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1.
Rev Med Virol ; 31(6): e2233, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33709529

RESUMO

Cellular receptors in human cytomegalovirus (HCMV) mother to child transmission play an important role in congenital infection. Placental trophoblast cells are a significant cell type in placental development, placental functional processes, and in HCMV transmission. Different cells within the placental floating and chorionic villi present alternate receptors for HCMV cell entry. Syncytiotrophoblasts present neonatal Fc receptors that bind and transport circulating maternal immunoglobulin G across the placental interface which can also be bound to HCMV virions, facilitating viral entry into the placenta and foetal circulation. Cytotrophoblast express HCMV receptors including integrin-α1ß1, integrin-αVß3, epidermal growth factor receptor and platelet-derived growth factor receptor alpha. The latter interacts with HCMV glycoprotein-H, glycoprotein-L and glycoprotein-O (gH/gL/gO) trimers (predominantly in placental fibroblasts) and the gH/gL/pUL128, UL130-UL131A pentameric complex in other placental cell types. The pentameric complex allows viral tropism of placental trophoblasts, endothelial cells, epithelial cells, leukocytes and monocytes. This review outlines HCMV ligands and target receptor proteins in congenital HCMV infection.


Assuntos
Citomegalovirus , Células Endoteliais , Interações Hospedeiro-Patógeno , Placenta/virologia , Feminino , Glicoproteínas , Humanos , Transmissão Vertical de Doenças Infecciosas , Gravidez
2.
Int J Mol Sci ; 23(19)2022 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-36233116

RESUMO

The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97-cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97-cyclin interactions. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97-cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. As a conclusion, the results provide evidence for the functional importance of pUL97-cyclin interaction. High selective pressure on the formation of pUL97-cyclin complexes was identified by the use of clinically relevant mutants.


Assuntos
Ciclina H , Citomegalovirus , Proteínas Virais , Aminoácidos/metabolismo , Ciclina H/genética , Ciclina H/metabolismo , Ciclina T/genética , Ciclina T/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Citomegalovirus/fisiologia , Marcadores Genéticos , Humanos , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Virais/genética , Replicação Viral/genética
3.
Int J Mol Sci ; 22(23)2021 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-34884662

RESUMO

Human cytomegalovirus (HCMV) is a major pathogenic herpesvirus that is prevalent worldwide and it is associated with a variety of clinical symptoms. Current antiviral therapy options do not fully satisfy the medical needs; thus, improved drug classes and drug-targeting strategies are required. In particular, host-directed antivirals, including pharmaceutical kinase inhibitors, might help improve the drug qualities. Here, we focused on utilizing PROteolysis TArgeting Chimeras (PROTACs), i.e., hetero-bifunctional molecules containing two elements, namely a target-binding molecule and a proteolysis-inducing element. Specifically, a PROTAC that was based on a cyclin-dependent kinase (CDK) inhibitor, i.e., CDK9-directed PROTAC THAL-SNS032, was analyzed and proved to possess strong anti-HCMV AD169-GFP activity, with values of EC50 of 0.030 µM and CC50 of 0.175 µM (SI of 5.8). Comparing the effect of THAL-SNS032 with its non-PROTAC counterpart SNS032, data indicated a 3.7-fold stronger anti-HCMV efficacy. This antiviral activity, as illustrated for further clinically relevant strains of human and murine CMVs, coincided with the mid-nanomolar concentration range necessary for a drug-induced degradation of the primary (CDK9) and secondary targets (CDK1, CDK2, CDK7). In addition, further antiviral activities were demonstrated, such as the inhibition of SARS-CoV-2 replication, whereas other investigated human viruses (i.e., varicella zoster virus, adenovirus type 2, and Zika virus) were found insensitive. Combined, the antiviral quality of this approach is seen in its (i) mechanistic uniqueness; (ii) future options of combinatorial drug treatment; (iii) potential broad-spectrum activity; and (iv) applicability in clinically relevant antiviral models. These novel data are discussed in light of the current achievements of anti-HCMV drug development.


