RESUMO
Cyclodextrins (CDs) are cyclic oligosaccharides; the most common CDs contain six, seven, or eight glucose units called α-CDs, ß-CDs, and γ-CDs, respectively. The use of CDs in biomedical research is increasing due to their ability to interact with membrane lipids as well as a wide variety of poorly water-soluble molecules. We assessed the impact of CD cavity size, occupancy, and substitutions on cytotoxicity and cholesterol homeostasis. The potency of CD-mediated cytotoxicity was in the order of ß-CDs, α-CDs, and γ-CDs. Substitutions with hydroxypropyl or carboxymethyl group attenuated cytotoxicity compared with the native CDs, whereas CDs substituted with methyl groups exhibited cytotoxicity that was similar to that of the native CDs. The lipid components in blood exerted remarkable hemolysis-alleviating effects in methyl-ß-CD-induced hemolysis. Occupancy of the CD cavity with cholesterol or a structurally related lipid molecule abrogated the cytotoxic capacity of the CDs. Interestingly, hydroxypropyl-γ-CD (HPγCD) was able to reduce intracellular cholesterol accumulation in Niemannâ»Pick disease type C (NPC) patient-derived fibroblasts as efficiently as HPßCD. Proteomic study indicated that HPßCD and HPγCD treatments altered the expression pattern of cellular proteins, suggesting that some of the CD-induced cellular proteins may play an important function in modulating intracellular cholesterol homeostasis.
Assuntos
Colesterol/metabolismo , Ciclodextrinas/química , Ciclodextrinas/farmacologia , Doença de Niemann-Pick Tipo C/metabolismo , 2-Hidroxipropil-beta-Ciclodextrina/química , 2-Hidroxipropil-beta-Ciclodextrina/farmacologia , Linhagem Celular , Sobrevivência Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Células Jurkat , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos de Membrana/metabolismo , Estrutura Molecular , Proteômica , Testes de Toxicidade , beta-Ciclodextrinas/química , beta-Ciclodextrinas/farmacologiaRESUMO
Among nonhuman primates, SIV-infected Asian pigtailed macaques (PM) are relatively more susceptible to infection and disease progression than SIV-infected rhesus macaques (RM). In addition, SIV-infected African natural hosts such as the sooty mangabeys (SM) are resistant to disease. The mechanisms associated with such species-related variable clinical outcomes remain ill-defined but hold the potential to provide insights into the underlying mechanisms surrounding HIV pathogenesis. Recent findings indicate that the expression of the heterodimeric gut homing integrin α4ß7 can influence both susceptibility and disease progression in RM. It was reasoned that differences in the frequencies/surface densities of α4ß7-expressing lymphocytes might contribute to the differences in the clinical outcome of SIV infection among NHPs. In this article, we report that CD4(+) T cells from PM constitutively express significantly higher levels of α4ß7 than RM or SM. Retinoic acid, a key regulator of α4ß7 expression, was paradoxically found at higher levels in the plasma of SM versus RM or PM. We also observed pairing of ß7 with αE (αEß7) on CD4(+) T cells in the peripheral blood of SM, but not PM or RM. Finally, the differential mean density of expression of α4ß7 in RM versus SM versus PM was predominantly dictated by species-specific sequence differences at the level of the ß7 promoters, as determined by in vitro reporter/promoter construct transfection studies. We propose that differences in the regulation and expression of α4ß7 may explain, in part, the differences in susceptibility and SIV disease progression in these NHP models.
