Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
bioRxiv ; 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38405935

RESUMO

The broad tissue distribution and cell tropism of human cytomegalovirus indicates that the virus successfully replicates in tissues with various nutrient environments. HCMV requires and reprograms central carbon metabolism for viral replication. However, many studies focus on reprogramming of metabolism in high nutrient conditions that do not recapitulate physiological nutrient environments in the body. In this study, we investigate how HCMV successfully replicates when nutrients are suboptimal. We limited glucose following HCMV infection to determine how glucose supports virus replication and how nutrients potentially present in the physiological environment contribute to successful glucose independent HCMV replication. Glucose is required for HCMV viral genome synthesis, viral protein production and glycosylation, and virus production. However, supplement of glucose-free cultures with uridine, ribose, or UDP-GlcNAc-metabolites that support upper glycolytic branches-resulted in partially restored viral genome synthesis and subsequent partial restoration of viral protein levels. Low levels of virus production were also restored. Supplementing lower glycolysis in glucose-free cultures using pyruvate had no effect on virus replication. These results indicate nutrients that support upper glycolytic branches like the pentose phosphate pathway and hexosamine pathway can compensate for glucose during HCMV replication to support low levels of virus production. More broadly, our findings suggest that HCMV could successfully replicate in diverse metabolic niches, including those in the body with low levels of glucose, through alternative nutrient usage.

2.
Nat Commun ; 14(1): 638, 2023 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-36746962

RESUMO

The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-Mitochondria contact sites (ERMCS) is a platform for critical cellular processes, particularly lipid synthesis. How contacts are remodeled and the impact of altered contacts on lipid metabolism remains poorly understood. We show that the p97 AAA-ATPase and its adaptor ubiquitin-X domain adaptor 8 (UBXD8) regulate ERMCS. The p97-UBXD8 complex localizes to contacts and its loss increases contacts in a manner that is dependent on p97 catalytic activity. Quantitative proteomics and lipidomics of ERMCS demonstrates alterations in proteins regulating lipid metabolism and a significant change in membrane lipid saturation upon UBXD8 deletion. Loss of p97-UBXD8 increased membrane lipid saturation via SREBP1 and the lipid desaturase SCD1. Aberrant contacts can be rescued by unsaturated fatty acids or overexpression of SCD1. We find that the SREBP1-SCD1 pathway is negatively impacted in the brains of mice with p97 mutations that cause neurodegeneration. We propose that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation.


Assuntos
Lipídeos de Membrana , Ubiquitina , Animais , Camundongos , Ubiquitina/metabolismo , Lipídeos de Membrana/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Metabolismo dos Lipídeos
3.
Cell Mol Bioeng ; 14(6): 597-612, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34900013

RESUMO

INTRODUCTION: Platelet activation by mechanical means such as shear stress exposure, is a vital driver of thrombotic risk in implantable blood-contacting devices used in the treatment of heart failure. Lipids are essential in platelets activation and have been studied following biochemical activation. However, little is known regarding lipid alterations occurring with mechanical shear-mediated platelet activation. METHODS: Here, we determined if shear-activation of platelets induced lipidome changes that differ from those associated with biochemically-mediated platelet activation. We performed high-resolution lipidomic analysis on purified platelets from four healthy human donors. For each donor, we compared the lipidome of platelets that were non-activated or activated by shear, ADP, or thrombin treatment. RESULTS: We found that shear activation altered cell-associated lipids and led to the release of lipids into the extracellular environment. Shear-activated platelets released 21 phospholipids and sphingomyelins at levels statistically higher than platelets activated by biochemical stimulation. CONCLUSIONS: We conclude that shear-mediated activation of platelets alters the basal platelet lipidome. Further, these alterations differ and are unique in comparison to the lipidome of biochemically activated platelets. Many of the released phospholipids contained an arachidonic acid tail or were phosphatidylserine lipids, which have known procoagulant properties. Our findings suggest that lipids released by shear-activated platelets may contribute to altered thrombosis in patients with implanted cardiovascular therapeutic devices. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00692-x.

