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1.
Methods Mol Biol ; 2244: 1-18, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555579

RESUMO

Human cytomegalovirus (HCMV) is a betaherpesvirus with a global seroprevalence of 60-90%. HCMV is the leading cause of congenital infections and poses a great health risk to immunocompromised individuals. Although HCMV infection is typically asymptomatic in the immunocompetent population, infection can result in mononucleosis and has also been associated with the development of certain cancers, as well as chronic inflammatory diseases such as various cardiovascular diseases. In immunocompromised patients, including AIDS patients, transplant recipients, and developing fetuses, HCMV infection is associated with increased rates of morbidity and mortality. Currently there is no vaccine for HCMV and there is a need for new pharmacological treatments. Ongoing research seeks to further define the complex aspects of HCMV pathogenesis, which could potentially lead to the generation of new therapeutics to mitigate the disease states associated with HCMV infection. The following chapter reviews the advancements in our understanding of HCMV pathogenesis in the immunocompetent and immunocompromised hosts.


Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Citomegalovirus/genética , Citomegalovirus/patogenicidade , DNA Viral/genética , Humanos , Hospedeiro Imunocomprometido/imunologia , Estudos Soroepidemiológicos
2.
Methods Mol Biol ; 2244: 343-363, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555595

RESUMO

Immunodeficient mice engrafted with human tissues provide a robust model for the in vivo investigation of human-restricted viruses such as human cytomegalovirus (HCMV). Several humanized mouse models have been developed and improved over the last 30 years. Here, we describe a protocol for the transplant of human hematopoietic stem cells with autologous fetal liver and thymic tissues into NOD.Cg-PrkdcscidIL2rγtm1Wjl mice to create a humanized bone marrow-liver-thymus model (huBLT) that can be infected with HCMV. The presence of human thymus allows the development of a functional human immune system, including HLA-restricted human T-cells and B-cells. Indeed, following infection, huBLT mice generate virus-specific CD4+ and CD8+ T-cell responses. Additionally, both HCMV-specific IgM and IgG B-cell responses can be detected. This huBLT model provides the first animal model to explore the adaptive human immune response to HCMV infection.


Assuntos
Citomegalovirus/imunologia , Modelos Animais de Doenças , Transplante de Células-Tronco Hematopoéticas/métodos , Imunidade Adaptativa/imunologia , Animais , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Citomegalovirus/metabolismo , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/imunologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID
3.
Methods Mol Biol ; 2244: 365-401, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555596

RESUMO

Human cytomegalovirus (HCMV) is a leading viral cause of congenital infections in the central nervous system (CNS) and may result in severe long-term sequelae. High rates of sequelae following congenital HCMV infection and insufficient antiviral therapy in the perinatal period makes the development of an HCMV-specific vaccine a high priority of modern medicine. Due to the species specificity of HCMV, animal models are frequently used to study CMV pathogenesis. Studies of murine cytomegalovirus (MCMV) infections of adult mice have played a significant role as a model of CMV biology and pathogenesis, while MCMV infection of newborn mice has been successfully used as a model of perinatal CMV infection. Newborn mice infected with MCMV have high levels of viremia during which the virus establishes a productive infection in most organs, coupled with a robust inflammatory response. Productive infection in the brain parenchyma during early postnatal period leads to an extensive nonnecrotizing multifocal widespread encephalitis characterized by infiltration of components of both innate and adaptive immunity. As a result, impairment in postnatal development of mouse cerebellum leads to long-term motor and sensor disabilities. This chapter summarizes current findings of rodent models of perinatal CMV infection and describes methods for analysis of perinatal MCMV infection in newborn mice.


Assuntos
Citomegalovirus/imunologia , Modelos Animais de Doenças , Animais , Animais Recém-Nascidos , Encéfalo/imunologia , Sistema Nervoso Central/virologia , Citomegalovirus/metabolismo , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/imunologia , Encefalite , Doenças Fetais , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Muromegalovirus/imunologia , Cultura Primária de Células
4.
Viruses ; 13(2)2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33567734

