RESUMO
The recent coronavirus disease 2019 (COVID-19) pandemic was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 is characterized by respiratory distress, multiorgan dysfunction and, in some cases, death. The virus is also responsible for post-COVID-19 condition (commonly referred to as 'long COVID'). SARS-CoV-2 is a single-stranded, positive-sense RNA virus with a genome of approximately 30 kb, which encodes 26 proteins. It has been reported to affect multiple pathways in infected cells, resulting, in many cases, in the induction of a 'cytokine storm' and cellular senescence. Perhaps because it is an RNA virus, replicating largely in the cytoplasm, the effect of SARS-Cov-2 on genome stability and DNA damage responses (DDRs) has received relatively little attention. However, it is now becoming clear that the virus causes damage to cellular DNA, as shown by the presence of micronuclei, DNA repair foci and increased comet tails in infected cells. This review considers recent evidence indicating how SARS-CoV-2 causes genome instability, deregulates the cell cycle and targets specific components of DDR pathways. The significance of the virus's ability to cause cellular senescence is also considered, as are the implications of genome instability for patients suffering from long COVID.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Síndrome de COVID-19 Pós-Aguda , Instabilidade Genômica , Dano ao DNARESUMO
Over the past few months there have been reports of severe acute hepatitis in several hundred, otherwise healthy, immunocompetent young children. Several deaths have been recorded and a relatively large proportion of the patients have needed liver transplants. Most of the cases, so far, have been seen in the UK and in North America, but it has also been reported in many other European countries, the Middle East and Asia. Most common viruses have been ruled out as a causative agent; hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis C virus (HCV) were not detected, nor were Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human immunodeficiency virus (HIV) in many cases. A small proportion of the children had been infected with SARS-CoV-2 but these seem to be in a minority; similarly, almost none of the children had been vaccinated against COVID-19. Significantly, many of the patients were infected with adenovirus 41 (HAdV-F41). Previously, HAdV-41 had not been linked to hepatitis and is usually considered to cause gastroenteritis in both immunocompetent and immunocompromised patients. In two most recent studies, adeno-associated virus 2 (AAV2) was detected in almost all patients, together with species C and F HAdVs and human herpesvirus 6B (HHV6B). Here, I discuss the possibility that a change in tropism of HAdV-41 and changes in AAV2 may be responsible for their links to acute hepatitis.
Assuntos
Infecções por Adenoviridae , COVID-19 , Infecções por Vírus Epstein-Barr , Hepatite , Parvovirinae , Criança , Humanos , Pré-Escolar , Adenoviridae , Herpesvirus Humano 4 , SARS-CoV-2 , Hepatite/complicaçõesRESUMO
It has long been established that group A human adenoviruses (HAdV-A12, -A18, and -A31) can cause tumors in newborn rodents, with tumorigenicity related to the presence of a unique spacer region located between conserved regions 2 and 3 within the HAdV-A12 early region 1A (E1A) protein. Group B adenoviruses are weakly oncogenic, whereas most of the remaining human adenoviruses are nononcogenic. In an attempt to understand better the relationship between the structure of the AdE1A spacer region and oncogenicity of HAdVs, the structures of synthetic peptides identical or very similar to the adenovirus 12 E1A spacer region were determined and found to be α-helical using nuclear magnetic resonance (NMR) spectroscopy. This contrasts significantly with some previous suggestions that this region is unstructured. Using available predictive algorithms, the structures of spacer regions from other E1As were also examined, and the extent of the predicted α-helix was found to correlate reasonably well with the tumorigenicity of the respective virus. We suggest that this may represent an as-yet-unknown binding site for a partner protein required for tumorigenesis.IMPORTANCE This research analyzed small peptides equivalent to a region within the human adenovirus early region 1A protein that confers, in part, tumor-inducing properties to various degrees on several viral strains in rats and mice. The oncogenic spacer region is α-helical, which contrasts with previous suggestions that this region is unstructured. The helix is characterized by a stretch of amino acids rich in alanine residues that are organized into a hydrophobic, or "water-hating," surface that is considered to form a major site of interaction with cellular protein targets that mediate tumor formation. The extent of α-helix in E1A from other adenovirus species can be correlated to a limited degree to the tumorigenicity of that virus. Some serotypes show significant differences in predicted structural propensity, suggesting that the amino acid type and physicochemical properties are also of importance.