4.
mBio ; 12(3)2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947752

RESUMO

Stress and virus infection regulate lipid metabolism. Human cytomegalovirus (HCMV) infection induces fatty acid (FA) elongation and increases the abundance of lipids with very-long-chain FA (VLCFA) tails. While reprogramming of metabolism can be stress related, the role of stress in HCMV reprogramming of lipid metabolism is poorly understood. In this study, we engineered cells to knock out protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) in the ER stress pathway and measured lipid changes using lipidomics to determine if PERK is needed for lipid changes associated with HCMV infection. In HCMV-infected cells, PERK promotes increases in the levels of phospholipids with saturated FA (SFA) and monounsaturated FA (MUFA) VLCFA tails. Further, PERK enhances FA elongase 7 (ELOVL7) protein levels, which elongates SFA and MUFA VLCFAs. Additionally, we found that increases in the elongation of polyunsaturated fatty acids (PUFAs) associated with HCMV infection were independent of PERK and that lipids with PUFA tails accumulated in HCMV-infected PERK knockout cells. Additionally, the protein levels of ELOVL5, which elongates PUFAs, are increased by HCMV infection through a PERK-independent mechanism. These observations show that PERK differentially regulates ELOVL7 and ELOVL5, creating a balance between the synthesis of lipids with SFA/MUFA tails and PUFA tails. Additionally, we found that PERK was necessary for virus replication and the infectivity of released viral progeny. Overall, our findings indicate that PERK-and, more broadly, ER stress-may be necessary for the membrane biogenesis needed to generate infectious HCMV virions.IMPORTANCE HCMV is a common herpesvirus that establishes lifelong persistent infections. While infection is asymptomatic in most people, HCMV causes life-threatening illnesses in immunocompromised people, including transplant recipients and cancer patients. Additionally, HCMV infection is a leading cause of congenital disabilities. HCMV replication relies on lipid synthesis. Here, we demonstrated that the ER stress mediator PERK controls FA elongation and the cellular abundance of several types of lipids following HCMV infection. Specifically, PERK promotes FA elongase 7 synthesis and phospholipids with saturated/monounsaturated very-long-chain FA tails. Overall, our study shows that PERK is an essential host factor that supports HCMV replication and promotes lipidome changes caused by HCMV infection.


Assuntos
Citomegalovirus/genética , Citomegalovirus/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos/metabolismo , Interações entre Hospedeiro e Microrganismos , Metabolismo dos Lipídeos , Células Cultivadas , Estresse do Retículo Endoplasmático , Fibroblastos/virologia , Humanos , Replicação Viral/fisiologia , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
5.
mBio ; 12(2)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758082

RESUMO

Human cytomegalovirus (HCMV) replication depends on the activities of several host regulators of metabolism. Hypoxia-inducible factor 1α (HIF1α) was previously proposed to support virus replication through its metabolic regulatory function. HIF1α protein levels rise in response to HCMV infection in nonhypoxic conditions, but its effect on HCMV replication was not investigated. We addressed the role of HIF1α in HCMV replication by generating primary human cells with HIF1α knocked out using CRISPR/Cas9. When HIF1α was absent, we found that HCMV replication was enhanced, showing that HIF1α suppresses viral replication. We used untargeted metabolomics to determine if HIF1α regulates metabolite concentrations in HCMV-infected cells. We discovered that in HCMV-infected cells, HIF1α suppresses intracellular and extracellular concentrations of kynurenine. HIF1α also suppressed the expression of indoleamine 2,3-dioxygenase 1 (IDO1), the rate-limiting enzyme in kynurenine synthesis. In addition to its role in tryptophan metabolism, kynurenine acts as a signaling messenger by activating aryl hydrocarbon receptor (AhR). Inhibiting AhR reduces HCMV replication, while activating AhR with an exogenous ligand increases virus replication. Moreover, we found that feeding kynurenine to cells promotes HCMV replication. Overall, our findings indicate that HIF1α reduces HCMV replication by regulating metabolism and metabolite signaling.IMPORTANCE Viruses, including human cytomegalovirus (HCMV), reprogram cellular metabolism using host metabolic regulators to support virus replication. Alternatively, in response to infection, the host can use metabolism to limit virus replication. Here, our findings show that the host uses hypoxia-inducible factor 1α (HIF1α) as a metabolic regulator to reduce HCMV replication. Further, we found that HIF1α suppresses kynurenine synthesis, a metabolite that can promote HCMV replication by signaling through the aryl hydrocarbon receptor (AhR). In infected cells, the rate-limiting enzyme in kynurenine synthesis, indoleamine 2,3-dioxygenase 1 (IDO1), is suppressed by a HIF1α-dependent mechanism. Our findings describe a functional connection between HIF1α, IDO1, and AhR that allows HIF1α to limit HCMV replication through metabolic regulation, advancing our understanding of virus-host interactions.


Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/patogenicidade , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Cinurenina/antagonistas & inibidores , Replicação Viral/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Interações entre Hospedeiro e Microrganismos , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Cinurenina/análise , Cinurenina/metabolismo , Metabolômica/métodos , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais
6.
Elife ; 92020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32910773

RESUMO

Human Cytomegalovirus (HCMV) infects over half the world's population, is a leading cause of congenital birth defects, and poses serious risks for immuno-compromised individuals. To expand the molecular knowledge governing virion maturation, we analysed HCMV virions using proteomics, and identified a significant proportion of host exosome constituents. To validate this acquisition, we characterized exosomes released from uninfected cells, and demonstrated that over 99% of the protein cargo was subsequently incorporated into HCMV virions during infection. This suggested a common membrane origin, and utilization of host exosome machinery for virion assembly and egress. Thus, we selected a panel of exosome proteins for knock down, and confirmed that loss of 7/9 caused significantly less HCMV production. Saliently, we report that VAMP3 is essential for viral trafficking and release of infectious progeny, in various HCMV strains and cell types. Therefore, we establish that the host exosome pathway is intrinsic for HCMV maturation, and reveal new host regulators involved in viral trafficking, virion envelopment, and release. Our findings underpin future investigation of host exosome proteins as important modulators of HCMV replication with antiviral potential.


Assuntos
Citomegalovirus/fisiologia , Exossomos/metabolismo , Interações Hospedeiro-Patógeno , Montagem de Vírus , Liberação de Vírus , Linhagem Celular , Exossomos/genética , Humanos , Transporte Proteico , Proteômica , Proteína 3 Associada à Membrana da Vesícula/genética , Proteínas Virais/metabolismo , Vírion/fisiologia , Replicação Viral
7.
Curr Opin Virol ; 39: 60-69, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31766001

RESUMO

Oncogenic viruses, like all viruses, relies on host metabolism to provide the metabolites and energy needed for virus replication. Many DNA tumor viruses and retroviruses will reprogram metabolism during infection. Additionally, some viral oncogenes may alter metabolism independent of virus replication. Virus infection and cancer development share many similarities regarding metabolic reprogramming as both processes demand increased metabolic activity to produce biomass: cell proliferation in the case of cancer and virion production in the case of infection. This review discusses the parallels in metabolic reprogramming between human oncogenic viruses and oncogenesis.