Assuntos
Expressão Gênica , Integrinas/genética , Especificidade da Espécie , Animais , Sítios de Ligação , Células Sanguíneas/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Cercocebus atys , Clonagem Molecular , Genes Reporter , Imunofenotipagem , Integrinas/classificação , Integrinas/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Macaca , Dados de Sequência Molecular , Filogenia , Primatas , Regiões Promotoras Genéticas , Ligação Proteica , Receptores CCR5/genética , Análise de Sequência de DNA , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/sangue , Tretinoína/sangue , Tretinoína/metabolismoRESUMO
The tripartite motif protein TRIM5α restricts particular retrovirus infections by binding to the incoming capsid and inhibiting the early stage of virus infection. The TRIM5α RING domain exhibits E3 ubiquitin ligase activity and assists the higher-order association of TRIM5α dimers, which promotes capsid binding. We characterized a panel of RING domain mutants of the rhesus monkey TRIM5α (TRIM5α(rh)) protein. The RING domain function that significantly contributed to retroviral restriction depended upon the restricted virus. The E3 ubiquitin ligase activity of the RING domain contributes to the potency of HIV-1 restriction. Nonetheless, TRIM5α(rh) mutants without detectable E3 ubiquitin ligase activity still blocked reverse transcription and inhibited HIV-1 infection at a moderate level. When TRIM5α(rh) capsid binding was weakened by substitution with a less efficient B30.2/SPRY domain, the promotion of higher-order association by the RING domain was more important to HIV-1 restriction than its E3 ubiquitin ligase activity. For the restriction of N-tropic murine leukemia virus (N-MLV) and equine infectious anemia virus (EIAV) infection, promotion of higher-order association represented the major contribution of the RING domain. Thus, both identity of the target virus and the B30.2/SPRY domain-mediated affinity for the viral capsid determine the relative contribution of the two known RING domain functions to TRIM5α restriction of retrovirus infection.
Assuntos
Capsídeo/metabolismo , HIV-1 , Vírus da Anemia Infecciosa Equina , Vírus da Leucemia Murina , Proteínas/metabolismo , Infecções por Retroviridae/metabolismo , Animais , Western Blotting , Linhagem Celular , Cães , Proteínas de Fluorescência Verde/metabolismo , Humanos , Macaca mulatta , Microscopia de Fluorescência , Proteínas/genética , Infecções por Retroviridae/genética , Especificidade da Espécie , Ubiquitina-Proteína Ligases/metabolismoRESUMO
TRIM32, which belongs to the tripartite motif (TRIM) protein family, has the RING finger, B-box, and coiled-coil domain structures common to this protein family, along with an additional NHL domain at the C terminus. TRIM32 reportedly functions as an E3 ligase for actin, a protein inhibitor of activated STAT y (PIASy), dysbindin, and c-Myc, and it has been associated with diseases such as muscular dystrophy and epithelial carcinogenesis. Here, we identify a new substrate of TRIM32 and propose a mechanism through which TRIM32 might regulate apoptosis. Our overexpression and knockdown experiments demonstrate that TRIM32 sensitizes cells to TNFα-induced apoptosis. The RING domain is necessary for this pro-apoptotic function of TRM32 as well as being responsible for its E3 ligase activity. TRIM32 colocalizes and directly interacts with X-linked inhibitor of apoptosis (XIAP), a well known cancer therapeutic target, through its coiled-coil and NHL domains. TRIM32 overexpression enhances XIAP ubiquitination and subsequent proteasome-mediated degradation, whereas TRIM32 knockdown has the opposite effect, indicating that XIAP is a substrate of TRIM32. In vitro reconstitution assay reveals that XIAP is directly ubiquitinated by TRIM32. Our novel results collectively suggest that TRIM32 sensitizes TNFα-induced apoptosis by antagonizing XIAP, an anti-apoptotic downstream effector of TNFα signaling. This function may be associated with TRIM32-mediated tumor suppressive mechanism.
Assuntos
Apoptose/efeitos dos fármacos , Domínios RING Finger , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Apoptose/genética , Sequência de Bases , Regulação para Baixo/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Especificidade por Substrato , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido , Ubiquitinação/efeitos dos fármacosRESUMO
Many viruses require the maintenance of lysosomal cholesterol homeostasis for a successful infection; however, the role of lysosomal cholesterol homeostasis in the alphaherpesvirus life cycle is not clear. Here we show that the lysosomal cholesterol transport inhibitor U18666A interferes with the replication of pseudorabies virus (PRV), a member of the alphaherpesvirus subfamily. The treatment with U18666A caused a significant reduction in the production of infectious virus particles. The U18666A treatment was shown to suppress the release of PRV particles. Pretreating PRV virions with U18666A did not affect virus production, whereas pretreating target cells with U18666A led to a substantial reduction in virus yield. Our previous study showed that two cyclodextrin derivatives, 2-hydroxypropyl-ß-cyclodextrin (HPßCD) and 2-hydroxypropyl-γ-cyclodextrin (HPγCD), can rescue the cholesterol accumulation defect in primary fibroblasts derived from a Niemann-Pick disease type C (NPC) patient. Here, we demonstrate that treatment with HPßCD or HPγCD not only rescues the U18666A-induced cholesterol accumulation but also rescues the U18666A-induced inhibition of PRV production. Collectively, our data suggest that U18666A interferes with PRV infection via altering cellular functions that are critical for the viral life cycle and may include lysosomal cholesterol homeostasis.