Assuntos
Adenovírus Humanos/genética , Adenovírus Humanos/patogenicidade , Carcinogênese/genética , DNA Intergênico/genética , Peptídeos/química , Adenovírus Humanos/metabolismo , Motivos de Aminoácidos , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , DNA Intergênico/química , DNA Intergênico/metabolismo , Células Eucarióticas/patologia , Células Eucarióticas/virologia , Interações Hospedeiro-Patógeno/genética , Humanos , Camundongos , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Peptídeos/genética , Peptídeos/metabolismo , Biossíntese de Proteínas , Conformação Proteica em alfa-Hélice , Ratos , Sorogrupo , Relação Estrutura-AtividadeRESUMO
Gammaherpesviruses establish lifelong latent infection in B lymphocytes and are the causative agent of several B-cell malignancies and lymphoproliferative disorders. While a quiescent latent infection allows these pathogens to evade immune detection, initiation of an alternative lifecycle stage, known as lytic replication, is an essential step in the production and dissemination of infectious progeny. Although cessation of cellular proliferation is an eventual consequence of lytic induction, exactly how gammaherpesviruses manipulate the cell cycle prior to amplification of viral DNA remains under debate. Here we show that the onset of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic reactivation in B cells leads to S-phase accumulation and that exit from G1 is required for efficient viral DNA replication. We also show that lytic replication leads to an S-phase-specific activation of the DNA damage response (DDR) that is abrogated when lytic replication is restricted to G0/G1. Finally, we observe that expression of early lytic viral genes results in cellular replication stress with increased stalling of DNA replication forks. Overall, we demonstrate that S-phase entry is important for optimal KSHV replication, that G1 arresting compounds are effective inhibitors of viral propagation, and that lytic-induced cell-cycle arrest could occur through the obstruction of cellular replication forks and subsequent activation of the DDR.
Assuntos
Replicação do DNA/genética , Herpesviridae/genética , Linfoma de Efusão Primária/virologia , Fase S/genética , Replicação Viral/genética , Linhagem Celular , DNA Viral/genética , Fase G1/genética , Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 8/genética , Humanos , Ativação Viral/genética , Latência Viral/genéticaRESUMO
DNA double-strand break (DSB) signaling and repair are critical for cell viability, and rely on highly coordinated pathways whose molecular organization is still incompletely understood. Here, we show that heterogeneous nuclear ribonucleoprotein U-like (hnRNPUL) proteins 1 and 2 play key roles in cellular responses to DSBs. We identify human hnRNPUL1 and -2 as binding partners for the DSB sensor complex MRE11-RAD50-NBS1 (MRN) and demonstrate that hnRNPUL1 and -2 are recruited to DNA damage in an interdependent manner that requires MRN. Moreover, we show that hnRNPUL1 and -2 stimulate DNA-end resection and promote ATR-dependent signaling and DSB repair by homologous recombination, thereby contributing to cell survival upon exposure to DSB-inducing agents. Finally, we establish that hnRNPUL1 and -2 function downstream of MRN and CtBP-interacting protein (CtIP) to promote recruitment of the BLM helicase to DNA breaks. Collectively, these results provide insights into how mammalian cells respond to DSBs.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Ribonucleoproteínas Nucleares Heterogêneas/fisiologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Hidrolases Anidrido Ácido , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Proteína Homóloga a MRE11 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Infection by most DNA viruses activates a cellular DNA damage response (DDR), which may be to the detriment or advantage of the virus. In the case of adenoviruses, they neutralize antiviral effects of DDR activation by targeting a number of proteins for rapid proteasome-mediated degradation. We have now identified a novel DDR protein, tankyrase 1 binding protein 1 (TNKS1BP1) (also known as Tab182), which is degraded during infection by adenovirus serotype 5 and adenovirus serotype 12. In both cases, degradation requires the action of the early region 1B55K (E1B55K) and early region 4 open reading frame 6 (E4orf6) viral proteins and is mediated through the proteasome by the action of cullin-based cellular E3 ligases. The degradation of Tab182 appears to be serotype specific, as the protein remains relatively stable following infection with adenovirus serotypes 4, 7, 9, and 11. We have gone on to confirm that Tab182 is an integral component of the CNOT complex, which has transcriptional regulatory, deadenylation, and E3 ligase activities. The levels of at least 2 other members of the complex (CNOT3 and CNOT7) are also reduced during adenovirus infection, whereas the levels of CNOT4 and CNOT1 remain stable. The depletion of Tab182 with small interfering RNA (siRNA) enhances the expression of early region 1A proteins (E1As) to