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1.
J Biol Chem ; 293(40): 15678-15690, 2018 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-30135209

RESUMO

Protein trafficking in the endosomal system involves the recognition of specific signals within the cytoplasmic domains (CDs) of transmembrane proteins by clathrin adaptors. One such signal is the phosphoserine acidic cluster (PSAC), the prototype of which is in the endoprotease furin. How PSACs are recognized by clathrin adaptors has been controversial. We reported previously that HIV-1 Vpu, which modulates cellular immunoreceptors, contains a PSAC that binds to the µ subunits of clathrin adaptor protein (AP) complexes. Here, we show that the CD of furin binds the µ subunits of AP-1 and AP-2 in a phosphorylation-dependent manner. Moreover, we identify a potential PSAC in a cytoplasmic loop of the cellular transmembrane Serinc3, an inhibitor of the infectivity of retroviruses. The two serines within the PSAC of Serinc3 are phosphorylated by casein kinase II and mediate interaction with the µ subunits in vitro The sites of these serines vary among mammals in a manner suggesting host-pathogen conflict, yet the Serinc3 PSAC seems dispensable for anti-HIV activity and for counteraction by HIV-1 Nef. The CDs of Vpu and furin and the PSAC-containing loop of Serinc3 each bind the µ subunit of AP-2 (µ2) with similar affinities, but they appear to utilize different basic regions on µ2. The Serinc3 loop requires a region previously reported to bind the acidic plasma membrane lipid phosphatidylinositol 4,5-bisphosphate. These data suggest that the PSACs within different proteins recognize different basic regions on the µ surface, providing the potential to inhibit the activity of viral proteins without necessarily affecting cellular protein trafficking.


Assuntos
Complexo 1 de Proteínas Adaptadoras/química , Complexo 2 de Proteínas Adaptadoras/química , Furina/química , HIV-1/genética , Proteínas de Neoplasias/química , Fosfosserina/química , Receptores de Superfície Celular/química , Complexo 1 de Proteínas Adaptadoras/genética , Complexo 1 de Proteínas Adaptadoras/metabolismo , Complexo 2 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Furina/genética , Furina/metabolismo , Expressão Gênica , HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/química , Proteínas do Vírus da Imunodeficiência Humana/genética , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Células Jurkat/metabolismo , Células Jurkat/virologia , Cinética , Mamíferos , Glicoproteínas de Membrana , Modelos Moleculares , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfosserina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo , Vírion/genética , Vírion/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/química , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética
2.
Sci Rep ; 8(1): 11118, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-30042514

RESUMO

The human T-cell leukemia virus type 1 (HTLV-1) is highly dependent on cell-to-cell interaction for transmission and productive infection. Cell-to-cell interactions through the virological synapse, biofilm-like structures and cellular conduits have been reported, but the relative contribution of each mechanism on HTLV-1 transmission still remains vastly unknown. The HTLV-1 protein p8 has been found to increase viral transmission and cellular conduits. Here we show that HTLV-1 expressing cells are interconnected by tunneling nanotubes (TNTs) defined as thin structures containing F-actin and lack of tubulin connecting two cells. TNTs connected HTLV-1 expressing cells and uninfected T-cells and monocytes and the viral proteins Tax and Gag localized to these TNTs. The HTLV-1 expressing protein p8 was found to induce TNT formation. Treatment of MT-2 cells with the nucleoside analog cytarabine (cytosine arabinoside, AraC) reduced number of TNTs and furthermore reduced TNT formation induced by the p8 protein. Intercellular transmission of HTLV-1 through TNTs provides a means of escape from recognition by the immune system. Cytarabine could represent a novel anti-HTLV-1 drug interfering with viral transmission.


Assuntos
Comunicação Celular/genética , Infecções por HTLV-I/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Nanotubos/química , Tubulina (Proteína)/genética , Citoesqueleto de Actina/efeitos dos fármacos , Actinas/genética , Comunicação Celular/imunologia , Citarabina/farmacologia , Produtos do Gene tax/genética , Infecções por HTLV-I/transmissão , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/efeitos dos fármacos , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Humanos , Sistema Imunitário , Células Jurkat/virologia , Leucemia de Células T/genética , Leucemia de Células T/patologia , Leucemia de Células T/virologia , Linfócitos T/imunologia , Proteínas Virais/genética
3.
Virus Res ; 249: 69-75, 2018 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-29550509

RESUMO

HIV infection is characterized by accumulation of proviral sequences within the human host genome. Integration of viral-derived DNA occurs at preferential loci, suggesting a site-specific crosstalk between viral sequences and human genes. We here describe a genome engineering workflow to generate models for HIV-1 infection that for the first time recapitulate proviral integration at selected genomic loci and provide unique tools to study effects of HIV proviral integration site choice. Using this workflow, we have derived two BACH2-HIV-1 reporter models that mimic largely latent integration in the clinically relevant BACH2 gene locus, which has been associated with recurrent integration and HIV-reservoir maintenance in chronically infected patients.


Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Provírus/fisiologia , Integração Viral , HIV-1/genética , Humanos , Células Jurkat/virologia , Modelos Biológicos , Provírus/genética
4.
Virulence ; 8(8): 1732-1743, 2017 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-28762863

RESUMO

Elucidation of mechanisms underlying the establishment, maintenance of and reactivation from HIV-1 latency is essential for the development of therapeutic strategies aimed at eliminating HIV-1 reservoirs. Microbial translocation, as a consequence of HIV-1-induced deterioration of host immune system, is known to result in a systemic immune activation and transient outbursts of HIV-1 viremia in chronic HIV-1 infection. How these microbes cause the robust HIV-1 reactivation remains elusive. Dendritic cells (DCs) have previously been shown to reactivate HIV-1 from latency; however, the precise role of DCs in reactivating HIV-1 from latently infected T-cell remains obscure. In this study, by using HIV-1 latently infected Jurkat T cells, we demonstrated that AIDS-associated pathogens as represented by Mycobacterium bovis (M. bovis) Bacillus Calmette-Guérin (BCG) and bacterial component lipopolysaccharide (LPS) were unable to directly reactivate HIV-1 from Jurkat T cells; instead, they mature DCs to secrete TNF-α to accomplish this goal. Moreover, we found that HIV-1 latently infected Jurkat T cells could also mature DCs and enhance their TNF-α production during co-culture in a CD40-CD40L-signaling-dependent manner. This in turn led to viral reactivation from Jurkat T cells. Our results reveal how DCs help AIDS-associated pathogens to trigger HIV-1 reactivation from latency.


Assuntos
Síndrome da Imunodeficiência Adquirida/metabolismo , Células Dendríticas/imunologia , HIV-1/fisiologia , Células Jurkat/virologia , Fator de Necrose Tumoral alfa/farmacologia , Latência Viral , Síndrome da Imunodeficiência Adquirida/genética , Síndrome da Imunodeficiência Adquirida/virologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Técnicas de Cocultura , Células Dendríticas/citologia , HIV-1/genética , Humanos , Células Jurkat/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Ativação Viral
5.
Hum Cell ; 30(2): 117-123, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28070874

RESUMO

In a previous study, we reported that an identical defective provirus had integrated into multiple sites of the genome of a representative human T-lymphotropic virus type 1 (HTLV-1) cell line, MT-2. A possible explanation for this may be the repeated infection of this defective provirus to a cell. Therefore, we attempted to determine whether a defective provirus could transmit during the co-culture of HTLV-1 uninfected human T-cell line, Jurkat, with MT-2 cells treated with mitomycin C. As a result, we established not only a cell line with the integration of one complete provirus, but also a cell line with the integration of one defective provirus. The rearrangement of the T-cell receptor -γ gene of these cell lines showed them to be derived from Jurkat cells. Both HTLV-1 Tax/Rex and HBZ RNA were detected in the cell line, which harbors a complete provirus. On the other hand, HBZ RNA and transcriptional product specific for the defective provirus were detected in the cell line, which harbors a defective HTLV-1 provirus only. These results suggested that a defective HTLV-1 provirus with large depletion of internal sequence could transmit to other cells. Moreover, the defective provirus can be transcriptionally active. This suggested the possibility that the defective HTLV-1 provirus found in the lymphocytes of HTLV-1 carriers and patients with adult T-cell leukemia may transmit to other T-cells in vivo. The results also suggested that defective provirus in HTLV-1 carriers could be functional and may play a role in leukemogenesis.


Assuntos
Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Células Jurkat/virologia , Integração Viral , Sequência de Bases , Linhagem Celular , Rearranjo Gênico do Linfócito T , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Humanos , Reação em Cadeia da Polimerase/métodos , Receptores de Antígenos de Linfócitos T gama-delta/genética
6.
Viruses ; 8(2)2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26848681

RESUMO

Influenza virus infection has a significant impact on public health, since it is a major cause of morbidity and mortality. It is not well-known whether influenza virus infection affects cell death and human immunodeficiency virus (HIV)-1 replication in HIV-1-infected patients. Using a lymphoma cell line, Jurkat, we examined the in vitro effects of pandemic influenza A (H1N1) virus (pH1N1) infection on cell death and HIV-1 RNA production in infected cells. We found that pH1N1 infection increased apoptotic cell death through Fas and Bax-mediated pathways in HIV-1-infected Jurkat cells. Infection with pH1N1 virus could promote HIV-1 RNA production by activating host transcription factors including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB), nuclear factor of activated T-cells (NFAT) and activator protein 1 (AP-1) through mitogen-activated protein kinases (MAPK) pathways and T-cell antigen receptor (TCR)-related pathways. The replication of HIV-1 latent infection could be reactivated by pH1N1 infection through TCR and apoptotic pathways. These data indicate that HIV-1 replication can be activated by pH1N1 virus in HIV-1-infected cells resulting in induction of cell death through apoptotic pathways.