Assuntos
Antivirais , Citomegalovirus , Inibidores de Proteínas Quinases , Animais , Humanos , Camundongos , Antivirais/farmacologia , Linhagem Celular , Quinase 9 Dependente de Ciclina , Citomegalovirus/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Inibidores de Proteínas Quinases/farmacologia , Replicação Viral/efeitos dos fármacos , Proteólise
4.
Artigo em Inglês | MEDLINE | ID: mdl-33077661

RESUMO

Congenital cytomegalovirus (HCMV) infection may cause significant fetal malformation, lifelong disease, and, in severe cases, fetal or neonatal death. Placental infection with HCMV is the major mechanism of mother-to-child transmission (MTCT) and fetal injury. Thus, any pharmaceutical antiviral interference to reduce viral load may reduce placental damage, MTCT, and fetal disease. However, there is currently no licensed HCMV antiviral for use during pregnancy. In this study, aciclovir and the HCMV-specific antivirals letermovir, maribavir, and cidofovir were compared with ganciclovir for antiviral effects in model systems of pregnancy, including first-trimester TEV-1 trophoblast cell cultures and third-trimester ex vivo placental explant histocultures. HCMV-infected trophoblasts at 7 days postinfection (dpi) showed an EC50 of 21 µM for aciclovir, 0.0007 µM for letermovir, 0.11 µM for maribavir, and 0.29 µM for cidofovir, relative to 0.42 µM for ganciclovir. Antivirals added at 10 µM showed no cytotoxic effects and did not affect trophoblast cell proliferation (P > 0.9999). Multiple-round HCMV replication measured at 7 dpi showed letermovir, maribavir, and cidofovir treatment inhibited immediate early, early, and true late viral protein expression as assayed on Western blots. Antiviral treatment of HCMV-infected placental explants showed significant inhibition (P < 0.05) of viral replication with letermovir (83.3%), maribavir (83.6%), cidofovir (89.3%), and ganciclovir (82.4%), but not aciclovir (P > 0.9999). In ex vivo model systems, recently trialed HCMV antivirals letermovir and maribavir were effective at inhibiting HCMV replication. They partly fulfil requirements for use as safe and effective therapeutics during pregnancy to control congenital HCMV. Clinical trials of these newer agents would assist assessment of their utility in pregnancy.


Assuntos
Antivirais , Infecções por Citomegalovirus , Antivirais/farmacologia , Antivirais/uso terapêutico , Criança , Citomegalovirus , Infecções por Citomegalovirus/tratamento farmacológico , Feminino , Humanos , Recém-Nascido , Transmissão Vertical de Doenças Infecciosas , Placenta , Gravidez , Replicação Viral
5.
Prenat Diagn ; 40(13): 1681-1692, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32271956

RESUMO

Congenital CMV is the most common congenital infection in the developed world. Infection results in congenital disease ranging from asymptomatic infection to severe neurodevelopmental impairment, and occasionally fetal or neonatal death. Fetal infection can occur through maternal-fetal transmission during primary maternal infection or maternal reactivation or re-infection. Awareness among maternal health care providers and parents is low. The prevention of maternal CMV infection currently relies on hygiene measures, with no effective CMV vaccine or prophylactic therapies. No licensed treatment options are available to prevent maternal-fetal transmission or fetal disease. Hyperimmunoglobulin and valaciclovir have been investigated for prevention of maternal-fetal transmission or fetal treatment, with some evidence supporting consideration of maternal administration of hyperimmunoglobulin or valaciclovir therapy in certain circumstances. This article outlines the clinical evidence regarding proven preventative behavioral measures and experimental hyperimmunoglobulin and valaciclovir therapies, that is structured around common questions asked by pregnant women about CMV infection. It is aimed to help maternity health care providers counsel prospective parents about congenital CMV disease and the preventative and therapeutic strategies currently available.


