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1.
Int J Mol Sci ; 22(20)2021 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-34681641

RESUMO

G-quadruplexes (G4s) are noncanonical nucleic acid structures involved in the regulation of key cellular processes, such as transcription and replication. Since their discovery, G4s have been mainly investigated for their role in cancer and as targets in anticancer therapy. More recently, exploration of the presence and role of G4s in viral genomes has led to the discovery of G4-regulated key viral pathways. In this context, employment of selective G4 ligands has helped to understand the complexity of G4-mediated mechanisms in the viral life cycle, and highlighted the possibility to target viral G4s as an emerging antiviral approach. Research in this field is growing at a fast pace, providing increasing evidence of the antiviral activity of old and new G4 ligands. This review aims to provide a punctual update on the literature on G4 ligands exploited in virology. Different classes of G4 binders are described, with emphasis on possible antiviral applications in emerging diseases, such as the current COVID-19 pandemic. Strengths and weaknesses of G4 targeting in viruses are discussed.


Assuntos
Antivirais/química , Quadruplex G , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/virologia , DNA Viral/química , DNA Viral/metabolismo , Humanos , Ligantes , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , RNA Viral/química , RNA Viral/metabolismo , SARS-CoV-2/isolamento & purificação , Viroses/tratamento farmacológico , Viroses/patologia
2.
Nat Struct Mol Biol ; 28(10): 779-788, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34556871

RESUMO

Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover the complete transcription initiation phase of the poxvirus vaccinia. In the pre-initiation complex, the heterodimeric early transcription factor VETFs/l adopts an arc-like shape spanning the polymerase cleft and anchoring upstream and downstream promoter elements. VETFI emerges as a TBP-like protein that inserts asymmetrically into the DNA major groove, triggers DNA melting, ensures promoter recognition and enforces transcription directionality. The helicase VETFs fosters promoter melting and the phospho-peptide domain (PPD) of vRNAP subunit Rpo30 enables transcription initiation. An unprecedented upstream promoter scrunching mechanism assisted by the helicase NPH-I probably fosters promoter escape and transition into elongation. Our structures shed light on unique mechanisms of poxviral gene expression and aid the understanding of thus far unexplained universal principles in transcription.


Assuntos
Iniciação da Transcrição Genética , Vírus Vaccinia/química , Vírus Vaccinia/genética , Proteínas Virais/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , DNA Helicases/química , DNA Helicases/genética , DNA Viral/química , DNA Viral/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Viral da Expressão Gênica , Células HeLa , Humanos , Modelos Moleculares , Regiões Promotoras Genéticas , Domínios Proteicos , Subunidades Proteicas , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteínas Virais/genética
3.
Cells ; 10(7)2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34359841

RESUMO

Seed transmission is an important factor in the epidemiology of plant pathogens. Geminiviruses are serious pests spread in tropical and subtropical regions. They are transmitted by hemipteran insects, but a few cases of transmission through seeds were recently reported. Here, we investigated the tomato seed transmissibility of the begomovirus tomato yellow leaf curl Sardinia virus (TYLCSV), one of the agents inducing the tomato yellow leaf curl disease, heavily affecting tomato crops in the Mediterranean area. None of the 180 seedlings originating from TYLCSV-infected plants showed any phenotypic alteration typical of virus infection. Moreover, whole viral genomic molecules could not be detected in their cotyledons and true leaves, neither by membrane hybridization nor by rolling-circle amplification followed by PCR, indicating that TYLCSV is not a seed-transmissible pathogen for tomato. Examining the localization of TYLCSV DNA in progenitor plants, we detected the virus genome by PCR in all vegetative and reproductive tissues, but viral genomic and replicative forms were found only in leaves, flowers and fruit flesh, not in seeds and embryos. Closer investigations allowed us to discover for the first time that these embryos were superficially contaminated by TYLCSV DNA but whole genomic molecules were not detectable. Therefore, the inability of TYLCSV genomic molecules to colonize tomato embryos during infection justifies the lack of seed transmissibility observed in this host.


Assuntos
Begomovirus/genética , DNA Viral/genética , Flores/virologia , Frutas/virologia , Genoma Viral , Lycopersicon esculentum/virologia , Folhas de Planta/virologia , Begomovirus/metabolismo , Begomovirus/patogenicidade , DNA Viral/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Frutas/genética , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Lycopersicon esculentum/genética , Lycopersicon esculentum/crescimento & desenvolvimento , Lycopersicon esculentum/metabolismo , Doenças das Plantas/virologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Plântula/virologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo
4.
Nat Commun ; 12(1): 4710, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354070

