RESUMO
Disparate enveloped viruses initiate infection by fusing with endosomes. However, the highly diverse and dynamic nature of endosomes impairs mechanistic studies of fusion and identification of sub-cellular sites supporting the nucleocapsid release. We took advantage of the extreme stability of avian retrovirus-receptor complexes at neutral pH and of acid-dependence of virus-endosome fusion to isolate the latter step from preceding asynchronous internalization/trafficking steps. Viruses were trapped within endosomes in the presence of NH4Cl. Removal of NH4Cl resulted in a quick and uniform acidification of all subcellular compartments, thereby initiating synchronous viral fusion. Single virus imaging demonstrated that fusion was initiated within seconds after acidification and often culminated in the release of the viral core from an endosome. Comparative studies of cells expressing either the transmembrane or GPI-anchored receptor isoform revealed that the transmembrane receptor delivered the virus to more fusion-permissive compartments. Thus the identity of endosomal compartments, in addition to their acidity, appears to modulate viral fusion. A more striking manifestation of the virus delivery to distinct compartments in the presence of NH4Cl was the viral core release into the cytosol of cells expressing the transmembrane receptor and into endosomes of cells expressing the GPI-anchored isoform. In the latter cells, the newly released cores exhibited restricted mobility and were exposed to a more acidic environment than the cytoplasm. These cores appear to enter into the cytosol after an additional slow temperature-dependent step. We conclude that the NH4Cl block traps the virus within intralumenal vesicles of late endosomes in cells expressing the GPI-anchored receptor. Viruses surrounded by more than one endosomal membrane release their core into the cytoplasm in two steps--fusion with an intralumenal vesicle followed by a yet unknown temperature-dependent step that liberates the core from late endosomes.
Assuntos
Vírus do Sarcoma Aviário/genética , Vírus do Sarcoma Aviário/metabolismo , Endossomos/virologia , Proteínas do Core Viral/metabolismo , Proteínas Virais de Fusão/metabolismo , Cloreto de Amônio/química , Animais , Compartimento Celular , Linhagem Celular , Chlorocebus aethiops , Endossomos/metabolismo , Células HEK293 , Humanos , Isoformas de Proteínas/biossíntese , Transporte Proteico , Proteínas dos Retroviridae/genética , Proteínas dos Retroviridae/metabolismo , Proteínas do Core Viral/genética , Proteínas Virais de Fusão/genética , Internalização do VírusRESUMO
In the initial step of integration, retroviral integrase (IN) introduces precise nicks in the degenerate, short inverted repeats at the ends of linear viral DNA. The scissile phosphodiester bond is located immediately 3' of a highly conserved CA/GT dinucleotide, usually 2 bp from the ends. These nicks create new recessed 3'-OH viral DNA ends that are required for joining to host cell DNA. Previous studies have indicated that unpairing, "fraying," of the viral DNA ends by IN contributes to end recognition or catalysis. Here, we report that end fraying can be detected independently of catalysis with both avian sarcoma virus (ASV) and human immunodeficiency virus type 1 (HIV-1) IN proteins by use of fluorescence resonance energy transfer (FRET). The results were indicative of an IN-induced intramolecular conformational change in the viral DNA ends (cis FRET). Fraying activity is tightly coupled to the DNA binding capabilities of these enzymes, as follows: an inhibitor effective against both IN proteins was shown to block ASV IN DNA binding and end fraying, with similar dose responses; ASV IN substitutions that reduced DNA binding also reduced end fraying activity; and HIV-1 IN DNA binding and end fraying were both undetectable in the absence of a metal cofactor. Consistent with our previous results, end fraying is sequence-independent, suggesting that the DNA terminus per se is a major structural determinant for recognition. We conclude that frayed ends represent a functional intermediate in which DNA termini can be sampled for suitability for endonucleolytic processing.