Assuntos
Infecções por Citomegalovirus/terapia , Terapias Fetais/métodos , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Complicações Infecciosas na Gravidez/terapia , Cuidado Pré-Natal/métodos , Antivirais/uso terapêutico , Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/transmissão , Feminino , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Fatores Imunológicos/uso terapêutico , Recém-Nascido , Masculino , Gravidez , Complicações Infecciosas na Gravidez/diagnóstico , Diagnóstico Pré-Natal/métodos , Valaciclovir/uso terapêutico
6.
Artigo em Inglês | MEDLINE | ID: mdl-31262766

RESUMO

Mutations in the cytomegalovirus UL97 kinase gene contribute to antiviral resistance. Mutations A594S and G598D from two clinical isolates were analyzed, and bacterial artificial chromosome (BAC)-engineered A594S recombinant cytomegalovirus exhibited a ganciclovir-resistant phenotype on plaque reduction. Viral replication was comparable to that of the wild type. Cell-based kinase activity and autophosphorylation of ectopically expressed proteins showed that mutants retained some kinase activity. This study showed that patient-derived cytomegalovirus with different ganciclovir sensitivities retained replication efficiency and exhibited some kinase activity in vitro.


Assuntos
Antivirais/farmacologia , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/enzimologia , Ganciclovir/farmacologia , Proteínas Quinases/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Citomegalovirus/genética , Farmacorresistência Viral/genética , Humanos , Mutação/genética , Fases de Leitura Aberta/genética , Fosforilação , Proteínas Quinases/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
7.
J Gen Virol ; 97(7): 1676-1685, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27145986

RESUMO

Nuclear egress of herpesvirus capsids through the nuclear envelope is mediated by the multimeric nuclear egress complex (NEC). The human cytomegalovirus (HCMV) core NEC is defined by an interaction between the membrane-anchored pUL50 and its nuclear co-factor pUL53, tightly associated through heterodimeric corecruitment to the nuclear envelope. Cellular proteins, such as p32/gC1qR, emerin and protein kinase C (PKC), are recruited by direct interaction with pUL50 for the multimeric extension of the NEC. As a functionally important event, the recruitment of both viral and cellular protein kinases leads to site-specific lamin phosphorylation and nuclear lamina disassembly. In this study, interaction domains within pUL50 for its binding partners were defined by co-immunoprecipitation. The interaction domain for pUL53 is located within the pUL50 N-terminus (residues 10-169), interaction domains for p32/gC1qR (100-358) and PKC (100-280) overlap in the central part of pUL50, and the interaction domain for emerin is located in the C-terminus (265-397). Moreover, expression and formation of core NEC proteins at the nuclear rim were consistently detected in cells permissive for productive HCMV replication, including two trophoblast-cell lines. Importantly, regular nuclear-rim formation of the core NEC was blocked by inhibition of cyclin-dependent kinase (CDK) activity. In relation to the recently published crystal structure of the HCMV core NEC, our findings result in a refined view of NEC assembly. In particular, we suggest that CDKs may play an important regulatory role in NEC formation during HCMV replication.


Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Citomegalovirus/metabolismo , Membrana Nuclear/virologia , Proteínas Virais/metabolismo , Liberação de Vírus/fisiologia , Replicação Viral/fisiologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Lâmina Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Mapas de Interação de Proteínas , Proteína Quinase C-alfa/metabolismo , Estrutura Terciária de Proteína
8.
J Gen Virol ; 97(1): 144-151, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26555090

RESUMO

Cyclin-dependent kinases (CDKs) are multifaceted regulators involved in the replication of human cytomegalovirus. Recently, we demonstrated an interaction of CDK9-cyclin T1 as well as viral CDK orthologue pUL97 with the viral regulator pUL69, thereby leading to pUL69-activating phosphorylation. Here, we demonstrate that colocalization and direct pUL69-cyclin T1 interaction is independent of viral strains and host cell types. In vitro phosphorylation of pUL69 by CDK9 or pUL97 did not occur in a single site-specific manner, but at multiple sites. The previously described fine-speckled nuclear aggregation of pUL69 was assigned to the late phase of viral replication. CDK inhibitors, including a novel inhibitor of the CDK-activating kinase CDK7, massively intensified this fine-speckled accumulation. Interestingly, we also observed spontaneous pUL69 accumulation in the absence of inhibitors at a lower frequency. These findings provide new insight into pUL69 kinase interregulation and emphasize the importance of pUL69 phosphorylation for correct intranuclear localization.


Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Citomegalovirus/fisiologia , Interações Hospedeiro-Patógeno , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Processamento de Proteína Pós-Traducional , Transativadores/metabolismo , Humanos , Fosforilação , Transporte Proteico
9.
Curr Opin Infect Dis ; 29(6): 615-624, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27607910

RESUMO

PURPOSE OF REVIEW: The purpose of this review is to assess the recent studies of therapy of pregnant women and neonates, aimed at preventing the consequences of congenital cytomegalovirus (CMV) infection. RECENT FINDINGS: A recent randomized controlled trial of treatment of CMV during pregnancy with hyperimmune globulin did not show significant efficacy in prevention of foetal infection and morbidity, although there was a trend towards improvement with treatment. Trials of antiviral therapy of the mother during pregnancy have involved small numbers only, confounded by ethical and practical difficulties, and further studies are needed to demonstrate whether or not antivirals are useful and well tolerated in this setting.Antiviral treatment of neonatal CMV acquired congenitally has been studied in well controlled trials and the antiviral valganciclovir has shown efficacy in reducing the more severe outcomes. Trials are ongoing of the use of antivirals in less severe disease, although results are likely to take several years. SUMMARY: Congenital CMV infection is the most frequent cause of congenital malformation in developed countries, with a symptomatic prevalence of 0.64% of all live births. Infection may result in neurodevelopmental delay, foetal or neonatal death, and most frequently, sensorineural hearing loss. Successful control of viral infections during pregnancy and in the newborn period is essential in reducing early and late morbidity and mortality. Control of congenital CMV infection may be via primary prevention methods such as reducing contact with the pathogen, improved hygiene - both for the pregnant mother and for the neonate, or secondary prevention via reduction of vertical transmission from mother to foetus and reduction in consequences of infection by treatment of infected pregnant women and infected neonates.


Assuntos
Infecções por Citomegalovirus/tratamento farmacológico , Complicações Infecciosas na Gravidez/tratamento farmacológico , Citomegalovirus , Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/transmissão , Feminino , Doenças Fetais/prevenção & controle , Humanos , Imunoglobulinas/uso terapêutico , Recém-Nascido , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Gravidez , Ensaios Clínicos Controlados Aleatórios como Assunto
10.
Mol Cell Proteomics ; 13(8): 2132-46, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24969177

RESUMO

Herpesviral capsids are assembled in the host cell nucleus before being translocated into the cytoplasm for further maturation. The crossing of the nuclear envelope represents a major event that requires the formation of the nuclear egress complex (NEC). Previous studies demonstrated that human cytomegalovirus (HCMV) proteins pUL50 and pUL53, as well as their homologs in all members of Herpesviridae, interact with each other at the nuclear envelope and form the heterodimeric core of the NEC. In order to characterize further the viral and cellular protein content of the multimeric NEC, the native complex was isolated from HCMV-infected human primary fibroblasts at various time points and analyzed using quantitative proteomics. Previously postulated components of the HCMV-specific NEC, as well as novel potential NEC-associated proteins such as emerin, were identified. In this regard, interaction and colocalization between emerin and pUL50 were confirmed by coimmunoprecipitation and confocal microscopy analyses, respectively. A functional validation of viral and cellular NEC constituents was achieved through siRNA-mediated knockdown experiments. The important role of emerin in NEC functionality was demonstrated by a reduction of viral replication when emerin expression was down-regulated. Moreover, under such conditions, reduced production of viral proteins and deregulation of viral late cytoplasmic maturation were observed. Combined, these data prove the functional importance of emerin as an NEC component, associated with pUL50, pUL53, pUL97, p32/gC1qR, and further regulatory proteins. Summarized, our findings provide the first proteomics-based characterization and functional validation of the HCMV-specific multimeric NEC.


