus (HCMV) infects the majority of the human population and represents the leading viral cause of congenital birth defects. HCMV utilizes the glycoproteins gHgLgO (Trimer) to bind to platelet-derived growth factor receptor alpha (PDGFRα) and transforming growth factor beta receptor 3 (TGFßR3) to gain entry into multiple cell types. This complex is targeted by potent neutralizing antibodies and represents an important candidate for therapeutics against HCMV. Here, we determine three cryogenic electron microscopy (cryo-EM) structures of the trimer and the details of its interactions with four binding partners: the receptor proteins PDGFRα and TGFßR3 as well as two broadly neutralizing antibodies. Trimer binding to PDGFRα and TGFßR3 is mutually exclusive, suggesting that they function as independent entry receptors. In addition, Trimer-PDGFRα interaction has an inhibitory effect on PDGFRα signaling. Our results provide a framework for understanding HCMV receptor engagement, neutralization, and the development of anti-viral strategies against HCMV.
Assuntos
Citomegalovirus/química , Glicoproteínas de Membrana/química , Proteínas do Envelope Viral/química , Internalização do Vírus , Microscopia Crioeletrônica , Citomegalovirus/fisiologia , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Proteoglicanas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/química , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas do Envelope Viral/metabolismoRESUMO
Characterizing cell surface receptors mediating viral infection is critical for understanding viral tropism and developing antiviral therapies. Nevertheless, due to challenges associated with detecting protein interactions on the cell surface, the host receptors of many human pathogens remain unknown. Here, we build a library consisting of most single transmembrane human receptors and implement a workflow for unbiased and high-sensitivity detection of receptor-ligand interactions. We apply this technology to elucidate the long-sought receptor of human cytomegalovirus (HCMV), the leading viral cause of congenital birth defects. We identify neuropilin-2 (Nrp2) as the receptor for HCMV-pentamer infection in epithelial/endothelial cells and uncover additional HCMV interactors. Using a combination of biochemistry, cell-based assays, and electron microscopy, we characterize the pentamer-Nrp2 interaction and determine the architecture of the pentamer-Nrp2 complex. This work represents an important approach to the study of host-pathogen interactions and provides a framework for understanding HCMV infection, neutralization, and the development of novel anti-HCMV therapies.
Assuntos
Infecções por Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Neuropilina-2/metabolismo , Receptores Virais/metabolismo , Anticorpos Neutralizantes/química , Membrana Celular/metabolismo , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Mapeamento de Epitopos , Feminino , Células HEK293 , Humanos , Conformação Proteica , Proteínas do Envelope Viral/metabolismo , Internalização do VírusRESUMO
Human cytomegalovirus (HCMV) can cause severe diseases in fetuses, newborns, and immunocompromised individuals. Currently, no vaccines are approved, and treatment options are limited. Here, we analyzed the human B cell response of four HCMV top neutralizers from a cohort of 9,000 individuals. By single-cell analyses of memory B cells targeting the pentameric and trimeric HCMV surface complexes, we identified vulnerable sites on the shared gH/gL subunits as well as complex-specific subunits UL128/130/131A and gO. Using high-resolution cryogenic electron microscopy, we revealed the structural basis of the neutralization mechanisms of antibodies targeting various binding sites. Moreover, we identified highly potent antibodies that neutralized a broad spectrum of HCMV strains, including primary clinical isolates, that outperform known antibodies used in clinical trials. Our study provides a deep understanding of the mechanisms of HCMV neutralization and identifies promising antibody candidates to prevent and treat HCMV infection.
