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We develop candidate HIV-1 vaccines, of which two components, ChAdOx1.tHIVconsv1 (C1) and ChAdOx1.HIVconsv62 (C62), are delivered by the simian adenovirus-derived vaccine vector ChAdOx1. Aberrant adenovirus RNA splicing involving transgene(s) coding for the SARS-CoV-2 spike was suggested as an aetiology of rare adverse events temporarily associated with the initial deployment of adenovirus-vectored vaccines during the COVID-19 pandemic. Here, to eliminate this theoretically plausible splicing phenomenon from the list of possible pathomechanisms for our HIV-1 vaccine candidates, we directly sequenced mRNAs in C1- and C62-infected nonpermissive MRC-5 and A549 and permissive HEK293 human cell lines. Our two main observations in nonpermissive human cells, which are most similar to those which become infected after the intramuscular administration of vaccines into human volunteers, were that (i) the dominant adenovirus vector-derived mRNAs were the expected transcripts coding for the HIVconsvX immunogens and (ii) atypical splicing events within the synthetic open reading frame of the two transgenes are rare. We conclude that inadvertent RNA splicing is not a safety concern for the two tested candidate HIV-1 vaccines.
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SARS-CoV-2 entry requires sequential cleavage of the spike glycoprotein at the S1/S2 and the S2' cleavage sites to mediate membrane fusion. SARS-CoV-2 has a polybasic insertion (PRRAR) at the S1/S2 cleavage site that can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture-adapted SARS-CoV-2 virus with an S1/S2 deletion, we show that the polybasic insertion endows SARS-CoV-2 with a selective advantage in lung cells and primary human airway epithelial cells, but impairs replication in Vero E6, a cell line used for passaging SARS-CoV-2. Using engineered spike variants and live virus competition assays and by measuring growth kinetics, we find that the selective advantage in lung and primary human airway epithelial cells depends on the expression of the cell surface protease TMPRSS2, which enables endosome-independent virus entry by a route that avoids antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin cleavage site was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals, unlike wild-type virus. Analysis of 100,000 SARS-CoV-2 sequences derived from patients and 24 human postmortem tissues showed low frequencies of naturally occurring mutants that harbour deletions at the polybasic site. Taken together, our findings reveal that the furin cleavage site is an important determinant of SARS-CoV-2 transmission.
Assuntos
COVID-19/transmissão , Furina/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , COVID-19/virologia , Catepsinas/metabolismo , Chlorocebus aethiops , Endossomos/metabolismo , Células Epiteliais , Furões , Humanos , Evasão da Resposta Imune , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sistema Respiratório/citologia , Sistema Respiratório/virologia , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Empacotamento do Genoma Viral , Internalização do Vírus , Replicação Viral , Eliminação de Partículas ViraisRESUMO
BACKGROUND: ChAdOx1 nCoV-19 is a recombinant adenovirus vaccine against SARS-CoV-2 that has passed phase III clinical trials and is now in use across the globe. Although replication-defective in normal cells, 28 kbp of adenovirus genes is delivered to the cell nucleus alongside the SARS-CoV-2 S glycoprotein gene. METHODS: We used direct RNA sequencing to analyse transcript expression from the ChAdOx1 nCoV-19 genome in human MRC-5 and A549 cell lines that are non-permissive for vector replication alongside the replication permissive cell line, HEK293. In addition, we used quantitative proteomics to study over time the proteome and phosphoproteome of A549 and MRC5 cells infected with the ChAdOx1 nCoV-19 vaccine. RESULTS: The expected SARS-CoV-2 S coding transcript dominated in all cell lines. We also detected rare S transcripts with aberrant splice patterns or polyadenylation site usage. Adenovirus vector transcripts were almost absent in MRC-5 cells, but in A549 cells, there was a broader repertoire of adenoviral gene expression at very low levels. Proteomically, in addition to S glycoprotein, we detected multiple adenovirus proteins in A549 cells compared to just one in MRC5 cells. CONCLUSIONS: Overall, the ChAdOx1 nCoV-19 vaccine's transcriptomic and proteomic repertoire in cell culture is as expected. The combined transcriptomic and proteomics approaches provide a detailed insight into the behaviour of this important class of vaccine using state-of-the-art techniques and illustrate the potential of this technique to inform future viral vaccine vector design.