Assuntos
Citomegalovirus/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteômica/métodos , Proteínas Virais/metabolismo , Animais , Fibroblastos/virologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos
11.
Aust N Z J Obstet Gynaecol ; 56(1): 9-18, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26391432

RESUMO

Human cytomegalovirus (CMV) is under-recognised, despite being the leading infectious cause of congenital malformation, affecting ~0.3% of Australian live births. Approximately 11% of infants born with congenital CMV infection are symptomatic, resulting in clinical manifestations, including jaundice, hepatosplenomegaly, petechiae, microcephaly, intrauterine growth restriction and death. Congenital CMV infection may cause severe long-term sequelae, including progressive sensorineural hearing loss and developmental delay in 40-58% of symptomatic neonates, and ~14% of initially asymptomatic infected neonates. Up to 50% of maternal CMV infections have nonspecific clinical manifestations, and most remain undetected unless specific serological testing is undertaken. The combination of serology tests for CMV-specific IgM, IgG and IgG avidity provide improved distinction between primary and secondary maternal infections. In pregnancies with confirmed primary maternal CMV infection, amniocentesis with CMV-PCR performed on amniotic fluid, undertaken after 21-22 weeks gestation, may determine whether maternofetal virus transmission has occurred. Ultrasound and, to a lesser extent, magnetic resonance imaging are valuable tools to assess fetal structural and growth abnormalities, although the absence of fetal abnormalities does not exclude fetal damage. Diagnosis of congenital CMV infection at birth or in the first 3 weeks of an infant's life is crucial, as this should prompt interventions for prevention of delayed-onset hearing loss and neurodevelopmental delay in affected infants. Prevention strategies should also target mothers because increased awareness and hygiene measures may reduce maternal infection. Recognition of the importance of CMV in pregnancy and in neonates is increasingly needed, particularly as therapeutic and preventive interventions expand for this serious problem.


Assuntos
Infecções por Citomegalovirus/congênito , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Complicações Infecciosas na Gravidez , Anormalidades Congênitas/diagnóstico , Anormalidades Congênitas/prevenção & controle , Anormalidades Congênitas/virologia , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/prevenção & controle , Infecções por Citomegalovirus/transmissão , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/prevenção & controle , Deficiências do Desenvolvimento/virologia , Feminino , Doenças Fetais/diagnóstico , Doenças Fetais/prevenção & controle , Doenças Fetais/virologia , Perda Auditiva Neurossensorial/congênito , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/prevenção & controle , Perda Auditiva Neurossensorial/virologia , Humanos , Recém-Nascido , Gravidez , Complicações Infecciosas na Gravidez/diagnóstico , Complicações Infecciosas na Gravidez/prevenção & controle
12.
Rev Med Virol ; 24(6): 420-33, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25316174

RESUMO

Human cytomegalovirus is the leading non-genetic cause of congenital malformation in developed countries. Congenital CMV may result in fetal and neonatal death or development of serious clinical sequelae. In this review, we identified evidence-based interventions for prevention of congenital CMV at the primary level (prevention of maternal infection), secondary level (risk reduction of fetal infection and disease) and tertiary level (risk reduction of infected neonates being affected by CMV). A systematic review of existing literature revealed 24 eligible studies that met the inclusion criteria. Prevention of maternal infection using hygiene and behavioural interventions reduced maternal seroconversion rates during pregnancy. However, evidence suggested maternal adherence to education on preventative behaviours was a limiting factor. Treatment of maternal CMV infection with hyperimmune globulin (HIG) showed some evidence for efficacy in prevention of fetal infection and fetal/neonatal morbidity with a reasonable safety profile. However, more robust clinical evidence is required before HIG therapy can be routinely recommended. Limited evidence also existed for the safety and efficacy of established CMV antivirals (valaciclovir, ganciclovir and valganciclovir) to treat neonatal consequences of CMV infection, but toxicity and lack of randomised clinical trial data remain major issues. In the absence of a licensed CMV vaccine or robust clinical evidence for anti-CMV therapeutics, patient education and behavioural interventions that emphasise adherence remain the best preventative strategies for congenital CMV. There is a strong need for further data on the use of HIG and other antivirals in pregnancy, as well as the development of less toxic, novel, antiviral agents.


