RESUMO
Nasopharyngeal carcinoma (NPC) is a type of head and neck cancer. As a neoplastic disorder, NPC is a highly malignant squamous cell carcinoma that is derived from the nasopharyngeal epithelium. NPC is radiosensitive; radiotherapy or radiotherapy combining with chemotherapy are the main treatment strategies. However, both modalities are usually accompanied by complications and acquired resistance to radiotherapy is a significant impediment to effective NPC therapy. Therefore, there is an urgent need to discover effective radio-sensitization and radio-resistance biomarkers for NPC. Recent studies have shown that Epstein-Barr virus (EBV)-encoded products, microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), which share several common signaling pathways, can function in radio-related NPC cells or tissues. Understanding these interconnected regulatory networks will reveal the details of NPC radiation sensitivity and resistance. In this review, we discuss and summarize the specific molecular mechanisms of NPC radio-sensitization and radio-resistance, focusing on EBV-encoded products, miRNAs, lncRNAs and circRNAs. This will provide a foundation for the discovery of more accurate, effective and specific markers related to NPC radiotherapy. EBVencoded products, miRNAs, lncRNAs and circRNAs have emerged as crucial molecules mediating the radio-susceptibility of NPC. This understanding will improve the clinical application of markers and inform the development of novel therapeutics for NPC.
Assuntos
Carcinoma Nasofaríngeo/radioterapia , Neoplasias Nasofaríngeas/radioterapia , Dano ao DNA , DNA de Neoplasias/efeitos da radiação , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/radioterapia , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Herpesvirus Humano 4/fisiologia , Humanos , MicroRNAs/genética , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/virologia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/virologia , RNA Circular/genética , RNA Longo não Codificante/genética , RNA Neoplásico/genética , RNA Viral/efeitos da radiação , Tolerância a Radiação/genética , Proteínas Virais/efeitos da radiaçãoRESUMO
Endolysins are lytic enzymes encoded by bacteriophage that represent an emerging class of protein therapeutics. Considering macromolecular thermoresistance correlates with shelf life, PlyG, a Bacillus anthracis endolysin, was thermally characterized to further evaluate its therapeutic potential. Results from a biophysical thermal analysis revealed full-length PlyG and its isolated domains comprised thermal denaturation temperatures exceeding 63°C. In the absence of reducing agent, PlyG was determined to be kinetically unstable, a finding hypothesized to be attributable to the chemical oxidation of cysteine and/or methionine residues. The presence of reducing agent kinetically stabilized the endolysin, with PlyG retaining at least ~50% residual lytic activity after being heated at temperatures up to 80°C and remaining enzymatically functional after being boiled. Furthermore, the endolysin had a kinetic half-life at 50°C and 55°C of 35 and 5.5h, respectively. PlyG represents a thermostable proteinaceous antibacterial with subsequent prolonged therapeutic shelf life expectancy.
Assuntos
Bacillus anthracis/enzimologia , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/efeitos da radiação , Proteínas Virais/química , Proteínas Virais/efeitos da radiação , Antibacterianos/química , Antibacterianos/efeitos da radiação , Estabilidade Enzimática , Cinética , Desnaturação Proteica/efeitos da radiação , Estabilidade Proteica , TemperaturaRESUMO
Much research has been dedicated to understanding the molecular basis of UV damage to biomolecules, yet many questions remain regarding the specific pathways involved. Here we describe a genome-mediated mechanism that causes site-specific virus protein cleavage upon UV irradiation. Bacteriophage MS2 was disinfected with 254 nm UV, and protein damage was characterized with ESI- and MALDI-based FT-ICR, Orbitrap, and TOF mass spectroscopy. Top-down mass spectrometry of the products identified the backbone cleavage site as Cys46-Ser47 in the virus capsid protein, a location of viral genome-protein interaction. The presence of viral RNA was essential to inducing backbone cleavage. The similar bacteriophage GA did not exhibit site-specific protein cleavage. Based on the major protein fragments identified by accurate mass analysis, a cleavage mechanism is proposed by radical formation. The mechanism involves initial oxidation of the Cys46 side chain followed by hydrogen atom abstraction from Ser47 C(α). Computational protein QM/MM studies confirmed the initial steps of the radical mechanism. Collectively, this study describes a rare incidence of genome-induced protein cleavage without the addition of sensitizers.