Assuntos
Vacinas contra COVID-19/efeitos adversos , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , COVID-19/metabolismo , COVID-19/prevenção & controle , COVID-19/virologia , Vacinas contra COVID-19/genética , Linhagem Celular , Células Cultivadas , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Poliadenilação , Proteômica/métodos , RNA Mensageiro , RNA Viral , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Transcrição GênicaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), uses the viral spike (S) protein for host cell attachment and entry. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2. Cleavage of S generates a polybasic Arg-Arg-Ala-Arg carboxyl-terminal sequence on S1, which conforms to a C-end rule (CendR) motif that binds to cell surface neuropilin-1 (NRP1) and NRP2 receptors. We used x-ray crystallography and biochemical approaches to show that the S1 CendR motif directly bound NRP1. Blocking this interaction by RNA interference or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection and may potentially provide a therapeutic target for COVID-19.
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Betacoronavirus/fisiologia , Neuropilina-1/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus , Motivos de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , COVID-19 , Células CACO-2 , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Furina/metabolismo , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/química , Neuropilina-1/genética , Pandemias , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Viral genomes have high gene densities and complex transcription strategies rendering transcriptome analysis through short-read RNA-seq approaches problematic. Adenovirus transcription and splicing is especially complex. We used long-read direct RNA sequencing to study adenovirus transcription and splicing during infection. This revealed a previously unappreciated complexity of alternative splicing and potential for secondary initiating codon usage. Moreover, we find that most viral transcripts tend to shorten polyadenylation lengths as infection progresses. Development of an open reading frame centric bioinformatics analysis pipeline provided a deeper quantitative and qualitative understanding of adenovirus's genetic potential. Across the viral genome adenovirus makes multiple distinctly spliced transcripts that code for the same protein. Over 11,000 different splicing patterns were recorded across the viral genome, most occurring at low levels. This low-level use of alternative splicing patterns potentially enables the virus to maximise its coding potential over evolutionary timescales.
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Adenovírus Humanos/genética , Processamento Alternativo/genética , Evolução Molecular , Transcriptoma , Sequência de Bases , Linhagem Celular , Uso do Códon , Biologia Computacional/métodos , Éxons , Fibroblastos/virologia , Perfilação da Expressão Gênica , Genoma Viral , Humanos , Poliadenilação , Regiões Promotoras Genéticas , RNA Viral/genética , RNA-SeqRESUMO
Zika virus (ZIKV) has reemerged in the population and caused unprecedented global outbreaks. Here, the transcriptomic consequences of ZIKV infection were studied systematically first in human peripheral blood CD14+ monocytes and monocyte-derived macrophages with high-density RNA sequencing. Analyses of the ZIKV genome revealed that the virus underwent genetic diversification, and differential mRNA abundance was found in host cells during infection. Notably, there was a significant change in the cellular response, with cross talk between monocytes and natural killer (NK) cells as one of the highly identified pathways. Immunophenotyping of peripheral blood from ZIKV-infected patients further confirmed the activation of NK cells during acute infection. ZIKV infection in peripheral blood cells isolated from healthy donors led to the induction of gamma interferon (IFN-γ) and CD107a-two key markers of NK cell function. Depletion of CD14+ monocytes from peripheral blood resulted in a reduction of these markers and reduced priming of NK cells during infection. This was complemented by the immunoproteomic changes observed. Mechanistically, ZIKV infection preferentially counterbalances monocyte and/or NK cell activity, with implications for targeted cytokine immunotherapies. IMPORTANCE ZIKV reemerged in recent years, causing outbreaks in many parts of the world. Alarmingly, ZIKV infection has been associated with neurological complications such as Guillain-Barré syndrome (GBS) in adults and congenital fetal growth-associated anomalies in newborns. Host peripheral immune cells are one of the first to interact with the virus upon successful transmission from an infected female Aedes mosquito. However, little is known about the role of these immune cells during infection. In this work, the immune responses of monocytes, known target cells of ZIKV infection, were investigated by high-density transcriptomics. The analysis saw a robust immune response being elicited. Importantly, it also divulged that monocytes prime NK cell activities during virus infection. Removal of monocytes during the infection changed the immune milieu, which in turn reduced NK cell stimulation. This study provides valuable insights into the pathobiology of the virus and allows for the possibility of designing novel targeted therapeutics.