Assuntos
Infecções por Citomegalovirus/prevenção & controle , Infecções por Citomegalovirus/transmissão , Citomegalovirus/fisiologia , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Complicações Infecciosas na Gravidez/prevenção & controle , Adulto , Antivirais/uso terapêutico , Citomegalovirus/efeitos dos fármacos , Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/tratamento farmacológico , Feminino , Humanos , Recém-Nascido , Masculino , Gravidez , Complicações Infecciosas na Gravidez/tratamento farmacológico , Complicações Infecciosas na Gravidez/virologia
13.
Commun Biol ; 7(1): 340, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38504123

RESUMO

Human cytomegalovirus (CMV) infection is the leading non-genetic aetiology of congenital malformation in developed countries, causing significant fetal neurological injury. This study investigated potential CMV pathogenetic mechanisms of fetal neural malformation using in vitro human cerebral organoids. Cerebral organoids were permissive to CMV replication, and infection dysregulated cellular pluripotency and differentiation pathways. Aberrant expression of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK), sonic hedgehog (SHH), pluripotency, neurodegeneration, axon guidance, hippo signalling and dopaminergic synapse pathways were observed in CMV-infected organoids using immunofluorescence and RNA-sequencing. Infection with CMV resulted in dysregulation of 236 Autism Spectrum Disorder (ASD)-related genes (p = 1.57E-05) and pathways. This notable observation suggests potential links between congenital CMV infection and ASD. Using DisGeNET databases, 103 diseases related to neural malformation or mental disorders were enriched in CMV-infected organoids. Cytomegalovirus infection-related dysregulation of key cerebral cellular pathways potentially provides important, modifiable pathogenetic mechanisms for congenital CMV-induced neural malformation and ASD.


Assuntos
Transtorno do Espectro Autista , Infecções por Citomegalovirus , Doenças Fetais , Feminino , Humanos , Citomegalovirus/fisiologia , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Proteínas Hedgehog/metabolismo , Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/metabolismo , Organoides/metabolismo
14.
Viruses ; 16(6)2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38932210

RESUMO

Human cytomegalovirus (CMV) infection is the leading non-genetic cause of congenital malformation in developed countries, causing significant fetal injury, and in some cases fetal death. The pathogenetic mechanisms through which this host-specific virus infects then damages both the placenta and the fetal brain are currently ill-defined. We investigated the CMV modulation of key signaling pathway proteins for these organs including dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) and Sonic Hedgehog (SHH) pathway proteins using human first trimester placental trophoblast (TEV-1) cells, primary human astrocyte (NHA) brain cells, and CMV-infected human placental tissue. Immunofluorescence demonstrated the accumulation and re-localization of SHH proteins in CMV-infected TEV-1 cells with Gli2, Ulk3, and Shh re-localizing to the CMV cytoplasmic virion assembly complex (VAC). In CMV-infected NHA cells, DYRK1A re-localized to the VAC and DYRK1B re-localized to the CMV nuclear replication compartments, and the SHH proteins re-localized with a similar pattern as was observed in TEV-1 cells. Western blot analysis in CMV-infected TEV-1 cells showed the upregulated expression of Rb, Ulk3, and Shh, but not Gli2. In CMV-infected NHA cells, there was an upregulation of DYRK1A, DYRK1B, Gli2, Rb, Ulk3, and Shh. These in vitro monoculture findings are consistent with patterns of protein upregulation and re-localization observed in naturally infected placental tissue and CMV-infected ex vivo placental explant histocultures. This study reveals CMV-induced changes in proteins critical for fetal development, and identifies new potential targets for CMV therapeutic development.