Assuntos
Anticolesterolemiantes , Herpesvirus Suídeo 1 , 2-Hidroxipropil-beta-Ciclodextrina/farmacologia , Androstenos/farmacologia , Animais , Anticolesterolemiantes/farmacologia , Colesterol , HumanosRESUMO
Tripartite motif (TRIM) protein TRIM5alpha has been shown to restrict human immunodeficiency virus, type 1 infection in Old World monkey cells at the early post-entry step by poorly understood mechanisms. Currently, the physiological function of TRIM5alpha is not known. In this study, we showed that transiently overexpressed TRIM5alpha causes a morphological change in HEK293T cells. A proteomics analysis of the protein complexes that were pulled down with hemagglutinin-tagged TRIM5alpha suggested that the heat shock protein 70 (Hsp70) may serve as a TRIM5alpha-binding partner. The interaction between Hsp70 and TRIM5alpha was confirmed by co-localization and co-immunoprecipitation assays. Co-expression of Hsp70 reversed the TRIM5alpha-induced morphological change in HEK293T cells. Another heat shock protein Hsc70 also bound to TRIM5alpha, but unlike Hsp70, Hsc70 was not able to reverse the TRIM5alpha-induced morphological change, suggesting that Hsp70 specifically reverses the morphological change caused by TRIM5alpha. Studies using a series of TRIM5alpha deletion mutants demonstrate that, although the PRYSPRY domain is critical for binding to Hsp70, the entire TRIM5alpha structure is necessary to induce the morphological change of cells. When the ATPase domain of Hsp70 was mutated, the mutated Hsp70 could not counteract the morphological change induced by TRIM5alpha, indicating that the catalytic activity of Hsp70 protein is important for this function. Co-expression of Hsp70 elevated the levels of TRIM5alpha in the detergent-soluble fraction with a concomitant decrease in the detergent-insoluble fraction. Together these results suggest that Hsp70 plays critical roles in the cellular management against the TRIM5alpha-induced cellular insults.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/metabolismo , Animais , Linhagem Celular , Forma Celular , Proteínas de Choque Térmico HSP70/genética , Humanos , Macaca mulatta , Modelos Moleculares , Ligação Proteica , Proteínas/genética , Proteoma/análise , Retroviridae/genética , Retroviridae/metabolismo , Ubiquitina-Proteína LigasesRESUMO
BACKGROUND: Enveloped viruses including the simian immunodeficiency virus (SIV) replicating within host cells acquire host proteins upon egress from the host cells. A number of studies have catalogued such host proteins, and a few have documented the potential positive and negative biological functions of such host proteins. The studies conducted herein utilized proteomic analysis to identify differences in the spectrum of host proteins acquired by a single source of SIV replicating within CD4+ T cells from disease resistant sooty mangabeys and disease susceptible rhesus macaques. RESULTS: While a total of 202 host derived proteins were present in viral preparations from CD4+ T cells from both species, there were 4 host-derived proteins that consistently and uniquely associated with SIV replicating within CD4+ T cells from rhesus macaques but not sooty mangabeys; and, similarly, 28 host-derived proteins that uniquely associated with SIV replicating within CD4+ T cells from sooty mangabeys, but not rhesus macaques. Of interest was the finding that of the 4 proteins uniquely present in SIV preparations from rhesus macaques was a 26 S protease subunit 7 (MSS1) that was shown to enhance HIV-1 'tat' mediated transactivation. Among the 28 proteins found in SIV preparations from sooty mangabeys included several