Assuntos
Citomegalovirus , Proteínas do Envelope Viral , Recém-Nascido , Humanos , Glicoproteínas de Membrana , Anticorpos Neutralizantes , Células B de Memória , Anticorpos Antivirais , Análise de Célula ÚnicaRESUMO
Human cytomegalovirus (HCMV) infection of monocytes is essential for viral dissemination and persistence. We previously identified that HCMV entry/internalization and subsequent productive infection of this clinically relevant cell type is distinct when compared to other infected cells. We showed that internalization and productive infection required activation of epidermal growth factor receptor (EGFR) and integrin/c-Src, via binding of viral glycoprotein B to EGFR, and the pentamer complex to ß1/ß3 integrins. To understand how virus attachment drives entry, we compared infection of monocytes with viruses containing the pentamer vs. those without the pentamer and then used a phosphoproteomic screen to identify potential phosphorylated proteins that influence HCMV entry and trafficking. The screen revealed that the most prominent pentamer-biased phosphorylated protein was the lipid- and protein-phosphatase phosphatase and tensin homolog (PTEN). PTEN knockdown with siRNA or PTEN inhibition with a PTEN inhibitor decreased pentamer-mediated HCMV entry, without affecting trimer-mediated entry. Inhibition of PTEN activity affected lipid metabolism and interfered with the onset of the endocytic processes required for HCMV entry. PTEN inactivation was sufficient to rescue pentamer-null HCMV from lysosomal degradation. We next examined dephosphorylation of a PTEN substrate Rab7, a regulator of endosomal maturation. Inhibition of PTEN activity prevented dephosphorylation of Rab7. Phosphorylated Rab7, in turn, blocked early endosome to late endosome maturation and promoted nuclear localization of the virus and productive infection.
Assuntos
Monócitos , Internalização do Vírus , Humanos , Células Cultivadas , Monócitos/metabolismo , Citomegalovirus/fisiologia , Receptores ErbB/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismoRESUMO
Herpesviruses modulate immune control to secure lifelong infection. The mechanisms Human Cytomegalovirus (HCMV) employs in this regard can reveal unanticipated aspects of cellular signaling involved in antiviral immunity. Here, we describe a novel relationship between the TGF-ß family cytokine BMP9 and HCMV infection. We identify a cross-talk between BMP9-induced and IFN receptor-mediated signaling, showing that BMP9 boosts the transcriptional response to and antiviral activity of IFNß, thereby enhancing viral restriction. We also show that BMP9 is secreted by human fibroblasts upon HCMV infection. However, HCMV infection impairs BMP9-induced enhancement of the IFNß response, indicating that this signaling role of BMP9 is actively targeted by HCMV. Indeed, transmembrane proteins US18 and US20, which downregulate type I BMP receptors, are necessary and sufficient to cause inhibition of BMP9-mediated boosting of the antiviral response to IFNß. HCMV lacking US18 and US20 is more sensitive to IFNß. Thus, HCMV has a mutually antagonistic relationship with BMP9, which extends the growing body of evidence that BMP signaling is an underappreciated modulator of innate immunity in response to viral infection.
Assuntos
Fator 2 de Diferenciação de Crescimento , Imunidade Inata , Humanos , Citocinas/metabolismo , Citomegalovirus/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Transdução de SinaisRESUMO
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
Viruses have evolved a multitude of mechanisms to combat barriers to productive infection in the host cell. Virally-encoded miRNAs are one such means to regulate host gene expression in ways that benefit the virus lifecycle. miRNAs are small non-coding RNAs that regulate protein expression but do not trigger the adaptive immune response, making them powerful tools encoded by viruses to regulate cellular processes. Diverse viruses encode for miRNAs but little sequence homology exists between miRNAs of different viral species. Despite this, common cellular pathways are targeted for regulation, including apoptosis, immune evasion, cell growth and differentiation. Herein we will highlight the viruses that encode miRNAs and provide mechanistic insight into how viral miRNAs aid in lytic and latent infection by targeting common cellular processes. We also highlight how viral miRNAs can mimic host cell miRNAs as well as how viral miRNAs have evolved to regulate host miRNA expression. These studies dispel the myth that viral miRNAs are subtle regulators of gene expression, and highlight the critical importance of viral miRNAs to the virus lifecycle.