Assuntos
Genoma Viral/efeitos da radiação , Levivirus/metabolismo , Levivirus/efeitos da radiação , Proteínas Virais/metabolismo , Proteínas Virais/efeitos da radiação , Levivirus/genética , Espectrometria de Massas , Raios Ultravioleta , Proteínas Virais/genéticaRESUMO
Adenoviruses are resistant to monochromatic, low-pressure (LP) UV disinfection--but have been shown to be susceptible to inactivation by polychromatic, medium-pressure (MP) UV--when assayed using cell culture infectivity. One possible explanation for the difference between UV lamp types is that the additional UV wavelengths emitted by MP UV enable it to cause greater damage to viral proteins than LP UV. The objective of this study was to examine protein damage in adenoviruses treated with LP and MP UV. Results show that MP UV is more effective at damaging viral proteins at high UV doses, though LP UV caused some damage as well. To our knowledge, this study is the first to investigate protein damage in UV-treated adenovirus, and the overview presented here is expected to provide a basis for further, more detailed work.
Assuntos
Adenoviridae/efeitos da radiação , Desinfecção/métodos , Raios Ultravioleta , Proteínas Virais/efeitos da radiação , Adenoviridae/genética , Eletroforese em Gel de Poliacrilamida , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Fluorescent proteins (FPs) based on green fluorescent protein (GFP) are widely used throughout cell biology to study protein dynamics, and have extensive use as reporters of virus infection and spread. However, FP-tagging of viruses is limited by the constraints of viral genome size resulting in FP loss through recombination events. To overcome this, we have engineered a smaller ( approximately 10 kDa) flavin-based alternative to GFP ( approximately 25 kDa) derived from the light, oxygen or voltage-sensing (LOV) domain of the plant blue light receptor, phototropin. Molecular evolution and Tobacco mosaic virus (TMV)-based expression screening produced LOV variants with improved fluorescence and photostability in planta. One variant in particular, designated iLOV, possessed photophysical properties that made it ideally suited as a reporter of subcellular protein localization in both plant and mammalian cells. Moreover, iLOV fluorescence was found to recover spontaneously after photobleaching and displayed an intrinsic photochemistry conferring advantages over GFP-based FPs. When expressed either as a cytosolic protein or as a viral protein fusion, iLOV functioned as a superior reporter to GFP for monitoring local and systemic infections of plant RNA viruses. iLOV, therefore, offers greater utility in FP-tagging of viral gene products and represents a viable alternative where functional protein expression is limited by steric constraints or genome size.
Assuntos
Flavoproteínas/análise , Proteínas Luminescentes/análise , Vírus de Plantas/fisiologia , Plantas/virologia , Proteínas Virais/análise , Animais , Criptocromos , Evolução Molecular Direcionada , Flavinas/química , Flavoproteínas/genética , Flavoproteínas/metabolismo , Flavoproteínas/efeitos da radiação , Fluorescência , Genes Reporter , Engenharia Genética , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/efeitos da radiação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Luminescentes/efeitos da radiação , Microscopia Confocal , Microscopia de Fluorescência , Oxigênio/metabolismo , Fotodegradação , Vírus de Plantas/genética , Vírus de Plantas/metabolismo , Proteínas Recombinantes de Fusão , Vírus do Mosaico do Tabaco/genética , Vírus do Mosaico do Tabaco/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/efeitos da radiaçãoRESUMO
The photodynamic inactivation of herpes simplex virus type 1 (HSV-1) by two phthalocyanines (Pcs), the cationic dye HOSi-PcOSi(CH3)2(CH2)3N+(CH3)3I-(Pc5) and the amphiphilic dye aluminum dibenzodisulfophthalocyanine hydroxide (AlN2SB2POH), has been compared with that by the anionic dye, Merocyanine 540 (Mc540). Both Pc derivatives demonstrate a remarkable virucidal activity upon light activation even 3 h after the onset of HSV-1 adsorption, while Mc540 is effective for only 30 min after adsorption. Since fusion and virus penetration are promoted by membrane glycoproteins, we have studied the damage to viral proteins following photodynamic treatment (PDT) of HSV-1 and its relation to inactivation. The effect of AlN2SB2POH PDT is assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Major changes are found in the protein profile of PDT-treated HSV-1. A reduced ability of specific antibodies to react with HSV-1 major envelope proteins is detected by employing the Western blot assay. In particular, we demonstrate the related changes of glycoprotein D (gD), a structural protein of the HSV envelope. Since the envelope proteins participate in viral entry into the host cell, these alterations to viral envelope proteins may impair their ability to participate in early events of viral entry, leading to reduced infectivity of HSV-1. In contrast, no significant changes in the proteins' electrophoretic mobility could be seen after PDT with Mc540 or with Pc5. When HSV-1 purified proteins are subjected to combined electrophoretic and electro osmotic forces on cellulose acetate, there is a shift in their cathode mobility, which may indicate changes in the protein mass and protein net charges following AlN2SB2POH photosensitization. There are only minor changes in the virus proteins, assayed as above, when HSV-1 is treated with Pc5.