RESUMO

Human cytomegalovirus (HCMV) is a ubiquitous double-stranded DNA virus belonging to the ß-subgroup of the herpesvirus family. After the initial infection, the virus establishes latency in poorly differentiated myeloid precursors from where it can reactivate at later times to cause recurrences. In immunocompetent subjects, primary HCMV infection is usually asymptomatic, while in immunocompromised patients, HCMV infection can lead to severe, life-threatening diseases, whose clinical severity parallels the degree of immunosuppression. The existence of a strict interplay between HCMV and the immune system has led many to hypothesize that HCMV could also be involved in autoimmune diseases (ADs). Indeed, signs of active viral infection were later found in a variety of different ADs, such as rheumatological, neurological, enteric disorders, and metabolic diseases. In addition, HCMV infection has been frequently linked to increased production of autoantibodies, which play a driving role in AD progression, as observed in systemic lupus erythematosus (SLE) patients. Documented mechanisms of HCMV-associated autoimmunity include molecular mimicry, inflammation, and nonspecific B-cell activation. In this review, we summarize the available literature on the various ADs arising from or exacerbating upon HCMV infection, focusing on the potential role of HCMV-mediated immune activation at disease onset.


Assuntos
Doenças Autoimunes/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Autoanticorpos/imunologia , Doenças Autoimunes/patologia , Doenças Autoimunes/virologia , Autoimunidade , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/patologia , Infecções por Citomegalovirus/virologia , Humanos , Hospedeiro Imunocomprometido , Inflamação , Doenças Vasculares/patologia
5.
Viruses ; 13(1)2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33419104

RESUMO

Hearing loss, one of the most prevalent chronic health conditions, affects around half a billion people worldwide, including 34 million children. The World Health Organization estimates that the prevalence of disabling hearing loss will increase to over 900 million people by 2050. Many cases of congenital hearing loss are triggered by viral infections during different stages of pregnancy. However, the molecular mechanisms by which viruses induce hearing loss are not sufficiently explored, especially cases that are of embryonic origins. The present review first describes the cellular and molecular characteristics of the auditory system development at early stages of embryogenesis. These developmental hallmarks, which initiate upon axial specification of the otic placode as the primary root of the inner ear morphogenesis, involve the stage-specific regulation of several molecules and pathways, such as retinoic acid signaling, Sonic hedgehog, and Wnt. Different RNA and DNA viruses contributing to congenital and acquired hearing loss are then discussed in terms of their potential effects on the expression of molecules that control the formation of the auditory and vestibular compartments following otic vesicle differentiation. Among these viruses, cytomegalovirus and herpes simplex virus appear to have the most effect upon initial molecular determinants of inner ear development. Moreover, of the molecules governing the inner ear development at initial stages, SOX2, FGFR3, and CDKN1B are more affected by viruses causing either congenital or acquired hearing loss. Abnormalities in the function or expression of these molecules influence processes like cochlear development and production of inner ear hair and supporting cells. Nevertheless, because most of such virus-host interactions were studied in unrelated tissues, further validations are needed to confirm whether these viruses can mediate the same effects in physiologically relevant models simulating otic vesicle specification and growth.


Assuntos
Citomegalovirus/isolamento & purificação , Orelha Interna/embriologia , Orelha Interna/virologia , Perda Auditiva/virologia , Simplexvirus/isolamento & purificação , Animais , Diferenciação Celular , Inibidor de Quinase Dependente de Ciclina p27/genética , Citomegalovirus/patogenicidade , Perda Auditiva/congênito , Humanos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Fatores de Transcrição SOXB1/genética , Transdução de Sinais , Simplexvirus/patogenicidade
6.
PLoS Pathog ; 17(1): e1009088, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33497413