Assuntos
Carcinogênese , Reprogramação Celular , Redes e Vias Metabólicas , Vírus Oncogênicos/fisiologia , Biomassa , Proliferação de Células , Hepacivirus/fisiologia , Vírus da Hepatite B/fisiologia , Herpesviridae/fisiologia , Humanos , Poliomavírus das Células de Merkel/fisiologia , Neoplasias/metabolismo , Papillomaviridae/fisiologia , Retroviridae , Vírion/metabolismo , Replicação Viral
8.
J Virol ; 93(21)2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31391267

RESUMO

Human cytomegalovirus (HCMV) replication requires host metabolism. Infection alters the activity in multiple metabolic pathways, including increasing fatty acid elongation and lipid synthesis. The virus-host interactions regulating the metabolic changes associated with replication are essential for infection. While multiple host factors, including kinases and transcription factors, important for metabolic changes that occur following HCMV infection have been identified, little is known about the viral factors required to alter metabolism. In this study, we tested the hypothesis that pUL37x1 is important for the metabolic remodeling that is necessary for HCMV replication using a combination of metabolomics, lipidomics, and metabolic tracers to measure fatty acid elongation. We observed that fibroblast cells infected with wild-type (WT) HCMV had levels of metabolites similar to those in cells infected with a mutant virus lacking the UL37x1 gene, subUL37x1. However, we found that relative to WT-infected cells, subUL37x1-infected cells had reduced levels of two host proteins that were previously demonstrated to be important for lipid metabolism during HCMV infection: fatty acid elongase 7 (ELOVL7) and the endoplasmic reticulum (ER) stress-related kinase PERK. Moreover, we observed that HCMV infection results in an increase in phospholipids with very-long-chain fatty acid tails (PL-VLCFAs) that contain 26 or more carbons in one of their two tails. The levels of many PL-VLCFAs were lower in subUL37x1-infected cells than in WT-infected cells. Overall, we conclude that although pUL37x1 is not necessary for network-wide metabolic changes associated with HCMV infection, it is important for the remodeling of a subset of metabolic changes that occur during infection.IMPORTANCE Human cytomegalovirus (HCMV) is a common pathogen that asymptomatically infects most people and establishes a lifelong infection. However, HCMV can cause end-organ disease that results in death in the immunosuppressed and is a leading cause of birth defects. HCMV infection depends on host metabolism, including lipid metabolism. However, the viral mechanisms for remodeling of metabolism are poorly understood. In this study, we demonstrate that the viral UL37x1 protein (pUL37x1) is important for infection-associated increases in lipid metabolism, including fatty acid elongation to produce very-long-chain fatty acids (VLCFAs). Furthermore, we found that HCMV infection results in a significant increase in phospholipids, particularly those with VLCFA tails (PL-VLCFAs). We found that pUL37x1 was important for the high levels of fatty acid elongation and PL-VLCFA accumulation that occur in HCMV-infected cells. Our findings identify a viral protein that is important for changes in lipid metabolism that occur following HCMV infection.


Assuntos
Infecções por Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Metabolismo dos Lipídeos , Linhagem Celular , Citomegalovirus/genética , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Elongases de Ácidos Graxos/metabolismo , Ácidos Graxos/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Proteínas Imediatamente Precoces/genética , Lipidômica , Metaboloma , Mutação , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Replicação Viral , eIF-2 Quinase/metabolismo
10.
Geroscience ; 39(3): 251-259, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28681110

RESUMO

Human cytomegalovirus (CMV) is one of the largest viruses known to cause human diseases. Chronic CMV infection, as defined by anti-CMV IgG serology, increases with age and is highly prevalent in older adults. It has complex biology with significant immunologic and health consequences. This article aims to summarize research findings presented at the 6th International Workshop on CMV and Immunosenescence that relate to advances in the areas of CMV tropism, latency, CMV manipulation of cell metabolism, and T cell memory inflation, as well as novel diagnostic evaluation and translational research of chronic CMV infection in older adults. Information summarized here represents the current state of knowledge in these important fields. Investigators have also identified a number of areas that deserve further and more in-depth investigation, including building more precise parallels between mouse CMV (mCMV) and human CMV (HCMV) research. It is hoped that this article will also stimulate engaging discussion on strategies and direction to advance the science to the next level.