Assuntos
Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Células Cultivadas , Genoma Viral , Interações Hospedeiro-Patógeno , Humanos , Interferon gama/imunologia , Células Matadoras Naturais/virologia , Ativação Linfocitária , Depleção Linfocítica , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Macrófagos/virologia , Monócitos/virologia , Proteômica , RNA Viral/genética , Análise de Sequência de RNA , Transcriptoma , Replicação Viral , Zika virus/genéticaRESUMO
Adenovirus has evolved strategies to usurp host-cell factors and machinery to facilitate its life cycle, including cell entry, replication, assembly and egress. Adenovirus continues, therefore, to be an important model system for investigating fundamental cellular processes. The role of adenovirus E1B-55k in targeting host-cell proteins that possess antiviral activity for proteasomal degradation is now well established. To expand our understanding of E1B-55k in regulating the levels of host-cell proteins, we performed comparative proteome analysis of wild-type, and E1B-55k-deletion, adenovirus-infected cancer cells. As such we performed quantitative MS/MS analysis to monitor protein expression changes affected by viral E1B-55k. We identified 5937 proteins, and of these, 69 and 58 proteins were down-regulated during wild-type and E1B-55k (dl1520) adenovirus infection, respectively. This analysis revealed that there are many, previously unidentified, cellular proteins subjected to degradation by adenovirus utilizing pathways independent of E1B-55k expression. Moreover, we found that ALCAM, EPHA2 and PTPRF, three cellular proteins that function in the regulation of cell-cell contacts, appeared to be degraded by E1B-55k/E4orf3 and/or E1B-55k/E4orf6 complexes. These molecules, like integrin α3 (a known substrate of E1B-55k/E4orf6), are critical regulators of cell signalling, cell adhesion and cell surface modulation, and their degradation during infection is, potentially, pertinent to adenovirus propagation. The data presented in this study illustrate the broad nature of protein down-regulation mediated by adenovirus.
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Infecções por Adenoviridae/patologia , Adenoviridae/crescimento & desenvolvimento , Proteínas E1B de Adenovirus/genética , Deleção de Genes , Interações Hospedeiro-Patógeno , Proteoma/análise , Adenoviridae/genética , Infecções por Adenoviridae/virologia , Linhagem Celular Tumoral , Humanos , Proteômica , Espectrometria de Massas em Tandem , Fatores de TempoRESUMO
The working model to describe the mechanisms used to replicate the cancer-associated virus Epstein-Barr virus (EBV) is partly derived from comparisons with other members of the Herpes virus family. Many genes within the EBV genome are homologous across the herpes virus family. Published transcriptome data for the EBV genome during its lytic replication cycle show extensive transcription, but the identification of the proteins is limited. We have taken a global proteomics approach to identify viral proteins that are expressed during the EBV lytic replication cycle. We combined an enrichment method to isolate cells undergoing EBV lytic replication with SILAC-labeling coupled to mass-spectrometry and identified viral and host proteins expressed during the OPEN ACCESS Pathogens 2015, 4 740 EBV lytic replication cycle. Amongst the most frequently identified viral proteins are two components of the DNA replication machinery, the single strand DNA binding protein BALF2, DNA polymerase accessory protein BMRF1 and both subunits of the viral ribonucleoside-diphosphate reductase enzyme (BORF2 and BaRF1). An additional 42 EBV lytic cycle proteins were also detected. This provides proteomic identification for many EBV lytic replication cycle proteins and also identifies post-translational modifications.
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We used SILAC (stable isotope labelling of amino acids in cell culture) and high-throughput quantitative MS mass spectrometry to analyse the protein composition of highly purified WT wild type adenoviruses, mutant adenoviruses lacking an internal protein component (protein V) and recombinant adenoviruses of the type commonly used in gene therapy, including one virus that had been used in a clinical trial. We found that the viral protein abundance and composition were consistent across all types of virus examined except for the virus lacking protein V, which also had reduced amounts of another viral core protein, protein VII. In all the samples analysed we found no evidence of consistent packaging or contamination with cellular proteins. We believe this technique is a powerful method to analyse the protein composition of this important gene therapy vector and genetically engineered or synthetic virus-like particles. The raw data have been deposited at proteomexchange, identifer PXD001120.