RESUMO

Cytomegaloviruses (CMVs) are generally unable to cross species barriers, in part because prolonged coevolution with one host species limits their ability to evade restriction factors in other species. However, the limitation in host range is incomplete. For example, rhesus CMV (RhCMV) can replicate in human cells, albeit much less efficiently than in rhesus cells. Previously we reported that the protein kinase R (PKR) antagonist encoded by RhCMV, rTRS1, has limited activity against human PKR but is nonetheless necessary and sufficient to enable RhCMV replication in human fibroblasts (HF). We now show that knockout of PKR in human cells or treatment with the eIF2B agonist ISRIB, which overcomes the translational inhibition resulting from PKR activation, augments RhCMV replication in HF, indicating that human PKR contributes to the inefficiency of RhCMV replication in HF. Serial passage of RhCMV in HF reproducibly selected for viruses with improved ability to replicate in human cells. The evolved viruses contain an inverted duplication of the terminal 6.8 kb of the genome, including rTRS1. The duplication replaces ~11.8 kb just downstream of an internal sequence element, pac1-like, which is very similar to the pac1 cleavage and packaging signal found near the terminus of the genome. Plaque-purified evolved viruses produced at least twice as much rTRS1 as the parental RhCMV and blocked the PKR pathway more effectively in HF. Southern blots revealed that unlike the parental RhCMV, viruses with the inverted duplication isomerize in a manner similar to HCMV and other herpesviruses that have internal repeat sequences. The apparent ease with which this duplication event occurs raises the possibility that the pac1-like site, which is conserved in Old World monkey CMV genomes, may serve a function in facilitating rapid adaptation to evolutionary obstacles.


Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/patogenicidade , Fibroblastos/virologia , Rearranjo Gênico , Genoma Viral , Replicação Viral , eIF-2 Quinase/metabolismo , Animais , Citomegalovirus/genética , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , Fibroblastos/metabolismo , Especificidade de Hospedeiro , Humanos , Macaca mulatta , eIF-2 Quinase/genética
7.
Int J Mol Sci ; 22(2)2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33430060

RESUMO

Human cytomegalovirus (HCMV) is a human pathogenic herpesvirus associated with a variety of clinical symptoms. Current antiviral therapy is not always effective, so that improved drug classes and drug-targeting strategies are needed. Particularly host-directed antivirals, including pharmaceutical kinase inhibitors (PKIs), may help to overcome problems of drug resistance. Here, we focused on utilizing a selection of clinically relevant PKIs and determined their anticytomegaloviral efficacies. Particularly, PKIs directed to host or viral cyclin-dependent kinases, i.e., abemaciclib, LDC4297 and maribavir, exerted promising profiles against human and murine cytomegaloviruses. The anti-HCMV in vitro activity of the approved anti-cancer drug abemaciclib was confirmed in vivo using our luciferase-based murine cytomegalovirus (MCMV) animal model in immunocompetent mice. To assess drug combinations, we applied the Bliss independence checkerboard and Loewe additivity fixed-dose assays in parallel. Results revealed that (i) both affirmative approaches provided valuable information on anti-CMV drug efficacies and interactions, (ii) the analyzed combinations comprised additive, synergistic or antagonistic drug interactions consistent with the drugs' antiviral mode-of-action, (iii) the selected PKIs, especially LDC4297, showed promising inhibitory profiles, not only against HCMV but also other α-, ß- and γ-herpesviruses, and specifically, (iv) the combination treatment with LDC4297 and maribavir revealed a strong synergism against HCMV, which might open doors towards novel clinical options in the near future. Taken together, this study highlights the potential of therapeutic drug combinations of current developmental/preclinical PKIs.


Assuntos
Infecções por Citomegalovirus/tratamento farmacológico , Farmacorresistência Viral/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Replicação Viral/genética , Aminopiridinas/farmacologia , Animais , Antivirais/farmacologia , Benzimidazóis/farmacologia , Linhagem Celular , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Combinação de Medicamentos , Ganciclovir/farmacologia , Humanos , Camundongos , Pirazóis/farmacologia , Ribonucleosídeos/farmacologia , Triazinas/farmacologia , Replicação Viral/efeitos dos fármacos
8.
Anticancer Res ; 40(10): 5909-5917, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988922

RESUMO

BACKGROUND/AIM: Cytomegalovirus (CMV) replication may cause life-threatening complications after allogeneic haematopoietic stem cell transplantation (allo-HSCT). The aim of the study was to characterize CMV events, and the outcome of letermovir (LTV) CMV prophylaxis. PATIENTS AND METHODS: In this retrospective analysis of patients treated with an allo-HSCT between 2010 and 2020, we determined plasma CMV events, as well as associated risk factors. RESULTS: We identified 423 patients who had undergone allo-HSCT between 2010 and 2020. CMV DNAemia was found in 130/423 (30.7%) of patients. CMV reactivation rate was significantly higher in patients with acute graft-versus-host disease, HLA mismatch, and CMV IgG seropositivity of donors and recipients. Among 42 patients receiving LTV prophylaxis those, 5 (11.9%) showed CMV DNAemia under LTV versus 87/353 (24.6%) in a control group. CONCLUSION: Despite the development of better approaches with weekly monitoring and early treatment initiation, CMV reactivations play an important role after allo-HSCT.