Assuntos
Vírus do Sarcoma Aviário/enzimologia , Pareamento de Bases , DNA Viral/química , Integrase de HIV/metabolismo , HIV-1/enzimologia , Vírus do Sarcoma Aviário/genética , Vírus do Sarcoma Aviário/metabolismo , Sequência de Bases , Domínio Catalítico , Coenzimas/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Transferência Ressonante de Energia de Fluorescência , Integrase de HIV/química , HIV-1/genética , HIV-1/metabolismo , Metais/metabolismo , Reprodutibilidade dos TestesRESUMO
Members of the Nedd4 family of E3 ubiquitin ligases bind the L domain in avian sarcoma virus (ASV) Gag and facilitate viral particle release. Translational fusion of ASV Gag with an L domain deletion (Deltap2b) to proteins that comprise ESCRT-I, -II, and -III (the endocytic sorting complexes required for transport) rescued both Gag ubiquitination and particle release from cells. The ESCRT-I factors Vps37C or Tsg101 were more effective in rescue of Gag/Deltap2b budding than the ESCRT-II factor Eap20 or the ESCRT-III component CHMP6. Thus ESCRT components can substitute for Nedd4 family members in ASV Gag release. Unlike wild type, ASV Gag/Deltap2b -ESCRT chimeras failed to co-immunoprecipitate with co-expressed hemagglutinin-tagged Nedd4, indicating that Nedd4 was not stably associated with these Gag fusions. Release of the Gag-ESCRT-I or -II fusions was inhibited by a dominant negative mutant of Vps4 ATPase similar to wild type ASV Gag. In contrast to ASV Gag, HIV-1 Gag containing an L domain inactivating mutation (P7L) was efficiently rescued by fusion to a component of ESCRT-III (Chmp6) but not ESCRT-II (Eap20). Depletion of the endogenous pool of Eap20 (ESCRT-II) had little effect on HIV-1 Gag release but blocked ASV Gag release. In contrast, depletion of the endogenous pool of Vps37C (ESCRT-I) had little effect on ASV but blocked HIV-1 Gag release. Furthermore, an N-terminal fragment of Chmp6 inhibited both HIV-1 and ASV Gag release in a dominant negative manner. Taken together, these results indicate that ASV and HIV-1 Gag utilize different combinations of ESCRT proteins to facilitate the budding process, although they share some common elements.
Assuntos
Vírus do Sarcoma Aviário/metabolismo , HIV-1/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sítios de Ligação , Transporte Biológico , Linhagem Celular , Endocitose , Produtos do Gene gag/metabolismo , Humanos , Modelos Biológicos , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Ubiquitina/química , Proteínas de Transporte Vesicular/químicaRESUMO
To identify phosphotyrosine-containing proteins essential for maintaining the transformed state, we studied the tyrosine phosphorylation profile of temperature-sensitive mutant of Rous sarcoma virus, tsNY68, infected cells (68N7). Shifting the temperature from 39 degrees C (nonpermissive) to 32 degrees C (permissive) markedly increased the expression of phosphotyrosine-containing cell membrane proteins of approximately 40kDa, as assessed by SDS-PAGE. Membrane and nuclear proteins were separated by two-dimensional gel electrophoresis and immunoblotted with anti-phosphotyrosine antibody. Proteins showing temperature-dependent changes in phosphorylation profile were subjected to in-gel digestion with trypsin and analyzed by mass spectrometry. Five proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A3, hnRNP A2, annexin II, phosphoglycerate mutase 1, and triosephosphate isomerase 1. hnRNP A3 was phosphorylated at serine residues and had both serine and tyrosine phosphorylated sites. These results suggest an important complementary role for proteomics in identifying molecular abnormalities associated with tumor progression that may be attractive candidates for tumor diagnosis.
Assuntos
Vírus do Sarcoma Aviário/metabolismo , Fosfoproteínas/química , Proteômica/métodos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , Peptídeos/química , Fosfotirosina/química , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Temperatura , Tripsina/farmacologiaRESUMO
Compared with the MHC of typical mammals, the chicken MHC is smaller and simpler, with only two class I genes found in the B12 haplotype. We make five points to show that there is a single-dominantly expressed class I molecule that can have a strong effect on MHC function. First, we find only one cDNA for two MHC haplotypes (B14 and B15) and cDNAs corresponding to two genes for the other six (B2, B4, B6, B12, B19, and B21). Second, we find, for the B4, B12, and B15 haplotypes, that one cDNA is at least 10-fold more abundant than the other. Third, we use 2D gel electrophoresis of class I molecules from pulse-labeled cells to show that there is only one heavy chain spot for the B4 and B15 haplotypes, and one major spot for the B12 haplotype. Fourth, we determine the peptide motifs for B4, B12, and B15 cells in detail, including pool sequences and individual peptides, and show that the motifs are consistent with the peptides binding to models of the class I molecule encoded by the abundant cDNA. Finally, having shown for three haplotypes that there is a single dominantly expressed class I molecule at the level of RNA, protein, and antigenic peptide, we show that the motifs can explain the striking MHC-determined resistance and susceptibility to Rous sarcoma virus. These results are consistent with the concept of a "minimal essential MHC" for chickens, in strong contrast to typical mammals.