Assuntos
Envelhecimento/imunologia , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Imunossenescência , Idoso , Animais , Anticorpos Antivirais/sangue , Biomarcadores/sangue , Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/epidemiologia , Humanos , Imunoglobulina G/sangue , Memória Imunológica/imunologia , Linfócitos T/imunologia , Tropismo/imunologia , Estados Unidos/epidemiologia , Latência Viral/imunologia
11.
Cell Rep ; 10(8): 1375-85, 2015 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-25732827

RESUMO

Human cytomegalovirus (HCMV) infection rewires host-cell metabolism, upregulating flux from glucose into acetyl-CoA to feed fatty acid metabolism, with saturated very-long-chain fatty acids (VLFCAs) required for production of infectious virion progeny. The human genome encodes seven elongase enzymes (ELOVL) that extend long-chain fatty acids into VLCFA. Here, we identify ELOVL7 as pivotal for HCMV infection. HCMV induces ELOVL7 by more than 150-fold. This induction is dependent on mTOR and SREBP-1. ELOVL7 knockdown or mTOR inhibition impairs HCMV-induced fatty acid elongation, HCMV particle release, and infectivity per particle. ELOVL7 overexpression enhances HCMV replication. During HCMV infection, mTOR activity is maintained by the viral protein pUL38. Expression of pUL38 is sufficient to induce ELOVL7, and pUL38-deficient virus is partially defective in ELOVL7 induction and fatty acid elongation. Thus, through its ability to modulate mTOR and SREBP-1, HCMV induces ELOVL7 to synthesize the saturated VLCFA required for efficient virus replication.


Assuntos
Acetiltransferases/metabolismo , Citomegalovirus/fisiologia , Ácidos Graxos/metabolismo , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/genética , Biocatálise , Linhagem Celular , Elongases de Ácidos Graxos , Humanos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/antagonistas & inibidores , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Virais/metabolismo , Vírion/fisiologia , Replicação Viral
12.
Proc Natl Acad Sci U S A ; 111(52): E5706-15, 2014 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-25512541

RESUMO

An shRNA-mediated screen of the 48 human nuclear receptor genes identified multiple candidates likely to influence the production of human cytomegalovirus in cultured human fibroblasts, including the estrogen-related receptor α (ERRα), an orphan nuclear receptor. The 50-kDa receptor and a 76-kDa variant were induced posttranscriptionally following infection. Genetic and pharmacological suppression of the receptor reduced viral RNA, protein, and DNA accumulation, as well as the yield of infectious progeny. In addition, RNAs encoding multiple metabolic enzymes, including enzymes sponsoring glycolysis (enolase 1, triosephosphate isomerase 1, and hexokinase 2), were reduced when the function of ERRα was inhibited in infected cells. Consistent with the effect on RNAs, a substantial number of metabolites, which are normally induced by infection, were either not increased or were increased to a reduced extent in the absence of normal ERRα activity. We conclude that ERRα is needed for the efficient production of cytomegalovirus progeny, and we propose that the nuclear receptor contributes importantly to the induction of a metabolic environment that supports optimal cytomegalovirus replication.


Assuntos
Infecções por Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Receptor alfa de Estrogênio/metabolismo , Replicação Viral/fisiologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Glicólise/genética , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Biossíntese de Proteínas/genética , RNA Viral/genética , RNA Viral/metabolismo , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
13.
J Virol ; 88(13): 7170-7, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24719425