Assuntos
Acetatos/administração & dosagem , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Quinazolinas/administração & dosagem , Adulto , Idoso , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/etiologia , Infecções por Citomegalovirus/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transplante Homólogo/efeitos adversos , Replicação Viral/efeitos dos fármacos
9.
PLoS Pathog ; 16(9): e1008855, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32986788

RESUMO

SAMHD1 is a host restriction factor that functions to restrict both retroviruses and DNA viruses, based on its nuclear deoxynucleotide triphosphate (dNTP) hydrolase activity that limits availability of intracellular dNTP pools. In the present study, we demonstrate that SAMHD1 expression was increased following human cytomegalovirus (HCMV) infection, with only a modest effect on infectious virus production. SAMHD1 was rapidly phosphorylated at residue T592 after infection by cellular cyclin-dependent kinases, especially Cdk2, and by the viral kinase pUL97, resulting in a significant fraction of phosho-SAMHD1 being relocalized to the cytoplasm of infected fibroblasts, in association with viral particles and dense bodies. Thus, our findings indicate that HCMV-dependent SAMHD1 cytoplasmic delocalization and inactivation may represent a potential novel mechanism of HCMV evasion from host antiviral restriction activities.


Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/patogenicidade , Infecções por Herpesviridae/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/genética , Antivirais/farmacologia , Quinases Ciclina-Dependentes/metabolismo , Citomegalovirus/genética , Citoplasma/metabolismo , Citoplasma/virologia , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosforilação , Replicação Viral/efeitos dos fármacos
10.
Rev Med Virol ; 30(5): e2144, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32671966

RESUMO

The significantly higher mortality rates seen in the elderly compared with young children during the coronavirus disease 2019 (Covid-19) pandemic is likely to be driven in part by an impaired immune response in older individuals. Cytomegalovirus (CMV) seroprevalence approaches 80% in the elderly. CMV has been shown to accelerate immune ageing by affecting peripheral blood T cell phenotypes and increasing inflammatory mediated cytokines such as IL-6. The elderly with pre-existing but clinically silent CMV infection may therefore be particularly susceptible to severe Covid-19 disease and succumb to a cytokine storm which may have been promoted by CMV. Here, we evaluate the potential role of CMV in those with severe Covid-19 disease and consider how this relationship can be investigated in current research studies.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/epidemiologia , Síndrome da Liberação de Citocina/epidemiologia , Infecções por Citomegalovirus/epidemiologia , Citomegalovirus/patogenicidade , Imunossenescência , Pandemias , Pneumonia Viral/epidemiologia , Fatores Etários , Idoso , Betacoronavirus/imunologia , Criança , Coinfecção , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/mortalidade , Infecções por Coronavirus/virologia , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/mortalidade , Síndrome da Liberação de Citocina/virologia , Citocinas/genética , Citocinas/imunologia , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/mortalidade , Infecções por Citomegalovirus/virologia , Progressão da Doença , Humanos , Pneumonia Viral/imunologia , Pneumonia Viral/mortalidade , Pneumonia Viral/virologia , Estudos Soroepidemiológicos , Índice de Gravidade de Doença , Análise de Sobrevida , Linfócitos T/imunologia , Linfócitos T/patologia , Linfócitos T/virologia
11.
Proc Natl Acad Sci U S A ; 117(31): 18771-18779, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690704

RESUMO

Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of intrinsic, innate, and adaptive viral immune evasion. Here, we employed multiplexed tandem mass tag-based proteomics to characterize host proteins targeted for degradation late during HCMV infection. This approach revealed that mixed lineage kinase domain-like protein (MLKL), a key terminal mediator of cellular necroptosis, was rapidly and persistently degraded by the minimally passaged HCMV strain Merlin but not the extensively passaged strain AD169. The strain Merlin viral inhibitor of apoptosis pUL36 was necessary and sufficient both to degrade MLKL and to inhibit necroptosis. Furthermore, mutation of pUL36 Cys131 abrogated MLKL degradation and restored necroptosis. As the same residue is also required for pUL36-mediated inhibition of apoptosis by preventing proteolytic activation of procaspase-8, we define pUL36 as a multifunctional inhibitor of both apoptotic and necroptotic cell death.