molecules associated with immune function such as CD2, CD3ε, TLR4, TLR9 and TNFR and a bioactive form of IL-13. CONCLUSIONS: The finding of 4 host proteins that are uniquely associated with SIV replicating within CD4+ T cells from disease susceptible rhesus macaques and 28 host proteins that are uniquely associated with SIV replicating within CD4+ T cells from disease resistant sooty mangabeys provide the foundation for determining the potential role of each of these unique host-derived proteins in contributing to the polarized clinical outcome in these 2 species of nonhuman primates.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Especificidade de Hospedeiro , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Vírus da Imunodeficiência Símia/fisiologia , Replicação Viral , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Cercocebus atys , Suscetibilidade a Doenças , Imunidade Inata , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologiaRESUMO
TRIM5alpha protein blocks retroviral replication at early postentry stage reducing the accumulation of reverse transcriptase products. TRIM5alpha proteins of Old World primates restrict HIV-1 infection whereas TRIM5alpha proteins of most New World monkeys restrict SIV(mac) infection. TRIM5alpha protein has a RING domain, B-box 2 domain, coiled-coil domain, and PRYSPRY domain. The PRYSPRY domain of TRIM5alpha determines viral specificity and restriction potency by mediating recognition of the retroviral capsid. The coiled-coil domain is essential for TRIM5alpha oligomerization, which contributes to binding avidity for the viral capsid. The RING domain and B-box 2 domain are required for efficient restriction activity of TRIM5alpha protein but the mechanisms remain to be defined.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas/fisiologia , Infecções por Retroviridae/prevenção & controle , Animais , Fatores de Restrição Antivirais , Proteínas de Transporte/química , Ciclofilina A/fisiologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/fisiologia , Humanos , Camundongos , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteínas/química , Proteínas/genética , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Internalização do VírusRESUMO
Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder caused by mutations in NPC1 and NPC2 genes that result in an accumulation of cholesterol in lysosomes. The majority of children with NPC die in adolescence. Currently, no FDA-approved therapies exist for NPC and the mechanisms of NPC disease are not fully understood. Our recent study and the reports from other laboratories showed that 2-hydroxypropyl-γ-cyclodextrin (HPγCD) alleviates cholesterol accumulation in NPC1-deficient cells in spite of its low binding affinity for cholesterol. In this study, we explored the cellular changes that are induced upon HPγCD treatment in NPC1 patient-derived fibroblasts. We show that HPγCD treatment increases lysosome-ER association and enhances autophagic activity. Our study indicates that HPγCD induces an activation of the transcription factor EB (TFEB), a master regulator of lysosomal functions and autophagy. Lysosome-ER association could potentially function as conduits for cholesterol transport from lysosomes to the ER. Accumulating evidence suggests a role for autophagy in rescuing the cholesterol accumulation in NPC and other degenerative diseases. Collectively, our findings suggest that HPγCD restores cellular homeostasis in NPC1-deficient cells via enhancing lysosomal dynamics and functions. Understanding the mechanisms of HPγCD-induced cellular pathways could contribute to effective NPC therapies.