Assuntos
Produtos do Gene env/efeitos dos fármacos , Indóis/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Simplexvirus/efeitos dos fármacos , Proteínas Virais/efeitos dos fármacos , Proteínas Virais/efeitos da radiação , Animais , Chlorocebus aethiops , Produtos do Gene env/efeitos da radiação , Isoindóis , Cinética , Luz , Pirimidinonas/farmacologia , Simplexvirus/fisiologia , Simplexvirus/efeitos da radiação , Células VeroRESUMO
The interaction of ultraviolet radiation and virus particles of Western Equine Encephalomyelitis Virus (WEE) and Newcastle Disease Virus (NDV) which have respectively RNA of positive (RNA+) and negative (RNA-) polarity as genomes, was studied using purified particles. The purified virus preparations were irradiated at a range from 1,000 to 6,000 joules per m2 with posterior analysis of their propagation in primary cell cultures of chicken embryos. It could be observed that a radiation dose of to 4,500 joules per m2 could induce 10(9) TCID50 per ml as minimal loss of titer for WEE virus and NDV. The hemagglutination assay was used as a toll to evaluate the alterations caused by UV radiation on the molecular arrangement of virus proteins. Alterations of the virus hemagglutinating activity were only observe when radiation levels higher than 6,000 joules per m2 were used. The results from hemolysis assays showed the importance of the loss of the envelope integrity and the damages to nucleoprotein structures during the inactivation process, when we used radiation doses higher than 6,000 joules per m2. This model of study can increase our comprehension of the radiation effects on the cell physiology and biological components of the cell membranes.
Assuntos
RNA/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Vírus da Doença de Newcastle/efeitos da radiação , Vírus da Encefalite Equina do Oeste/efeitos da radiação , Hemaglutinação , Testes de Hemaglutinação , Hemólise , Proteínas Virais/efeitos da radiação , Vírus da Doença de Newcastle/isolamento & purificação , Vírus da Encefalite Equina do Oeste/isolamento & purificaçãoRESUMO
The papillomavirus E2 protein is a DNA binding protein that regulates viral transcription and replication. E2 binds DNA as a dimer. Recent crystallographic data for E2 complexed to DNA revealed that novel peptide structures in E2 mediated dimerization and DNA binding. To identify important features of these motifs we have used limited proteolysis and urea denaturation as biochemical probes for structure, applying these techniques to E2 alone, E2 bound to DNA, cross-linked products, and mutants that were targeted at Trp360, a contact point along the dimer interface. DNA binding stabilized E2 structure, shifting the point at which it denatures from 5 to 7.6 M urea. In contrast, Trp360 mutant proteins, while dimeric, were more sensitive to denaturation by urea when bound to DNA. The most striking results came from uv cross-linking studies in which Trp360 was targeted as the site of cross-linking. Ultraviolet cross-linking dramatically increased the resistance of E2 to proteolysis regardless of the protease tested and with no deleterious effect on the affinity of E2 for DNA. Cross-linking through Cys356 with bismaleimidohexane did not promote stabilization. The ability to stabilize or destabilize E2 by Trp360-targeted modifications demonstrates the importance of the Trp360-Trp360 interaction, which may represent a general feature of the beta-barrel motif.