Assuntos
Apoptose/fisiologia , Citomegalovirus , Necroptose/fisiologia , Proteínas Virais/metabolismo , Células Cultivadas , Citomegalovirus/química , Citomegalovirus/metabolismo , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Humanos , Ligação Proteica , Proteólise
12.
Med Sci (Paris) ; 36(4): 367-375, 2020 Apr.
Artigo em Francês | MEDLINE | ID: mdl-32356713

RESUMO

Human cytomegalovirus (HCMV) is an important ubiquitous opportunistic pathogen that belongs to the betaherpesviridae. Primary HCMV infection is generally asymptomatic in immunocompetent individuals. In contrast, HCMV infection causes serious disease in immunocompromised patients and is the leading cause of congenital viral infection. Although they are effective, the use of conventional molecules is limited by the emergence of resistance and by their toxicity. New antivirals targeting other replication steps and inducing fewer adverse effects are therefore needed. During HCMV replication, DNA packaging is performed by the terminase complex, which cleaves DNA to package the virus genome into the capsid. With no counterpart in mammalian cells, these terminase proteins are ideal targets for highly specific antivirals. A new terminase inhibitor, letermovir, recently proved effective against HCMV in phase III clinical trials. However, its mechanism of action is unclear and it has no significant activity against other herpesvirus or non-human CMV.


Assuntos
Antivirais/uso terapêutico , Infecções por Citomegalovirus/tratamento farmacológico , Endodesoxirribonucleases/antagonistas & inibidores , Terapia de Alvo Molecular/métodos , Citomegalovirus/patogenicidade , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/imunologia , Endodesoxirribonucleases/fisiologia , Humanos , Hospedeiro Imunocomprometido , Terapia de Alvo Molecular/tendências , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/fisiologia , Montagem de Vírus/efeitos dos fármacos , Montagem de Vírus/fisiologia , Replicação Viral/efeitos dos fármacos
13.
PLoS Pathog ; 16(5): e1008476, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32384127

RESUMO

Cytomegaloviruses (order Herpesvirales) display remarkable species-specificity as a result of long-term co-evolution with their mammalian hosts. Human cytomegalovirus (HCMV) is exquisitely adapted to our species and displays high genetic diversity. We leveraged information on inter-species divergence of primate-infecting cytomegaloviruses and intra-species diversity of clinical isolates to provide a genome-wide picture of HCMV adaptation across different time-frames. During adaptation to the human host, core viral genes were commonly targeted by positive selection. Functional characterization of adaptive mutations in the primase gene (UL70) indicated that selection favored amino acid replacements that decrease viral replication in human fibroblasts, suggesting evolution towards viral temperance. HCMV intra-species diversity was largely governed by immune system-driven selective pressure, with several adaptive variants located in antigenic domains. A significant excess of positively selected sites was also detected in the signal peptides (SPs) of viral proteins, indicating that, although they are removed from mature proteins, SPs can contribute to viral adaptation. Functional characterization of one of these SPs indicated that adaptive variants modulate the timing of cleavage by the signal peptidase and the dynamics of glycoprotein intracellular trafficking. We thus used evolutionary information to generate experimentally-testable hypotheses on the functional effect of HCMV genetic diversity and we define modulators of viral phenotypes.


Assuntos
Adaptação Biológica/genética , Infecções por Citomegalovirus/genética , Citomegalovirus/genética , Adaptação Fisiológica/genética , Animais , Evolução Biológica , Citomegalovirus/metabolismo , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/metabolismo , Evolução Molecular , Glicoproteínas/metabolismo , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Filogenia , Especificidade da Espécie , Proteínas Virais/metabolismo
14.
Nat Struct Mol Biol ; 27(6): 581-588, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32451488

RESUMO

TENT4 enzymes generate 'mixed tails' of diverse nucleotides at 3' ends of RNAs via nontemplated nucleotide addition to protect messenger RNAs from deadenylation. Here we discover extensive mixed tailing in transcripts of hepatitis B virus (HBV) and human cytomegalovirus (HCMV), generated via a similar mechanism exploiting the TENT4-ZCCHC14 complex. TAIL-seq on HBV and HCMV RNAs revealed that TENT4A and TENT4B are responsible for mixed tailing and protection of viral poly(A) tails. We find that the HBV post-transcriptional regulatory element (PRE), specifically the CNGGN-type pentaloop, is critical for TENT4-dependent regulation. HCMV uses a similar pentaloop, an interesting example of convergent evolution. This pentaloop is recognized by the sterile alpha motif domain-containing ZCCHC14 protein, which in turn recruits TENT4. Overall, our study reveals the mechanism of action of PRE, which has been widely used to enhance gene expression, and identifies the TENT4-ZCCHC14 complex as a potential target for antiviral therapeutics.


