RESUMO
Well-controlled repair mechanisms are involved in the maintenance of genomic stability, and their failure can precipitate DNA abnormalities and elevate tumor risk. In addition, the tumor microenvironment, enriched with factors inducing oxidative stress and affecting cell cycle checkpoints, intensifies DNA damage when repair pathways falter. Recent research has unveiled associations between certain bacteria, including Mycoplasmas, and various cancers, and the causative mechanism(s) are under active investigation. We previously showed that Mycoplasma fermentans DnaK, an HSP70 family chaperone protein, hampers the activity of proteins like PARP1 and p53, crucial for genomic integrity. Moreover, our analysis of its interactome in human cancer cell lines revealed DnaK's engagement with several components of DNA-repair machinery. Finally, in vivo experiments performed in our laboratory using a DnaK knock-in mouse model generated by our group demonstrated that DnaK exposure led to increased DNA copy number variants, indicative of genomic instability. We present here evidence that expression of DnaK is linked to increased i) incidence of tumors in vivo upon exposure to urethane, a DNA damaging agent; ii) spontaneous DNA damage ex vivo; and iii) expression of proinflammatory cytokines ex vivo, variations in reactive oxygen species levels, and increased ß-galactosidase activity across tissues. Moreover, DnaK was associated with increased centromeric instability. Overall, these findings highlight the significance of Mycoplasma DnaK in the etiology of cancer and other genetic disorders providing a promising target for prevention, diagnostics, and therapeutics.
Assuntos
Proteínas de Bactérias , Proteínas de Choque Térmico HSP70 , Mycoplasma , Neoplasias , Animais , Humanos , Camundongos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA , Dano ao DNA , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Mycoplasma/fisiologia , Neoplasias/metabolismo , Neoplasias/microbiologia , Neoplasias/patologia , Microambiente TumoralRESUMO
The human microbiota affects critical cellular functions, although the responsible mechanism(s) is still poorly understood. In this regard, we previously showed that Mycoplasma fermentans DnaK, an HSP70 chaperone protein, hampers the activity of important cellular proteins responsible for DNA integrity. Here, we describe a novel DnaK knock-in mouse model generated in our laboratory to study the effect of M. fermentans DnaK expression in vivo. By using an array-based comparative genomic hybridization assay, we demonstrate that exposure to DnaK was associated with a higher number of DNA copy number variants (CNVs) indicative of unbalanced chromosomal alterations, together with reduced fertility and a high rate of fetal abnormalities. Consistent with their implication in genetic disorders, one of these CNVs caused a homozygous Grid2 deletion, resulting in an aberrant ataxic phenotype that recapitulates the extensive biallelic deletion in the Grid2 gene classified in humans as autosomal recessive spinocerebellar ataxia 18. Our data highlight a connection between components of the human urogenital tract microbiota, namely Mycoplasmas, and genetic abnormalities in the form of DNA CNVs, with obvious relevant medical, diagnostic, and therapeutic implications.
Assuntos
Variações do Número de Cópias de DNA , Infecções por Mycoplasma , Mycoplasma fermentans/genética , Homozigoto , Infecções por Mycoplasma/genética , Infecções por Mycoplasma/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BLRESUMO
AIDS-defining cancers declined after combined antiretroviral therapy (cART) introduction, but lymphomas are still elevated in HIV type 1 (HIV-1)-infected patients. In particular, non-Hodgkin's lymphomas (NHLs) represent the majority of all AIDS-defining cancers and are the most frequent cause of death in these patients. We have recently demonstrated that amino acid (aa) insertions at the HIV-1 matrix protein p17 COOH-terminal region cause protein destabilization, leading to conformational changes. Misfolded p17 variants (vp17s) strongly impact clonogenic B cell growth properties that may contribute to B cell lymphomagenesis as suggested by the significantly higher frequency of detection of vp17s with COOH-terminal aa insertions in plasma of HIV-1-infected patients with NHL. Here, we expand our previous observations by assessing the prevalence of vp17s in large retrospective cohorts of patients with and without lymphoma. We confirm the significantly higher prevalence of vp17s in lymphoma patients than in HIV-1-infected individuals without lymphoma. Analysis of 3,990 sequences deposited between 1985 and 2017 allowed us to highlight a worldwide increasing prevalence of HIV-1 mutants expressing vp17s over time. Since genomic surveillance uncovered a cluster of HIV-1 expressing a B cell clonogenic vp17 dated from 2011 to 2019, we conclude that aa insertions can be fixed in HIV-1 and that mutant viruses displaying B cell clonogenic vp17s are actively spreading.