a limited extent during adenovirus infection, but the depletion of CNOT1 is particularly advantageous to the virus and results in a marked increase in the expression of adenovirus early proteins. In addition, the depletion of Tab182 and CNOT1 results in a limited increase in the viral DNA level during infection. We conclude that the cellular CNOT complex is a previously unidentified major target for adenoviruses during infection.IMPORTANCE Adenoviruses target a number of cellular proteins involved in the DNA damage response for rapid degradation. We have now shown that Tab182, which we have confirmed to be an integral component of the mammalian CNOT complex, is degraded following infection by adenovirus serotypes 5 and 12. This requires the viral E1B55K and E4orf6 proteins and is mediated by cullin-based E3 ligases and the proteasome. In addition to Tab182, the levels of other CNOT proteins are also reduced during adenovirus infection. Thus, CNOT3 and CNOT7, for example, are degraded, whereas CNOT4 and CNOT1 are not. The siRNA-mediated depletion of components of the complex enhances the expression of adenovirus early proteins and increases the concentration of viral DNA produced during infection. This study highlights a novel protein complex, CNOT, which is targeted for adenovirus-mediated protein degradation. To our knowledge, this is the first time that the CNOT complex has been identified as an adenoviral target.
Assuntos
Infecções por Adenoviridae/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/química , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Adenoviridae/imunologia , Adenoviridae/patogenicidade , Infecções por Adenoviridae/virologia , Proteínas Culina/metabolismo , Exorribonucleases , Células HEK293 , Células HeLa , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Repressoras , SorogrupoRESUMO
Double-strand breaks (DSBs) in DNA are recognized by the Ku70/80 heterodimer and the MRE11-RAD50-NBS1 (MRN) complex and result in activation of the DNA-PK and ATM kinases, which play key roles in regulating the cellular DNA damage response (DDR). DNA tumor viruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) are known to interact extensively with the DDR during the course of their replicative cycles. Here we show that during lytic amplification of KSHV DNA, the Ku70/80 heterodimer and the MRN complex consistently colocalize with viral genomes in replication compartments (RCs), whereas other DSB repair proteins form foci outside RCs. Depletion of MRE11 and abrogation of its exonuclease activity negatively impact viral replication, while in contrast, knockdown of Ku80 and inhibition of the DNA-PK enzyme, which are involved in nonhomologous end joining (NHEJ) repair, enhance amplification of viral DNA. Although the recruitment of DSB-sensing proteins to KSHV RCs is a consistent occurrence across multiple cell types, activation of the ATM-CHK2 pathway during viral replication is a cell line-specific event, indicating that recognition of viral DNA by the DDR does not necessarily result in activation of downstream signaling pathways. We have also observed that newly replicated viral DNA is not associated with cellular histones. Since the presence and modification of these DNA-packaging proteins provide a scaffold for docking of multiple DNA repair factors, the absence of histone deposition may allow the virus to evade localization of DSB repair proteins that would otherwise have a detrimental effect on viral replication.IMPORTANCE Tumor viruses are known to interact with machinery responsible for detection and repair of double-strand breaks (DSBs) in DNA, although detail concerning how Kaposi's sarcoma-associated herpesvirus (KSHV) modulates these cellular pathways during its lytic replication phase was previously lacking. By undertaking a comprehensive assessment of the localization of DSB repair proteins during KSHV replication, we have determined that a DNA damage response (DDR) is directed to viral genomes but is distinct from the response to cellular DNA damage. We also demonstrate that although recruitment of the MRE11-RAD50-NBS1 (MRN) DSB-sensing complex to viral genomes and activation of the ATM kinase can promote KSHV replication, proteins involved in nonhomologous end joining (NHEJ) repair restrict amplification of viral DNA. Overall, this study extends our understanding of the virus-host interactions that occur during lytic replication of KSHV and provides a deeper insight into how the DDR is manipulated during viral infection.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 8/fisiologia , Proteínas Nucleares/metabolismo , Sarcoma de Kaposi/metabolismo , Ativação Viral/fisiologia , Hidrolases Anidrido Ácido , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Enzimas Reparadoras do DNA/genética , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Proteína Homóloga a MRE11 , Proteínas Nucleares/genética , Sarcoma de Kaposi/genéticaRESUMO