RESUMO

Cyanophage S-2L is known to profoundly alter the biophysical properties of its DNA by replacing all adenines (A) with 2-aminoadenines (Z), which still pair with thymines but with a triple hydrogen bond. It was recently demonstrated that a homologue of adenylosuccinate synthetase (PurZ) and a dATP triphosphohydrolase (DatZ) are two important pieces of the metabolism of 2-aminoadenine, participating in the synthesis of ZTGC-DNA. Here, we determine that S-2L PurZ can use either dATP or ATP as a source of energy, thereby also depleting the pool of nucleotides in dATP. Furthermore, we identify a conserved gene (mazZ) located between purZ and datZ genes in S-2L and related phage genomes. We show that it encodes a (d)GTP-specific diphosphohydrolase, thereby providing the substrate of PurZ in the 2-aminoadenine synthesis pathway. High-resolution crystal structures of S-2L PurZ and MazZ with their respective substrates provide a rationale for their specificities. The Z-cluster made of these three genes - datZ, mazZ and purZ - was expressed in E. coli, resulting in a successful incorporation of 2-aminoadenine in the bacterial chromosomal and plasmidic DNA. This work opens the possibility to study synthetic organisms containing ZTGC-DNA.


Assuntos
DNA Bacteriano/genética , Genes Virais , Siphoviridae/genética , 2-Aminopurina/análogos & derivados , 2-Aminopurina/metabolismo , Adenilossuccinato Sintase/química , Adenilossuccinato Sintase/genética , Adenilossuccinato Sintase/metabolismo , Bacteriófagos , Pareamento de Bases , Cristalografia por Raios X , DNA Bacteriano/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Desoxiadenosinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Viral , Redes e Vias Metabólicas , Modelos Moleculares , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Podoviridae/classificação , Podoviridae/genética , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Siphoviridae/classificação , Eletricidade Estática , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
5.
PLoS One ; 16(8): e0256357, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34428230

RESUMO

Torquetenovirus (TTV) is present in biological fluids from healthy individuals and measurement of its titer is used to assess immune status in individuals with chronic infections and after transplants. We assessed if the titer of TTV in saliva varied with the presence of SARS-CoV-2 in the nasopharynx and could be a marker of COVID-19 status. Saliva from 91 individuals positive for SARS-CoV-2 in nasal-oropharyngeal samples, and from 126 individuals who were SARS-CoV-2-negative, all with mild respiratory symptoms, were analyzed. Both groups were similar in age, gender, symptom duration and time after symptom initiation when saliva was collected. Titers of TTV and SARS-CoV-2 were assessed by gene amplification. Loss of smell (p = 0.0001) and fever (p = 0.0186) were more prevalent in SARS-CoV-2-positive individuals, while sore throat (p = 0.0001), fatigue (p = 0.0037) and diarrhea (p = 0.0475) were more frequent in the SARS-CoV-2 negative group. The saliva TTV and nasal-oropharyngeal SARS-CoV-2 titers were correlated (p = 0.0085). The TTV level decreased as symptoms resolved in the SARS-CoV-2 infected group (p = 0.0285) but remained unchanged in the SARS-CoV-2 negative controls. In SARS-CoV-2 positive subjects who provided 2-4 saliva samples and in which TTV was initially present, the TTV titer always decreased over time as symptoms resolved. We propose that sequential TTV measurement in saliva is potentially useful to assess the likelihood of symptom resolution in SARS-CoV-2-positive individuals and to predict prognosis.


Assuntos
Biomarcadores/análise , COVID-19/diagnóstico , Saliva/virologia , Torque teno virus/isolamento & purificação , Adulto , COVID-19/virologia , DNA Viral/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Reação em Cadeia da Polimerase , Prognóstico , SARS-CoV-2/isolamento & purificação , Torque teno virus/genética
6.
Biochemistry ; 60(37): 2795-2809, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34464102

RESUMO

The geminivirus replication protein, Rep, has long been recognized as a high-value target for control of geminivirus infections as this protein is highly conserved and essential for viral replication and proliferation. In addition, inhibition of viral replication has been pursued through various antiviral strategies with varying degrees of success, including inhibitory peptides that target Rep. While much effort has centered around sequence characterization of the Rep protein and inhibitory peptides, detailed structural analysis has been missing. This study computationally investigated the presence of common structural features within these inhibitory peptides and if these features could inform if a particular peptide will bind Rep and/or interfere with viral replication. Molecular dynamics simulations of the inhibitory peptide library showed that simply possessing stable structural features does not inform interference of viral replication regardless of the binding of Rep. Additionally, nearly all known Rep inhibitory peptides sample a conserved ß-sheet structural motif, possibly informing structure-function relationships in binding Rep. In particular, two peptides (A22 and A64) characterized by this structural motif were computationally docked against a wide variety of geminivirus Rep proteins to determine a mechanism of action. Computational docking revealed these peptides utilize a common Rep protein sequence motif for binding, HHN-x1/2-Q. The results identified residues in both Rep and the inhibitory peptides that play a significant role in the interaction, establishing the foundation for a rational structure-based design approach for the construction of both broadly reactive and geminivirus species-specific inhibitors.