Assuntos
Linfócitos B , Antígenos HIV , HIV-1 , Linfoma Relacionado a AIDS , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Linfócitos B/virologia , Variação Genética , Antígenos HIV/genética , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Linfoma Relacionado a AIDS/epidemiologia , Linfoma Relacionado a AIDS/virologia , Prevalência , Estudos Retrospectivos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
BACKGROUND: Chemotherapy is a primary treatment for cancer, but its efficacy is often limited by cancer-associated bacteria (CAB) that impair tumor suppressor functions. Our previous research found that Mycoplasma fermentans DnaK, a chaperone protein, impairs p53 activities, which are essential for most anti-cancer chemotherapeutic responses. METHODS: To investigate the role of DnaK in chemotherapy, we treated cancer cell lines with M. fermentans DnaK and then with commonly used p53-dependent anti-cancer drugs (cisplatin and 5FU). We evaluated the cells' survival in the presence or absence of a DnaK-binding peptide (ARV-1502). We also validated our findings using primary tumor cells from a novel DnaK knock-in mouse model. To provide a broader context for the clinical significance of these findings, we investigated human primary cancer sequencing datasets from The Cancer Genome Atlas (TCGA). We identified F. nucleatum as a CAB carrying DnaK with an amino acid composition highly similar to M. fermentans DnaK. Therefore, we investigated the effect of F. nucleatum DnaK on the anti-cancer activity of cisplatin and 5FU. RESULTS: Our results show that both M. fermentans and F. nucleatum DnaKs reduce the effectiveness of cisplatin and 5FU. However, the use of ARV-1502 effectively restored the drugs' anti-cancer efficacy. CONCLUSIONS: Our findings offer a practical framework for designing and implementing novel personalized anti-cancer strategies by targeting specific bacterial DnaKs in patients with poor response to chemotherapy, underscoring the potential for microbiome-based personalized cancer therapies.
Assuntos
Antineoplásicos , Neoplasias , Animais , Camundongos , Humanos , Cisplatino , Proteína Supressora de Tumor p53 , Fluoruracila , BactériasRESUMO
The COVID-19 pandemic triggered an unparalleled pursuit of vaccines to induce specific adaptive immunity, based on virus-neutralizing antibodies and T cell responses. Although several vaccines have been developed just a year after SARS-CoV-2 emerged in late 2019, global deployment will take months or even years. Meanwhile, the virus continues to take a severe toll on human life and exact substantial economic costs. Innate immunity is fundamental to mammalian host defense capacity to combat infections. Innate immune responses, triggered by a family of pattern recognition receptors, induce interferons and other cytokines and activate both myeloid and lymphoid immune cells to provide protection against a wide range of pathogens. Epidemiological and biological evidence suggests that the live-attenuated vaccines (LAV) targeting tuberculosis, measles, and polio induce protective innate immunity by a newly described form of immunological memory termed "trained immunity." An LAV designed to induce adaptive immunity targeting a particular pathogen may also induce innate immunity that mitigates other infectious diseases, including COVID-19, as well as future pandemic threats. Deployment of existing LAVs early in pandemics could complement the development of specific vaccines, bridging the protection gap until specific vaccines arrive. The broad protection induced by LAVs would not be compromised by potential antigenic drift (immune escape) that can render viruses resistant to specific vaccines. LAVs might offer an essential tool to "bend the pandemic curve," averting the exhaustion of public health resources and preventing needless deaths and may also have therapeutic benefits if used for postexposure prophylaxis of disease.
Assuntos
COVID-19/prevenção & controle , Imunidade Inata , Pandemias/prevenção & controle , Vacinas/imunologia , Imunidade Adaptativa , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Imunidade Heteróloga , Memória Imunológica , SARS-CoV-2/imunologia , Vacinas Atenuadas/imunologiaRESUMO
The HIV-1 matrix protein p17 (p17) is a pleiotropic molecule impacting on different cell types. Its interaction with many cellular proteins underlines the importance of the viral protein as a major determinant of human specific adaptation. We previously showed the proangiogenic capability of p17. Here, by integrating functional analysis and receptor binding, we identify a functional epitope that displays molecular mimicry with human erythropoietin (EPO) and promotes angiogenesis through common beta chain receptor (ßCR) activation. The functional EPO-like epitope was found to be present in the matrix protein of HIV-1 ancestors SIV originated in chimpanzees (SIVcpz) and gorillas (SIVgor) but not in that of HIV-2 and its ancestor SIVsmm from sooty mangabeys. According to biological data, evolution of the EPO-like epitope showed a clear differentiation between HIV-1/SIVcpz-gor and HIV-2/SIVsmm branches, thus highlighting this epitope on p17 as a divergent signature discriminating HIV-1 and HIV-2 ancestors. P17 is known to enhance HIV-1 replication. Similarly to other ßCR ligands, p17 is capable of attracting and activating HIV-1 target cells and promoting a proinflammatory microenvironment. Thus, it is tempting to speculate that acquisition of an epitope on the matrix proteins of HIV-1 ancestors capable of triggering ßCR may have represented a critical step to enhance viral aggressiveness and early human-to-human SIVcpz/gor dissemination. The hypothesis that the p17/ßCR interaction and ßCR abnormal stimulation may also play a role in sustaining chronic activation and inflammation, thus marking the difference between HIV-1 and HIV-2 in term of pathogenicity, needs further investigation.