BACKGROUND: The Ccr4-Not complex (CNOT complex in mammals) is a unique and highly conserved complex with numerous cellular functions. Until now, there has been relatively little known about the importance of the CNOT complex subunits in the DNA damage response (DDR) in mammalian cells. CNOT4 is a subunit of the complex with E3 ubiquitin ligase activity that interacts transiently with the CNOT1 subunit. Here, we attempt to investigate the role of human CNOT4 subunit in the DDR in human cells. MATERIAL AND METHODS: In this study, cell viability in the absence of CNOT4 was assessed using a Cell Titer-Glo Luminescence assay up to 4 days post siRNA transfection. In a further experiment, CNOT4-depleted HeLa cells were exposed to 3Gy ionizing radiation (IR). Ataxia telangiectasia-mutated (ATM) and ATM Rad3-related (ATR) signaling pathways were then investigated by western blotting for phosphorylated substrates. In addition, foci formation of histone 2A family member X (γH2AX), replication protein A (RPA), TP53 binding protein 1 (53BP1), and DNA repair protein RAD51 homolog 1 was also determined by immunofluorescence microscopy comparing control and CNOT4-depleted HeLa cells 0, 8, and 24 h post IR treatment. RESULTS: Our results from cell viability assays showed a significant reduction of cell growth activity at 24 (P value 0.02) and 48 h (P value 0.002) post siRNA. Western blot analysis showed slightly reduced or slightly delayed DDR signaling in CNOT4-depleted HeLa cells after IR. More significantly, we observed increased formation of γH2AX, RPA, 53BP1, and RAD51 foci after IR in CNOT4-depleted cells compared with the control cells. CONCLUSION: We conclude that depletion of CNOT4 affects various aspects of the cellular response to DNA damage.
Assuntos
Proteínas de Ciclo Celular , Radiação Ionizante , Animais , Humanos , Células HeLa , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Reparo do DNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fosforilação , Mamíferos/genética , Mamíferos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Oncogenic viruses contribute to 15% of global human cancers. To achieve that, virus-encoded oncoproteins deregulate cellular transcription, antagonize common cellular pathways, and thus drive cell transformation. Notably, adenoviruses were the first human viruses proven to induce cancers in diverse animal models. Over the past decades, human adenovirus (HAdV)-mediated oncogenic transformation has been pivotal in deciphering underlying molecular mechanisms. Key adenovirus oncoproteins, encoded in early regions 1 (E1) and 4 (E4), co-ordinate these processes. Among the different adenovirus species, the most extensively studied HAdV-C5 displays lower oncogenicity than HAdV-A12. A complete understanding of the different HAdV-A12 and HAdV-C5 oncoproteins in virus-mediated cell transformation, as summarized here, is relevant for adenovirus research and offers broader insights into viral transformation and oncogenesis.
Assuntos
Adenovírus Humanos , Humanos , Adenovírus Humanos/genética , Adenovírus Humanos/fisiologia , Animais , Oncogenes , Transformação Celular Viral , Neoplasias/virologia , Neoplasias/genética , Proteínas Oncogênicas/metabolismo , Proteínas Oncogênicas/genética , Carcinogênese/genéticaRESUMO
Increased proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs) is recognised as a universal hallmark of pulmonary arterial hypertension (PAH), in part related to the association with reduced pyruvate dehydrogenase (PDH) activity, resulting in decreased oxidative phosphorylation of glucose and increased aerobic glycolysis (Warburg effect). Perhexiline is a well-recognised carnitine palmitoyltransferase-1 (CPT1) inhibitor used in cardiac diseases, which reciprocally increases PDH activity, but is associated with variable pharmacokinetics related to polymorphic variation of the cytochrome P450-2D6 (CYP2D6) enzyme, resulting in the risk of neuro and hepatotoxicity in 'slow metabolisers' unless blood levels are monitored and dose adjusted. We have previously reported that a novel perhexiline fluorinated derivative (FPER-1) has the same therapeutic profile as perhexiline but is not metabolised by CYP2D6, resulting in more predictable pharmacokinetics than the parent drug. We sought to investigate the effects of perhexiline and FPER-1 on PDH flux in PASMCs from patients with PAH. We first confirmed that PAH PASMCs exhibited increased cell proliferation, enhanced phosphorylation of AKTSer473, ERK 1/2Thr202/Tyr204 and PDH-E1αSer293, indicating a Warburg effect when compared to healthy PASMCs. Pre-treatment with perhexiline or FPER-1 significantly attenuated PAH PASMC proliferation in a concentration-dependent manner and suppressed the activation of the AKTSer473 but had no effect on the ERK pathway. Perhexiline and FPER-1 markedly activated PDH (seen as dephosphorylation of PDH-E1αSer293), reduced glycolysis, and upregulated mitochondrial respiration in these PAH PASMCs as detected by Seahorse analysis. However, both perhexiline and FPER-1 did not induce apoptosis as measured by caspase 3/7 activity. We show for the first time that both perhexiline and FPER-1 may represent therapeutic agents for reducing cell proliferation in human PAH PASMCs, by reversing Warburg physiology.