Assuntos
Astrócitos , Infecções por Citomegalovirus , Citomegalovirus , Proteínas Hedgehog , Placenta , Proteínas Tirosina Quinases , Transdução de Sinais , Humanos , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Citomegalovirus/fisiologia , Gravidez , Placenta/virologia , Placenta/metabolismo , Astrócitos/virologia , Astrócitos/metabolismo , Feminino , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Infecções por Citomegalovirus/virologia , Infecções por Citomegalovirus/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fosforilação , Trofoblastos/virologia , Trofoblastos/metabolismo , Quinases Dyrk , Linhagem Celular , Células Cultivadas
15.
J Gen Virol ; 94(Pt 11): 2495-2503, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23939977

RESUMO

Human cytomegalovirus (CMV) infects monocytes and other haematopoietic progenitor cells which then act as reservoirs for latency and virus dissemination. The chemokine CCL2 (monocyte chemotactic protein-1 or MCP-1) exhibits potent chemotactic activity for monocytes and is a likely target for CMV-induced immunomodulation. In this study, we demonstrate CMV modulates CCL2 expression in MRC-5 fibroblasts with multiplicity-dependent kinetics, where CCL2 is upregulated during early stage infection, followed by CCL2 inhibition at late stage infection. This CMV-induced CCL2 modulation was dependent upon virus replication, as UV-inactivated virus did not elicit any changes in CCL2 levels. Dual immunofluorescence staining showed CMV strains AD169, purified AD169, Merlin, FIX WT (FLAG-US28/WT) and pUS28-deficient FIX (FIX-ΔUS28) all induced upregulation of CCL2 primarily within infected cells. Focal upregulation of CCL2 within FIX-ΔUS28-infected cells demonstrated intracellular CCL2 accumulation was independent of CCL2 sequestration by the CMV-encoded chemokine receptor US28. Infection with purified virus confirmed CMV-induced CCL2 upregulation was not due to any CCL2-inducing factors contained within non-purified virus stocks. The CMV-induced CCL2 expression kinetics occurred concurrently with modulation of the CCL2 transcriptional activators NF-κB, interferon regulatory factor 3 and cytokine IFN-ß, independent of virus strain, and with the establishment of viral replication compartments within infected cell nuclei. This is the first report to our knowledge to demonstrate CMV modulation of CCL2 expression within infected cells during viral replication. This immune modulation may facilitate virus dissemination, establishment of latency and pathogenesis of CMV-induced host disease.


Assuntos
Quimiocina CCL2/metabolismo , Citomegalovirus/patogenicidade , Fibroblastos/virologia , Regulação da Expressão Gênica , Replicação Viral/fisiologia , Linhagem Celular , Quimiocina CCL2/genética , Citomegalovirus/fisiologia , Humanos , Imunomodulação , Cinética
16.
Virus Res ; 335: 199200, 2023 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-37591314

RESUMO

Human cytomegalovirus (HCMV) infection is shaped by a tightly regulated interplay between viral and cellular proteins. Distinct kinase activities, such as the viral cyclin-dependent kinase ortholog (vCDK) pUL97 and cellular CDK7 are both crucial for efficient viral replication. Previously, we reported that both kinases, vCDK/pUL97 and CDK7, interact with cyclin H, thereby achieving an enhanced level of kinase activity and overall functionality in viral replication. Here we provide a variety of novel results, as generated on a methodologically extended basis, and present a concept for the codetermination of viral replication efficiency through these kinase activities: (i) cyclin H expression, in various human cell types, is substantially upregulated by strains of HCMV including the clinically relevant HCMV Merlin; (ii) vCDK/pUL97 interacts with human cyclin H in both HCMV-infected and plasmid-transfected cell systems; (iii) a doxycycline-inducible shRNA-dependent knock-down (KD) of cyclin H significantly reduces pUL97 activity (qSox in vitro kinase assay); (iv) accordingly, pUL97 in vitro kinase activity is seen significantly increased upon addition of recombinant cyclin H; (v) as a point of specific importance, human CDK7 activity shows an increase by vCDK/pUL97-mediated trans-stimulation (whereas pUL97 is not stimulated by CDK7); (vi) phosphosite-specific antibodies indicate an upregulated CDK7 phosphorylation upon HCMV infection, as mediated through a pUL97-specific modulatory effect (i.e. shown by pUL97 inhibitor treatment or pUL97-deficient viral mutant); (vii) finally, an efficient KD of cyclin H in primary fibroblasts generally results in an impaired HCMV replication efficiency as measured on protein and genomic levels. These results show evidence for the codetermination of viral replication by vCDK/pUL97, cyclin H and CDK7, thus supporting the specific importance of cyclin H as a central regulatory factor, and suggesting novel targeting options for antiviral drugs.


