RESUMO
Hepatitis B virus (HBV) core protein (HBc) plays many roles in the HBV life cycle, such as regulation of transcription, RNA encapsidation, reverse transcription, and viral release. To accomplish these functions, HBc interacts with many host proteins and undergoes different post-translational modifications (PTMs). One of the most common PTMs is ubiquitination, which was shown to change the function, stability, and intracellular localization of different viral proteins, but the role of HBc ubiquitination in the HBV life cycle remains unknown. Here, we found that HBc protein is post-translationally modified through K29-linked ubiquitination. We performed a series of co-immunoprecipitation experiments with wild-type HBc, lysine to arginine HBc mutants and wild-type ubiquitin, single lysine to arginine ubiquitin mutants, or single ubiquitin-accepting lysine constructs. We observed that HBc protein could be modified by ubiquitination in transfected as well as infected hepatoma cells. In addition, ubiquitination predominantly occurred on HBc lysine 7 and the preferred ubiquitin chain linkage was through ubiquitin-K29. Mass spectrometry (MS) analyses detected ubiquitin protein ligase E3 component N-recognin 5 (UBR5) as a potential E3 ubiquitin ligase involved in K29-linked ubiquitination. These findings emphasize that ubiquitination of HBc may play an important role in HBV life cycle.
Assuntos
Vírus da Hepatite B/genética , Processamento de Proteína Pós-Traducional/genética , Ubiquitinação/genética , Proteínas Virais/genética , Arginina/genética , Carcinoma Hepatocelular/genética , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células Hep G2 , Hepatite B/genética , Humanos , Lisina/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
In this paper, correlation analysis of protein and mRNA levels in the soil dwelling bacteria Streptomyces coelicolor (S. coelicolor M145) is presented during development of the population as it grew in liquid medium using three biological and two technical replicates, measured during exponential growth, and its entry into the stationary phase. The proteome synthesis time series are compared with the gene expression time series measured previously under identical experimental conditions. Results reveal that about one third of protein/mRNA synthesis profiles are well correlated while another third are correlated negatively. Functional analysis of the highly correlated groups is presented. Based on numerical simulation, the negative correlation between protein and mRNA is shown to be caused by the difference between the rate of translation and protein degradation.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteoma/metabolismo , RNA Mensageiro/metabolismo , Streptomyces coelicolor/crescimento & desenvolvimento , Transcriptoma , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Proteoma/análise , RNA Mensageiro/genética , Solo/química , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismoRESUMO
BACKGROUND: Progression rates from initial HIV-1 infection to advanced AIDS vary significantly among infected individuals. A distinct subgroup of HIV-1-infected individuals-termed viremic non-progressors (VNP) or controllers-do not seem to progress to AIDS, maintaining high CD4+ T cell counts despite high levels of viremia for many years. Several studies have evaluated multiple host factors, including immune activation, trying to elucidate the atypical HIV-1 disease progression in these patients; however, limited work has been done to characterize viral factors in viremic controllers. METHODS: We analyzed HIV-1 isolates from three VNP individuals and compared the replicative fitness, near full-length HIV-1 genomes and intra-patient HIV-1 genetic diversity with viruses from three typical (TP) and one rapid (RP) progressor individuals. RESULTS: Viremic non-progressors and typical patients were infected for >10 years (range 10-17 years), with a mean CD4+ T-cell count of 472 cells/mm3 (442-529) and 400 cells/mm3 (126-789), respectively. VNP individuals had a less marked decline in CD4+ cells (mean -0.56, range -0.4 to -0.7 CD4+/month) than TP patients (mean -10.3, -8.2 to -13.1 CD4+/month). Interestingly, VNP individuals carried viruses with impaired replicative fitness, compared to HIV-1 isolates from the TP and RP patients (p < 0.05, 95% CI). Although analyses of the near full-length HIV-1 genomes showed no clear patterns of single-nucleotide polymorphisms (SNP) that could explain the decrease in replicative fitness, both the number of SNPs and HIV-1 population diversity correlated inversely with the replication capacity of the viruses (r = -0.956 and r = -0.878, p < 0.01, respectively). CONCLUSION: It is likely that complex multifactorial parameters govern HIV-1 disease progression in each individual, starting with the infecting virus (phenotype, load, and quasispecies diversity) and the intrinsic ability of the host to respond to the infection. Here we analyzed a subset of viremic controller patients and demonstrated that similar to the phenomenon observed in patients with a discordant response to antiretroviral therapy (i.e., high CD4+ cell counts with detectable plasma HIV-1 RNA load), reduced viral replicative fitness seems to be linked to slow disease progression in these antiretroviral-naïve individuals.