Assuntos
MicroRNAs , Vírus , MicroRNAs/genética , MicroRNAs/metabolismo , Vírus/genética , Vírus/metabolismo , Diferenciação Celular , Processamento de Proteína Pós-Traducional , Expressão Gênica , Regulação Viral da Expressão Gênica/genética , Regulação da Expressão GênicaRESUMO
Unlike most RNA and DNA viruses that broadly stimulate mRNA decay and interfere with host gene expression, human cytomegalovirus (HCMV) extensively remodels the host translatome without producing an mRNA decay enzyme. By performing a targeted loss-of-function screen in primary human fibroblasts, we here identify the host CCR4-NOT deadenylase complex members CNOT1 and CNOT3 as unexpected pro-viral host factors that selectively regulate HCMV reproduction. We find that the scaffold subunit CNOT1 is specifically required for late viral gene expression and genome-wide host responses in CCR4-NOT-disrupted cells. By profiling poly(A)-tail lengths of individual HCMV and host mRNAs using nanopore direct RNA sequencing, we reveal poly(A)-tails of viral messages to be markedly longer than those of cellular mRNAs and significantly less sensitive to CCR4-NOT disruption. Our data establish that mRNA deadenylation by host CCR4-NOT is critical for productive HCMV replication and define a new mechanism whereby herpesvirus infection subverts cellular mRNA metabolism to remodel the gene expression landscape of the infected cell. Moreover, we expose an unanticipated host factor with potential to become a therapeutic anti-HCMV target.
Assuntos
Infecções por Herpesviridae , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR4/genética , Receptores CCR4/metabolismoRESUMO
Herpesviruses are ubiquitous, genetically diverse DNA viruses, with long-term presence in humans associated with infrequent but significant pathology. Human leukocyte antigen (HLA) class I presents intracellularly derived peptide fragments from infected tissue cells to CD8+ T and natural killer cells, thereby directing antiviral immunity. Allotypes of highly polymorphic HLA class I are distinguished by their peptide binding repertoires. Because this HLA class I variation is a major determinant of herpesvirus disease, we examined if sequence diversity of virus proteins reflects evasion of HLA presentation. Using population genomic data from EpsteinBarr virus (EBV), human cytomegalovirus (HCMV), and VaricellaZoster virus, we tested whether diversity differed between the regions of herpesvirus proteins that can be recognized, or not, by HLA class I. Herpesviruses exhibit lytic and latent infection stages, with the latter better enabling immune evasion. Whereas HLA binding peptides of lytic proteins are conserved, we found that EBV and HCMV proteins expressed during latency have increased peptide sequence diversity. Similarly, latent, but not lytic, herpesvirus proteins have greater population structure in HLA binding than nonbinding peptides. Finally, we found patterns consistent with EBV adaption to the local HLA environment, with less efficient recognition of EBV isolates by high-frequency HLA class I allotypes. Here, the frequency of CD8+ T cell epitopes inversely correlated with the frequency of HLA class I recognition. Previous analyses have shown that pathogen-mediated natural selection maintains exceptional polymorphism in HLA residues that determine peptide recognition. Here, we show that HLA class I peptide recognition impacts diversity of globally widespread pathogens.
Assuntos
Herpesviridae , Antígenos de Histocompatibilidade Classe I , Peptídeos , Variação Genética , Herpesviridae/genética , Herpesviridae/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Peptídeos/genéticaRESUMO
Replication-dependent (RD) histones are deposited onto human cytomegalovirus (HCMV) genomes at the start of infection. We examined how HCMV affects the de novo production of RD histones and found that viral infection blocked the accumulation of RD histone mRNAs that normally occurs during the S phase. Furthermore, RD histone mRNAs present in HCMV-infected cells did not undergo the unique 3' processing required for their normal nuclear export and translation. The protein that orchestrates processing in the nucleus, stem loopbinding protein (SLBP), was found predominantly in the cytoplasm, and RD histone proteins were not de novo synthesized in HCMV-infected cells. Intriguingly, however, we found that SLBP was required for the efficient synthesis and assembly of infectious progeny virions. We conclude that HCMV infection attenuates RD histone mRNA accumulation and processing and the de novo protein synthesis of the RD histones, while utilizing SLBP for an alternative purpose to support infectious virion production.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Histonas , Replicação Viral , Divisão Celular , Citomegalovirus/genética , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/virologia , Replicação do DNA , Histonas/metabolismo , HumanosRESUMO
Fundamental to mammalian intrinsic and innate immune defenses against pathogens is the production of Type I and Type II interferons, such as IFN-ß and IFN-γ, respectively. The comparative effects of IFN classes on the cellular proteome, protein interactions, and virus restriction within cell types that differentially contribute to immune defenses are needed for understanding immune signaling. Here, a multilayered proteomic analysis, paired with biochemical and molecular virology assays, allows distinguishing host responses to IFN-ß and IFN-γ and associated antiviral impacts during infection with several ubiquitous human viruses. In differentiated macrophage-like monocytic cells, we classified proteins upregulated by IFN-ß, IFN-γ, or pro-inflammatory LPS. Using parallel reaction monitoring, we developed a proteotypic peptide library for shared and unique ISG signatures of each IFN class, enabling orthogonal confirmation of protein alterations. Thermal proximity coaggregation analysis identified the assembly and maintenance of IFN-induced protein interactions. Comparative proteomics and cytokine responses in macrophage-like monocytic cells and primary keratinocytes provided contextualization of their relative capacities to restrict virus production during infection with herpes simplex virus type-1, adenovirus, and human cytomegalovirus. Our findings demonstrate how IFN classes induce distinct ISG abundance and interaction profiles that drive antiviral defenses within cell types that differentially coordinate mammalian immune responses.