Assuntos
Geminiviridae/enzimologia , Geminiviridae/metabolismo , Replicação Viral/fisiologia , Motivos de Aminoácidos/genética , Sequência de Aminoácidos/genética , DNA Helicases/metabolismo , DNA Viral/metabolismo , Geminiviridae/genética , Peptídeos/metabolismo , Ligação Proteica/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/ultraestrutura , Replicação Viral/genética
7.
Biomed Res Int ; 2021: 9957440, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34212044

RESUMO

Objective: To investigate the expression of microRNA-122 (miR-122) in the progression of chronic hepatitis B virus- (HBV-) infected liver diseases, thus determining the role of serum miR-122 as a marker of HBV-caused liver injury. Methods: Sera were collected from patients with different stages of HBV infection (n = 63) and healthy volunteers (n = 11). And the serum miR-122 levels were detected using RT-qPCR. Moreover, an analysis was applied for identifying the specific correlation of the miR-122 level with HBV DNA, HBeAg, and ALT levels. After liver biopsy, Ishak scoring was utilized for evaluation of the fibrosis stage and the histological activity index (HAI). Results: We confirmed, in the serum, increased miR-122 expression in HBV-infected patients and its highest expression in chronic HBV carriers, based on such comparison between the healthy controls and patients. The correlation analysis results were taken as confirmation of the positive relationship of miR-122 with HBV DNA (r = 0.354, P = 0.005) and ALT (r = 0.331, P = 0.009). But no correlation of this molecule with HBeAg levels was found (P = 0.187). In comparison with the HBeAg-negative patients, serum miR-122 expression showed an increase in the HBeAg-positive patients (P = 0.001). miR-122 expression, in addition, was of a significant correlation with HAI, but not with the liver fibrosis score. Conclusion: The peak of the serum miR-122 expression normally occurs in the early stage of the progression from the HBV carrier phase to chronic hepatitis to cirrhosis. This molecule can be considered as a marker for evaluation of HBV-caused liver injury.


Assuntos
Regulação Viral da Expressão Gênica , Vírus da Hepatite B , Hepatite B Crônica/sangue , Cirrose Hepática/sangue , MicroRNAs/sangue , Adulto , Biomarcadores/sangue , Biópsia , DNA Viral/metabolismo , Progressão da Doença , Marcadores Genéticos , Antígenos E da Hepatite B/biossíntese , Hepatite B Crônica/virologia , Humanos , Fígado/patologia , Cirrose Hepática/virologia , Hepatopatias/genética , Hepatopatias/virologia , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Soro , Carga Viral , Adulto Jovem
8.
PLoS One ; 16(7): e0250051, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34197460

RESUMO

Wolbachia are endosymbionts of numerous arthropod and some nematode species, are important for their development and if present can cause distinct phenotypes of their hosts. Prophage DNA has been frequently detected in Wolbachia, but particles of Wolbachia bacteriophages (phage WO) have been only occasionally isolated. Here, we report the characterization and isolation of a phage WO of the southern ground cricket, Allonemobius socius, and provided the first whole-genome sequence of phage WO from this arthropod family outside of Asia. We screened A. socius abdomen DNA extracts from a cricket population in eastern Missouri by quantitative PCR for Wolbachia surface protein and phage WO capsid protein and found a prevalence of 55% and 50%, respectively, with many crickets positive for both. Immunohistochemistry using antibodies against Wolbachia surface protein showed many Wolbachia clusters in the reproductive system of female crickets. Whole-genome sequencing using Oxford Nanopore MinION and Illumina technology allowed for the assembly of a high-quality, 55 kb phage genome containing 63 open reading frames (ORF) encoding for phage WO structural proteins and host lysis and transcriptional manipulation. Taxonomically important regions of the assembled phage genome were validated by Sanger sequencing of PCR amplicons. Analysis of the nucleotides sequences of the ORFs encoding the large terminase subunit (ORF2) and minor capsid (ORF7) frequently used for phage WO phylogenetics showed highest homology to phage WOAu of Drosophila simulans (94.46% identity) and WOCin2USA1 of the cherry fruit fly, Rhagoletis cingulata (99.33% identity), respectively. Transmission electron microscopy examination of cricket ovaries showed a high density of phage particles within Wolbachia cells. Isolation of phage WO revealed particles characterized by 40-62 nm diameter heads and up to 190 nm long tails. This study provides the first detailed description and genomic characterization of phage WO from North America that is easily accessible in a widely distributed cricket species.