Assuntos
Eritropoetina/genética , Antígenos HIV/metabolismo , HIV-1/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Células Cultivadas , Epitopos/imunologia , Eritropoetina/metabolismo , Evolução Molecular , Antígenos HIV/genética , Soropositividade para HIV , HIV-1/genética , HIV-2 , Humanos , Mimetismo Molecular , Vírus da Imunodeficiência Símia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
BACKGROUND: Studies of molecular mechanisms underlying tumor cell signaling highlighted a critical role for kinases in carcinogenesis and cancer progression. To this regard, protein kinases regulates a number of critical cellular pathways by adding phosphate groups to specific substrates. For this reason, their involvement in the complex interactions between the human microbiota and cancer cells to determine therapy and tumor progression outcome is becoming increasingly relevant. Mycoplasmas are components of the normal human microbiota, and several species have also been associated to human diseases, including certain cancers. It is also important to note that Mycoplasmas and their proteins are a component of the common tumor microenvironment. In addition, several epidemiological, in vivo and in vitro studies indicate a close involvement of Mycoplasmas in cellular transformation and cancer progression. METHODS: In this study, we investigate the effect of exogenous Mycoplasma DnaK on kinases activity by treating in vitro four different eukaryotic cancer cell lines, namely lung and prostate cancer, colon adenocarcinoma, and neuroblastoma. Phosphorylation of kinases and specific substrates was measured at 20 and 60 min. RESULTS: Kinome analysis of our data indicates that Mycoplasma DnaK promotes the dysregulation of the activity of specific kinases and their substrates, with a known involvement in carcinogenesis and cancer progression. CONCLUSIONS: Given the similarity in structure and amino acid composition of this protein with other bacterial DnaKs we provide a novel mechanism whereby components of the human microbiota and present in the tumor microenvironment are able to deregulate phosphorylation events occurring during carcinogenesis and cancer progression.
Assuntos
Carcinogênese , Transformação Celular Neoplásica , Linhagem Celular , Humanos , Masculino , Fosforilação , Proteínas Quinases/metabolismo , Microambiente TumoralRESUMO
Dendritic cell (DC)-based cancer immunotherapy has achieved modest clinical benefits, but several technical hurdles in DC preparation, activation, and cancer/testis antigen (CTA) delivery limit its broad applications. Here, we report the development of immortalized and constitutively activated human primary blood dendritic cell lines (ihv-DCs). The ihv-DCs are a subset of CD11c+/CD205+ DCs that constitutively display costimulatory molecules. The ihv-DCs can be genetically modified to express human telomerase reverse transcriptase (hTERT) or the testis antigen MAGEA3 in generating CTA-specific cytotoxic T lymphocytes (CTLs). In an autologous setting, the HLA-A2+ ihv-DCs that present hTERT antigen prime autologous T cells to generate hTERT-specific CTLs, inducing cytolysis of hTERT-expressing target cells in an HLA-A2-restricted manner. Remarkably, ihv-DCs that carry two allogeneic HLA-DRB1 alleles are able to prime autologous T cells to proliferate robustly in generating HLA-A2-restricted, hTERT-specific CTLs. The ihv-DCs, which are engineered to express MAGEA3 and high levels of 4-1BBL and MICA, induce simultaneous production of both HLA-A2-restricted, MAGEA3-specific CTLs and NK cells from HLA-A2+ donor peripheral blood mononuclear cells. These cytotoxic lymphocytes suppress lung metastasis of A549/A2.1 lung cancer cells in NSG mice. Both CTLs and NK cells are found to infiltrate lung as well as lymphoid tissues, mimicking the in vivo trafficking patterns of cytotoxic lymphocytes. This approach should facilitate the development of cell-based immunotherapy for human lung cancer.