Assuntos
Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/metabolismo , gama-Ciclodextrinas/farmacologia , Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Células Cultivadas , Criança , Feminino , Humanos , Lactente , Masculino , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Proteínas de Transporte Vesicular/genéticaRESUMO
Niemann-Pick type C (NPC) disease is a fatal hereditary neurodegenerative disorder characterized by a massive accumulation of cholesterol in lysosomes and late endosomes due to a defect in intracellular cholesterol trafficking. Dysfunction in intracellular cholesterol trafficking is responsible for about 50 rare inherited lysosomal storage disorders including NPC. The lysosomal proteins NPC1 and NPC2 play a crucial role in trafficking of cholesterol from late endosomes and lysosomes to other cellular compartments. However, the detailed mechanisms of cholesterol trafficking at the late endosomes/lysosomes (LE/LY) are poorly understood. Studies showed that 2-hydroxypropyl-ß-cyclodextrin (HPßCD) alleviates the cholesterol accumulation defect in animal model and has been approved for a phase 2b/3 clinical trial for NPC. HPßCD is known to bind cholesterol; however, the mechanisms how HPßCD mediates the exit of cholesterol from the LE/LY compartments are still unknown. Further, another cyclodextrin (CD) derivative, 2-hydroxypropyl-γ-cyclodextrin (HPγCD), was shown to reduce intracellular cholesterol accumulation in NPC patient cells and NPC mice model. Herein, we identified a number of candidate proteins differentially expressed in NPC patient-derived cells compared to cells derived from a healthy donor using a proteomic approach. Interestingly, both HPßCD and HPγCD treatments modulated the expression of most of these NPC-specific proteins. Data showed that treatment with both CDs induces the expression of the lysosome-associated membrane protein 1 (LAMP-1) in NPC patient-derived cells. Remarkably, LAMP-1 overexpression in HeLa cells rescued U18666A-induced cholesterol accumulation suggesting a role of LAMP-1 in cholesterol trafficking. We propose that HPßCD and HPγCD facilitate cholesterol export from the LE/LY compartments via the LAMP-1 protein, which may play a crucial role in cholesterol trafficking at the LE/LY compartments when there is no functional NPC1 protein. Together, this study uncovers new cellular mechanisms for cholesterol trafficking, which will contribute to development of novel therapeutic approaches for lysosomal storage diseases.
Assuntos
2-Hidroxipropil-beta-Ciclodextrina/farmacologia , Colesterol/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/metabolismo , gama-Ciclodextrinas/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisossomos , Transporte Proteico/efeitos dos fármacos , Proteínas/metabolismo , Proteômica/métodos , beta-Ciclodextrinas/farmacologiaRESUMO
Human immunodeficiency virus (HIV) infection and its associated diseases continue to increase despite the progress in our understanding of HIV biology and the availability of a number of antiretroviral drugs. Adherence is a significant factor in the success of HIV therapy and current HIV treatment regimens require a combination of antiviral drugs to be taken at least daily for the remainder of a patient's life. A drug delivery system that allows sustained drug delivery could reduce the medical burden and costs associated with medication nonadherence. Here, we describe a novel supramolecular assembly or matrix that contains an anionic polymer hyaluronic acid, cationic polymer poly-l-lysine, and anionic oligosaccharide sulfobutylether-beta-cyclodextrin. HIV reverse transcriptase inhibitors Zidovudine and Lamivudine were successfully encapsulated into the polymer assembly in a noncovalent manner. The physicochemical properties and antiviral activity of the polymer assemblies were studied. The results of this study suggest that the supramolecular assemblies loaded with HIV drugs exert potent antiviral activity and allow sustained drug release. A novel drug delivery formulation such as the one described here could facilitate our efforts to reduce the morbidity and mortality associated with HIV infections and could be utilized in the design of therapeutic approaches for other diseases.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Terapia Antirretroviral de Alta Atividade/métodos , Materiais Biocompatíveis , Ácido Hialurônico/química , Fármacos Anti-HIV/química , Células Cultivadas , Preparações de Ação Retardada , Sistemas de Liberação de Medicamentos , HIV-1/efeitos dos fármacos , Humanos , Lamivudina/administração & dosagem , Lamivudina/química , Polilisina/química , Zidovudina/administração & dosagem , Zidovudina/química , beta-CiclodextrinasRESUMO
The innate immune response is a host defense mechanism against infection by viruses and bacteria. Type I interferons (IFNα/ß) play a crucial role in innate immunity. If not tightly regulated under normal conditions and during immune responses, IFN production can become aberrant, leading to inflammatory and autoimmune diseases. In this study, we identified TRIM11 (tripartite motif containing 11) as a novel negative regulator of IFNß production. Ectopic expression of TRIM11 decreased IFNß promoter activity induced by poly (I:C) stimulation or overexpression of RIG-I (retinoic acid-inducible gene-I) signaling cascade components RIG-IN (constitutively active form of RIG-I), MAVS (mitochondrial antiviral signaling protein), or TBK1 (TANK-binding kinase-1). Conversely, TRIM11 knockdown enhanced IFNß promoter activity induced by these stimuli. Moreover, TRIM11 overexpression inhibited the phosphorylation and dimerization of IRF3 and expression of IFNß mRNA. By contrast, TRIM11 knockdown increased the IRF3 phosphorylation and IFNß mRNA expression. We also found that TRIM11 and TBK1, a key kinase that phosphorylates IRF3 in the RIG-I pathway, interacted with each other through CC and CC2 domain, respectively. This interaction was enhanced in the presence of the TBK1 adaptor proteins, NAP1 (NF-κB activating kinase-associated protein-1), SINTBAD (similar to NAP1 TBK1 adaptor) or TANK (TRAF family member-associated NF-κB activator). Consistent with its inhibitory role in RIG-I-mediated IFNß signaling, TRIM11 overexpression enhanced viral infectivity, whereas TRIM11 knockdown produced the opposite effect. Collectively, our results suggest that TRIM11 inhibits RIG-I-mediated IFNß production by targeting the TBK1 signaling complex.