Assuntos
Vírus do Sarcoma Aviário/genética , Genes MHC Classe I , Peptídeos/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Vírus do Sarcoma Aviário/metabolismo , Galinhas , DNA Complementar/metabolismo , Eletroforese em Gel Bidimensional , Citometria de Fluxo , Genes Dominantes , Haplótipos , Modelos Moleculares , Dados de Sequência Molecular , Doenças das Aves Domésticas/virologia , Sarcoma Aviário/virologia , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
Eukaryotic cells target mRNAs to the nonsense-mediated mRNA decay (NMD) pathway when translation terminates within the coding region. In mammalian cells, this is presumably due to a downstream signal deposited during pre-mRNA splicing. In contrast, unspliced retroviral RNA undergoes NMD in chicken cells when premature termination codons (PTCs) are present in the gag gene. Surprisingly, deletion of a 401-nt 3' UTR sequence immediately downstream of the normal gag termination codon caused this termination event to be recognized as premature. We termed this 3' UTR region the Rous sarcoma virus (RSV) stability element (RSE). The RSE also stabilized the viral RNA when placed immediately downstream of a PTC in the gag gene. Deletion analysis of the RSE indicated a smaller functional element. We conclude that this 3' UTR sequence stabilizes termination codons in the RSV RNA, and termination codons not associated with such an RSE sequence undergo NMD.
Assuntos
Regiões 3' não Traduzidas/genética , Vírus do Sarcoma Aviário/genética , Códon sem Sentido/genética , Códon de Terminação/genética , Estabilidade de RNA/genética , Animais , Vírus do Sarcoma Aviário/metabolismo , Células Cultivadas , Embrião de Galinha , Genes Virais/genética , Biossíntese de Proteínas , RNA/genética , TransfecçãoRESUMO
Avian sarcoma and leukosis virus subgroup A (ASLV-A) entry is mediated by interactions between the viral glycoprotein EnvA and its cognate receptor Tva. Previously, some interesting mutants of ASLV-A have been selected by others which can use chicken Tva, but not quail Tva, for efficient entry. The mutant phenotypes are caused by two point mutations within the surface subunit of EnvA (S. L. Holmen, D. C. Melder, and M. J. Federspiel, J. Virol. 75:726-737, 2001). In this study, we have shown that the altered receptor specificity maps to the LDL-A module of Tva. Further, we have identified two residues in the chicken LDL-A module that allow more efficient viral entry by the mutant viruses. These results demonstrate that the altered receptor specificity of the mutant viruses is determined by specific interactions with residues in the LDL-A module of Tva.
Assuntos
Aminoácidos/metabolismo , Vírus da Leucose Aviária/metabolismo , Proteínas Aviárias/metabolismo , Vírus do Sarcoma Aviário/metabolismo , Receptores Virais/metabolismo , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Galinhas , Interações Hidrofóbicas e Hidrofílicas , Estrutura Terciária de Proteína , Receptores Virais/química , Receptores Virais/genéticaRESUMO
The functionally exchangeable L domains of HIV-1 and Rous sarcoma virus (RSV) Gag bind Tsg101 and Nedd4, respectively. Tsg101 and Nedd4 function in endocytic trafficking, and studies show that expression of Tsg101 or Nedd4 fragments interfere with release of HIV-1 or RSV Gag, respectively, as virus-like particles (VLPs). To determine whether functional exchangeability reflects use of the same trafficking pathway, we tested the effect on RSV Gag release of co-expression with mutated forms of Vps4, Nedd4 and Tsg101. A dominant-negative mutant of Vps4A, an AAA ATPase required for utilization of endosomal sorting proteins that was shown previously to interfere with HIV-1 budding, also inhibited RSV Gag release, indicating that RSV uses the endocytic trafficking machinery, as does HIV. Nedd4 and Tsg101 interacted in the presence or absence of Gag and, through its binding of Nedd4, RSV Gag interacted with Tsg101. Deletion of the N-terminal region of Tsg101 or the HECT domain of Nedd4 did not prevent interaction; however, three-dimensional spatial imaging suggested that the interaction of RSV Gag with full-length Tsg101 and N-terminally truncated Tsg101 was not the same. Co-expression of RSV Gag with the Tsg101 C-terminal fragment interfered with VLP release minimally; however, a significant fraction of the released VLPs was tethered to each other. The results suggest that, while Tsg101 is not required for RSV VLP release, alterations in the protein interfere with VLP budding/fission events. We conclude that RSV and HIV-1 Gag direct particle release through independent ESCRT-mediated pathways that are linked through Tsg101-Nedd4 interaction.