Assuntos
Aptidão Genética , Infecções por HIV/virologia , Sobreviventes de Longo Prazo ao HIV , HIV-1/isolamento & purificação , HIV-1/fisiologia , Replicação Viral , Adulto , Estudos de Coortes , Variação Genética , Genoma Viral , HIV-1/classificação , HIV-1/genética , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNARESUMO
Phosphatidylinositol 4-kinase IIIß (PI4KB) is indispensable for the replication of various positive-sense single stranded RNA viruses, which hijack this cellular enzyme to remodel intracellular membranes of infected cells to set up the functional replication machinery. Therefore, the inhibition of this PI4K isoform leads to the arrest of viral replication. Here, we report on the synthesis of novel PI4KB inhibitors, which were rationally designed based on two distinct structural types of inhibitors that bind in the ATP binding side of PI4KB. These "hybrids" not only excel in outstanding inhibitory activity but also show high selectivity to PI4KB compared to other kinases. Thus, these compounds exert selective nanomolar or even subnanomolar activity against PI4KB as well as profound antiviral effect against hepatitis C virus, human rhinovirus, and coxsackievirus B3. Our crystallographic analysis unveiled the exact position of the side chains and explains their extensive contribution to the inhibitory activity.
Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Antivirais/química , Antivirais/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Desenho de Fármacos , Células HeLa , Humanos , Estrutura MolecularRESUMO
We report on an extensive structure-activity relationship study of novel PI4K IIIß inhibitors. The purine derivative of the potent screening hit T-00127-HEV1 has served as a suitable starting point for a thorough investigation of positions 8 and 2. While position 8 of the purine scaffold can only bear a small substituent to maintain the inhibitory activity, position 2 is opened for extensive modification and can accommodate even substituted phenyl rings without the loss of PI4K IIIß inhibitory activity. These empirical observations nicely correlate with the results of our docking study, which suggests that position 2 directs towards solution and can provide the necessary space for the interaction with remote residues of the enzyme, whereas the cavity around position 8 is strictly limited. The obtained compounds have also been subjected to antiviral screening against a panel of (+)ssRNA viruses.
Assuntos
Antivirais/farmacologia , Enterovirus Humano B/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Rhinovirus/efeitos dos fármacos , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Purinas/síntese química , Purinas/química , Relação Estrutura-AtividadeRESUMO
Phosphatidylinositol 4-kinase IIIß is a cellular lipid kinase pivotal to pathogenesis of various RNA viruses. These viruses hijack the enzyme in order to modify the structure of intracellular membranes and use them for the construction of functional replication machinery. Selective inhibitors of this enzyme are potential broad-spectrum antiviral agents, as inhibition of this enzyme results in the arrest of replication of PI4K IIIß-dependent viruses. Herein, we report a detailed study of novel selective inhibitors of PI4K IIIß, which exert antiviral activity against a panel of single-stranded positive-sense RNA viruses. Our crystallographic data show that the inhibitors occupy the binding site for the adenine ring of the ATP molecule and therefore prevent the phosphorylation reaction.
Assuntos
Antivirais/química , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Trifosfato de Adenosina/química , Antivirais/síntese química , Antivirais/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Etilenodiaminas/síntese química , Etilenodiaminas/química , Etilenodiaminas/farmacologia , Células HeLa , Humanos , Imidazóis/síntese química , Imidazóis/química , Imidazóis/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Simulação de Acoplamento Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/química , Conformação Proteica , Pirazóis/síntese química , Pirazóis/química , Pirazóis/farmacologia , Piridinas/síntese química , Piridinas/química , Piridinas/farmacologia , Pirimidinas/síntese química , Pirimidinas/química , Pirimidinas/farmacologia , Vírus de RNA/efeitos dos fármacos , Relação Estrutura-Atividade , Triazinas/síntese química , Triazinas/química , Triazinas/farmacologiaRESUMO
2'-Deoxy-5-ethynyluridine (EdU) has been previously shown to be a cell poison whose toxicity depends on the particular cell line. The reason is not known. Our data indicates that different efficiency of EdU incorporation plays an important role. The EdU-mediated toxicity was elevated by the inhibition of 2'-deoxythymidine 5'-monophosphate synthesis. EdU incorporation resulted in abnormalities of the cell cycle including the slowdown of the S phase and a decrease in DNA synthesis. The slowdown but not the cessation of the first cell division after EdU administration was observed in all of the tested cell lines. In HeLa cells, a 10 µM EdU concentration led to the cell death in the 100% of cells probably due to the activation of an intra S phase checkpoint in the subsequent S phase. Our data also indicates that this EdU concentration induces interstrand DNA crosslinks in HeLa cells. We suppose that these crosslinks are the primary DNA damage resulting in cell death. According to our results, the EdU-mediated toxicity is further increased by the inhibition of thymidylate synthase by EdU itself at its higher concentrations.