Assuntos
Regulação da Expressão Gênica/imunologia , Herpesvirus Humano 1/imunologia , Interferon beta/genética , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína Ligases/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Chlorocebus aethiops , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/imunologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Interferon beta/imunologia , Camundongos , NF-kappa B/genética , NF-kappa B/imunologia , Fosforilação/efeitos dos fármacos , Poli I-C/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Serina-Treonina Quinases/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Imunológicos , Transdução de Sinais , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/imunologia , Células VeroRESUMO
Old World monkey TRIM5alpha proteins are known to block the replication of human immunodeficiency virus and other retroviruses in a species-specific fashion. In this report, we show that specific forms of simian TRIM5alpha proteins can restrict herpes simplex virus (HSV) infection. To define the effect of TRIM5alpha on HSV replication, we examined HSV infection in HeLa cell lines that stably express simian and human orthologs of TRIM5alpha proteins. We demonstrated that several simian TRIM5alpha proteins can restrict HSV replication, with the TRIM5alpha protein of rhesus macaques showing the strongest inhibition of HSV infection. We also found that the level of the inhibition of virus replication was viral strain-specific. TRIM5alpha is likely to inhibit HSV at the early stage of infection; however, at later times of infection, the levels of TRIM5alpha are significantly decreased. Thus, some TRIM5alpha proteins exhibit antiviral effects that extend beyond retroviral infections, but HSV may be able to reduce this restriction by reducing TRIM5alpha levels during the later phases of virus replication. Our results also argue that TRIM5alpha is only part of the reduced level of HSV replication in rhesus macaques, which are known to be less susceptible to HSV infection than other primates.
Assuntos
Herpes Simples/virologia , Simplexvirus/fisiologia , Replicação Viral/fisiologia , Animais , Linhagem Celular , Fibroblastos/virologia , Regulação Viral da Expressão Gênica , Células HeLa/virologia , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 2/metabolismo , Humanos , Macaca mulatta/virologia , Fatores de Transcrição/fisiologia , Proteínas Virais/biossínteseRESUMO
Host cells respond to viral infection by many mechanisms, including the production of type I interferons which act in a paracrine and autocrine manner to induce the expression of antiviral interferon-stimulated genes (ISGs). Viruses have evolved means to inhibit interferon signaling to avoid induction of the innate immune response. Herpes simplex virus 1 (HSV-1) has several mechanisms to inhibit type I interferon production, the activities of ISGs, and the interferon signaling pathway itself. We report that the inhibition of the Jak/STAT pathway by HSV-1 requires viral gene expression and that viral immediate-early protein ICP27 plays a role in downregulating STAT-1 phosphorylation and in preventing the accumulation of STAT-1 in the nucleus. We also show that expression of ICP27 by transfection causes an inhibition of IFN-induced STAT-1 nuclear accumulation. Therefore, ICP27 is necessary and sufficient for at least some of the effects of HSV infection on STAT-1.