Assuntos
Citomegalovirus/genética , Vírus da Hepatite B/genética , Interações Hospedeiro-Patógeno/fisiologia , RNA Viral/metabolismo , Linhagem Celular , Citomegalovirus/patogenicidade , Vírus da Hepatite B/patogenicidade , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Filogenia , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , RNA Viral/química
15.
Proc Natl Acad Sci U S A ; 117(23): 12961-12968, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32444487

RESUMO

Viral immune evasion is currently understood to focus on deflecting CD8 T cell recognition of infected cells by disrupting antigen presentation pathways. We evaluated viral interference with the ultimate step in cytotoxic T cell function, the death of infected cells. The viral inhibitor of caspase-8 activation (vICA) conserved in human cytomegalovirus (HCMV) and murine CMV (MCMV) prevents the activation of caspase-8 and proapoptotic signaling. We demonstrate the key role of vICA from either virus, in deflecting antigen-specific CD8 T cell-killing of infected cells. vICA-deficient mutants, lacking either UL36 or M36, exhibit greater susceptibility to CD8 T cell control than mutants lacking the set of immunoevasins known to disrupt antigen presentation via MHC class I. This difference is evident during infection in the natural mouse host infected with MCMV, in settings where virus-specific CD8 T cells are adoptively transferred. Finally, we identify the molecular mechanism through which vICA acts, demonstrating the central contribution of caspase-8 signaling at a point of convergence of death receptor-induced apoptosis and perforin/granzyme-dependent cytotoxicity.


Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Evasão da Resposta Imune , Linfócitos T Citotóxicos/imunologia , Animais , Apoptose/imunologia , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular , Técnicas de Cocultura , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/virologia , Modelos Animais de Doenças , Fibroblastos , Granzimas/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Camundongos , Camundongos Knockout , Muromegalovirus/genética , Muromegalovirus/imunologia , Muromegalovirus/metabolismo , Mutagênese , Perforina/genética , Perforina/metabolismo , Receptores de Morte Celular/metabolismo , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/metabolismo , Imagem com Lapso de Tempo , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/metabolismo
16.
Sci Rep ; 10(1): 8530, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444790

RESUMO

A better understanding of the mechanisms underlying cell tropisms and the efficiency of viral infection is critical for the development of vaccines and antiviral drugs for viral diseases. In this study, we worked on the entry mechanisms of guinea pig cytomegalovirus and found that endogenous expression of a combination of two components (GP131 and GP133) of the pentameric glycoprotein complex, which is required for non-fibroblast cell tropisms, enhanced viral infection more than 10-fold. In addition, D138A alteration in GP131 increased this enhancement by an additional 10-fold. Although differences in the efficiency of viral infection among various cell types are usually explained by differences in viral entry or traffic processes, our experimental evidences dismissed such possibilities. Instead, our findings that i) endogenous expression of GP131 and GP133 after nuclear delivery of viral DNA still enhanced infection and ii) an HDAC inhibitor overcame the need of the endogenous expression led us to hypothesize a novel mechanism that controls the efficiency of viral infection through the activation of gene expression from viral DNA delivered to the nuclei. Further studies of this unexpected phenomena warrant to understand novel but also general mechanisms for cell tropisms of viral infection and determinants that control infection efficiency.


Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/patogenicidade , Células Epiteliais/virologia , Glicoproteínas/metabolismo , Proteínas Virais/metabolismo , Animais , Células Cultivadas , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/metabolismo , Células Epiteliais/metabolismo , Glicoproteínas/química , Glicoproteínas/genética , Cobaias , Multimerização Proteica , Proteínas Virais/química , Proteínas Virais/genética , Internalização do Vírus
17.
Medicine (Baltimore) ; 99(17): e19765, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32332615

RESUMO

BACKGROUND: Congenital cytomegalovirus (CMV) disease, a common mother-to-child infection, can lead to neurological sequelae. Some clinical trials have shown that oral valganciclovir (VGCV) can improve hearing and neurodevelopmental impairment in infants with congenital CMV disease. However, VGCV has neither been approved in Japan nor other countries as a treatment for this disease by the government health insurance. METHODS: This study is a non-randomized, prospective, open-label, multicenter, single-arm clinical trial and will include subjects meeting the following criteria: confirmation of positive CMV-DNA amplification in urine by an in vitro diagnostic test within 21 days of age; congenital CMV disease with one or more central nervous system disorders-microcephaly, hydrocephalus or ventricular enlargement, periventricular calcification, cortical hypoplasia or white matter injury, retinal choroiditis, and abnormal auditory brainstem response (ABR); and infants within 2 months of age with a gestational age ≥32 weeks at birth and weighing ≥1800 g at the time of registration. Subjects will be orally administered 16 mg/kg VGCV twice daily for 6 months. The target number of cases for enrollment between February 3, 2020 and July 31, 2021 is 25. Primary endpoint is the change in whole blood CMV loads before and after 6 months of treatment. The important secondary endpoint is the change in ABR (both best and total ear hearing assessments) before and after 6 months of treatment. The safety endpoints are adverse events and drug side effects. DISCUSSION: To the best of our knowledge, this multicenter, open-label, single-arm study will be the first well-designed clinical trial to evaluate the efficacy of oral VGCV in infants with congenital CMV diseases. The findings will reveal the efficacy and safety of oral VGCV treatments and enable the approval of oral VGCV as a treatment for infants with congenital CMV disease by the government health insurance of Japan.


Assuntos
Protocolos Clínicos , Infecções por Citomegalovirus/tratamento farmacológico , Valganciclovir/normas , Administração Oral , Antivirais/normas , Antivirais/uso terapêutico , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/patogenicidade , Feminino , Humanos , Lactente , Recém-Nascido , Transmissão Vertical de Doença Infecciosa , Japão , Masculino , Estudos Prospectivos , Valganciclovir/uso terapêutico
18.
Int J Mol Sci ; 21(5)2020 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-32121406

RESUMO

Human cytomegalovirus (HCMV) infections are wide-spread among the general population with manifestations ranging from asymptomatic to severe developmental disabilities in newborns and life-threatening illnesses in individuals with a compromised immune system. Nearly all current drugs suffer from one or more limitations, which emphasizes the critical need to develop new approaches and new molecules. We reasoned that a 'poly-pharmacy' approach relying on simultaneous binding to multiple receptors involved in HCMV entry into host cells could pave the way to a more effective therapeutic outcome. This work presents the study of a synthetic, small molecule displaying pleiotropicity of interactions as a competitive antagonist of viral or cell surface receptors including heparan sulfate proteoglycans and heparan sulfate-binding proteins, which play important roles in HCMV entry and spread. Sulfated pentagalloylglucoside (SPGG), a functional mimetic of heparan sulfate, inhibits HCMV entry into human foreskin fibroblasts and neuroepithelioma cells with high potency. At the same time, SPGG exhibits no toxicity at levels as high as 50-fold more than its inhibition potency. Interestingly, cell-ELISA assays showed downregulation in HCMV immediate-early gene 1 and 2 (IE 1&2) expression in presence of SPGG further supporting inhibition of viral entry. Finally, HCMV foci were observed to decrease significantly in the presence of SPGG suggesting impact on viral spread too. Overall, this work offers the first evidence that pleiotropicity, such as demonstrated by SPGG, may offer a new poly-therapeutic approach toward effective inhibition of HCMV.