Assuntos
Adenovírus Humanos/química , Adenovírus Humanos/genética , Adenovírus Humanos/isolamento & purificação , Aminoácidos/análise , Linhagem Celular , Células HEK293 , Células HeLa , Humanos , Marcação por Isótopo , Mutação , Proteômica , Espectrometria de Massas em Tandem , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Disease caused by dengue virus is a global health concern with up to 390 million individuals infected annually worldwide. There are no vaccines or antiviral compounds available to either prevent or treat dengue disease which may be fatal. To increase our understanding of the interaction of dengue virus with the host cell, we analyzed changes in the proteome of human A549 cells in response to dengue virus type 2 infection using stable isotope labelling in cell culture (SILAC) in combination with high-throughput mass spectrometry (MS). Mock and infected A549 cells were fractionated into nuclear and cytoplasmic extracts before analysis to identify proteins that redistribute between cellular compartments during infection and reduce the complexity of the analysis. We identified and quantified 3098 and 2115 proteins in the cytoplasmic and nuclear fractions respectively. Proteins that showed a significant alteration in amount during infection were examined using gene enrichment, pathway and network analysis tools. The analyses revealed that dengue virus infection modulated the amounts of proteins involved in the interferon and unfolded protein responses, lipid metabolism and the cell cycle. The SILAC-MS results were validated for a select number of proteins over a time course of infection by Western blotting and immunofluorescence microscopy. Our study demonstrates for the first time the power of SILAC-MS for identifying and quantifying novel changes in cellular protein amounts in response to dengue virus infection.
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Vírus da Dengue/fisiologia , Dengue/metabolismo , Proteoma/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Dengue/virologia , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem , Proteínas Virais/metabolismoRESUMO
Identification of proteins by tandem mass spectrometry requires a reference protein database, but these are only available for model species. Here we demonstrate that, for a non-model species, the sequencing of expressed mRNA can generate a protein database for mass spectrometry-based identification. This combination of high-throughput sequencing and protein identification technologies allows detection of genes and proteins. We use human cells infected with human adenovirus as a complex and dynamic model to demonstrate the robustness of this approach. Our proteomics informed by transcriptomics (PIT) technique identifies >99% of over 3,700 distinct proteins identified using traditional analysis that relies on comprehensive human and adenovirus protein lists. We show that this approach can also be used to highlight genes and proteins undergoing dynamic changes in post-transcriptional protein stability.
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Proteoma/química , Proteômica/métodos , Transcriptoma , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Arginina/metabolismo , Células CHO , Isótopos de Carbono , Cromatografia Líquida , Cricetinae , Cricetulus , Bases de Dados de Proteínas , Células HeLa , Humanos , Lisina/metabolismo , Isótopos de Nitrogênio , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de Proteína/métodos , Software , Espectrometria de Massas em Tandem/métodosRESUMO
Adenoviral infection induces nucleoplasmic redistribution of a nucleolar nucleophosmin 1/NPM1/B23.1. NPM1 is preferentially localized in the nucleoli of normal cells, whereas it is also present at the nuclear matrix in cancer cells. However, the biological roles of NPM1 during infection are unknown. Here, by analyzing a pV-deletion mutant, Ad5-dV/TSB, we demonstrate that pV promotes the NPM1 translocation from the nucleoli to the nucleoplasm in normal cells, and the NPM1 translocation is correlated with adenoviral replication. Lack of pV causes a dramatic reduction of adenoviral replication in normal cells, but not cancer cells, and Ad5-dV/TSB was defective in viral assembly in normal cells. NPM1 knockdown inhibits adenoviral replication, suggesting an involvement of NPM1 in adenoviral biology. Further, we show that NPM1 interacts with empty adenovirus particles which are an intermediate during virion maturation by immunoelectron microscopy. Collectively, these data implicate that pV participates in a process of viral assembly through NPM1.