Assuntos
Citotoxicidade Imunológica/imunologia , Células Dendríticas/imunologia , Leucócitos Mononucleares/citologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/prevenção & controle , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Apoptose , Proliferação de Células , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/transplante , Humanos , Interferon gama/metabolismo , Neoplasias Pulmonares/secundário , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Engenharia Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We isolated a strain of human mycoplasma that promotes lymphomagenesis in SCID mice, pointing to a p53-dependent mechanism similar to lymphomagenesis in uninfected p53-/- SCID mice. Additionally, mycoplasma infection in vitro reduces p53 activity. Immunoprecipitation of p53 in mycoplasma-infected cells identified several mycoplasma proteins, including DnaK, a member of the Hsp70 chaperon family. We focused on DnaK because of its ability to interact with proteins. We demonstrate that mycoplasma DnaK interacts with and reduces the activities of human proteins involved in critical cellular pathways, including DNA-PK and PARP1, which are required for efficient DNA repair, and binds to USP10 (a key p53 regulator), impairing p53-dependent anticancer functions. This also reduced the efficacy of anticancer drugs that depend on p53 to exert their effect. mycoplasma was detected early in the infected mice, but only low copy numbers of mycoplasma DnaK DNA sequences were found in some primary and secondary tumors, pointing toward a hit-and-run/hide mechanism of transformation. Uninfected bystander cells took up exogenous DnaK, suggesting a possible paracrine function in promoting malignant transformation, over and above cells infected with the mycoplasma. Phylogenetic amino acid analysis shows that other bacteria associated with human cancers have similar DnaKs, consistent with a common mechanism of cellular transformation mediated through disruption of DNA-repair mechanisms, as well as p53 dysregulation, that also results in cancer-drug resistance. This suggests that the oncogenic properties of certain bacteria are DnaK-mediated.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , Carcinogênese/genética , Transformação Celular Neoplásica/genética , Chaperonas Moleculares/genética , Mycoplasma/genética , Adenosina Trifosfatases/classificação , Animais , Antineoplásicos/uso terapêutico , Proteínas de Bactérias/classificação , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Reparo do DNA , DNA Bacteriano/genética , Proteína Quinase Ativada por DNA/metabolismo , Modelos Animais de Doenças , Genes Bacterianos/genética , Células HCT116 , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Linfoma/genética , Linfoma/microbiologia , Linfoma/patologia , Camundongos , Camundongos SCID , Chaperonas Moleculares/classificação , Mycoplasma/patogenicidade , Infecções por Mycoplasma/microbiologia , Mycoplasma fermentans/genética , Mycoplasma fermentans/patogenicidade , Oncogenes , Filogenia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Análise de Sequência , Análise de Sequência de Proteína , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina Tiolesterase/metabolismoRESUMO
Several species of mycoplasmas, including Mycoplasma fermentans, are associated with certain human cancers. We previously isolated and characterized in our laboratory a strain of human mycoplasma M. fermentans subtype incognitus (MF-I1) able to induce lymphoma in a Severe Combined Immuno-Deficient (SCID) mouse model, and we demonstrated that its chaperone protein, DnaK, binds and reduces functions of human poly-ADP ribose polymerase-1 (PARP1) and ubiquitin carboxyl-terminal hydrolase protein-10 (USP10), which are required for efficient DNA repair and proper p53 activities, respectively. We also showed that other bacteria associated with human cancers (including Mycoplasmapneumoniae, Helicobacterpylori, Fusobacteriumnucleatum, Chlamydiathrachomatis, and Chlamydia pneumoniae) have closely related DnaK proteins, indicating a potential common mechanism of cellular transformation. Here, we quantify dnaK mRNA copy number by RT-qPCR analysis in different cellular compartments following intracellular MF-I1 infection of HCT116 human colon carcinoma cells. DnaK protein expression in infected cells was also detected and quantified by Western blot. The amount of viable intracellular mycoplasma reached a steady state after an initial phase of growth and was mostly localized in the cytoplasm of the invaded cells, while we detected a logarithmically increased number of viable extracellular bacteria. Our data indicate that, after invasion, MF-I1 is able to establish a chronic intracellular infection. Extracellular replication was more efficient while MF-I1 cultured in cell-free axenic medium showed a markedly reduced growth rate. We also identified modifications of important regulatory regions and heterogeneous lengths of dnaK mRNA transcripts isolated from intracellular and extracellular MF-I1. Both characteristics were less evident in dnaK mRNA transcripts isolated from MF-I1 grown in cell-free axenic media. Taken together, our data indicate that MF-I1, after establishing a chronic infection in eukaryotic cells, accumulates different forms of dnaK with efficient RNA turnover.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Mycoplasma fermentans/genética , Células Cultivadas , Células HCT116 , Humanos , Mutação , Infecções por Mycoplasma/microbiologiaRESUMO