Assuntos
Quinases Ciclina-Dependentes , Citomegalovirus , Humanos , Antivirais , Ciclina H , Quinases Ciclina-Dependentes/genética , Citomegalovirus/genética , Fosforilação
19.
Placenta ; 112: 62-65, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34298423

RESUMO

Human cytomegalovirus congenital infection is the leading non-genetic cause of fetal malformation in developed countries. There are currently no safe antivirals for use during pregnancy. Placental trophoblast cells specifically secrete exosomes containing miRNA from the chromosome 19 miRNA cluster (C19MC) which confer viral resistance to recipient cells. We show the highly expressed C19MC miRNA miR-517a-3p inhibits HCMV replication and viral protein expression in both fibroblast and trophoblast cell cultures (71.6% and 50.4% inhibition of HCMV DNA at 7 days post infection respectively; p < 0.05). This naturally occurring molecule has potential for opening-up antiviral therapeutic strategies for pregnancy.


Assuntos
Citomegalovirus , MicroRNAs/fisiologia , Complicações Infecciosas na Gravidez/imunologia , Células Cultivadas , Feminino , Interações Hospedeiro-Patógeno , Humanos , Gravidez , Replicação Viral
20.
Sci Rep ; 10(1): 1082, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974453

RESUMO

Human cytomegalovirus (CMV) is the leading non-genetic cause of fetal malformation in developed countries. CMV placental infection is a pre-requisite for materno-fetal transmission of virus, and fetal infection. We investigated the roles of the viral pentameric complex gH/gL/pUL128-pUL131A, and cellular platelet-derived growth factor receptor-α (PDGFRα) for CMV infection in first trimester extravillous-derived (SGHPL-4) and villous-derived (HTR-8/SVneo) trophoblast cells. Infection with four CMV clinical and laboratory strains (Merlin, TB40E, Towne, AD169), and Merlin deletion mutants of UL128-, UL130-, and UL131A-genes, showed a cell type-dependent requirement of the viral pentameric complex for infection of trophoblast cells. The viral pentameric complex was essential for infection of villous trophoblasts, but non-essential for extravillous trophoblasts. Blocking of PDGFRα in extravillous trophoblasts, which naturally express PDGFRα, inhibited entry of pentameric complex-deficient CMV strains, but not the entry of pentameric positive CMV strains. Transient expression of PDGFRα in villous trophoblasts, which are naturally deficient in PDGFRα, promoted the entry of CMV strains lacking gH/gL/pUL128-pUL131A, but had no effect on entry of pentameric positive CMV strains. These results suggest PDGFRα is an important cell receptor for entry of CMV mutant strains lacking gH/gL/pUL128-pUL131A complexes in some placental cells, suggesting these entry pathways could be potential antiviral targets.


Assuntos
Infecções por Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Complicações Infecciosas na Gravidez/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Trofoblastos/metabolismo , Internalização do Vírus , Linhagem Celular , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Feminino , Humanos , Placenta/citologia , Placenta/metabolismo , Placenta/virologia , Gravidez , Complicações Infecciosas na Gravidez/genética , Complicações Infecciosas na Gravidez/virologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Trofoblastos/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
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