Assuntos
Vírus do Sarcoma Aviário/metabolismo , Proteínas de Ligação a DNA/metabolismo , Produtos do Gene gag/metabolismo , HIV-1/metabolismo , Transporte Proteico , Fatores de Transcrição/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte , Produtos do Gene gag/genética , Hemaglutininas/metabolismo , Humanos , Ubiquitina-Proteína Ligases Nedd4 , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , ATPases Vacuolares Próton-Translocadoras , Proteínas de Transporte VesicularRESUMO
Two rat kidney cell lines transformed by two strains of ASV virus were investigated. It was demonstrated that these two lines (1) showed density-independent growth, (2) had a decreased requirement for serum in the culture medium, (3) had the ability to grow in a chemically defined medium (without serum), and the rate of this growth had increased with the increase in starting density of cells, and (4) had the ability of anchrage-independent growth, even without serum. These results confirmed autostimulation of growth of W12 and GCA cells. It was also shown that the crude conditioned media contained autocrine growth factors, which could be extracted with 1M acetic acid. The extracts (AEs) stimulated the growth of the parental cells and NRK-49F cells almost as well as 5% calf serum and the extraction resulted in several-fold purification of mitogenic substances. These substances were not only specific to parental lines, but also stimulated growth of other transformed lines and normal NRK-49F cells. Extracts from the conditioned media of W12 and GCA cells intensified the rate of anchorage-independent growth in the concentration-dependent manner. In AE-W12, two peaks of mitogenic activity were detected (F1, F2) and similarly in AE-GCA (F3, F4). Fractions F2 (approximately 8 kDa), F3 (approximately 25 kDa) and F4 (approximately 12 kDa) were thermostable but F1 (approximately 45 kDa) was thermolabile. All four fractions were sensitive to trypsin and DTT treatment, and were acid-stable. Using ELISA kit it was shown that W12 and GCA cells released TGFbeta1 and GCA cells released very small quantities of bFGF. These results confirmed the autocrine regulation of growth in both cell lines.
Assuntos
Vírus do Sarcoma Aviário/fisiologia , Transformação Celular Viral , Proteínas Oncogênicas Virais/metabolismo , Fatores de Crescimento Transformadores/metabolismo , Animais , Vírus do Sarcoma Aviário/metabolismo , Adesão Celular , Linhagem Celular Transformada/metabolismo , Proliferação de Células/efeitos dos fármacos , Transformação Celular Viral/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados , Relação Dose-Resposta a Droga , Substâncias de Crescimento , Mitógenos/metabolismo , Proteínas Oncogênicas Virais/farmacologia , Ratos , Fatores de Tempo , Fatores de Crescimento Transformadores/farmacologia , Células Tumorais CultivadasRESUMO
The Rous sarcoma virus (RSV) Gag polyprotein undergoes transient nuclear trafficking as an intrinsic part of the virus assembly pathway. Nuclear export of Gag is crucial for the efficient production of viral particles and is accomplished through the action of a leptomycin B (LMB)-dependent nuclear export signal (NES) in the p10 domain (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc. Natl. Acad. Sci. USA 99:3944-3949, 2002). We have now mapped the nuclear export activity to the C-terminal portion of the p10 sequence and identified the four hydrophobic amino acids within this region that comprise a leucine-rich NES. Alteration of these hydrophobic residues resulted in the accumulation of Gag proteins within the nucleus and a budding defect greater than that obtained with LMB treatment of cells expressing the wild-type Gag protein (Scheifele et al., Proc. Natl. Acad. Sci. USA 99:3944-3949, 2002). In addition, export of Gag from the nucleus was found to be a rate-limiting step in virus-like particle production. Consistent with a role for the NES sequence in viral replication, this cluster of hydrophobic residues in p10 is conserved across a wide range of avian retroviruses. Furthermore, naturally occurring substitutions within this region in related viruses maintained nuclear export activity and remained sensitive to the activity of LMB. Using gain-of-function approaches, we found that the hydrophobic motif in p10 was sufficient to promote the nuclear export of a heterologous protein and was positionally independent within the Gag polyprotein. Finally, the export pathway was further defined by the ability of specific nucleoporin inhibitors to prevent the egress of Gag from the nucleus, thereby identifying additional cellular mediators of RSV replication.