Assuntos
Apoptose , Coinfecção/fisiopatologia , Infecções por HIV/fisiopatologia , HIV-1/fisiologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/fisiopatologia , Células Jurkat/citologia , Animais , Linhagem Celular , Embrião de Galinha , Coinfecção/genética , Coinfecção/metabolismo , Coinfecção/virologia , Saúde Global , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/genética , Influenza Humana/metabolismo , Influenza Humana/virologia , Células Jurkat/metabolismo , Células Jurkat/virologia , NF-kappa B/genética , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo
7.
Clin Epigenetics ; 8: 19, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26900410

RESUMO

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) latency represents the major barrier to virus eradication in infected individuals because cells harboring latent HIV-1 provirus are not affected by current antiretroviral therapy (ART). We previously demonstrated that DNA methylation of HIV-1 long terminal repeat (5' LTR) restricts HIV-1 reactivation and, together with chromatin conformation, represents an important mechanism of HIV-1 latency maintenance. Here, we explored the new issue of temporal development of DNA methylation in latent HIV-1 5' LTR. RESULTS: In the Jurkat CD4(+) T cell model of latency, we showed that the stimulation of host cells contributed to de novo DNA methylation of the latent HIV-1 5' LTR sequences. Consecutive stimulations of model CD4(+) T cell line with TNF-α and PMA or with SAHA contributed to the progressive accumulation of 5' LTR DNA methylation. Further, we showed that once established, the high DNA methylation level of the latent 5' LTR in the cell line model was a stable epigenetic mark. Finally, we explored the development of 5' LTR DNA methylation in the latent reservoir of HIV-1-infected individuals who were treated with ART. We detected low levels of 5' LTR DNA methylation in the resting CD4(+) T cells of the group of patients who were treated for up to 3 years. However, after long-term ART, we observed an accumulation of 5' LTR DNA methylation in the latent reservoir. Importantly, within the latent reservoir of some long-term-treated individuals, we uncovered populations of proviral molecules with a high density of 5' LTR CpG methylation. CONCLUSIONS: Our data showed the presence of 5' LTR DNA methylation in the long-term reservoir of HIV-1-infected individuals and implied that the transient stimulation of cells harboring latent proviruses may contribute, at least in part, to the methylation of the HIV-1 promoter.


Assuntos
Metilação de DNA , Infecções por HIV/genética , Repetição Terminal Longa de HIV/genética , HIV-1/genética , Provírus/genética , Latência Viral/genética , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Linhagem Celular/virologia , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Células Jurkat/virologia , Masculino , Provírus/fisiologia , Fatores de Tempo , Latência Viral/fisiologia
8.
Transfusion ; 55(6): 1256-62, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25645088

RESUMO

BACKGROUND: A new Marseilleviridae virus family member, giant blood Marseille-like (GBM) virus, was recently reported in persons from France in the serum of an infant with adenitis, in the blood of 4% of healthy blood donors, and in 9% of multiply transfused thalassemia patients. These results suggested the presence of a nucleocytoplasmic large DNA virus potentially transmissible by blood product transfusion. STUDY DESIGN AND METHODS: To investigate this possibility we tested the plasma from 113 US blood donors and 74 multiply transfused Cameroon patients for GBM viral DNA using highly sensitive polymerase chain reaction (PCR) assays. RESULTS: GBM DNA was not detected by nested PCR in any of these 187 human specimens. CONCLUSIONS: Further testing is required to confirm the occurrence of human GBM virus infections.