BACKGROUND: With the aim of providing a dynamic evaluation of the effects of basic environmental parameters on COVID-19-related death rate, we assessed the correlation between average monthly high temperatures and population density, with death/rate (monthly number of deaths/1 M people) for the months of March (start of the analysis and beginning of local epidemic in most of the Western World, except in Italy where it started in February) and April 2020 (continuation of the epidemic). Different geographical areas of the Northern Hemisphere in the United States and in Europe were selected in order to provide a wide range among the different parameters. The death rates were gathered from an available dataset. As a further control, we also included latitude, as a proxy for temperature. METHODS: Utilizing a publicly available dataset, we retrieved data for the months of March and April 2020 for 25 areas in Europe and in the US. We computed the monthly number of deaths/1 M people of confirmed COVID-19 cases and calculated the average monthly high temperatures and population density for all these areas. We determined the correlation between number of deaths/1 M people and the average monthly high temperatures, the latitude and the population density. RESULTS: We divided our analysis in two parts: analysis of the correlation among the different variables in the month of March and subsequent analysis in the month of April. The differences were then evaluated. In the month of March there was no statistical correlation between average monthly high temperatures of the considered geographical areas and number of deaths/1 M people. However, a statistically significant inverse correlation became significant in the month of April between average monthly high temperatures (p = 0.0043) and latitude (p = 0.0253) with number of deaths/1 M people. We also observed a statistically significant correlation between population density and number of deaths/1 M people both in the month of March (p = 0.0297) and in the month of April (p = 0.0116), when three areas extremely populated (NYC, Los Angeles and Washington DC) were included in the calculation. Once these three areas were removed, the correlation was not statistically significant (p = 0.1695 in the month of March, and p = 0.7076 in the month of April). CONCLUSIONS: The number of COVID-19-related deaths/1 M people was essentially the same during the month of March for all the geographical areas considered, indicating essentially that the infection was circulating quite uniformly except for Lombardy, Italy, where it started earlier. Lockdown measures were implemented between the end of March and beginning of April, except for Italy which started March 9th. We observed a strong, statistically significant inverse correlation between average monthly high temperatures with the number of deaths/1 M people. We confirmed the data by analyzing the correlation with the latitude, which can be considered a proxy for high temperature. Previous studies indicated a negative effect of high climate temperatures on Sars-COV-2 spreading. Our data indicate that social distancing measure are more successful in the presence of higher average monthly temperatures in reducing COVID-19-related death rate, and a high level of population density seems to negatively impact the effect of lockdown measures.
Assuntos
Infecções por Coronavirus/mortalidade , Meio Ambiente , Mortalidade , Pneumonia Viral/mortalidade , Temperatura , Betacoronavirus/fisiologia , COVID-19 , Infecções por Coronavirus/epidemiologia , District of Columbia/epidemiologia , Monitoramento Ambiental/métodos , Europa (Continente)/epidemiologia , Geografia , Humanos , Itália/epidemiologia , Los Angeles/epidemiologia , Cidade de Nova Iorque/epidemiologia , Pandemias , Pneumonia Viral/epidemiologia , Densidade Demográfica , SARS-CoV-2 , Comportamento SocialRESUMO
BACKGROUND: The new Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was first detected in Wuhan (China) in December of 2019 is responsible for the current global pandemic. Phylogenetic analysis revealed that it is similar to other betacoronaviruses, such as SARS-CoV and Middle-Eastern Respiratory Syndrome, MERS-CoV. Its genome is â¼ 30 kb in length and contains two large overlapping polyproteins, ORF1a and ORF1ab that encode for several structural and non-structural proteins. The non-structural protein 1 (nsp1) is arguably the most important pathogenic determinant, and previous studies on SARS-CoV indicate that it is both involved in viral replication and hampering the innate immune system response. Detailed experiments of site-specific mutagenesis and in vitro reconstitution studies determined that the mechanisms of action are mediated by (a) the presence of specific amino acid residues of nsp1 and (b) the interaction between the protein and the host's small ribosomal unit. In fact, substitution of certain amino acids resulted in reduction of its negative effects. METHODS: A total of 17,928 genome sequences were obtained from the GISAID database (December 2019 to July 2020) from patients infected by SARS-CoV-2 from different areas around the world. Genomes alignment was performed using MAFFT (REFF) and the nsp1 genomic regions were identified using BioEdit and verified using BLAST. Nsp1 protein of SARS-CoV-2 with and without deletion have been subsequently modelled using I-TASSER. RESULTS: We identified SARS-CoV-2 genome sequences, from several Countries, carrying a previously unknown deletion of 9 nucleotides in position 686-694, corresponding to the AA position 241-243 (KSF). This deletion was found in different geographical areas. Structural prediction modelling suggests an effect on the C-terminal tail structure. CONCLUSIONS: Modelling analysis of a newly identified deletion of 3 amino acids (KSF) of SARS-CoV-2 nsp1 suggests that this deletion could affect the structure of the C-terminal region of the protein, important for regulation of viral replication and negative effect on host's gene expression. In addition, substitution of the two amino acids (KS) from nsp1 of SARS-CoV was previously reported to revert loss of interferon-alpha expression. The deletion that we describe indicates that SARS-CoV-2 is undergoing profound genomic changes. It is important to: (i) confirm the spreading of this particular viral strain, and potentially of strains with other deletions in the nsp1 protein, both in the population of asymptomatic and pauci-symptomatic subjects, and (ii) correlate these changes in nsp1 with potential decreased viral pathogenicity.
Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Deleção de Sequência , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Betacoronavirus/patogenicidade , COVID-19 , Doenças Transmissíveis Emergentes/virologia , Infecções por Coronavirus/epidemiologia , Frequência do Gene , Genoma Viral , Geografia , Humanos , Lisina/genética , Modelos Moleculares , Pandemias/estatística & dados numéricos , Fenilalanina/genética , Pneumonia Viral/epidemiologia , Domínios Proteicos/genética , SARS-CoV-2 , Serina/genética , Proteínas não Estruturais Virais/química , Virulência/genética , Replicação Viral/genéticaRESUMO
BACKGROUND: SARS-CoV-2 is a RNA coronavirus responsible for the pandemic of the Severe Acute Respiratory Syndrome (COVID-19). RNA viruses are characterized by a high mutation rate, up to a million times higher than that of their hosts. Virus mutagenic capability depends upon several factors, including the fidelity of viral enzymes that replicate nucleic acids, as SARS-CoV-2 RNA dependent RNA polymerase (RdRp). Mutation rate drives viral evolution and genome variability, thereby enabling viruses to escape host immunity and to develop drug resistance. METHODS: We analyzed 220 genomic sequences from the GISAID database derived from patients infected by SARS-CoV-2 worldwide from December 2019 to mid-March 2020. SARS-CoV-2 reference genome was obtained from the GenBank database. Genomes alignment was performed using Clustal Omega. Mann-Whitney and Fisher-Exact tests were used to assess statistical significance. RESULTS: We characterized 8 novel recurrent mutations of SARS-CoV-2, located at positions 1397, 2891, 14408, 17746, 17857, 18060, 23403 and 28881. Mutations in 2891, 3036, 14408, 23403 and 28881 positions are predominantly observed in Europe, whereas those located at positions 17746, 17857 and 18060 are exclusively present in North America. We noticed for the first time a silent mutation in RdRp gene in England (UK) on February 9th, 2020 while a different mutation in RdRp changing its amino acid composition emerged on February 20th, 2020 in Italy (Lombardy). Viruses with RdRp mutation have a median of 3 point mutations [range: 2-5], otherwise they have a median of 1 mutation [range: 0-3] (p value < 0.001). CONCLUSIONS: These findings suggest that the virus is evolving and European, North American and Asian strains might coexist, each of them characterized by a different mutation pattern. The contribution of the mutated RdRp to this phenomenon needs to be investigated. To date, several drugs targeting RdRp enzymes are being employed for SARS-CoV-2 infection treatment. Some of them have a predicted binding moiety in a SARS-CoV-2 RdRp hydrophobic cleft, which is adjacent to the 14408 mutation we identified. Consequently, it is important to study and characterize SARS-CoV-2 RdRp mutation in order to assess possible drug-resistance viral phenotypes. It is also important to recognize whether the presence of some mutations might correlate with different SARS-CoV-2 mortality rates.
Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Evolução Molecular , Genoma Viral/genética , Mutação , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , RNA Polimerase Dependente de RNA/genética , Adulto , Ásia/epidemiologia , COVID-19 , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/mortalidade , Farmacorresistência Viral/genética , Europa (Continente)/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , América do Norte/epidemiologia , Oceania/epidemiologia , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/mortalidade , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2RESUMO
The Zika virus (ZIKV) causes microcephaly and the Guillain-Barré syndrome. Little is known about how ZIKV causes these conditions or which ZIKV viral protein(s) is responsible for the associated ZIKV-induced cytopathic effects, including cell hypertrophy, growth restriction, cell-cycle dysregulation, and cell death. We used fission yeast for the rapid, global functional analysis of the ZIKV genome. All 14 proteins or small peptides were produced under an inducible promoter, and we measured the intracellular localization and the specific effects on ZIKV-associated cytopathic activities of each protein. The subcellular localization of each ZIKV protein was in overall agreement with its predicted protein structure. Five structural and two nonstructural ZIKV proteins showed various levels of cytopathic effects. The expression of these ZIKV proteins restricted cell proliferation, induced hypertrophy, or triggered cellular oxidative stress leading to cell death. The expression of premembrane protein (prM) resulted in cell-cycle G1 accumulation, whereas membrane-anchored capsid (anaC), membrane protein (M), envelope protein (E), and nonstructural protein 4A (NS4A) caused cell-cycle G2/M accumulation. A mechanistic study revealed that NS4A-induced cellular hypertrophy and growth restriction were mediated specifically through the target of rapamycin (TOR) cellular stress pathway involving Tor1 and type 2A phosphatase activator Tip41. These findings should provide a reference for future research on the prevention and treatment of ZIKV diseases.
Assuntos
Genoma Viral/genética , Schizosaccharomyces/virologia , Proteínas não Estruturais Virais/genética , Zika virus/genética , Ciclo Celular/genética , Morte Celular/genética , Proliferação de Células/genética , Estudo de Associação Genômica Ampla/métodos , Hipertrofia/genética , Proteínas de Membrana/genética , Estresse Oxidativo/genética , Regiões Promotoras Genéticas/genética , Infecção por Zika virus/virologiaRESUMO
A complete picture of HIV antigenicity during early replication is needed to elucidate the full range of options for controlling infection. Such information is frequently gained through analyses of isolated viral envelope antigens, host CD4 receptors, and cognate antibodies. However, direct examination of viral particles and virus-cell interactions is now possible via advanced microscopy techniques and reagents. Using such methods, we recently determined that CD4-induced (CD4i) transition state epitopes in the HIV surface antigen, gp120, while not exposed on free particles, rapidly become immunoreactive upon virus-cell binding. Here, we use 3D direct stochastic optical reconstruction microscopy (dSTORM) to show that certain CD4i epitopes specific to transition state structures are exposed across the surface of cell-bound virions, thus explaining their immunoreactivity. Moreover, such structures and their marker epitopes are dispersed to regions of virions distal to CD4 contact. We further show that the appearance and positioning of distal CD4i exposures is partially dependent on Gag maturation and intact matrix-gp41 interactions within the virion. Collectively, these observations provide a unique perspective of HIV during early replication. These features may define unique insights for understanding how humoral responses target virions and for developing related antiviral countermeasures.
Assuntos
Epitopos/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Vírion/imunologia , Ligação Viral , Antígenos CD4/metabolismo , Contagem de Linfócito CD4 , Linhagem Celular , Epitopos/química , Anticorpos Anti-HIV/imunologia , Antígenos HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/química , Humanos , Vírion/química , Vírion/metabolismoRESUMO
Studies of the human microbiome have elucidated an array of complex interactions between prokaryotes and their hosts. However, precise bacterial pathogen-cancer relationships remain largely elusive, although several bacteria, particularly those establishing persistent intra-cellular infections, like mycoplasmas, can alter host cell cycles, affect apoptotic pathways, and stimulate the production of inflammatory substances linked to DNA damage, thus potentially promoting abnormal cell growth and transformation. Consistent with this idea, in vivo experiments in several chemically induced or genetically deficient mouse models showed that germ-free conditions reduce colonic tumor formation. We demonstrate that mycoplasma DnaK, a chaperone protein belonging to the Heath shock protein (Hsp)-70 family, binds Poly-(ADP-ribose) Polymerase (PARP)-1, a protein that plays a critical role in the pathways involved in recognition of DNA damage and repair, and reduces its catalytic activity. It also binds USP10, a key p53 regulator, reducing p53 stability and anti-cancer functions. Finally, we showed that bystander, uninfected cells take up exogenous DnaK-suggesting a possible paracrine function in promoting cellular transformation, over and above direct mycoplasma infection. We propose that mycoplasmas, and perhaps certain other bacteria with closely related DnaK, may have oncogenic activity, mediated through the inhibition of DNA repair and p53 functions, and may be involved in the initiation of some cancers but not necessarily involved nor necessarily even be present in later stages.