RESUMO
Early region 1A (E1A) of human adenovirus (HAdV) has been the focus of over 30 years of investigation and is required for the oncogenic capacity of HAdV in rodents. Alternative splicing of the E1A transcript generates mRNAs encoding multiple E1A proteins. The 55-residue (55R) E1A protein, which is encoded by the 9S mRNA, is particularly interesting due to the unique properties it displays relative to all other E1A isoforms. 55R E1A does not contain any of the conserved regions (CRs) present in the other E1A isoforms. The C-terminal region of the 55R E1A protein contains a unique sequence compared to all other E1A isoforms, which results from a frameshift generated by alternative splicing. The 55R E1A protein is thought to be produced preferentially at the late stages of infection. Here we report the first study to directly investigate the function of the species C HAdV 55R E1A protein during infection. Polyclonal rabbit antibodies (Abs) have been generated that are capable of immunoprecipitating HAdV-2 55R E1A. These Abs can also detect HAdV-2 55R E1A by immunoblotting and indirect immunofluorescence assay. These studies indicate that 55R E1A is expressed late and is localized to the cytoplasm and to the nucleus. 55R E1A was able to activate the expression of viral genes during infection and could also promote productive replication of species C HAdV. 55R E1A was also found to interact with the S8 component of the proteasome, and knockdown of S8 was detrimental to viral replication dependent on 55R E1A.
Assuntos
Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Adenovírus Humanos/genética , RNA Mensageiro/química , RNA Viral/química , Adenosina Trifosfatases/metabolismo , Proteínas E1A de Adenovirus/imunologia , Adenovírus Humanos/imunologia , Sequência de Aminoácidos , Anticorpos Antivirais/imunologia , Linhagem Celular , Núcleo Celular/metabolismo , Inibição de Contato , Citoplasma/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Dados de Sequência Molecular , Ligação Proteica , Transporte Proteico , Transcrição Gênica , Replicação Viral/genéticaRESUMO
The ability of adenovirus early region proteins, E1B-55K and E4orf6, to usurp control of cellular ubiquitin ligases and target proteins for proteasome-dependent degradation during infection is well established. Here we show that the E4 gene product, E4orf3 can, independently of E1B-55K and E4orf6, target the transcriptional corepressor transcriptional intermediary factor 1γ (TIF1γ) for proteasome-mediated degradation during infection. Initial mass spectrometric studies identified TIF1 family members-TIF1α, TIF1ß, and TIF1γ-as E1B-55K-binding proteins in both transformed and infected cells, but analyses revealed that, akin to TIF1α, TIF1γ is reorganized in an E4orf3-dependent manner to promyelocytic leukemia protein-containing nuclear tracks during infection. The use of a number of different adenovirus early region mutants identified the specific and sole requirement for E4orf3 in mediating TIF1γ degradation. Further analyses revealed that TIF1γ is targeted for degradation by a number of divergent human adenoviruses, suggesting that the ability of E4orf3 to regulate TIF1γ expression is evolutionarily conserved. We also determined that E4orf3 does not utilize the Cullin-based ubiquitin ligases, CRL2 and CRL5, or the TIF1α ubiquitin ligase in order to promote TIF1γ degradation. Further studies suggested that TIF1γ possesses antiviral activity and limits adenovirus early and late gene product expression during infection. Indeed, TIF1γ knockdown accelerates the adenovirus-mediated degradation of MRE11, while TIF1γ overexpression delays the adenovirus-mediated degradation of MRE11. Taken together, these studies have identified novel adenovirus targets and have established a new role for the E4orf3 protein during infection.