Assuntos
Adenoviridae/metabolismo , Proteínas Nucleares/metabolismo , Proteínas do Core Viral/metabolismo , Montagem de Vírus/efeitos dos fármacos , Adenoviridae/genética , Linhagem Celular , Nucléolo Celular/metabolismo , Proteínas Nucleares/genética , Nucleofosmina , Proteínas do Core Viral/genética , Proteínas do Core Viral/farmacologia , Replicação ViralRESUMO
Oncolytic adenoviruses represent a promising therapeutic medicine for human cancer therapy, but successful translation into human clinical trials requires careful evaluation of their viral characteristics. While the function of adenovirus proteins has been analyzed in detail, the dynamics of adenovirus infection remain largely unknown due to technological constraints that prevent adequate tracking of adenovirus particles after infection. Fluorescence labeling of adenoviral particles is one new strategy designed to directly analyze the dynamic processes of viral infection in virus-host cell interactions. We hypothesized that the double labeling of an adenovirus with fluorescent proteins would allow us to properly analyze intracellular viruses and the fate of viral proteins in a live analysis of an adenovirus as compared to single labeling. Thus, we generated a fluorescently labeled adenovirus with both a red fluorescent minor capsid protein IX (pIX) [pIX monomeric red fluorescent protein 1 (mRFP1)] and a green fluorescent minor core protein V (pV) [pV enhanced green fluorescent protein (EGFP)], resulting in Ad5-IX-mRFP1-E3-V-EGFP. The fluorescent signals for pIX-mRFP1 and pV-EGFP were detected within 10 min in living cells. However, a growth curve analysis of Ad5-IX-mRFP1-E3-V-EGFP showed an approximately 150-fold reduced production of the viral progeny at 48 h postinfection as compared to adenovirus type 5. Interestingly, pIX-mRFP1 and pV-EGFP were initially localized in the cytoplasm and nucleolus, respectively, at 18 h postinfection. These proteins were observed in the nucleus during the late stage of infection, and relocalization of the proteins was observed in an adenoviral-replication-dependent manner. These results indicate that simultaneous detection of adenoviruses using dual-fluorescent proteins is suitable for real-time analysis, including identification of infected cells and monitoring of viral spread, which will be required for a complete evaluation of oncolytic adenoviruses.
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Adenoviridae/metabolismo , Proteínas do Capsídeo/metabolismo , Corantes Fluorescentes/metabolismo , Proteínas do Core Viral/metabolismo , Adenoviridae/fisiologia , Adenoviridae/ultraestrutura , Linhagem Celular , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinética , Proteínas Luminescentes/metabolismo , Transporte Proteico , Coloração e Rotulagem , Frações Subcelulares/metabolismo , Vírion/ultraestrutura , Replicação Viral , Proteína Vermelha FluorescenteRESUMO
Adenoviruses replicate primarily in the host cell nucleus, and it is well established that adenovirus infection affects the structure and function of host cell nucleoli in addition to coding for a number of nucleolar targeted viral proteins. Here we used unbiased proteomics methods, including high throughput mass spectrometry coupled with stable isotope labeling by amino acids in cell culture (SILAC) and traditional two-dimensional gel electrophoresis, to identify quantitative changes in the protein composition of the nucleolus during adenovirus infection. Two-dimensional gel analysis revealed changes in six proteins. By contrast, SILAC-based approaches identified 351 proteins with 24 proteins showing at least a 2-fold change after infection. Of those, four were previously reported to have aberrant localization and/or functional relevance during adenovirus infection. In total, 15 proteins identified as changing in amount by proteomics methods were examined in infected cells using confocal microscopy. Eleven of these proteins showed altered patterns of localization in adenovirus-infected cells. Comparing our data with the effects of actinomycin D on the nucleolar proteome revealed that adenovirus infection apparently specifically targets a relatively small subset of nucleolar antigens at the time point examined.
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Adenoviridae/crescimento & desenvolvimento , Nucléolo Celular/metabolismo , Proteínas Nucleares/análise , Proteômica/métodos , Adenoviridae/fisiologia , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/virologia , Dactinomicina/farmacologia , Eletroforese em Gel Bidimensional , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Marcação por Isótopo/métodos , Espectrometria de Massas , Inibidores da Síntese de Ácido Nucleico/farmacologia , Proteoma/análiseRESUMO
Previous reports have shown that adenovirus recruits nucleolar protein upstream-binding factor (UBF) into adenovirus DNA replication centres. Here, we report that despite having a different mode of viral DNA replication, herpes simplex virus type 1 (HSV-1) also recruits UBF into viral DNA replication centres. Moreover, as with adenovirus, enhanced green fluorescent protein-tagged fusion proteins of UBF inhibit viral DNA replication. We propose that UBF is recruited to the replication compartments to aid replication of HSV-1 DNA. In addition, this is a further example of the role of nucleolar components in viral life cycles.