RESUMO

UNLABELLED: During virion maturation, the Rous sarcoma virus (RSV) capsid protein is cleaved from the Gag protein as the proteolytic intermediate CA-SP. Further trimming at two C-terminal sites removes the spacer peptide (SP), producing the mature capsid proteins CA and CA-S. Abundant genetic and structural evidence shows that the SP plays a critical role in stabilizing hexameric Gag interactions that form immature particles. Freeing of CA-SP from Gag breaks immature interfaces and initiates the formation of mature capsids. The transient persistence of CA-SP in maturing virions and the identification of second-site mutations in SP that restore infectivity to maturation-defective mutant viruses led us to hypothesize that SP may play an important role in promoting the assembly of mature capsids. This study presents a biophysical and biochemical characterization of CA-SP and its assembly behavior. Our results confirm cryo-electron microscopy (cryo-EM) structures reported previously by Keller et al. (J. Virol. 87:13655-13664, 2013, doi:10.1128/JVI.01408-13) showing that monomeric CA-SP is fully capable of assembling into capsid-like structures identical to those formed by CA. Furthermore, SP confers aggressive assembly kinetics, which is suggestive of higher-affinity CA-SP interactions than observed with either of the mature capsid proteins. This aggressive assembly is largely independent of the SP amino acid sequence, but the formation of well-ordered particles is sensitive to the presence of the N-terminal ß-hairpin. Additionally, CA-SP can nucleate the assembly of CA and CA-S. These results suggest a model in which CA-SP, once separated from the Gag lattice, can actively promote the interactions that form mature capsids and provide a nucleation point for mature capsid assembly. IMPORTANCE: The spacer peptide is a documented target for antiretroviral therapy. This study examines the biochemical and biophysical properties of CA-SP, an intermediate form of the retrovirus capsid protein. The results demonstrate a previously unrecognized activity of SP in promoting capsid assembly during maturation.


Assuntos
Proteínas do Capsídeo/química , Capsídeo/metabolismo , Fragmentos de Peptídeos/química , Vírus do Sarcoma de Rous/fisiologia , Montagem de Vírus , Sequência de Aminoácidos , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
14.
Proc Natl Acad Sci U S A ; 110(51): E5006-15, 2013 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-24297925

RESUMO

Herpes simplex virus 1 (HSV-1) infection triggers specific metabolic changes in its host cell. To explore the interactions between cellular metabolism and HSV-1 infection, we performed an siRNA screen of cellular metabolic genes, measuring their effect on viral replication. The screen identified multiple enzymes predicted to influence HSV-1 replication, including argininosuccinate synthetase 1 (AS1), which consumes aspartate as part of de novo arginine synthesis. Knockdown of AS1 robustly enhanced viral genome replication and the production of infectious virus. Using high-resolution liquid chromatography-mass spectrometry, we found that the metabolic phenotype induced by knockdown of AS1 in human fibroblasts mimicked multiple aspects of the metabolic program observed during HSV-1 infection, including an increase in multiple nucleotides and their precursors. Together with the observation that AS1 protein and mRNA levels decrease during wild-type infection, this work suggests that reduced AS1 activity is partially responsible for the metabolic program induced by infection.


Assuntos
Argininossuccinato Sintase/metabolismo , Fibroblastos/enzimologia , Herpes Simples/enzimologia , Herpesvirus Humano 1/fisiologia , Replicação Viral/fisiologia , Animais , Argininossuccinato Sintase/genética , Chlorocebus aethiops , Fibroblastos/patologia , Fibroblastos/virologia , Técnicas de Silenciamento de Genes , Genoma Viral/fisiologia , Herpes Simples/genética , Herpes Simples/patologia , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Vero
15.
PLoS Pathog ; 9(5): e1003333, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23696731

RESUMO

Human cytomegalovirus hijacks host cell metabolism, increasing the flux of carbon from glucose to malonyl-CoA, the committed precursor to fatty acid synthesis and elongation. Inhibition of acetyl-CoA carboxylase blocks the production of progeny virus. To probe further the role of fatty acid metabolism during infection, we performed an siRNA screen to identify host cell metabolic enzymes needed for the production of infectious cytomegalovirus progeny. The screen predicted that multiple long chain acyl-CoA synthetases and fatty acid elongases are needed during infection, and the levels of RNAs encoding several of these enzymes were upregulated by the virus. Roles for acyl-CoA synthetases and elongases during infection were confirmed by using small molecule antagonists. Consistent with a role for these enzymes, mass spectrometry-based fatty acid analysis with ¹³C-labeling revealed that malonyl-CoA is consumed by elongases to produce very long chain fatty acids, generating an approximately 8-fold increase in C26-C34 fatty acid tails in infected cells. The virion envelope was yet further enriched in C26-C34 saturated fatty acids, and elongase inhibitors caused the production of virions with lower levels of these fatty acids and markedly reduced infectivity. These results reveal a dependence of cytomegalovirus on very long chain fatty acid metabolism.