Assuntos
Infecções por Adenoviridae/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Adenovírus Humanos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/virologia , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/genética , Adenovírus Humanos/genética , Linhagem Celular , Humanos , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
Activation of the cellular DNA damage response is detrimental to adenovirus (Ad) infection. Ad has therefore evolved a number of strategies to inhibit ATM- and ATR-dependent signaling pathways during infection. Recent work suggests that the Ad5 E4orf3 protein prevents ATR activation through its ability to mislocalize the MRN complex. Here we provide evidence to indicate that Ad12 has evolved a different strategy from Ad5 to inhibit ATR. We show that Ad12 utilizes a CUL2/RBX1/elongin C-containing ubiquitin ligase to promote the proteasomal degradation of the ATR activator protein topoisomerase-IIbeta-binding protein 1 (TOPBP1). Ad12 also uses this complex to degrade p53 during infection, in contrast to Ad5, which requires a CUL5-based ubiquitin ligase. Although Ad12-mediated degradation of p53 is dependent upon both E1B-55K and E4orf6, Ad12-mediated degradation of TOPBP1 is solely dependent on E4orf6. We propose that Ad12 E4orf6 has two principal activities: to recruit the CUL2-based ubiquitin ligase and to act as substrate receptor for TOPBP1. In support of the idea that Ad12 E4orf6 specifically prevents ATR activation during infection by targeting TOPBP1 for degradation, we demonstrate that Ad12 E4orf6 can inhibit the ATR-dependent phosphorylation of CHK1 in response to replication stress. Taken together, these data provide insights into how Ad modulates ATR signaling pathways during infection.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Virais/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Adenoviridae/fisiologia , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/genética , Elonguina , Imunofluorescência , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Microscopia Confocal , Mutação , Proteínas Nucleares/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/genéticaRESUMO
Over 100 human adenoviruses (HAdVs) have been isolated and allocated to seven species, A-G. Species F comprises two members-HAdV-F40 and HAdV-F41. As their primary site of infection is the gastrointestinal tract they have been termed, with species A, enteric adenoviruses. HAdV-F40 and HAdV-F41 are a common cause of gastroenteritis and diarrhoea in children. Partly because of difficulties in propagating the viruses in the laboratory, due to their restrictions on growth in many cell lines, our knowledge of the properties of individual viral proteins is limited. However, the structure of HAdV-F41 has recently been determined by cryo-electron microscopy. The overall structure is similar to those of HAdV-C5 and HAdV-D26 although with some differences. The sequence and arrangement of the hexon hypervariable region 1 (HVR1) and the arrangement of the C-terminal region of protein IX differ. Variations in the penton base and hexon HVR1 may play a role in facilitating infection of intestinal cells by HAdV-F41. A unique feature of HAdV-F40 and F41, among human adenoviruses, is the presence and expression of two fibre genes, giving long and short fibre proteins. This may also contribute to the tropism of these viruses. HAdV-F41 has been linked to a recent outbreak of severe acute hepatitis "of unknown origin" in young children. Further investigation has shown a very high prevalence of adeno-associated virus-2 in the liver and/or plasma of some cohorts of patients. These observations have proved controversial as HAdV-F41 had not been reported to infect the liver and AAV-2 has generally been considered harmless.
Assuntos
Infecções por Adenovirus Humanos , Adenovírus Humanos , Hepatite A , Hepatite , Humanos , Criança , Pré-Escolar , Adenovírus Humanos/genética , Virulência , Microscopia Crioeletrônica , Infecções por Adenovirus Humanos/epidemiologia , FilogeniaRESUMO
The mammalian Ccr4-Not complex, carbon catabolite repression 4 (Ccr4)-negative on TATA-less (Not), is a large, highly conserved, multifunctional assembly of proteins that acts at different cellular levels to regulate gene expression. It is involved in the control of the cell cycle, chromatin modification, activation and inhibition of transcription initiation, control of transcription elongation, RNA export, and nuclear RNA surveillance; the Ccr4-Not complex also plays a central role in the regulation of mRNA decay. Growing evidence suggests that gene transcription has a vital role in shaping the landscape of genome replication and is also a potent source of replication stress and genome instability. Here, we have examined the effects of the inactivation of the Ccr4-Not complex, via the depletion of the scaffold subunit CNOT1, on DNA replication and genome integrity in mammalian cells. In CNOT1-depleted cells, the elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which, together with R-loop accumulation, results in replication fork slowing, DNA damage, and senescence. Furthermore, we have shown that the stability of TBP mRNA increases in the absence of CNOT1, which may explain its elevated protein expression in CNOT1-depleted cells. Finally, we have shown the activation of mitogen-activated protein kinase signalling as evidenced by ERK1/2 phosphorylation in the absence of CNOT1, which may be responsible for the observed cell cycle arrest at the border of G1/S.