Assuntos
Doadores de Sangue , Infecções por Vírus de DNA/virologia , Vírus de DNA/isolamento & purificação , DNA Viral/sangue , Reação em Cadeia da Polimerase/métodos , Viremia/virologia , Acanthamoeba/virologia , Adulto , Anticorpos Antivirais/sangue , Camarões/epidemiologia , Primers do DNA , Infecções por Vírus de DNA/sangue , Infecções por Vírus de DNA/epidemiologia , Infecções por Vírus de DNA/transmissão , Vírus de DNA/genética , Vírus de DNA/imunologia , França/epidemiologia , Humanos , Células Jurkat/virologia , Masculino , Pessoa de Meia-Idade , Fases de Leitura Aberta/genética , Plasma/virologia , Sensibilidade e Especificidade , Soro/virologia , Reação Transfusional , Estados Unidos/epidemiologia , Proteínas Virais/sangue , Proteínas Virais/genética , Viremia/sangue , Viremia/epidemiologia , Cultura de Vírus
9.
In Vitro Cell Dev Biol Anim ; 50(5): 453-63, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24442370

RESUMO

Cell fusion occurs in physiological and pathological conditions and plays a role in regulation of cell fate. The analysis of cell population dynamics and cell cycle in cell-cell fusion experiments is necessary to determine changes in the quantitative equilibrium of cell populations and to identify potential bystander effects. Here, using cocultures of Jurkat HIV-1 envelope expressing cells and CD4(+) cells as a model system and flow cytometry for the analysis, the number, viability, and cell cycle status of the populations participating in fusion were determined. In 3-day cocultures, a sustained reduction of the number of CD4(+) cells was observed while they showed high viability and normal cell cycle progression; fusion, but not inhibition of proliferation or death, accounted for their decrease. In contrast, the number of Env(+) cells decreased in cocultures due to fusion, death, and an inherent arrest at G1. Most of syncytia formed in the first 6 h of coculture showed DNA synthesis activity, indicating that the efficient recruitment of proliferating cells contributed to amplify the removal of CD4(+) cells by syncytia formation. Late in cocultures, approximately 50% of syncytia were viable and a subpopulation still underwent DNA synthesis, even when the recruitment of additional cells was prevented by the addition of the fusion inhibitor T-20, indicating that a population of syncytia may progress into the cell cycle. These results show that the quantitative analysis of cellular outcomes of cell-cell fusion can be performed by flow cytometry.


Assuntos
Ciclo Celular/genética , Proliferação de Células/genética , Citometria de Fluxo , Células Gigantes/citologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/virologia , Fusão Celular , Técnicas de Cocultura , DNA/biossíntese , Células Gigantes/metabolismo , HIV-1/metabolismo , Humanos , Técnicas In Vitro , Células Jurkat/metabolismo , Células Jurkat/virologia , Proteínas do Envelope Viral/metabolismo
10.
J Cell Physiol ; 228(12): 2305-13, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23696271

RESUMO

HIV-1 infection and replication are affected by host factors. Recent studies demonstrate that molecules from apoptotic pathways regulate HIV-1 replication. Therefore, studies on effects of host factors that maintain host cell survival and influence HIV-1 replication are critical to understanding the mechanisms of HIV-1 replicative cycle. Using the susceptible Jurkat cell line, CD4(+) T cells, and peripheral blood mononuclear cells (PBMCs), we studied the role of FLIP, an inhibitor of caspase-8, in HIV-1 production. Full length cellular FLIP (cFLIP) inhibited HIV-1 replication in these cells. cFLIP upregulated the expression of viral restriction factors, such as TRIM5, Apobec3G, and Bst2/tetherin, decreased nuclear factor 1C expression and inactivated ERK and p38 induced by HIV-1 in Jurkat cells. cFLIP blocked the trafficking of gp120 and Gag p24 capsid protein into lipid rafts with inhibition of Tsg101 and Alix in ESCRT signaling pathway. cFLIP also promoted Bst2/tetherin trafficking into lipid rafts. These results indicate that cFLIP may inhibit the HIV-1 replication cycle at multiple steps, including viral RNA release, transcription, traffic and assembly. We also found that cFLIP expression downregulated Fas expression and inactivated FADD in the Fas-mediated apoptotic pathway. The inactivated FADD also inhibited HIV-1 replication.


Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/biossíntese , Linfócitos T CD4-Positivos/virologia , Replicação do DNA/genética , HIV-1/genética , Células Jurkat/virologia , Leucócitos Mononucleares/virologia , Replicação Viral/genética , Desaminase APOBEC-3G , Antígenos CD/genética , Antígenos CD/metabolismo , Fatores de Restrição Antivirais , Apoptose/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Regulação para Baixo , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Proteína do Núcleo p24 do HIV/genética , Proteína do Núcleo p24 do HIV/metabolismo , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Humanos , Células Jurkat/metabolismo , Leucócitos Mononucleares/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/virologia , Fatores de Transcrição NFI/genética , Fatores de Transcrição NFI/metabolismo , Transdução de Sinais , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Regulação para Cima
11.
J Steroid Biochem Mol Biol ; 138: 63-71, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23542659