Assuntos
Coenzima A Ligases/metabolismo , Infecções por Citomegalovirus/metabolismo , Citomegalovirus/metabolismo , Ácidos Graxos/biossíntese , Malonil Coenzima A/metabolismo , Vírion/metabolismo , Linhagem Celular , Coenzima A Ligases/genética , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Ácidos Graxos/genética , Humanos , Malonil Coenzima A/genética , RNA Interferente Pequeno , Vírion/genética
16.
J Virol ; 84(13): 6377-86, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20427531

RESUMO

Retrovirus assembly is driven by polymerization of the Gag polyprotein as nascent virions bud from host cells. Gag is then processed proteolytically, releasing the capsid protein (CA) to assemble de novo inside maturing virions. CA has N-terminal and C-terminal domains (NTDs and CTDs, respectively) whose folds are conserved, although their sequences are divergent except in the 20-residue major homology region (MHR) in the CTD. The MHR is thought to play an important role in assembly, and some mutations affecting it, including the F167Y substitution, are lethal. A temperature-sensitive second-site suppressor mutation in the NTD, A38V, restores infectivity. We have used cryoelectron tomography to investigate the morphotypes of this double mutant. Virions produced at the nonpermissive temperature do not assemble capsids, although Gag is processed normally; moreover, they are more variable in size than the wild type and have fewer glycoprotein spikes. At the permissive temperature, virions are similar in size and spike content as in the wild type and capsid assembly is restored, albeit with altered polymorphisms. The mutation F167Y-A38V (referred to as FY/AV in this paper) produces fewer tubular capsids than wild type and more irregular polyhedra, which tend to be larger than in the wild type, containing approximately 30% more CA subunits. It follows that FY/AV CA assembles more efficiently in situ than in the wild type and has a lower critical concentration, reflecting altered nucleation properties. However, its infectivity is lower than that of the wild type, due to a 4-fold-lower budding efficiency. We conclude that the wild-type CA protein sequence represents an evolutionary compromise between competing requirements for optimization of Gag assembly (of the immature virion) and CA assembly (in the maturing virion).


Assuntos
Proteínas do Capsídeo/genética , Mutação de Sentido Incorreto , Vírus do Sarcoma de Rous/fisiologia , Vírus do Sarcoma de Rous/ultraestrutura , Supressão Genética , Vírion/ultraestrutura , Montagem de Vírus , Animais , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Viabilidade Microbiana , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Vírus do Sarcoma de Rous/genética
17.
J Mol Biol ; 389(2): 438-51, 2009 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-19361521

RESUMO

In maturing retroviral virions, CA protein assembles to form a capsid shell that is essential for infectivity. The structure of the two folded domains [N-terminal domain (NTD) and C-terminal domain (CTD)] of CA is highly conserved among various retroviruses, and the capsid assembly pathway, although poorly understood, is thought to be conserved as well. In vitro assembly reactions with purified CA proteins of the Rous sarcoma virus (RSV) were used to define factors that influence the kinetics of capsid assembly and provide insights into underlying mechanisms. CA multimerization was triggered by multivalent anions providing evidence that in vitro assembly is an electrostatically controlled process. In the case of RSV, in vitro assembly was a well-behaved nucleation-driven process that led to the formation of structures with morphologies similar to those found in virions. Isolated RSV dimers, when mixed with monomeric protein, acted as efficient seeds for assembly, eliminating the lag phase characteristic of a monomer-only reaction. This demonstrates for the first time the purification of an intermediate on the assembly pathway. Differences in the intrinsic tryptophan fluorescence of monomeric protein and the assembly-competent dimer fraction suggest the involvement of the NTD in the formation of the functional dimer. Furthermore, in vitro analysis of well-characterized CTD mutants provides evidence for assembly dependence on the second domain and suggests that the establishment of an NTD-CTD interface is a critical step in capsid assembly initiation. Overall, the data provide clear support for a model whereby capsid assembly within the maturing virion is dependent on the formation of a specific nucleating complex that involves a CA dimer and is directed by additional virion constituents.