Assuntos
Transporte Ativo do Núcleo Celular , Vírus do Sarcoma Aviário/metabolismo , Produtos do Gene gag/metabolismo , Sinais de Localização Nuclear , Sequência de Aminoácidos , Animais , Linhagem Celular , Carioferinas/metabolismo , Dados de Sequência Molecular , Codorniz , Receptores Citoplasmáticos e Nucleares/metabolismo , Vírion/metabolismo , Proteína Exportina 1RESUMO
The cytopathic effect (CPE) seen with some subgroups of avian sarcoma and leukosis virus (ASLV) is associated with viral Env activation of the death-promoting activity of TVB (a tumor necrosis factor receptor-related receptor that is most closely related to mammalian TNF-related apoptosis-inducing ligand [TRAIL] receptors) and with viral superinfection leading to unintegrated viral DNA (UVD) accumulation, which is presumed to activate a cellular DNA damage response. In this study, we employed cells that express signaling-deficient ASLV receptors to demonstrate that an ASLV CPE can be uncoupled from the death-promoting functions of the TVB receptor. However, these cell-killing events were associated with much higher levels of viral superinfection and DNA accumulation than those seen when the virus used signaling-competent TVB receptors. These findings suggest that a putative cellular DNA damage response that is activated by UVD accumulation might act in concert with the death-signaling pathways activated by Env-TVB interactions to trigger cell death. Such a model is consistent with the well-established synergy that exists between TRAIL-signaling pathways and DNA damage responses which is currently being exploited in cancer therapy regimens.
Assuntos
Vírus da Leucose Aviária/metabolismo , Vírus do Sarcoma Aviário/metabolismo , Morte Celular/fisiologia , Receptores Virais/fisiologia , Vírus da Leucose Aviária/genética , Vírus do Sarcoma Aviário/genética , Sequência de Bases , Adesão Celular , Linhagem Celular , Efeito Citopatogênico Viral , Dano ao DNA , Primers do DNA , Citometria de Fluxo , Produtos do Gene env/metabolismo , Humanos , Reação em Cadeia da PolimeraseRESUMO
Viral fusion proteins catalyze merger of viral and cell membranes through a series of steps that have not yet been well defined. To elucidate the mechanism of virus entry, we have imaged fusion between single virions bearing avian sarcoma and leukosis virus (ASLV) envelope glycoprotein (Env) and the cell membrane. Viral particles were labeled with a lipophilic dye and with palmitylated enhanced YFP that was incorporated into the inner leaflet of the viral membrane. When individual virions were bound to target cells expressing cognate receptors, they transferred their lipids and contents only when exposed to low, but not neutral, pH. These data are consistent with the proposed two-step mechanism of ASLV entry that involves receptor-priming followed by low pH activation. Most importantly, lipid mixing commonly occurred before formation of a small fusion pore that was quickly and sensitively detected by pH-dependent changes in palmitylated enhanced YFP fluorescence. Nascent fusion pores were metastable and irreversibly closed, remained small, or fully enlarged, permitting nucleocapsid delivery into the cytosol. These findings strongly imply that hemifusion and a small pore are the key intermediates of ASLV fusion. When added before low pH treatment, a peptide designed to prevent Env from folding into a final helical-bundle conformation abolished virus-cell fusion and infection. Therefore, we conclude that, after receptor-activation, Env undergoes low pH-dependent refolding into a six-helix bundle and, in doing so, sequentially catalyzes hemifusion, fusion pore opening, and enlargement.
Assuntos
Vírus do Sarcoma Aviário/metabolismo , Membrana Celular/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/metabolismo , Proteínas Virais de Fusão/ultraestrutura , Vírion/metabolismo , Linhagem Celular , Corantes Fluorescentes , Vetores Genéticos , Humanos , Concentração de Íons de Hidrogênio , Microscopia Confocal , Microscopia de Fluorescência , Dobramento de Proteína , TransfecçãoRESUMO
The MA domain of retroviral Gag proteins mediates association with the host cell membrane during assembly. The biochemical nature of this interaction is not well understood. We have used an in vitro flotation assay to directly measure Rous sarcoma virus (RSV) MA-membrane interaction in the absence of host cell factors. The association of purified MA and MA-containing proteins with liposomes of defined composition was electrostatic in nature and depended upon the presence of a biologically relevant concentration of negatively charged lipids. A mutant MA protein known to be unable to promote Gag membrane association and budding in vivo failed to bind to liposomes. These results were supported by computational modeling. The intrinsic affinity of RSV MA for negatively charged membranes appears insufficient to promote efficient plasma membrane binding during assembly. However, an artificially dimerized form of MA bound to liposomes by at least an order of magnitude more tightly than monomeric MA. This result suggests that the clustering of MA domains, via Gag-Gag interactions during virus assembly, drives membrane association in vivo.