RESUMO

BACKGROUND: The HIV epidemic is expanding worldwide with an increasing number of distinct viral subtypes and circulating recombinant forms (CRFs). Out of 34 million adults living with HIV and AIDS, women account for one half of all HIV-1 infections worldwide. These gender differences in HIV pathogenesis may be attributed to sex hormones. Little is known about the role of sex hormone effects on HIV Subtypes pathogenesis. The aim of our study was to determine sex hormone effects on replication and transmissibility of HIV subtypes. METHODS: Peripheral blood mononuclear cells (PBMC) and monocyte derived dendritic cells (MDDC) from male and female donors were infected with HIV subtypes A-D and CRF02_AG, CRF01_AE, MN (lab adapted), Group-O, Group-N and HIV-2 at a concentration of 5ng/ml of p24 or p27. Virus production was evaluated by measuring p24 and p27 levels in culture supernatants. Similar experiments were carried out in the presence of physiological concentrations of sex steroid hormones. R5/X4 expressions measured by flow cytometry and transmissibility was evaluated by transfer of HIV from primary dendritic cells (DC) to autologous donor PBMC. RESULTS: Our results from primary PBMC and MDDC from male and female donors indicate in the absence of physiological concentrations of hormone treatment virus production was observed in three clusters; high replicating virus (subtype B and C), moderate replicative virus (subtype A, D, CRF01_AE, Group_N) and least replicative virus (strain MN). However, dose of sex steroid hormone treatment influenced HIV replication and transmission kinetics in PBMC, DCs and cell lines. Such effects were inconsistent between donors and HIV subtypes. Sex hormone effects on HIV entry receptors (CCR5/CXCR4) did not correlate with virus production. CONCLUSIONS: Subtypes B and C showed higher replication in PBMC from males and females and were transmitted more efficiently through DC to male and female PBMC compared with other HIV-1 subtypes, HIV-1 Group O and HIV-2. These findings are consistent with increased worldwide prevalence of subtype B and C compared to other subtypes. Sex steroid hormones had variable effect on replication or transmission of different subtypes. These findings suggest that subtype, gender and sex hormones may play a crucial role in the replication and transmission of HIV.


Assuntos
Hormônios Esteroides Gonadais/farmacologia , Infecções por HIV/transmissão , HIV-1/patogenicidade , Células Cultivadas , Células Dendríticas/virologia , Estrogênios/farmacologia , Feminino , Infecções por HIV/metabolismo , HIV-1/efeitos dos fármacos , Humanos , Células Jurkat/virologia , Leucócitos Mononucleares/virologia , Masculino , Progesterona/farmacologia , Receptores CCR5/metabolismo , Testosterona/farmacologia , Replicação Viral/efeitos dos fármacos
12.
Nat Commun ; 3: 1303, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23250433

RESUMO

Ligand polyvalency is a powerful modulator of protein-receptor interactions. Host-pathogen infection interactions are often mediated by glycan ligand-protein interactions, yet its interrogation with very high copy number ligands has been limited to heterogenous systems. Here we report that through the use of nested layers of multivalency we are able to assemble the most highly valent glycodendrimeric constructs yet seen (bearing up to 1,620 glycans). These constructs are pure and well-defined single entities that at diameters of up to 32 nm are capable of mimicking pathogens both in size and in their highly glycosylated surfaces. Through this mimicry these glyco-dendri-protein-nano-particles are capable of blocking (at picomolar concentrations) a model of the infection of T-lymphocytes and human dendritic cells by Ebola virus. The high associated polyvalency effects (ß>10(6), ß/N ~10(2)-10(3)) displayed on an unprecedented surface area by precise clusters suggest a general strategy for modulation of such interactions.


Assuntos
Glicoproteínas/uso terapêutico , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Nanopartículas/uso terapêutico , Moléculas de Adesão Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/virologia , Ebolavirus , Glicoproteínas/química , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Células Jurkat/efeitos dos fármacos , Células Jurkat/virologia , Lectinas Tipo C/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Monócitos/virologia , Nanopartículas/química , Receptores de Superfície Celular/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/virologia
13.
Uirusu ; 62(1): 121-8, 2012 Jun.
Artigo em Japonês | MEDLINE | ID: mdl-23189832

RESUMO

Identification of a specific viral receptor is important for understanding the virus infection mechanism. I identified P-selectin glycoprotein ligand-1 (PSGL-1) as one of the functional receptors for enterovirus 71 (EV71), a pathogen that causes hand, foot, and mouth disease. PSGL-1, which belongs to the sialomucin family, is a transmembrane protein mainly expressed on leukocytes. Tyrosine sulfation in the N-terminal region of PSGL-1 is critical for PSGL-1's capacity to bind EV71. The identification of EV71 receptors provides important mechanistic information about viral entry into cells and helps us understand viral pathogenesis and develop new anti-viral strategies.