Assuntos
Bacteriófagos/genética , Genoma Viral , Gryllidae/microbiologia , Animais , Bacteriófagos/classificação , Bacteriófagos/isolamento & purificação , Proteínas do Capsídeo/genética , DNA Bacteriano/química , DNA Bacteriano/metabolismo , DNA Viral/química , DNA Viral/metabolismo , Feminino , Gryllidae/virologia , Proteínas de Membrana/genética , Missouri , Fases de Leitura Aberta/genética , Filogenia , Sequenciamento Completo do Genoma , Wolbachia/genética , Wolbachia/isolamento & purificação , Wolbachia/virologia
9.
Am J Pathol ; 191(10): 1787-1804, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34197777

RESUMO

Although pathologies associated with acute virus infections have been extensively studied, the effects of long-term latent virus infections are less well understood. Human cytomegalovirus, which infects 50% to 80% of humans, is usually acquired during early life and persists in a latent state for the lifetime. The purpose of this study was to determine whether systemic murine cytomegalovirus (MCMV) infection acquired early in life disseminates to and becomes latent in the eye and if ocular MCMV can trigger in situ inflammation and occurrence of ocular pathology. This study found that neonatal infection of BALB/c mice with MCMV resulted in dissemination of virus to the eye, where it localized principally to choroidal endothelia and pericytes and less frequently to the retinal pigment epithelium (RPE) cells. MCMV underwent ocular latency, which was associated with expression of multiple virus genes and from which MCMV could be reactivated by immunosuppression. Latent ocular infection was associated with significant up-regulation of several inflammatory/angiogenic factors. Retinal and choroidal pathologies developed in a progressive manner, with deposits appearing at both basal and apical aspects of the RPE, RPE/choroidal atrophy, photoreceptor degeneration, and neovascularization. The pathologies induced by long-term ocular MCMV latency share features of previously described human ocular diseases, such as age-related macular degeneration.


Assuntos
Envelhecimento/patologia , Corioide/patologia , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Muromegalovirus/fisiologia , Retina/patologia , Indutores da Angiogênese/metabolismo , Animais , Animais Recém-Nascidos , Antígenos Virais/metabolismo , Corioide/diagnóstico por imagem , Corioide/ultraestrutura , Corioide/virologia , DNA Viral/metabolismo , Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/diagnóstico por imagem , Interações Hospedeiro-Patógeno , Imunossupressão , Inflamação/patologia , Camundongos Endogâmicos BALB C , Muromegalovirus/genética , Fagócitos/patologia , Retina/diagnóstico por imagem , Retina/ultraestrutura , Retina/virologia , Epitélio Pigmentado da Retina/diagnóstico por imagem , Epitélio Pigmentado da Retina/patologia , Tomografia de Coerência Óptica , Ativação Viral
10.
J Virol ; 95(19): e0100921, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34287039

RESUMO

Porcine circovirus type 2 (PCV2) causes several disease syndromes in grower pigs. PCV2 infection triggers endoplasmic reticulum (ER) stress, autophagy, and oxidative stress, all of which support PCV2 replication. We have recently reported that nuclear HMGB1 is an anti-PCV2 factor by binding to viral genomic DNA. However, how PCV2 manipulates host cell responses to favor its replication has not been explored. Here, we demonstrate that PCV2 infection increased expression of ERO1α, generation of reactive oxygen species (ROS), and nucleocytoplasmic migration of HMGB1 via protein kinase R-like endoplasmic reticulum kinase (PERK) activation in PK-15 cells. Inhibition of PERK or ERO1α repressed ROS production in PCV2-infected cells and increased HMGB1 retention within nuclei. These findings indicate that PCV2-induced activation of the PERK-ERO1α axis would lead to enhanced generation of ROS sufficient to decrease HMGB1 retention in the nuclei, thus derepressing viral DNA from HMGB1 sequestration. The viral Rep and Cap proteins were able to induce PERK-ERO1α-mediated ROS accumulation. Cysteine residues 107 and 305 of Rep or 108 of Cap played important roles in PCV2-induced PERK activation and distribution of HMGB1. Of the mutant viruses, only the mutant PCV2 with substitution of all three cysteine residues failed to activate PERK with reduced ROS generation and decreased nucleocytoplasmic migration of HMGB1. Collectively, this study offers novel insight into the mechanism of enhanced viral replication in which PCV2 manipulates ER to perturb its redox homeostasis via the PERK-ERO1α axis, and the ER-sourced ROS from oxidative folding is sufficient to reduce HMGB1 retention in the nuclei-hence the release of HMGB1-bound viral DNA for replication. IMPORTANCE Considering the fact that clinical porcine circovirus-associated diseases (PCVAD) mostly results from activation of latent PCV2 infection by confounding factors such as coinfection or environmental stresses, we propose that such confounding factors might impose oxidative stress to the animals, where PCV2 in infected cells might utilize the elevated reactive oxygen species (ROS) to promote HMGB1 migration out of nuclei in favor of its replication. An animal infection model with a particular stressor could be approached with or without antioxidant treatment to examine the relationship among the stressor, ROS level, HMGB1 distribution in target tissues, virus replication, and severity of PCVAD. This will help decide the use of antioxidants in the feeding regime on pig farms that suffer from PCVAD. Further investigation could examine if similar strategies are employed by DNA viruses, such as PCV3 and BFDV and if there is cross talk among endoplasmic reticulum (ER) stress, autophagy/mitophagy, and mitochondrial-sourced ROS in favor of PCV2 replication.