Assuntos
Repressão Catabólica , Fatores de Transcrição , Animais , Fatores de Transcrição/metabolismo , RNA/metabolismo , RNA Mensageiro/genética , Instabilidade Genômica , Mamíferos/metabolismoRESUMO
The adenovirus C5 E1B-55K protein is crucial for viral replication and is expressed early during infection. It can interact with E4orf6 to form a complex that functions as a ubiquitin E3 ligase. This complex targets specific cellular proteins and marks them for ubiquitination and, predominantly, subsequent proteasomal degradation. E1B-55K interacts with various proteins, with p53 being the most extensively studied, although identifying binding sites has been challenging. To explain the diverse range of proteins associated with E1B-55K, we hypothesized that other binding partners might recognize the simple p53 binding motif (xWxxxPx). In silico analyses showed that many known E1B-55K binding proteins possess this amino acid sequence; therefore, we investigated whether other xWxxxPx-containing proteins also bind to E1B-55K. Our findings revealed that many cellular proteins, including ATR, CHK1, USP9, and USP34, co-immunoprecipitate with E1B-55K. During adenovirus infection, several well-characterized E1B-55K binding proteins and newly identified interactors, including CSB, CHK1, and USP9, are degraded in a cullin-dependent manner. Notably, certain binding proteins, such as ATR and USP34, remain undegraded during infection. Structural predictions indicate no conservation of structure around the proposed binding motif, suggesting that the interaction relies on the correct arrangement of tryptophan and proline residues.
Assuntos
Infecções por Adenoviridae , Proteínas E4 de Adenovirus , Adenovírus Humanos , Humanos , Adenoviridae/metabolismo , Proteínas E1B de Adenovirus/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Infecções por Adenoviridae/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas E4 de Adenovirus/genética , Proteínas E4 de Adenovirus/metabolismo , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismoRESUMO
It is clear that a number of host-cell factors facilitate virus replication and, conversely, a number of other factors possess inherent antiviral activity. Research, particularly over the last decade or so, has revealed that there is a complex inter-relationship between viral infection and the host-cell DNA-damage response and repair pathways. There is now a realization that viruses can selectively activate and/or repress specific components of these host-cell pathways in a temporally coordinated manner, in order to promote virus replication. Thus, some viruses, such as simian virus 40, require active DNA-repair pathways for optimal virus replication, whereas others, such as adenovirus, go to considerable lengths to inactivate some pathways. Although there is ever-increasing molecular insight into how viruses interact with host-cell damage pathways, the precise molecular roles of these pathways in virus life cycles is not well understood. The object of this review is to consider how DNA viruses have evolved to manage the function of three principal DNA damage-response pathways controlled by the three phosphoinositide 3-kinase (PI3K)-related protein kinases ATM, ATR and DNA-PK and to explore further how virus interactions with these pathways promote virus replication.
Assuntos
Dano ao DNA , Reparo do DNA , Vírus de DNA/genética , Transdução de Sinais/genética , Replicação Viral/genética , Vírus de DNA/metabolismo , HumanosRESUMO
Adenovirus type 5 (Ad5) inactivates the host cell DNA damage response by facilitating the degradation of Mre11, DNA ligase IV, and p53. In the case of p53, this is achieved through polyubiquitylation by Ad5E1B55K and Ad5E4orf6, which recruit a Cul5-based E3 ubiquitin ligase. Recent evidence indicates that this paradigm does not apply to other adenovirus serotypes, since Ad12, but not Ad5, causes the degradation of TOPBP1 through the action of E4orf6 alone and a Cul2-based E3 ubiquitin ligase. We now have extended these studies to adenovirus groups A to E. While infection by Ad4, Ad5, and Ad12 (groups E, C, and A, respectively) cause the degradation of Mre11, DNA ligase IV, and p53, infection with Ad3, Ad7, Ad9, and Ad11 (groups B1, B1, D, and B2, respectively) only affects DNA ligase IV levels. Indeed, Ad3, Ad7, and Ad11 cause the marked accumulation of p53. Despite this, MDM2 levels were very low following infection with all of the viruses examined here, regardless of whether they increase p53 expression. In addition, we found that only Ad12 causes the degradation of TOPBP1, and, like Ad5, Ad4 recruits a Cul5-based E3 ubiquitin ligase to degrade p53. Surprisingly, Mre11 and DNA ligase IV degradation do not appear to be significantly affected in Ad4-, Ad5-, or Ad12-infected cells depleted of Cul2 or Cul5, indicating that E1B55K and E4orf6 recruit multiple ubiquitin ligases to target cellular proteins. Finally, although Mre11 is not degraded by Ad3, Ad7, Ad9, and Ad11, no viral DNA concatemers could be detected. We suggest that group B and D adenoviruses have evolved mechanisms based on the loss of DNA ligase IV and perhaps other unknown molecules to disable the host cell DNA damage response to promote viral replication.