Assuntos
Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/efeitos dos fármacos , Glucosídeos/farmacologia , Proteoglicanas de Heparan Sulfato/genética , Ésteres do Ácido Sulfúrico/farmacologia , Células Cultivadas , Citomegalovirus/genética , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Fibroblastos/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Recém-Nascido , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
19.
J Gen Virol ; 101(4): 426-439, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32068527

RESUMO

Congenital cytomegalovirus (cCMV) is a leading cause of birth defects. The guinea pig is the only small cCMV animal model. Guinea pig cytomegalovirus (GPCMV) encodes similar glycoprotein complexes to human CMV (HCMV) including gB and the gH-based pentamer complex (PC). In HCMV, both gB and PC are neutralizing antibody antigens. The relevance of GPCMV PC for virus tropism and vaccine target remains controversial. A novel guinea pig placental amniotic sac epithelial (GPASE) cell-line did not express viral cell receptor platelet derived growth factor receptor alpha (PDGFRA) and resulted in requirement for the PC for GPCMV infection unless PDGFRA was ectopically expressed. High titer anti-gB sera from a GPCMV gB vaccine study was evaluated for GPCMV neutralizing capability on GPASE cells in comparison to convalescent sera from GPCMV(PC+) or GPCMV(PC-) infected animals. Anti-gB sera neutralized fibroblast infection but was less effective compared to anti-GPCMV(PC-), which had antibodies to gH/gL. However, both anti-GPCMV(PC-) and anti-gB sera similarly had reduced neutralizing capability on GPASE and renal epithelial cells in comparison to anti-GPCMV(PC+) sera, which had additional antibodies to PC. Overall, results demonstrate the importance of the PC for GPCMV tropism to various cell types that lack PDGFRA expression and the limited ability of anti-gB sera to neutralize GPCMV on non-fibroblast cells despite the essential nature of gB glycoprotein.


Assuntos
Âmnio/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/genética , Glicoproteínas/metabolismo , Placenta/imunologia , Proteínas do Envelope Viral/metabolismo , Vacinas Virais/imunologia , Âmnio/citologia , Âmnio/metabolismo , Âmnio/virologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linhagem Celular , Citomegalovirus/metabolismo , Citomegalovirus/patogenicidade , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , Feminino , Técnicas de Inativação de Genes , Cobaias , Mutação , Testes de Neutralização , Placenta/citologia , Placenta/metabolismo , Placenta/virologia , Gravidez , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Tropismo Viral
20.
PLoS One ; 15(2): e0228900, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32053638

RESUMO

BACKGROUND: CMV infection of the fetus or neonate can lead to devastating disease, and there are no effective prevention strategies to date. Vitamin D is a potent immunomodulator, supports antiviral immune responses, and plays an important role in placental immunity. METHODS: Retrospective cohort study to evaluate the impact of low maternal vitamin D on congenital and early postnatal transmission of CMV among HIV-infected, non-breastfeeding women and their HIV exposed but negative infants from an urban HIV clinic. Vitamin D panel was performed on stored maternal plasma obtained near time of delivery. Infant CMV testing at 0-6 months included urine and oral cultures, and/or serum polymerase chain reaction testing. RESULTS: Cohort included 340 mother-infant pairs (births 1991-2014). Among 38 infants (11%) with a CMV+ test between 0-6 months, 4.7% (14/300) had congenital CMV transmission (CMV+ test 0-3 weeks), and 7.6% (24/315) had peri/postnatal CMV (CMV+ test >3 weeks-6 months). Women with lower calcitriol (1,25-dihydroxyvitamin D), the active form of vitamin D, were more likely to have an infant with congenital (OR 12.2 [95% CI 1.61-92.2] P = 0.02) and peri/postnatal (OR 9.84 [95% CI 2.63-36.8] P = 0.0007) infections in multivariate analyses, independent of maternal HIV viral load and CD4 count. CONCLUSION: This study demonstrates an association between inadequate maternal calcitriol during pregnancy and increased congenital and early postnatal acquisition of CMV among non-breastfeeding women with HIV and their HIV negative infants.


Assuntos
Infecções por Citomegalovirus/transmissão , Transmissão Vertical de Doença Infecciosa/prevenção & controle , Deficiência de Vitamina D/metabolismo , Adulto , Antivirais/uso terapêutico , Contagem de Linfócito CD4 , Estudos de Coortes , Citomegalovirus/genética , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/imunologia , DNA Viral/sangue , Feminino , Infecções por HIV/complicações , Humanos , Lactente , Recém-Nascido , Plasma/virologia , Gravidez , Complicações Infecciosas na Gravidez/tratamento farmacológico , Estudos Retrospectivos , Carga Viral , Vitamina D/metabolismo , Deficiência de Vitamina D/fisiopatologia
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