Assuntos
Núcleo Celular/metabolismo , Circovirus/fisiologia , DNA Viral/metabolismo , Retículo Endoplasmático/metabolismo , Proteína HMGB1/metabolismo , Oxirredutases/metabolismo , eIF-2 Quinase/metabolismo , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Cisteína/metabolismo , Replicação do DNA , Ativação Enzimática , Espécies Reativas de Oxigênio/metabolismo , Suínos , Regulação para Cima , Proteínas Virais/metabolismo , Replicação Viral
11.
J Biol Chem ; 297(3): 100999, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34303704

RESUMO

High yields of RNA are routinely prepared following the two-step approach of high-yield in vitro transcription using T7 RNA polymerase followed by extensive purification using gel separation or chromatographic methods. We recently demonstrated that in high-yield transcription reactions, as RNA accumulates in solution, T7 RNA polymerase rebinds and extends the encoded RNA (using the RNA as a template), resulting in a product pool contaminated with longer-than-desired, (partially) double-stranded impurities. Current purification methods often fail to fully eliminate these impurities, which, if present in therapeutics, can stimulate the innate immune response with potentially fatal consequences. In this work, we introduce a novel in vitro transcription method that generates high yields of encoded RNA without double-stranded impurities, reducing the need for further purification. Transcription is carried out at high-salt conditions to eliminate RNA product rebinding, while promoter DNA and T7 RNA polymerase are cotethered in close proximity on magnetic beads to drive promoter binding and transcription initiation, resulting in an increase in overall yield and purity of only the encoded RNA. A more complete elimination of double-stranded RNA during synthesis will not only reduce overall production costs, but also should ultimately enable therapies and technologies that are currently being hampered by those impurities.


Assuntos
DNA Viral/genética , RNA Polimerases Dirigidas por DNA/metabolismo , RNA/isolamento & purificação , Sais/química , Transcrição Genética , Proteínas Virais/metabolismo , Bacteriófago T7/genética , DNA Viral/metabolismo , Regiões Promotoras Genéticas , RNA/biossíntese
12.
Cell Physiol Biochem ; 55(S2): 71-88, 2021 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-34242500

RESUMO

Psychological stress is an important factor involved in disease manifestations of human papillomavirus (HPV) infection, and it can participate in HPV-associated carcinogenesis. The impact or effect which stress can have (exert) depends on a person's genetic pool, experiences and behaviors. Due to inconsistencies in some study results, this issue remains a subject of research. Concerning the course of HPV manifestations, it has been observed that a higher number of life stressors in at least the previous 6 months, the absence of social support and the types of personal coping mechanisms employed, all influence HPV progression. In women with cervical dysplasia, a connection between greater stress experiences and dysregulation of specific immune responses has been observed. Once HPV enters a cell via the α6 integrin there are three possible sequences: latent infection, subclinical infection, and clinically manifest disease. HPV proliferation in differentiated epithelial cells induces morphologically cytopathic changes (koilocytosis, epidermal thickening, hyperplasia, hyperkeratosis). Oncogenic transformation requires the integration of the virus genome into the host genome. In doing so, DNA in the E1 region of E2 breaks down, leading to transcription disorders of E6 and E7. For the formation of irreversible malignancy, the following sequence is necessary: initial expression of E6 and E7 genes followed by suppression of apoptosis and the stabile expression of E6 and E7 proteins that protect transformed cells from apoptosis. A successful immune response is characterized by a strong, local cell-mediated immune response. Several factors are important for the regression of HPV manifestation/infection, among which is psychological stress which can prolong the duration and severity of HPV disease. Stress hormones may reactivate latent tumor viruses, stimulate viral oncogene expression, and inhibit antiviral host responses. In the regression of HPV infection, increased activity of Th1 cells was observed. However, during psychosocial stress, a decrease in the Th1 type of immune response is seen, and there is a shift towards a Th2 response. Understanding perceived stress and biological changes in stress, as well as the evaluation of immune parameters, gives researchers a better picture of how stress influences HPV infections and how to improve disease management and outcomes.