Assuntos
Inflamação/genética , Chaperonas Moleculares/genética , Infecções por Mycoplasma/genética , Mycoplasma/genética , Neoplasias/genética , Apoptose/genética , Transformação Celular Neoplásica/genética , Dano ao DNA/genética , Reparo do DNA/genética , Humanos , Inflamação/microbiologia , Inflamação/patologia , Mycoplasma/patogenicidade , Infecções por Mycoplasma/microbiologia , Neoplasias/patologia , Poli(ADP-Ribose) Polimerase-1/genética , Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/genéticaRESUMO
Extracellular vesicles (EVs) released by various cells are small phospholipid membrane-enclosed entities that can carry miRNA. They are now central to research in many fields of biology because they seem to constitute a new system of cell-cell communication. Physical and chemical characteristics of many EVs, as well as their biogenesis pathways, resemble those of retroviruses. Moreover, EVs generated by virus-infected cells can incorporate viral proteins and fragments of viral RNA, being thus indistinguishable from defective (noninfectious) retroviruses. EVs, depending on the proteins and genetic material incorporated in them, play a significant role in viral infection, both facilitating and suppressing it. Deciphering the mechanisms of EV-cell interactions may facilitate the design of EVs that inhibit viral infection and can be used as vehicles for targeted drug delivery.
Assuntos
Vesículas Extracelulares/fisiologia , Viroses/etiologia , Desaminase APOBEC-3G/fisiologia , Animais , Exossomos/fisiologia , Humanos , MicroRNAs/fisiologia , Vírion/fisiologia , Viroses/terapiaRESUMO
HIV-1 infection is associated with increased risk for B-cell lymphomas. How HIV infection promotes the development of lymphoma is unclear, but it may involve chronic B-cell activation, inflammation, and/or impaired immunity, possibly leading to a loss of control of oncogenic viruses and reduced tumor immunosurveillance. We hypothesized that HIV structural proteins may contribute to lymphomagenesis directly, because they can persist long term in lymph nodes in the absence of viral replication. The HIV-1 transgenic mouse Tg26 carries a noninfectious HIV-1 provirus lacking part of the gag-pol region, thus constituting a model for studying the effects of viral products in pathogenesis. Approximately 15% of Tg26 mice spontaneously develop leukemia/lymphoma. We investigated which viral proteins are associated with the development of leukemia/lymphoma in the Tg26 mouse model, and performed microarray analysis on RNA from spleen and lymph nodes to identify potential mechanisms of lymphomagenesis. Of the viral proteins examined, only expression of HIV-1 matrix protein p17 was associated with leukemia/lymphoma development and was highly expressed in bone marrow before disease. The tumor cells resembled pro-B cells, and were CD19+IgM-IgD-CD93+CD43+CD21-CD23-VpreB+CXCR4+ Consistent with the pro-B-cell stage of B-cell development, microarray analysis revealed enrichment of transcripts, including Rag1, Rag2, CD93, Vpreb1, Vpreb3, and Igll1 We confirmed RAG1 expression in Tg26 tumors, and hypothesized that HIV-1 matrix protein p17 may directly induce RAG1 in B cells. Stimulation of human activated B cells with p17 enhanced RAG1 expression in three of seven donors, suggesting that intracellular signaling by p17 may lead to genomic instability and transformation.
Assuntos
Antígenos HIV/genética , Linfoma de Células B/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Animais , Linfócitos B/metabolismo , Medula Óssea/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Antígenos HIV/metabolismo , HIV-1/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Linfonodos/metabolismo , Linfoma de Células B/metabolismo , Camundongos Transgênicos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
HIV necessitates host factors for successful completion of its life cycle. Mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that forms two complexes, mTORC1 and mTORC2. Rapamycin is an allosteric inhibitor of mTOR that selectively inhibits mTORC1. Rapamycin interferes with viral entry of CCR5 (R5)-tropic HIV and with basal transcription of the HIV LTR, potently inhibiting replication of R5 HIV but not CXCR4 (X4)-tropic HIV in primary cells. The recently developed ATP-competitive mTOR kinase inhibitors (TOR-KIs) inhibit both mTORC1 and mTORC2. Using INK128 as a prototype TOR-KI, we demonstrate potent inhibition of both R5 and X4 HIV in primary lymphocytes (EC50 < 50 nM), in the absence of toxicity. INK128 inhibited R5 HIV entry by reducing CCR5 levels. INK128 also inhibited both basal and induced transcription of HIV genes, consistent with inhibition of mTORC2, whose activity is critical for phosphorylation of PKC isoforms and, in turn, induction of NF-κB. INK128 enhanced the antiviral potency of the CCR5 antagonist maraviroc, and had favorable antiviral interactions with HIV inhibitors of reverse transcriptase, integrase and protease. In humanized mice, INK128 decreased plasma HIV RNA by >2 log10 units and partially restored CD4/CD8 cell ratios. Targeting of cellular mTOR with INK128 (and perhaps others TOR-KIs) provides a potential strategy to inhibit HIV, especially in patients with drug resistant HIV strains.