Assuntos
Proteômica , Humanos , Proteômica/métodos , Inflamação/virologia , Inflamação/imunologia , Interferon gama/imunologia , Interferon beta/metabolismo , Interferon beta/imunologia , Interferon beta/genética , Imunidade Inata , Queratinócitos/virologia , Queratinócitos/imunologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Replicação Viral/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/virologia , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Citomegalovirus/imunologia , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 1/imunologia , Interferons/imunologia , Interferons/metabolismo , Interferons/genética , Lipopolissacarídeos/farmacologia , Monócitos/imunologia , Monócitos/virologia , Monócitos/metabolismo , Monócitos/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , ProteomaRESUMO
Latent human cytomegalovirus (hCMV) infection can pose a serious threat of reactivation and disease occurrence in immune-compromised individuals. Although T cells are at the core of the protective immune response to hCMV infection, a detailed characterization of different T cell subsets involved in hCMV immunity is lacking. Here, in an unbiased manner, we characterized over 8000 hCMV-reactive peripheral memory T cells isolated from seropositive human donors, at a single-cell resolution by analysing their single-cell transcriptomes paired with the T cell antigen receptor (TCR) repertoires. The hCMV-reactive T cells were highly heterogeneous and consisted of different developmental and functional memory T cell subsets such as, long-term memory precursors and effectors, T helper-17, T regulatory cells (TREGs) and cytotoxic T lymphocytes (CTLs) of both CD4 and CD8 origin. The hCMV-specific TREGs, in addition to being enriched for molecules known for their suppressive functions, showed enrichment for the interferon response signature gene sets. The hCMV-specific CTLs were of two types, the pre-effector- and effector-like. The co-clustering of hCMV-specific CD4-CTLs and CD8-CTLs in both pre-effector as well as effector clusters suggest shared transcriptomic signatures between them. The huge TCR clonal expansion of cytotoxic clusters suggests a dominant role in the protective immune response to CMV. The study uncovers the heterogeneity in the hCMV-specific memory T cells revealing many functional subsets with potential implications in better understanding of hCMV-specific T cell immunity. The data presented can serve as a knowledge base for designing vaccines and therapeutics.
Assuntos
Linfócitos T CD8-Positivos , Infecções por Citomegalovirus , Citomegalovirus , Células T de Memória , Receptores de Antígenos de Linfócitos T , Análise de Célula Única , Linfócitos T Citotóxicos , Transcriptoma , Humanos , Citomegalovirus/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/genética , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Células T de Memória/imunologia , Células T de Memória/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Perfilação da Expressão Gênica , Linfócitos T CD4-Positivos/imunologiaRESUMO
Natural killer (NK) cells are innate lymphocytes that participate in immune responses against virus-infected cells and tumors. As a countermeasure, viruses and tumors employ strategies to evade NK-cell-mediated immunosurveillance. In this review, we examine immune evasion strategies employed by viruses, focusing on examples from human CMV and severe acute respiratory syndrome coronavirus 2. We explore selected viral evasion mechanisms categorized into three classes: (1) providing ligands for the inhibitory receptor NKG2A, (2) downregulating ligands for the activating receptor NKG2D, and (3) inducing the immunosuppressive cytokine transforming growth factor (TGF)-ß. For each class, we draw parallels between immune evasion by viruses and tumors, reviewing potential opportunities for overcoming evasion in cancer therapy. We suggest that in-depth investigations of host-pathogen interactions between viruses and NK cells will not only deepen our understanding of viral immune evasion but also shed light on how NK cells counter such evasion attempts. Thus, due to the parallels of immune evasion by viruses and tumors, we propose that insights gained from antiviral NK-cell responses may serve as valuable lessons that can be leveraged for designing future cancer immunotherapies.