Assuntos
Papillomaviridae/fisiologia , Infecções por Papillomavirus/psicologia , Estresse Psicológico , Carcinogênese , DNA Viral/genética , DNA Viral/metabolismo , Células Epiteliais/citologia , Células Epiteliais/virologia , Humanos , Sistema Nervoso/metabolismo , Sistema Nervoso/virologia , Papillomaviridae/genética , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/patologia , Sistema Hipófise-Suprarrenal/metabolismo , Sistema Hipófise-Suprarrenal/virologia
13.
Mol Cell ; 81(15): 3145-3159.e7, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34214465

RESUMO

Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a ∼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.


Assuntos
Bacteriófago T7/genética , DNA Viral/química , Periplasma/química , Proteínas do Core Viral/química , Biologia Computacional , Microscopia Crioeletrônica , Citoplasma/química , DNA Viral/metabolismo , Bicamadas Lipídicas/metabolismo , Periplasma/genética , Periplasma/metabolismo , Podoviridae/química , Podoviridae/genética , Proteínas do Core Viral/metabolismo
14.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298953

RESUMO

A novel siphovirus, vB_PagS_MED16 (MED16) was isolated in Lithuania using Pantoea agglomerans strain BSL for the phage propagation. The double-stranded DNA genome of MED16 (46,103 bp) contains 73 predicted open reading frames (ORFs) encoding proteins, but no tRNA. Our comparative sequence analysis revealed that 26 of these ORFs code for unique proteins that have no reliable identity when compared to database entries. Based on phylogenetic analysis, MED16 represents a new genus with siphovirus morphology. In total, 35 MED16 ORFs were given a putative functional annotation, including those coding for the proteins responsible for virion morphogenesis, phage-host interactions, and DNA metabolism. In addition, a gene encoding a preQ0 DNA deoxyribosyltransferase (DpdA) is present in the genome of MED16 and the LC-MS/MS analysis indicates 2'-deoxy-7-amido-7-deazaguanosine (dADG)-modified phage DNA, which, to our knowledge, has never been experimentally validated in genomes of Pantoea phages. Thus, the data presented in this study provide new information on Pantoea-infecting viruses and offer novel insights into the diversity of DNA modifications in bacteriophages.


Assuntos
DNA Viral , Genoma Viral , Guanosina , Fases de Leitura Aberta , Pantoea/virologia , Siphoviridae , Proteínas Virais , DNA Viral/genética , DNA Viral/metabolismo , Guanosina/análogos & derivados , Guanosina/química , Guanosina/metabolismo , Siphoviridae/genética , Siphoviridae/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
15.
Int J Mol Sci ; 22(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299262

RESUMO

NK cells play crucial roles in defending against persistent HBV. However, NK cells present dysfunction in chronic hepatitis B virus (CHB) infection, and the associated mechanism is still not fully understood. Except for the regulatory receptors, NK cells could also be regulated by the surface and intracellular pattern recognition receptors (PRRs). In the present study, we found that the level of the adaptor of DNA sensor STING in NK cells was significantly decreased in HBeAg-negative CHB patients, and it was positively associated with the degranulation ability of NK cells. Compared to NK cells from healthy donors, NK cells from HBeAg-negative CHB patients displayed a lower responsiveness to cGAMP stimulation. Further investigation showed that HBsAg could inhibit the STING expression in NK cells and suppress the response of NK cells to cGAMP. Significantly, STAT3 was identified to be a transcription factor that directly regulated STING transcription by binding to the promoter. In addition, STAT3 positively regulated the STING associated IFN-α response of NK cells. These findings suggested that STING is an important adaptor in NK cell recognition and activation, while HBsAg disturbs NK cell function by the STAT3-STING axis, providing a new mechanism of NK disability in HBeAg-negative CHB infection.


Assuntos
Antígenos de Superfície da Hepatite B/metabolismo , Células Matadoras Naturais/metabolismo , Proteínas de Membrana/metabolismo , Adulto , DNA Viral/metabolismo , Feminino , Hepatite B/imunologia , Hepatite B/metabolismo , Antígenos de Superfície da Hepatite B/imunologia , Antígenos E da Hepatite B/imunologia , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/imunologia , Hepatite B Crônica/metabolismo , Humanos , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Fator de Transcrição STAT3/metabolismo , Carga Viral , Replicação Viral
16.
J Virol ; 95(17): e0055521, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34105995