Assuntos
Proteínas do Capsídeo/fisiologia , Capsídeo/química , Vírus do Sarcoma de Rous/fisiologia , Montagem de Vírus , Cinética , Multimerização Proteica , Retroviridae , Eletricidade Estática
18.
Nature ; 457(7230): 694-8, 2009 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-19194444

RESUMO

For a retrovirus such as HIV to be infectious, a properly formed capsid is needed; however, unusually among viruses, retrovirus capsids are highly variable in structure. According to the fullerene conjecture, they are composed of hexamers and pentamers of capsid protein (CA), with the shape of a capsid varying according to how the twelve pentamers are distributed and its size depending on the number of hexamers. Hexamers have been studied in planar and tubular arrays, but the predicted pentamers have not been observed. Here we report cryo-electron microscopic analyses of two in-vitro-assembled capsids of Rous sarcoma virus. Both are icosahedrally symmetric: one is composed of 12 pentamers, and the other of 12 pentamers and 20 hexamers. Fitting of atomic models of the two CA domains into the reconstructions shows three distinct inter-subunit interactions. These observations substantiate the fullerene conjecture, show how pentamers are accommodated at vertices, support the inference that nucleation is a crucial morphologic determinant, and imply that electrostatic interactions govern the differential assembly of pentamers and hexamers.


Assuntos
Capsídeo/metabolismo , Capsídeo/ultraestrutura , Vírus do Sarcoma de Rous/química , Vírus do Sarcoma de Rous/ultraestrutura , Montagem de Vírus , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/ultraestrutura , Microscopia Crioeletrônica , HIV/química , HIV/genética , HIV/ultraestrutura , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/ultraestrutura , Mutação , Polimorfismo Genético , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Vírus do Sarcoma de Rous/genética , Eletricidade Estática
19.
J Virol ; 82(12): 5951-61, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18400856

RESUMO

During retroviral maturation, the CA protein undergoes dramatic structural changes and establishes unique intermolecular interfaces in the mature capsid shell that are different from those that existed in the immature precursor. The most conserved region of CA, the major homology region (MHR), has been implicated in both immature and mature assembly, although the precise contribution of the MHR residues to each event has been largely undefined. To test the roles of specific MHR residues in mature capsid assembly, an in vitro system was developed that allowed for the first-time formation of Rous sarcoma virus CA into structures resembling authentic capsids. The ability of CA to assemble organized structures was destroyed by substitutions of two conserved hydrophobic MHR residues and restored by second-site suppressors, demonstrating that these MHR residues are required for the proper assembly of mature capsids in addition to any role that these amino acids may play in immature particle assembly. The defect caused by the MHR mutations was identified as an early step in the capsid assembly process. The results provide strong evidence for a model in which the hydrophobic residues of the MHR control a conformational reorganization of CA that is needed to initiate capsid assembly and suggest that the formation of an interdomain interaction occurs early during maturation.


Assuntos
Proteínas do Capsídeo/química , Capsídeo/fisiologia , Retroviridae/química , Montagem de Vírus , Sequência de Aminoácidos , Substituição de Aminoácidos , Capsídeo/ultraestrutura , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/isolamento & purificação , Proteínas do Capsídeo/ultraestrutura , Dicroísmo Circular , Interações Hidrofóbicas e Hidrofílicas , Modelos Químicos , Dados de Sequência Molecular , Desnaturação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Retroviridae/genética , Vírus do Sarcoma de Rous/química , Vírus do Sarcoma de Rous/genética , Homologia de Sequência de Aminoácidos , Espectrofotometria Ultravioleta , Termodinâmica , Triptofano/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...