Assuntos
Células Matadoras Naturais , Neoplasias , Humanos , Monitorização Imunológica , Evasão da Resposta Imune , Neoplasias/terapia , Neoplasias/metabolismo , ImunoterapiaRESUMO
Human Cytomegalovirus (HCMV) infection is life-threatening for immunocompromised patients. Quantitative molecular assays on whole blood or plasma are the gold standard for the diagnosis of invasive HCMV infection and for monitoring antiviral treatment in individuals at risk of HCMV disease. For these reasons, an accurate standardization toward the WHO 1st International Standard among different centers and diagnostic kits represents an effort for better clinical management of HCMV-positive patients. Herein, we evaluate, for the first time, the performance of a new transcription-mediated amplification (TMA) assay versus quantitative polymerase chain reaction (qPCR) chemistry, used as a routine method, on whole blood samples. A total of 755 clinical whole blood specimens were collected and tested simultaneously with TMA and qPCR assays. The data showed a qualitative agreement of 99.27% for positive quantified samples and 89.39% for those undetected between the two tested methods. Evaluation of viremia in positive samples highlighted a good correlation between TMA and qPCR chemistries in terms of International Units (ΔLog10 IU/mL: -0.29 ± 0.40). The TMA assay showed a significant correlation with qPCR in patients monitored for up to 3 months, thus allowing an accurate assessment of viremia in transplant patients. Therefore, TMA chemistry showed good agreement with qPCR testing, used as a current diagnostic routine. It also offers important advantages, such as FDA approval on plasma and In Vitro Diagnostic (IVD) on both plasma and whole blood, automated workflow with minimal hands-on time, and random access loading, thus enabling a rapid and reliable diagnostic in HCMV-infected patients. IMPORTANCE: In this paper, we describe the clinical performance of a novel transcription-mediated amplification (TMA) assay for the detection and quantification of human Cytomegalovirus (HCMV) DNA from whole blood samples. This is a pivotal analysis in immunocompromised patients [transplanted, HIV-positive, and Hematopoietic Stem Cell (HSC) recipients], and molecular tests with high sensitivity and specificity are necessary to evaluate the HCMV viral load in these patients. To our knowledge, this is the first in-depth evaluation of TMA chemistry for HCMV diagnosis on whole blood samples. Moreover, also technical aspects of this assay make it suitable for clinical diagnostics.