RESUMO

Three prime repair exonuclease 1 (TREX1) is the most abundant 3'→5' exonuclease in mammalian cells. It has been suggested that TREX1 degrades HIV-1 DNA to enable the virus to evade the innate immune system. However, the exact role of TREX1 during early steps of HIV-1 infection is not clearly understood. In this study, we report that HIV-1 infection is associated with upregulation, perinuclear accumulation, and nuclear localization of TREX1. However, TREX1 overexpression did not affect reverse transcription or nuclear entry of the virus. Surprisingly, HIV-1 DNA integration was increased in TREX1-overexpressing cells, suggesting a role of the exonuclease in the post-nuclear entry step of infection. Accordingly, preintegration complexes (PICs) extracted from TREX1-overexpressing cells retained higher levels of DNA integration activity. TREX1 depletion resulted in reduced levels of proviral integration, and PICs formed in TREX1-depleted cells retained lower DNA integration activity. Addition of purified TREX1 to PICs also enhanced DNA integration activity, suggesting that TREX1 promotes HIV-1 integration by stimulating PIC activity. To understand the mechanism, we measured TREX1 exonuclease activity on substrates containing viral DNA ends. These studies revealed that TREX1 preferentially degrades the unprocessed viral DNA, but the integration-competent 3'-processed viral DNA remains resistant to degradation. Finally, we observed that TREX1 addition stimulates the activity of HIV-1 intasomes assembled with the unprocessed viral DNA but not that of intasomes containing the 3'-processed viral DNA. These biochemical analyses provide a mechanism by which TREX1 directly promotes HIV-1 integration. Collectively, our study demonstrates that HIV-1 infection upregulates TREX1 to facilitate viral DNA integration. IMPORTANCE Productive HIV-1 infection is dependent on a number of cellular factors. Therefore, a clear understanding of how the virus exploits the cellular machinery will identify new targets for inhibiting HIV-1 infection. The three prime repair exonuclease 1 (TREX1) is the most active cellular exonuclease in mammalian cells. It has been reported that TREX1 prevents accumulation of HIV-1 DNA and enables the virus to evade the host innate immune response. Here, we show that HIV-1 infection results in the upregulation, perinuclear accumulation, and nuclear localization of TREX1. We also provide evidence that TREX1 promotes HIV-1 integration by preferentially degrading viral DNAs that are incompatible with chromosomal insertion. These observations identify a novel role of TREX1 in a post-nuclear entry step of HIV-1 infection.


Assuntos
DNA Viral/metabolismo , Exodesoxirribonucleases/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Imunidade Inata/imunologia , Fosfoproteínas/metabolismo , Integração Viral , Replicação Viral , Núcleo Celular , DNA Viral/genética , Exodesoxirribonucleases/genética , Células HEK293 , Infecções por HIV/genética , Células HeLa , Humanos , Fosfoproteínas/genética
17.
Viruses ; 13(5)2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068736

RESUMO

Deoxyuridine in DNA has recently been in the focus of research due to its intriguing roles in several physiological and pathophysiological situations. Although not an orthodox DNA base, uracil may appear in DNA via either cytosine deamination or thymine-replacing incorporations. Since these alterations may induce mutation or may perturb DNA-protein interactions, free living organisms from bacteria to human contain several pathways to counteract uracilation. These efficient and highly specific repair routes uracil-directed excision repair initiated by representative of uracil-DNA glycosylase families. Interestingly, some bacteriophages exist with thymine-lacking uracil-DNA genome. A detailed understanding of the strategy by which such phages can replicate in bacteria where an efficient repair pathway functions for uracil-excision from DNA is expected to reveal novel inhibitors that can also be used for biotechnological applications. Here, we also review the several potential biotechnological applications already implemented based on inhibitors of uracil-excision repair, such as Crispr-base-editing and detection of nascent uracil distribution pattern in complex genomes.


Assuntos
DNA Viral/química , DNA Viral/genética , Uracila , Vírus/genética , Bacteriófagos/efeitos dos fármacos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Biotecnologia , DNA Viral/metabolismo , Desenvolvimento de Medicamentos , Humanos , Modelos Moleculares , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Conformação Proteica , Relação Estrutura-Atividade , Uracila/química , Uracila-DNA Glicosidase/química , Uracila-DNA Glicosidase/metabolismo , Vírus/efeitos dos fármacos , Vírus/metabolismo
18.
Adv Virus Res ; 109: 163-199, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33934827

RESUMO

The vertebrate innate immune system confers host cells with mechanisms to protect against both evolutionarily ancient pathogens and newly emerging pathogenic strains. Innate immunity relies on the host cell's ability to distinguish between self and pathogen-derived molecules. To achieve this, the innate immune system uses germline encoded receptors called pattern recognition receptors (PRRs), which recognize various molecular signatures, including nucleic acids, proteins, lipids, glycans and glycolipids. Among these molecules, the recognition of pathogenic, mislocalized, or damaged DNA by cellular protein receptors, commonly called DNA sensors, represents a major surveillance pathway for initiating immune signaling. The ability of cells to temporally regulate DNA sensor activation and subsequent signal termination is critical for effective immune signaling. These same mechanisms are also co-opted by pathogens to promote their replication. Therefore, there is significant interest in understanding DNA sensor regulatory networks during microbial infections and autoimmune disease. One emerging aspect of DNA sensor regulation is through post-translational modifications (PTMs), including phosphorylation, acetylation, ubiquitination, ADP-ribosylation, SUMOylation, methylation, deamidation, glutamylation. In this chapter, we discuss how PTMs have been shown to positively or negatively impact DNA sensor functions via diverse mechanisms, including direct regulation of enzymatic activity, protein-protein and protein-DNA interactions, protein translocations and protein turnover. In addition, we highlight the ability of virus-induced PTMs to promote immune evasion. We also discuss the recent evidence linking PTMs on DNA sensors with human diseases and more broadly, highlight promising directions for future research on PTM-mediated regulation of DNA sensor-dependent immune signaling.