Assuntos
Herpesvirus Humano 1/fisiologia , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Replicação Viral , Replicação do DNA , DNA Viral/metabolismo , Células HeLa , HumanosRESUMO
Adenovirus infection subverts nucleolar structure and function. B23 is a nucleolar protein present in two isoforms (B23.1 and B23.2) and both isoforms have been identified as stimulatory factors for adenovirus DNA replication. Here, it is demonstrated that the two isoforms of B23, B23.1 and B23.2, interact and co-localize differently with viral DNA replication proteins pTP and DBP in adenovirus-infected cells. Thus, the mechanism by which the two proteins stimulate viral DNA replication is likely to differ. These data also demonstrate the importance of testing both isoforms of B23 for interactions with viral proteins and nucleic acids.
Assuntos
Infecções por Adenoviridae/metabolismo , Adenoviridae/fisiologia , DNA Viral/biossíntese , Proteínas Nucleares/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Infecções por Adenoviridae/virologia , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Células HeLa , Humanos , Ligação ProteicaRESUMO
Adenoviruses target their double-stranded DNA genome and its associated core proteins to the interphase nucleus; this core structure then enters through the nuclear pore complex. We have used digitonin permeabilized cell import assays to study the cellular import factors involved in nuclear entry of virus DNA and the core proteins, protein V and protein VII. We show that inhibition of transportin results in aberrant localization of protein V and that transportin is necessary for protein V to accumulate in the nucleolus. Furthermore, inhibition of transportin results in inhibition of protein VII and DNA import, whereas disruption of the classical importin alpha-importin beta import pathway has little effect. We show that mature protein VII has different import preferences from the precursor protein, preVII from which it is derived by proteolytic processing. While bacterially expressed glutathione S-transferase (GST)-preVII primarily utilizes the pathway mediated by importin alpha-importin beta, bacterially expressed GST-VII favours the transportin pathway. This is significant because while preVII is important during viral replication and assembly only mature VII is available during viral DNA import to a newly infected cell. Our results implicate transportin as a key import receptor for the nuclear localization of adenovirus core.
Assuntos
Adenovírus Humanos/metabolismo , Núcleo Celular/metabolismo , DNA Viral/metabolismo , Carioferinas/fisiologia , Proteínas do Core Viral/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Células HeLa , HumanosRESUMO
This chapter describes the techniques used to study nucleolar-localized proteins. The chapter starts with cloning of viral proteins for expression in mammalian cells as fusion proteins to well-characterized tags such as enhanced green fluorescence protein (EGFP). This follows on to techniques for transient expression in mammalian cells and immunofluorescence techniques used to examine subcellular localization. Finally there is guidance on the types of antigens and metabolic features of the nucleolus that can be used as markers to confirm that the protein in question is indeed localized in the nucleolus and determine whether it affects gross rRNA synthesis.
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Adenoviridae/metabolismo , Nucléolo Celular/metabolismo , Proteínas do Core Viral/metabolismo , Clonagem Molecular , Imunofluorescência , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Fases de Leitura AbertaAssuntos
Nucléolo Celular/genética , Ribossomos/genética , Ribossomos/fisiologia , Transcrição Gênica , Animais , Ciclo Celular , Nucléolo Celular/fisiologia , Cromossomos , Humanos , Modelos Biológicos , Neoplasias/genética , Malformações do Sistema Nervoso/genética , Ribossomos/virologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
When human adenovirus infects human cells there is disruption of rRNA biogenesis. This report examines the effect of adenovirus infection on the nucleolar protein, upstream binding factor (UBF) which plays a major role in regulating rRNA synthesis. We determined that early after infection, UBF associates with the replication of viral DNA, preferentially associating with the ends of the linear viral genome, and that addition of anti-UBF serum to in vitro replication assays markedly reduced viral DNA replication. Regions of UBF important to these observations are also established. Interestingly, sequestering the majority of UBF from the nucleolus did not lead to the ablation of rRNA synthesis or the sequestration of RNA pol I. In infected cells the bulk of RNA synthesis was RNA pol I associated and distinct from the location of most of the detectable UBF. We propose that UBF plays a role in viral DNA replication, further strengthening the role of nucleolar antigens in the adenovirus life cycle.