Assuntos
Infecções por Citomegalovirus , Viremia , Humanos , Reação em Cadeia da Polimerase/métodos , Citomegalovirus/genética , Hospedeiro Imunocomprometido , DNA Viral/genéticaRESUMO
Viperin is a multifunctional interferon-inducible protein that is directly induced in cells by human cytomegalovirus (HCMV) infection. The viral mitochondrion-localized inhibitor of apoptosis (vMIA) interacts with viperin at the early stages of infection and translocates it from the endoplasmic reticulum to the mitochondria, where viperin modulates the cellular metabolism to increase viral infectivity. Viperin finally relocalizes to the viral assembly compartment (AC) at late stages of infection. Despite the importance of vMIA interactions with viperin during viral infection, their interacting residues are unknown. In the present study, we showed that cysteine residue 44 (Cys44) of vMIA and the N-terminal domain (amino acids [aa] 1 to 42) of viperin are necessary for their interaction and for the mitochondrial localization of viperin. In addition, the N-terminal domain of mouse viperin, which is structurally similar to that of human viperin, interacted with vMIA. This indicates that the structure, rather than the sequence composition, of the N-terminal domain of viperin, is required for the interaction with vMIA. Recombinant HCMV, in which Cys44 of vMIA was replaced by an alanine residue, failed to translocate viperin to the mitochondria at the early stages of infection and inefficiently relocalized it to the AC at late stages of infection, resulting in the impairment of viperin-mediated lipid synthesis and a reduction in viral replication. These data indicate that Cys44 of vMIA is therefore essential for the intracellular trafficking and function of viperin to increase viral replication. Our findings also suggest that the interacting residues of these two proteins are potential therapeutic targets for HCMV-associated diseases. IMPORTANCE Viperin traffics to the endoplasmic reticulum (ER), mitochondria, and viral assembly compartment (AC) during human cytomegalovirus (HCMV) infection. Viperin has antiviral activity at the ER and regulates cellular metabolism at the mitochondria. Here, we show that Cys44 of HCMV vMIA protein and the N-terminal domain (aa 1 to 42) of viperin are necessary for their interaction. Cys44 of vMIA also has a critical role for viperin trafficking from the ER to the AC via the mitochondria during viral infection. Recombinant HCMV expressing a mutant vMIA Cys44 has impaired lipid synthesis and viral infectivity, which are attributed to mislocalization of viperin. Cys44 of vMIA is essential for the trafficking and function of viperin and may be a therapeutic target for HCMV-associated diseases.
Assuntos
Proteínas Imediatamente Precoces , Proteína Viperina , Proteínas Virais , Viroses , Animais , Humanos , Camundongos , Cisteína/metabolismo , Citomegalovirus/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Lipídeos , Mitocôndrias/metabolismo , Viroses/metabolismo , Proteína Viperina/metabolismo , Proteínas Virais/metabolismoRESUMO
Human cytomegalovirus (HCMV) is a leading cause of congenital birth defects. Though the underlying mechanisms remain poorly characterized, mouse models of congenital CMV infection have demonstrated that the neuronal migration process is damaged. In this study, we evaluated the effects of HCMV infection on connexin 43 (Cx43), a crucial adhesion molecule mediating neuronal migration. We show in multiple cellular models that HCMV infection downregulated Cx43 posttranslationally. Further analysis identified the immediate early protein IE1 as the viral protein responsible for the reduction of Cx43. IE1 was found to bind the Cx43 C terminus and promote Cx43 degradation through the ubiquitin-proteasome pathway. Deletion of the Cx43-binding site in IE1 rendered it incapable of inducing Cx43 degradation. We validated the IE1-induced loss of Cx43 in vivo by introducing IE1 into the fetal mouse brain. Noteworthily, ectopic IE1 expression induced cortical atrophy and neuronal migration defects. Several lines of evidence suggest that these damages result from decreased Cx43, and restoration of Cx43 levels partially rescued IE1-induced interruption of neuronal migration. Taken together, the results of our investigation reveal a novel mechanism of HCMV-induced neural maldevelopment and identify a potential intervention target. IMPORTANCE Congenital CMV (cCMV) infection causes neurological sequelae in newborns. Recent studies of cCMV pathogenesis in animal models reveal ventriculomegaly and cortical atrophy associated with impaired neural progenitor cell (NPC) proliferation and migration. In this study, we investigated the mechanisms underlying these NPC abnormalities. We show that Cx43, a critical adhesion molecule mediating NPC migration, is downregulated by HCMV infection in vitro and HCMV-IE1 in vivo. We provide evidence that IE1 interacts with the C terminus of Cx43 to promote its ubiquitination and consequent degradation through the proteasome. Moreover, we demonstrate that introducing IE1 into mouse fetal brains led to neuronal migration defects, which was associated with Cx43 reduction. Deletion of the Cx43-binding region in IE1 or ectopic expression of Cx43 rescued the IE1-induced migration defects in vivo. Our study provides insight into how cCMV infection impairs neuronal migration and reveals a target for therapeutic interventions.