Assuntos
DNA Viral/metabolismo , Imunidade Inata , Processamento de Proteína Pós-Traducional , Transdução de Sinais/imunologia , Técnicas Biossensoriais , Livros , Interações Hospedeiro-Patógeno , Humanos , Espectrometria de Massas , Fosforilação , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/imunologia , Transdução de Sinais/genética , Vírus/imunologia , Vírus/patogenicidade
19.
PLoS Pathog ; 17(5): e1009580, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33974675

RESUMO

Human papillomaviruses (HPVs) utilize an atypical mode of nuclear import during cell entry. Residing in the Golgi apparatus until mitosis onset, a subviral complex composed of the minor capsid protein L2 and viral DNA (L2/vDNA) is imported into the nucleus after nuclear envelope breakdown by associating with mitotic chromatin. In this complex, L2 plays a crucial role in the interactions with cellular factors that enable delivery and ultimately tethering of the viral genome to mitotic chromatin. To date, the cellular proteins facilitating these steps remain unknown. Here, we addressed which cellular proteins may be required for this process. Using label-free mass spectrometry, biochemical assays, microscopy, and functional virological assays, we discovered that L2 engages a hitherto unknown protein complex of Ran-binding protein 10 (RanBP10), karyopherin alpha2 (KPNA2), and dynein light chain DYNLT3 to facilitate transport towards mitotic chromatin. Thus, our study not only identifies novel cellular interactors and mechanism that facilitate a poorly understood step in HPV entry, but also a novel cellular transport complex.


Assuntos
Alphapapillomavirus/fisiologia , Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , DNA Viral/metabolismo , Genoma Viral/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Infecções por Papillomavirus/virologia , Transporte Ativo do Núcleo Celular , Alphapapillomavirus/genética , Proteínas do Capsídeo/genética , Cromatina/genética , Dineínas/genética , Dineínas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Proteínas Associadas aos Microtúbulos/genética , Mitose , Internalização do Vírus , alfa Carioferinas/genética , alfa Carioferinas/metabolismo
20.
J Virol ; 95(15): e0049521, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34011543

RESUMO

During retrovirus infection, a histone-free DNA copy of the viral RNA genome is synthesized and rapidly loaded with nucleosomes de novo upon nuclear entry. The potential role of viral accessory proteins in histone loading onto retroviral DNAs has not been extensively investigated. The p12 protein of Moloney murine leukemia virus (MMLV) is a virion protein that is critical for tethering the incoming viral DNA to host chromatin in the early stages of infection. Infection by virions containing a mutant p12 (PM14) defective in chromatin tethering results in the formation of viral DNAs that do not accumulate in the nucleus. In this report, we show that viral DNAs of these mutants are not loaded with histones. Moreover, the DNA genomes delivered by mutant p12 show prolonged association with viral structural proteins nucleocapsid (NC) and capsid (CA). The histone-poor viral DNA genomes do not become associated with the host RNA polymerase II machinery. These findings provide insights into fundamental aspects of retroviral biology, indicating that tethering to host chromatin by p12 and retention in the nucleus are required to allow loading of histones onto the viral DNA. IMPORTANCE Incoming retroviral DNAs are rapidly loaded with nucleosomal histones upon entry into the nucleus and before integration into the host genome. The entry of murine leukemia virus DNA into the nucleus occurs only upon dissolution of the nuclear membrane in mitosis, and retention in the nucleus requires the action of a viral protein, p12, which tethers the DNA to host chromatin. Data presented here show that the tethering activity of p12 is required for the loading of histones onto the viral DNA. p12 mutants lacking tethering activity fail to acquire histones, retain capsid and nucleocapsid proteins, and are poorly transcribed. The work defines a new requirement for a viral protein to allow chromatinization of viral DNA.


Assuntos
Proteínas do Capsídeo/metabolismo , Produtos do Gene gag/genética , Histonas/metabolismo , Vírus da Leucemia Murina de Moloney/crescimento & desenvolvimento , Vírus da Leucemia Murina de Moloney/metabolismo , Capsídeo/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , DNA Viral/metabolismo , Genoma Viral/genética , Células HEK293 , Células HeLa , Humanos , Vírus da Leucemia Murina de Moloney/genética , Montagem de Vírus/genética
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