Assuntos
Lipossomos/metabolismo , Fosfoproteínas/metabolismo , Proteínas da Matriz Viral/metabolismo , Vírus do Sarcoma Aviário/metabolismo , Vírus do Sarcoma Aviário/fisiologia , Membrana Celular/metabolismo , Desenho Assistido por Computador , Modelos Moleculares , Montagem de VírusRESUMO
The pivotal role of kinases in signal transduction and cellular regulation has lent them considerable appeal as pharmacological targets across a broad spectrum of pathologies. Since the discovery that the v-Src oncogene encoded a protein kinase in 1978, kinases have remained a focus of research for pharmaceutical laboratories and academic groups alike. Many have sought to develop orally available low molecular weight synthetic kinase modulators (predominantly inhibitors) and thus capitalize on the links between aberrant regulation and disease. This interest in kinases as drug targets was fueled in recent years by the success of several kinase inhibitors in the clinic, primarily Gleevec for the treatment of chronic myelogenous leukemia and Iressa for the treatment of advanced non-small cell lung cancer. This review focuses on the development of small molecule drugs, most of them binding in or close to the ATP binding pocket. After some general considerations regarding the selection of a particular kinase for drug discovery, we will discuss the encouraging lessons learned from some of the kinase inhibitors currently in various stages of development. The majority of this review is dedicated to a detailed description and discussion of the various assay formats currently being employed for high throughput screening.
Assuntos
Desenho de Fármacos , Biblioteca de Peptídeos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Vírus do Sarcoma Aviário/metabolismo , Sítios de Ligação , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Neoplasias Pulmonares/patologia , Programas de Rastreamento , Inibidores de Proteínas Quinases/síntese química , Proteínas Quinases/efeitos dos fármacosRESUMO
Retroviral late domains (L domains) are short amino acid sequences in the Gag protein that facilitate the process of budding. L domains act by recruiting the ESCRT complexes, which normally function in the formation of multivesicular bodies. The PTAP late domain of human immunodeficiency virus (HIV) is believed to specifically recruit this machinery by binding the ESCRT protein TSG101. It was recently demonstrated that expression of a C-terminal fragment of TSG101 (TSG-3') blocked the budding of both PTAP-dependent and PPPY-dependent retroviruses. We show here that TSG-3' expression leads to the formation of large spherical entities that we call TICS (TSG-3'-induced cellular structures) in the cytoplasm. Rous sarcoma virus (RSV) and murine leukemia virus (MLV) Gag proteins are selectively recruited to these structures, but HIV type 1 Gag is completely excluded. Experiments with various HIV and RSV vector constructs as well as HIV and RSV chimeras suggest that recruitment to the TICS is late domain independent and does not involve recognition of any single amino acid sequence. TICS appear to have no limiting membrane and do not colocalize with markers for any membranous cellular compartment. Wild-type TSG101 is also recruited to TICS, but most other ESCRT proteins are excluded. These structures are similar in nature to aggresomes, colocalize with the aggresome marker GFP-250, and are highly enriched in ubiquitin but in other ways do not fully meet the description of aggresomes. We conclude that the block to retroviral budding by TSG-3' may be the result of its sequestration of Gag, depletion of free TSG101, or depletion of free ubiquitin.