Assuntos
Enterovirus Humano A/metabolismo , Enterovirus Humano A/patogenicidade , Glicoproteínas de Membrana/metabolismo , Animais , Enterovirus Humano A/fisiologia , Síndrome Mão-Pé/virologia , Humanos , Células Jurkat/virologia , Leucócitos , Glicoproteínas de Membrana/isolamento & purificação , Glicoproteínas de Membrana/fisiologia , Camundongos , Ligação Proteica , Tirosina/análogos & derivados , Replicação Viral
14.
Virology ; 432(2): 444-51, 2012 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-22832123

RESUMO

The HIV-1 accessory protein Vpr induces G2 cell cycle arrest and apoptosis. Previous studies indicate that the induction of G2-arrest requires the localization of Vpr to the nuclear envelope. Here we show that treatment of Vpr-expressing HeLa cells with the caspase 3 inhibitor Z-DEVD-fmk induced accumulation of Vpr at the nuclear lamina, while other proteins or structures of the nuclear envelope were not influenced. Furthermore, Z-DEVD-fmk enhances the Vpr-mediated G2-arrest that even occurred in HIV-1(NL4-3)-infected T-cells. Mutation of Pro-35, which is important for the integrity of helix-α1 in Vpr, completely abrogated the Z-DEVD-fmk-mediated accumulation of Vpr at the nuclear lamina and the enhancement of G2-arrest. As expected, inhibition of caspase 3 reduced the induction of apoptosis by Vpr. Taken together, we could show that besides its role in Vpr-mediated apoptosis induction caspase 3 influences the localization of Vpr at the nuclear envelope and thereby augments the Vpr-induced G2-arrest.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Membrana Nuclear/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Apoptose , Caspase 3/genética , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/fisiologia , Sobrevivência Celular , HIV-1/metabolismo , Células HeLa/virologia , Humanos , Células Jurkat/virologia , Transfecção , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/farmacologia
15.
PLoS Pathog ; 8(3): e1002609, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22479182

RESUMO

The HIV-1 accessory protein Vpu counteracts tetherin (BST-2/CD317) by preventing its incorporation into virions, reducing its surface expression, and ultimately promoting its degradation. Here we characterize a putative trafficking motif, EXXXLV, in the second alpha helix of the subtype-B Vpu cytoplasmic tail as being required for efficient tetherin antagonism. Mutation of this motif prevents ESCRT-dependent degradation of tetherin/Vpu complexes, tetherin cell surface downregulation, but not its physical interaction with Vpu. Importantly, this motif is required for efficient cell-free virion release from CD4+ T cells, particularly after their exposure to type-1 interferon, indicating that the ability to reduce surface tetherin levels and promote its degradation is important to counteract restriction under conditions that the virus likely encounters in vivo. Vpu EXXXLV mutants accumulate with tetherin at the cell surface and in endosomal compartments, but retain the ability to bind both ß-TrCP2 and HRS, indicating that this motif is required for a post-binding trafficking event that commits tetherin for ESCRT-dependent degradation and prevents its transit to the plasma membrane and viral budding zones. We further found that while Vpu function is dependent on clathrin, and the entire second alpha helix of the Vpu tail can be functionally complemented by a clathrin adaptor binding peptide derived from HIV-1 Nef, none of the canonical clathrin adaptors nor retromer are required for this process. Finally we show that residual activity of Vpu EXXXLV mutants requires an intact endocytic motif in tetherin, suggesting that physical association of Vpu with tetherin during its recycling may be sufficient to compromise tetherin activity to some degree.


Assuntos
Antígenos CD/metabolismo , Endossomos/metabolismo , HIV-1/patogenicidade , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Interferons/farmacologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Proteínas Virais Reguladoras e Acessórias/metabolismo , Antígenos CD/imunologia , Citoplasma/metabolismo , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Regulação Viral da Expressão Gênica , Células HEK293/metabolismo , Células HEK293/virologia , HIV-1/efeitos dos fármacos , HIV-1/metabolismo , Células HeLa/metabolismo , Células HeLa/virologia , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Células Jurkat/metabolismo , Células Jurkat/virologia , Transporte Proteico , Proteínas Virais Reguladoras e Acessórias/genética , Vírion/metabolismo , Montagem de Vírus , Liberação de Vírus/efeitos dos fármacos , Replicação Viral
16.
J Gen Virol ; 93(Pt 2): 330-340, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22012462