Assuntos
Conexina 43 , Infecções por Citomegalovirus , Citomegalovirus , Proteínas Imediatamente Precoces , Animais , Humanos , Recém-Nascido , Camundongos , Conexina 43/genética , Conexina 43/metabolismo , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Gliomas are the most prevalent and devastating primary malignant brain tumors in adults. Despite substantial advances in understanding glioma biology, there have been no regulatory drug approvals in the US since bevacizumab in 2009 and tumor treating fields in 2011. Recent phase III clinical trials have failed to meet their prespecified therapeutic primary endpoints, highlighting the need for novel therapies. The poor prognosis of glioma patients, resistance to chemo-radiotherapy, and the immunosuppressive tumor microenvironment underscore the need for the development of novel therapies. Gene therapy-based immunotherapeutic strategies that couple the ability of the host immune system to specifically kill glioma cells and develop immunological memory have shown remarkable progress. Two adenoviral vectors expressing Ad-HSV1-TK/GCV and Ad-Flt3L have shown promising preclinical data, leading to FDA approval of a non-randomized, phase I open-label, first in human trial to test safety, cytotoxicity, and immune-stimulatory efficiency in high-grade glioma patients (NCT01811992). This review provides a thorough overview of immune-stimulatory gene therapy highlighting recent advancements, potential drawbacks, future directions, and recommendations for future implementation of clinical trials.
Assuntos
Neoplasias Encefálicas , Glioma , Animais , Humanos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/patologia , Roedores/genética , Adenoviridae/genética , Glioma/genética , Glioma/terapia , Glioma/patologia , Terapia Genética , Timidina Quinase/genética , Vetores Genéticos/genética , Microambiente TumoralRESUMO
Once inside the host cell, DNA viruses must overcome the physical barrier posed by the nuclear envelope to establish a successful infection. The mechanism underlying this process remains unclear. Here, we show that the herpesvirus exploits the immune adaptor stimulator of interferon genes (STING) to facilitate nuclear import of the viral genome. Following the entry of the viral capsid into the cell, STING binds the viral capsid, mediates capsid docking to the nuclear pore complex via physical interaction, and subsequently enables accumulation of the viral genome in the nucleus. Silencing STING in human cytomegalovirus (HCMV)-susceptible cells inhibited nuclear import of the viral genome and reduced the ensuing viral gene expression. Overexpressing STING increased the host cell's susceptibility to HCMV and herpes simplex virus 1 by improving the nuclear delivery of viral DNA at the early stage of infection. These observations suggest that the proviral activity of STING is conserved and exploited by the herpesvirus family. Intriguingly, in monocytes, which act as latent reservoirs of HCMV, STING deficiency negatively regulated the establishment of HCMV latency and reactivation. Our findings identify STING as a proviral host factor regulating latency and reactivation of herpesviruses.
Assuntos
Citomegalovirus/fisiologia , DNA Viral/metabolismo , Genoma Viral/fisiologia , Proteínas de Membrana/metabolismo , Replicação Viral/fisiologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , DNA Viral/genética , Regulação Viral da Expressão Gênica , Proteínas de Membrana/genética , Interferência de RNA , RNA Interferente Pequeno , Internalização do VírusRESUMO
Viruses modulate mitochondrial processes during infection to increase biosynthetic precursors and energy output, fueling virus replication. In a surprising fashion, although it triggers mitochondrial fragmentation, the prevalent pathogen human cytomegalovirus (HCMV) increases mitochondrial metabolism through a yet-unknown mechanism. Here, we integrate molecular virology, metabolic assays, quantitative proteomics, and superresolution confocal microscopy to define this mechanism. We establish that the previously uncharacterized viral protein pUL13 is required for productive HCMV replication, targets the mitochondria, and functions to increase oxidative phosphorylation during infection. We demonstrate that pUL13 forms temporally tuned interactions with the mitochondrial contact site and cristae organizing system (MICOS) complex, a critical regulator of cristae architecture and electron transport chain (ETC) function. Stimulated emission depletion superresolution microscopy shows that expression of pUL13 alters cristae architecture. Indeed, using live-cell Seahorse assays, we establish that pUL13 alone is sufficient to increase cellular respiration, not requiring the presence of other viral proteins. Our findings address the outstanding question of how HCMV targets mitochondria to increase bioenergetic output and expands the knowledge of the intricate connection between mitochondrial architecture and ETC function.