Assuntos
Vírus do Sarcoma Aviário/crescimento & desenvolvimento , Estruturas Citoplasmáticas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Produtos do Gene gag/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Vírus do Sarcoma Aviário/genética , Vírus do Sarcoma Aviário/metabolismo , Células Cultivadas , Galinhas , Estruturas Citoplasmáticas/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , HIV-1/genética , Substâncias Macromoleculares/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transfecção , Ubiquitina/análiseRESUMO
Rous sarcoma virus (RSV) budding requires an interaction of the L domain within the p2b region of Gag with cellular Nedd4-family E3 ubiquitin protein ligases. Members of our laboratories previously demonstrated that overexpression of a fragment of the chicken Nedd4-like protein (LDI-1 WW) inhibits Gag release in a dominant-negative manner (A. Kikonyogo, F. Bouamr, M. L. Vana, Y. Xiang, A. Aiyar, C. Carter, and J. Leis, Proc. Natl. Acad. Sci. USA 98:11199-11204, 2001). We have now identified the complete 3' end of LDI-1 and determined that it has a C-terminal ubiquitin ligase HECT domain, similar to other Nedd4 family members. While overexpression of the full-length LDI-1 clone (LDI-1 FL) had little effect on Gag budding, an LDI-1 FL mutant with a substitution in the HECT domain catalytic site blocked Gag release, similar to LDI-1 WW. The coexpression of Gag and hemagglutinin-tagged ubiquitin (HA-Ub) resulted in the detection of mono- and polyubiquitinated forms of Gag in cells and mostly monoubiquitinated Gag in virus-like particles (VLPs). When the Nedd4-binding site (L domain) was deleted, ubiquitinated Gag was not detected. Interestingly, the release of Gag with ubiquitin covalently linked to the C terminus (Gag-Ub) was still blocked by LDI-1 WW. To understand the mechanism of this inhibition, we examined cells expressing Gag and LDI-1 WW by electron microscopy. In the presence of LDI-1 WW, VLPs were found in electron-dense inclusion bodies in the cytoplasm of transfected cells. In contrast, when cells that coexpressed Gag-Ub and LDI-1 WW were examined, inclusion bodies were detected but did not contain VLPs. These results indicate that the ubiquitination of Gag is dependent upon Nedd4 binding to the L domain and suggest that Nedd4 has additional functions during RSV release besides the ubiquitination of Gag.
Assuntos
Vírus do Sarcoma Aviário/crescimento & desenvolvimento , Regulação Viral da Expressão Gênica , Produtos do Gene gag/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Vírus do Sarcoma Aviário/genética , Vírus do Sarcoma Aviário/metabolismo , Células COS , Linhagem Celular , Chlorocebus aethiops , Complexos Endossomais de Distribuição Requeridos para Transporte , Produtos do Gene gag/química , Humanos , Camundongos , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Ubiquitina-Proteína Ligases Nedd4 , Coelhos , Ubiquitina-Proteína Ligases/genética , Vírion/metabolismoRESUMO
The subgroup A to E avian sarcoma and leukosis viruses (ASLVs) are highly related and are thought to have evolved from a common ancestor. These viruses use distinct cell surface proteins as receptors to gain entry into avian cells. Chickens have evolved resistance to infection by the ASLVs. We have identified the mutations responsible for the block to virus entry in chicken lines resistant to infection by subgroup A ASLVs [ASLV(A)]. The tva genetic locus determines the susceptibility of chicken cells to ASLV(A) viruses. In quail, the ASLV(A) susceptibility allele tva(s) encodes two forms of the Tva receptor; these proteins are translated from alternatively spliced mRNAs. The normal cellular function of the Tva receptor is unknown; however, the extracellular domain contains a 40-amino-acid, cysteine-rich region that is homologous to the ligand binding region of the low-density lipoprotein receptor (LDLR) proteins. The chicken tva(s) cDNAs had not yet been fully characterized; we cloned the chicken tva cDNAs from two lines of subgroup A-susceptible chickens, line H6 and line 0. Two types of chicken tva(s) cDNAs were obtained. These cDNAs encode a longer and shorter form of the Tva receptor homologous to the Tva forms in quail. Two different defects were identified in cDNAs cloned from two different ASLV(A)-resistant inbred chickens, line C and line 7(2). Line C tva(r) contains a single base pair substitution, resulting in a cysteine-to-tryptophan change in the LDLR-like region of Tva. This mutation drastically reduces the binding affinity of Tva(R) for the ASLV(A) envelope glycoproteins. Line 7(2) tva(r2) contains a 4-bp insertion in exon 1 that causes a change in the reading frame, which blocks expression of the Tva receptor.