Assuntos
Herpesvirus Humano 1/patogenicidade , Proteínas Imediatamente Precoces/metabolismo , Interferon Tipo I/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Núcleo Celular/metabolismo , Chlorocebus aethiops , Regulação para Baixo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Proteínas Imediatamente Precoces/genética , Interferon Tipo I/genética , Interferon-alfa , Mutação , Fosforilação , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Transfecção , Células VeroRESUMO
The retroviral restriction factors, TRIM5alpha and TRIMCyp, consist of RING and B-box 2 domains separated by a coiled coil from carboxy-terminal domains. These carboxy-terminal domains (the B30.2(SPRY) domain in TRIM5alpha and the cyclophilin A domain in TRIMCyp) recognize the retroviral capsid. Here we show that some B-box 2 changes in TRIM5alpha, but not in TRIMCyp, resulted in decreased human immunodeficiency virus (HIV-1) capsid binding. The phenotypic effects of these B-box 2 changes on the restriction of retroviral infection depended on the potency of restriction and the affinity of the TRIM5alpha interaction with the viral capsid, two properties specified by the B30.2(SPRY) domain. Thus, some alterations in the TRIM5alpha B-box 2 domain apparently affect the orientation or conformation of the B30.2(SPRY) domain, influencing capsid recognition.
Assuntos
Proteínas do Capsídeo/metabolismo , Proteínas de Transporte/metabolismo , HIV-1/imunologia , Vírus da Leucemia Murina de Moloney/imunologia , Proteínas/metabolismo , Vírus da Imunodeficiência Símia/imunologia , Animais , Fatores de Restrição Antivirais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Células HeLa , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/genética , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
The host cell factors TRIM5alpha(hu) and Fv-1 restrict N-tropic murine leukemia virus (N-MLV) infection at an early postentry step before or after reverse transcription, respectively. Interestingly, the identity of residue 110 of the MLV capsid determines susceptibility to both TRIM5alpha(hu) and Fv-1. In this study, we investigate the fate of the MLV capsid in cells expressing either the TRIM5alpha(hu) or Fv-1 restriction factor. The expression of TRIM5alpha(hu), but not Fv-1, specifically promoted the premature conversion of particulate N-MLV capsids within infected cells to soluble capsid proteins. The TRIM5alpha(hu)-mediated disassembly of particulate N-MLV capsids was dependent upon residue 110 of the viral capsid. Furthermore, the deletion or disruption of TRIM5alpha(hu) domains necessary for potent N-MLV restriction completely abrogated the disappearance of particulate N-MLV capsids observed with wild-type TRIM5alpha(hu). These results suggest that premature disassembly of the viral capsid contributes to the restriction of N-MLV infection by TRIM5alpha(hu), but not by Fv-1.
Assuntos
Proteínas de Transporte/fisiologia , Vírus da Leucemia Murina/fisiologia , Substituição de Aminoácidos , Animais , Fatores de Restrição Antivirais , Sequência de Bases , Capsídeo/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Primers do DNA/genética , Humanos , Vírus da Leucemia Murina/genética , Vírus da Leucemia Murina/patogenicidade , Camundongos , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
In owl monkeys, the typical retroviral restriction factor of primates, TRIM5alpha, is replaced by TRIMCyp. TRIMCyp consists of the TRIM5 RING, B-box 2 and coiled-coil domains, as well as the intervening linker regions, fused with cyclophilin A. TRIMCyp restricts infection of retroviruses, such as human immunodeficiency virus (HIV-1) and feline immunodeficiency virus (FIV), with capsids that can bind cyclophilin A. The TRIM5 coiled coil promotes the trimerization of TRIMCyp. Here we show that cyclophilin A that is oligomeric as a result of fusion with a heterologous multimer exhibits substantial antiretroviral activity. The addition of the TRIM5 RING, B-box 2 and Linker 2 to oligomeric cyclophilin A generated a protein with antiretroviral activity approaching that of wild-type TRIMCyp. Multimerization increased the binding of cyclophilin A to the HIV-1 capsid, promoting accelerated uncoating of the capsid and restriction of infection.