RESUMO

Herpesvirus saimiri (HVS), a T-lymphotropic monkey herpesvirus, induces fulminant T-cell lymphoma in non-natural primate hosts. In addition, it can immortalize human T-cells in vitro. HVS tyrosine kinase-interacting protein (Tip) is an essential viral gene required for T-cell transformation both in vitro and in vivo. In this study, we found that Tip interacts with the STAT6 transcription factor and induces phosphorylation of STAT6 in T-cells. The interaction with STAT6 requires the Tyr(127) residue and Lck-binding domain of Tip, which are indispensable for interleukin (IL)-2-independent T-cell transformation by HVS. It was also demonstrated that Tip induces nuclear translocation of STAT6, as well as activation of STAT6-dependent transcription in Jurkat T-cells. Interestingly, the phosphorylated STAT6 mainly colocalized with vesicles containing Tip within T-cells, but was barely detectable in the nucleus. However, nuclear translocation of phospho-STAT6 and transcriptional activation of STAT6 by IL-4 stimulation were not affected significantly in T-cells expressing Tip. Collectively, these findings suggest that the constitutive activation of STAT6 by Tip in T-cells may contribute to IL-2-independent T-cell transformation by HVS.


Assuntos
Transformação Celular Viral , Herpesvirus Saimiriíneo 2/patogenicidade , Células Jurkat/imunologia , Células Jurkat/virologia , Fosfoproteínas/metabolismo , Fator de Transcrição STAT6/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Humanos , Mapeamento de Interação de Proteínas , Transcrição Gênica
17.
Virol J ; 8: 443, 2011 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-21933387

RESUMO

BACKGROUND: Xenotropic Murine Leukemia Virus-related (XMRV) virus is a recently identified mouse gammaretrovirus that has the ability to infect certain human cells. In this study, we investigated the susceptibility of primary neuronal cell types to infection with XMRV. FINDINGS: We observed that the human primary progenitors, progenitor-derived neurons, and progenitor-derived astrocytes supported XMRV multiplication. Interestingly, both progenitors and progenitor-derived neurons were more susceptible compared with progenitor-derived astrocytes. In addition, XMRV-infected Jurkat cells were able to transmit infection to neuronal cells. CONCLUSIONS: These data suggest that neuronal cells are susceptible for XMRV infection.


Assuntos
Astrócitos/virologia , Suscetibilidade a Doenças , Células Jurkat/virologia , Células-Tronco Neurais/virologia , Neurônios/virologia , Infecções por Retroviridae/virologia , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/genética , Animais , Astrócitos/citologia , Diferenciação Celular , Humanos , Imuno-Histoquímica , Células Jurkat/citologia , Masculino , Camundongos , Células-Tronco Neurais/citologia , Neurônios/citologia , Cultura Primária de Células , Neoplasias da Próstata/virologia , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Retroviridae/imunologia , Infecções por Retroviridae/metabolismo , Infecções por Retroviridae/transmissão , Células Tumorais Cultivadas , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/metabolismo , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/patogenicidade
18.
J Leukoc Biol ; 86(5): 1171-8, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19641037

RESUMO

Lymphocyte trafficking is a multistep, intricate process and involves a number of host factors such as integrins and chemokine receptors on lymphocytes, adhesion molecules on endothelial cells, and chemokines present in the local microenvironment. Previous studies have shown that HIV-1 Nef inhibits T cell chemotaxis in response to the physiological ligand SDF-1alpha [( 1) ]. In this study, we aimed to gain a better understanding of the inhibitory mechanisms and to define the molecular determinants of HIV-1 Nef for this phenotype. We showed that HIV-1 Nef inhibited transwell and transendothelial migration of T cells. Specifically, HIV-1 Nef protein impaired T cell chemotaxis toward SDF-1alpha without altering CXCR4 expression. Moreover, we showed that HIV-1 Nef protein down-modulated LFA-1 expression on T lymphocytes and diminished adhesion and polarization of T lymphocytes and as a result, led to decreased migration across the endothelium. Furthermore, we showed that the myristoylation site and DeltaSD domain played important roles in Nef-mediated inhibition of transwell and transendothelial migration and polarization of T lymphocytes; however, different sites or domains were needed for Nef-mediated LFA-1 down-modulation and impaired adhesion of T lymphocyte. Taken together, these results demonstrated that HIV-1 Nef inhibited T lymphocyte migration at multiple steps and suggest that membrane localization and intracellular signaling events likely contribute to the inhibitory effects of Nef on T cell migration and subsequently, the pathobiology of the HIV-1 Nef protein.


Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , HIV-1/patogenicidade , Linfócitos T/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/fisiologia , Síndrome da Imunodeficiência Adquirida/virologia , Movimento Celular , Quimiotaxia de Leucócito , Primers do DNA , Endotélio Vascular/imunologia , Endotélio Vascular/virologia , Citometria de Fluxo , Amplificação de Genes , Genes Reporter , HIV-1/genética , Humanos , Células Jurkat/virologia , Mutação , Mapeamento por Restrição , Transdução de Sinais , Linfócitos T/fisiologia , Linfócitos T/virologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia
19.
Blood ; 114(5): 1016-25, 2009 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-19494354

RESUMO

Human T-lymphotropic virus type 1 (HTLV-1) spreads directly between lymphocytes and other cells via a specialized cell-cell contact, termed the virological synapse. The formation of the virological synapse is accompanied by the orientation of the microtubule-organizing center (MTOC) in the infected T cell toward the cell contact region with the noninfected target cell. We previously demonstrated that the combination of intracellular Tax protein expression and the stimulation of the intercellular adhesion molecule-1 (ICAM-1) on the cell surface is sufficient to trigger MTOC polarization in the HTLV-1-infected T cell. However, the mechanism by which Tax and ICAM-1 cause the MTOC polarization is not fully understood. Here we show that the presence of Tax at the MTOC region and its ability to stimulate cyclic AMP-binding protein-dependent pathways are both required for MTOC polarization in the HTLV-1-infected T cell at the virological synapse. Furthermore, we show that the MTOC polarization induced by ICAM-1 engagement depends on activation of the Ras-MEK-ERK signaling pathway. Our findings indicate that efficient MTOC polarization at the virological synapse requires Tax-mediated stimulation of T-cell activation pathways in synergy with ICAM-1 cross-linking. The results also reveal differences in the signaling pathways used to trigger MTOC polarization between the immunologic synapse and the virological synapse.


Assuntos
Produtos do Gene tax/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Molécula 1 de Adesão Intercelular/fisiologia , Centro Organizador dos Microtúbulos/ultraestrutura , Transdução de Sinais/fisiologia , Linfócitos T/virologia , Internalização do Vírus , Polaridade Celular/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Cicloeximida/farmacologia , Citocalasina B/farmacologia , Produtos do Gene tax/genética , Genes pX , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Células Jurkat/virologia , Fusão de Membrana/fisiologia , Centro Organizador dos Microtúbulos/fisiologia , Mutação de Sentido Incorreto , Nocodazol/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Transporte Proteico , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/fisiologia , Linfócitos T/ultraestrutura , Moduladores de Tubulina/farmacologia , Ubiquitinação
20.
Blood ; 113(12): 2732-41, 2009 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-18945959

RESUMO

Exosomes are secreted cellular vesicles that can be internalized by dendritic cells (DCs), contributing to antigen-specific naive CD4(+) T-cell activation. Here, we demonstrate that human immunodeficiency virus type 1 (HIV-1) can exploit this exosome antigen-dissemination pathway intrinsic to mature DCs (mDCs) for mediating trans-infection of T lymphocytes. Capture of HIV-1, HIV-1 Gag-enhanced green fluorescent protein (eGFP) viral-like particles (VLPs), and exosomes by DCs was up-regulated upon maturation, resulting in localization within a CD81(+) compartment. Uptake of VLPs or exosomes could be inhibited by a challenge with either particle, suggesting that the expression of common determinant(s) on VLP or exosome surface is necessary for internalization by mDCs. Capture by mDCs was insensitive to proteolysis but blocked when virus, VLPs, or exosomes were produced from cells treated with sphingolipid biosynthesis inhibitors that modulate the lipid composition of the budding particles. Finally, VLPs and exosomes captured by mDCs were transmitted to T lymphocytes in an envelope glycoprotein-independent manner, underscoring a new potential viral dissemination pathway.


Assuntos
Células Dendríticas/virologia , Endocitose/fisiologia , Exossomos/fisiologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Microdomínios da Membrana/fisiologia , Internalização do Vírus , Apresentação de Antígeno , Antígenos CD/análise , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/virologia , Ceramidas/biossíntese , Ceramidas/fisiologia , Células Dendríticas/imunologia , Endocitose/efeitos dos fármacos , Exocitose/fisiologia , Exossomos/química , Fumonisinas/farmacologia , Proteínas de Fluorescência Verde/análise , Humanos , Células Jurkat/efeitos dos fármacos , Células Jurkat/virologia , Rim , Lipídeos de Membrana/análise , Lipídeos de Membrana/fisiologia , Microdomínios da Membrana/química , Pronase/farmacologia , Tetraspanina 28 , Produtos do Gene gag do Vírus da Imunodeficiência Humana/análise
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