Assuntos
Infecções por Adenoviridae/genética , Infecções por Adenoviridae/metabolismo , Adenoviridae/fisiologia , Dano ao DNA , Adenoviridae/classificação , Adenoviridae/genética , Infecções por Adenoviridae/enzimologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Ligases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Especificidade da Espécie , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The largest E1A isoform of human adenovirus (Ad) includes a C-4 zinc finger domain within conserved region 3 (CR3) that is largely responsible for activating transcription of the early viral genes. CR3 interacts with multiple cellular factors, but its mechanism of action is modeled primarily on the basis of the mechanism for the prototype E1A protein of human Ad type 5. We expanded this model to include a representative member from each of the six human Ad subgroups. All CR3 domains tested were capable of transactivation. However, there were dramatic differences in their levels of transcriptional activation. Despite these functional variations, the interactions of these representative CR3s with known cellular transcriptional regulators revealed only modest differences. Four common cellular targets of all representative CR3s were identified: the proteasome component human Sug1 (hSug1)/S8, the acetyltransferases p300/CREB binding protein (CBP), the mediator component mediator complex subunit 23 (MED23) protein, and TATA binding protein (TBP). The first three factors appear to be critical for CR3 function. RNA interference against human TBP showed no significant reduction in transactivation by any CR3 tested. These results indicate that the cellular factors previously shown to be important for transactivation by Ad5 CR3 are similarly bound by the E1A proteins of other types. This was confirmed experimentally using a transcriptional squelching assay, which demonstrated that the CR3 regions of each Ad type could compete with Ad5 CR3 for limiting factors. Interestingly, a mutant of Ad5 CR3 (V147L) was capable of squelching wild-type Ad5 CR3, despite its failure to bind TBP, MED23, p300/CBP-associated factor (pCAF), or p300/CBP, suggestive of the possibility that an additional as yet unidentified cellular factor is required for transactivation by E1A CR3.
Assuntos
Infecções por Adenoviridae/genética , Infecções por Adenoviridae/virologia , Adenoviridae/classificação , Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Adenoviridae/patogenicidade , Infecções por Adenoviridae/metabolismo , Animais , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/virologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Imunofluorescência , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular , Luciferases/metabolismo , Complexo Mediador/genética , Complexo Mediador/metabolismo , Camundongos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Ativação Transcricional , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
The anaphase-promoting complex/cyclosome (APC/C) is a multicomponent E3 ubiquitin ligase that, by targeting protein substrates for 26S proteasome-mediated degradation through ubiquitination, coordinates the temporal progression of eukaryotic cells through mitosis and the subsequent G1 phase of the cell cycle. Other functions of the APC/C are, however, less well defined. Here we show that two APC/C components, APC5 and APC7, interact directly with the coactivators CBP and p300 through protein-protein interaction domains that are evolutionarily conserved in adenovirus E1A. This interaction stimulates intrinsic CBP/p300 acetyltransferase activity and potentiates CBP/p300-dependent transcription. We also show that APC5 and APC7 suppress E1A-mediated transformation in a CBP/p300-dependent manner, indicating that these components of the APC/C may be targeted during cellular transformation. Furthermore, we establish that CBP is required in APC/C function; specifically, gene ablation of CBP by RNA-mediated interference markedly reduces the E3 ubiquitin ligase activity of the APC/C and the progression of cells through mitosis. Taken together, our results define discrete roles for the APC/C-CBP/p300 complexes in growth regulation.