Assuntos
Citotoxinas/toxicidade , Dano ao DNA , Desoxiuridina/análogos & derivados , Inibidores Enzimáticos/toxicidade , Timidilato Sintase/antagonistas & inibidores , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citotoxinas/metabolismo , DNA/biossíntese , DNA/genética , DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , Desoxiuridina/metabolismo , Desoxiuridina/toxicidade , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Fase S/efeitos dos fármacos , Tetra-Hidrofolatos/biossíntese , Timidina/metabolismo , Timidina/farmacologia , Timidina Monofosfato/metabolismoRESUMO
The study describes the method of a sensitive detection of double-stranded DNA molecules in situ. It is based on the oxidative attack on the deoxyribose moiety by copper(I) in the presence of oxygen. We have shown previously that the oxidative attack leads to the formation of frequent gaps in DNA. Here we have demonstrated that the gaps can be utilized as the origins for an efficient synthesis of complementary labeled strands by DNA polymerase I and that such enzymatic detection of the double-stranded DNA is a sensitive approach enabling in-situ detection of both the nuclear and mitochondrial genomes in formaldehyde-fixed human cells.
Assuntos
Núcleo Celular/genética , DNA Mitocondrial/genética , DNA/genética , DNA Polimerase I/metabolismo , Células HeLa , HumanosRESUMO
5-Bromo-2'-deoxyuridine (BrdU) and 2'-deoxy-5-ethynyluridine (EdU) are widely used as markers of replicated DNA. While BrdU is detected using antibodies, the click reaction typically with fluorescent azido-dyes is used for EdU localisation. We have performed an analysis of ten samples of antibodies against BrdU with respect to their reactivity with EdU. Except for one sample all the others evinced reactivity with EdU. A high level of EdU persists in nuclear DNA even after the reaction of EdU with fluorescent azido-dyes if the common concentration of dye is used. Although a ten-time increase of azido-dye concentration resulted in a decrease of the signal provided by anti-BrdU antibodies, it also resulted in a substantial increase of the non-specific signal. We have shown that this unwanted reactivity is effectively suppressed by non-fluorescent azido molecules. In this respect, we have tested two protocols of the simultaneous localisation of incorporated BrdU and EdU. They differ in the mechanism of the revelation of incorporated BrdU for the reaction with antibodies. The first one was based on the use of hydrochloric acid, the second one on the incubation of samples with copper(I) ions. The use of hydrochloric acid resulted in a significant increase of the non-specific signal. In the case of the second method, no such effect was observed.
Assuntos
Anticorpos/imunologia , Bromodesoxiuridina/imunologia , Desoxiuridina/análogos & derivados , Microscopia de Fluorescência , Anticorpos/química , Afinidade de Anticorpos , Transporte Biológico , Biotinilação , Bromodesoxiuridina/química , Bromodesoxiuridina/metabolismo , Reações Cruzadas/imunologia , DNA/química , DNA/metabolismo , Desoxiuridina/química , Desoxiuridina/imunologia , Desoxiuridina/metabolismo , Corantes Fluorescentes , Células HeLa , Humanos , Coloração e RotulagemRESUMO
A new method of the light microscopy detection of BrdU-labeled DNA in situ is described. It is based on the oxidative attack at the deoxyribose moiety by copper(I) in the presence of oxygen, which leads to the abstraction of hydrogen atom from deoxyribose culminating in the elimination of the nucleobase, scission of the nucleic-acid strand and formation of frequent gaps. The gaps allow the reaction of the antibodies with the commonly used markers of replication (e.g. 5-bromo-2'-deoxyuridine), which are otherwise masked. The method developed makes it possible to detect nuclear and mitochondrial DNA replication efficiently. In most cases, it does not inhibit effective protein detections and in addition enables simultaneous localization of newly-synthesized RNA. The alternative presently-used methods result in protein denaturation and/or extensive DNA cleavage followed by the DNA-bound proteins peeling off.