Assuntos
Fármacos Anti-HIV/farmacologia , Ciclofilina A/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Proteínas/farmacologia , Fatores de Transcrição/farmacologia , Sequência de Aminoácidos , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Aotidae , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Ciclofilina A/química , Ciclofilina A/genética , Ciclofilina A/metabolismo , Dimerização , HIV-1/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The tripartite motif (TRIM) protein, TRIM5alpha, restricts some retroviruses, including human immunodeficiency virus (HIV-1), from infecting the cells of particular species. TRIM proteins contain RING, B-box, coiled-coil and, in some cases, B30.2(SPRY) domains. We investigated the properties of human TRIM family members closely related to TRIM5. These TRIM proteins, like TRIM5alpha, assembled into homotrimers and co-localized in the cytoplasm with TRIM5alpha. TRIM5alpha turned over more rapidly than related TRIM proteins. TRIM5alpha, TRIM34 and TRIM6 associated with HIV-1 capsid-nucleocapsid complexes assembled in vitro; the TRIM5alpha and TRIM34 interactions with these complexes were dependent on their B30.2(SPRY) domains. Only TRIM5alpha potently restricted infection by the retroviruses studied; overexpression of TRIM34 resulted in modest inhibition of simian immunodeficiency virus (SIV(mac)) infection. In contrast to the other TRIM genes examined, TRIM5 exhibited evidence of positive selection. The unique features of TRIM5alpha among its TRIM relatives underscore its special status as an antiviral factor.
Assuntos
Proteínas de Transporte/metabolismo , HIV-1/fisiologia , Nucleocapsídeo/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Fatores de Restrição Antivirais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Citoplasma/química , Cães , HIV-1/imunologia , Células HeLa , Humanos , Macaca mulatta , Proteínas de Membrana/metabolismo , Filogenia , Ligação Proteica , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
Cyclophilin A (Cyp A) binds the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein and contributes to the early events in virus replication in some cells. The retroviral restriction factor TRIM5alpha can inhibit the early, post-entry phase of infection by associating with the incoming viral capsid. Cyp A has been proposed to prevent restriction factor binding in human cells, thus enhancing HIV-1 infectivity, and to potentiate restriction of HIV-1 in monkey cells. Here we show that the positive effects of Cyp A-CA binding on HIV-1 infectivity do not depend on human TRIM5alpha. Disruption of Cyp A binding to CA partially relieved the block to HIV-1 infection imposed by several TRIM5alpha variants, but Cyp A-CA binding was not absolutely required for TRIM5alpha antiviral activity. Inhibition of Cyp A function by cyclosporine significantly decreased the efficiency of TRIM5alpha-mediated restriction only when the restricted virus capsid interacted with Cyp A.
Assuntos
Proteínas de Transporte/metabolismo , Ciclofilina A/metabolismo , HIV-1/metabolismo , Animais , Fatores de Restrição Antivirais , Proteínas de Transporte/genética , Linhagem Celular , Ciclosporina/farmacologia , Cães , Inativação Gênica , Haplorrinos , Humanos , Imunossupressores/farmacologia , Ligação Proteica , RNA Interferente Pequeno , Tacrolimo/metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Replicação ViralRESUMO
TRIM5alpha and TRIMCyp are retroviral restriction factors that, like other members of the tripartite motif (TRIM) family, contain RING, B-box 2 and coiled-coil domains. We found that both proteins are rapidly turned over, with half-lives of 50-60 min. Polyubiquitylation and rapid degradation of TRIM5alpha depended upon intact RING and B-box 2 domains. A chimera consisting of monkey TRIM5alpha with a RING domain of human TRIM21 exhibited a half-life of 210 min, yet potently restricted human immunodeficiency virus; therefore, rapid turnover of TRIM5alpha is not required for its antiretroviral activity. TRIM5alpha forms cytoplasmic bodies that contain other polyubiquitylated proteins, heat shock proteins and dynein, and thus resemble aggresome precursors. Consistent with this interpretation, proteasomal inhibitors triggered the formation of TRIM5alpha(rh)-containing aggresomes in a microtubule-dependent manner. Thus, TRIM5alpha levels in the cell are maintained by continuous synthesis and rapid proteasome-mediated degradation, imbalances in which result in the formation of pre-aggresomal cytoplasmic bodies.