Assuntos
Vírus da Leucose Aviária/patogenicidade , Vírus do Sarcoma Aviário/patogenicidade , Galinhas/imunologia , Mutação , Receptores Virais/genética , Sequência de Aminoácidos , Animais , Leucose Aviária/imunologia , Leucose Aviária/virologia , Vírus da Leucose Aviária/metabolismo , Proteínas Aviárias , Vírus do Sarcoma Aviário/metabolismo , Sequência de Bases , Células Cultivadas , Embrião de Galinha , Galinhas/virologia , Dados de Sequência Molecular , Codorniz , Receptores Virais/química , Receptores Virais/metabolismo , Sarcoma Aviário/imunologia , Sarcoma Aviário/virologia , Análise de Sequência de DNARESUMO
The fusion protein of avian sarcoma and leukosis virus is likely to fold into a six-helix bundle as part of its final configuration. A peptide, R99, inhibits fusion, probably by binding into the grooves of the triple-stranded coiled coil that becomes the central core of the six-helix bundle. The stages at which the envelope protein (Env) of avian sarcoma and leukosis virus subgroup A folds into a bundle during low pH-induced fusion were determined. Effector cells expressing Env were bound to target cells expressing the cognate receptor Tva, and intermediates of fusion were created. R99 was added and the extent of fusion inhibition was used to distinguish between a prebundle state with exposed grooves and a state in which the grooves were no longer exposed. The native conformation of Env was not sensitive to R99. But adding a soluble form of Tva to effector cells conferred sensitivity. Acidic pH applied at low temperature created an intermediate state of local hemifusion. Surprisingly, R99 caused these locally hemifused membranes to separate. This indicates that the grooves of Env were still exposed, that prebundle configurations of Env stabilized hemifused states, and that binding of R99 altered the conformation of Env. In the presence of an inhibitory lipid that blocks fusion before hemifusion, applying low pH at 37 degrees C created an intermediate in which R99 was without effect. This suggests that the six-helix bundle can form before hemifusion and that subsequent conformational changes, such as formation of the trimeric hairpin, are responsible for pore formation and/or growth.
Assuntos
Vírus do Sarcoma Aviário/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Produtos do Gene env/metabolismo , Fusão de Membrana/efeitos dos fármacos , Fusão de Membrana/fisiologia , Peptídeos/farmacologia , Células 3T3 , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Camundongos , Modelos Biológicos , Conformação Proteica/efeitos dos fármacos , Dobramento de Proteína , Estrutura Secundária de Proteína/efeitos dos fármacosRESUMO
The Rous sarcoma virus (RSV) negative regulator of splicing (NRS) is an RNA element that represses splicing and promotes polyadenylation of viral RNA. The NRS acts as a pseudo 5' splice site (ss), and serine-arginine (SR) proteins, U1snRNP, and U6 small nuclear ribonucleoproteins (snRNPs) are implicated in its function. The NRS also efficiently binds U11 snRNP of the U12-dependent splicing pathway, which is interesting, because U11 binds only poorly to authentic substrates that lack a U12-type 3' splice site. It is of considerable interest to understand how the low abundance U11 snRNP binds the NRS so well. Here we show that U11 can bind the NRS as a mono-snRNP in vitro and that a G-rich element located downstream of the U11 site is required for efficient binding. Mutational analyses indicated that two of four G tracts in this region were important for optimal U11 binding and that the G-rich region did not function indirectly by promoting U1 snRNP binding to an overlapping site. Surprisingly, inactivation of U2 snRNP also decreased U11 binding to the NRS. The NRS harbors a branch point-like/pyrimidine tract sequence (BP/Py) just upstream of the U1/U11 site that is characteristic of 3' splice sites. Deletion of this region decreased U2 and U11 binding, and deletion of the G-rich region also reduced U2 binding. The G element, but not the BP/Py sequence, was also required for U11 binding to the NRS in vivo as assessed by minor class splicing from the NRS to a minor class 3'ss from the P120 gene. These results indicate that efficient U11 binding to the isolated NRS involves at least two elements in addition to the U11 consensus sequence and may have implications for U11 binding to authentic splicing substrates.
Assuntos
Retroviridae/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Arginina/química , Vírus do Sarcoma Aviário/metabolismo , Sequência de Bases , Sítios de Ligação , Biotinilação , Linhagem Celular , Núcleo Celular/metabolismo , Análise Mutacional de DNA , Deleção de Genes , Humanos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Serina/química , Transcrição Gênica , TransfecçãoRESUMO
We used enzymatic digestion and mass spectrometry to identify the sites of glycosylation on the SU component of the Avian Sarcoma/Leukosis virus (ASLV) Envelope Glycoprotein (Subgroup A). The analysis was done with an SU(A)-rIgG fusion protein that binds the cognate receptor (Tva) specifically. PNGase F removed all the carbohydrate from the SU(A)-rIgG fusion. PNGase F is specific for N-linked carbohydrates; this shows that all the carbohydrate on SU(A) is N-linked. There are 10 modified aspargines in SU(A) (N17, N59, N80, N97, N117, N196, N230, N246, N254, and N330). All conform to the consensus site for N-linked glycosylation NXS/T. There is one potential glycosylation site (N236) that is not modified. Removing most of the carbohydrate from the mature SU(A)-rIgG by PNGase F treatment greatly reduces the ability of the protein to bind Tva, suggesting that carbohydrate may play a direct role in receptor binding.