Assuntos
Papillomavirus Bovino 1/metabolismo , Proteínas de Ligação a DNA/metabolismo , Triptofano , Proteínas Virais/metabolismo , Sequência de Bases , Sítios de Ligação , Quimotripsina , Primers do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/efeitos da radiação , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/metabolismo , Fragmentos de Peptídeos/isolamento & purificação , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efeitos da radiação , Tripsina/metabolismo , Raios Ultravioleta , Proteínas Virais/genética , Proteínas Virais/efeitos da radiaçãoRESUMO
Human rhinoviruses (HRVs) and encephalomyocarditis virus (EMCV) belong to different genera of the picornavirus family, but the translation of the RNAs of both viruses is by the same mechanism, that is, internal ribosome entry. In rabbit reticulocyte lysates this translation initiation is efficient for mRNAs bearing the EMCV 5' untranslated region (5' UTR), but very inefficient for mRNAs bearing the HRV 5' UTR, unless factors from HeLa cells are added. The copurification of the HeLa cell translation stimulatory activity with proteins which can be specifically cross-linked to the HRV 5' UTR by u.v. irradiation has been examined. Both the EMCV and HRV 5' UTRs can be cross-linked to a 58/60K protein doublet present in HeLa cell extracts in higher amounts than in reticulocyte lysates, which is shown to be very similar, if not identical to the polypyrimidine tract binding protein (PTB) previously identified as a component of a multi-subunit complex necessary for pre-mRNA splicing. However, the activity in HeLa cell extracts that specifically stimulates translation initiation on mRNAs with the HRV 5' UTR does not copurify with the majority of the 58/60K protein present in these extracts, but copurifies with a minor fraction of these proteins and with a 97K protein which can be cross-linked to the HRV 5' UTR but not to the EMCV 5' UTR, and which is absent from reticulocyte lysates. It is proposed that the specific translation initiation stimulatory activity found in HeLa cells is due to a high M(r) complex containing the 97K polypeptide and PTB.
Assuntos
Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Rhinovirus/metabolismo , Spliceossomos/metabolismo , Proteínas Virais/biossíntese , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica , Células HeLa , Humanos , Cinética , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/efeitos da radiação , Plasmídeos , Coelhos , Reticulócitos/metabolismo , Rhinovirus/genética , Transcrição Gênica , Raios Ultravioleta , Proteínas Virais/isolamento & purificação , Proteínas Virais/efeitos da radiaçãoRESUMO
We have previously identified a novel 8 bp sequence (UV-responsive element, URE: TGACAACA) present in the regulatory region of polyoma DNA that interacts with protein factors induced in rat fibroblast cells by exposure to UV light. In the present study, we demonstrate through competitive binding assays that this sequence is distinct from the partially homologous AP1 and CRE target sequences. The proteins that bind to the URE appear to have transcriptional activity in UV-exposed rat fibroblasts. In addition, the URE appears to play a role in promoting the replication of polyoma DNA as determined through two different experimental approaches. Together, these findings suggest that the URE is a novel DNA binding element that interacts with proteins involved in the transcription and replication of polyoma sequences.
Assuntos
Replicação do DNA/genética , DNA Viral/genética , Polyomavirus/genética , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Gênica/genética , Sequência de Bases , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/efeitos da radiação , Regulação Viral da Expressão Gênica/genética , Regulação Viral da Expressão Gênica/efeitos da radiação , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/genética , Polyomavirus/efeitos da radiação , Proteínas Recombinantes de Fusão/genética , Proteínas Virais/genética , Proteínas Virais/efeitos da radiaçãoRESUMO
The UV-irradiation was found to induce formation of the RNA-protein cross-links and intraviral RNA chain breaks in the particles of flexuous potato virus X (PVX). Using the technique developed previously for the rod-like tobacco mosaic virus (TMV), the quantum yields of RNA-protein cross-links and intraviral RNA polynucleotide chain breaks formation in the PVX were determined and found to be more or less close to those found for the intraviral TMV RNA.
Assuntos
Vírus de Plantas/metabolismo , RNA Viral/metabolismo , Raios Ultravioleta , Proteínas Virais/metabolismo , Vírion/metabolismo , RNA Viral/efeitos da radiação , Solanum tuberosum , Proteínas Virais/efeitos da radiaçãoRESUMO
When purified, [35S]methionine-labeled vesicular stomatitis virus (VSV) was exposed to ultraviolet light, an irradiation-induced change in the viral proteins was detected by SDS-polyacrylamide gel electrophoresis and immunoblotting. With dose of uv irradiation in the same range as that required to inactivate VSV leader RNA, a loss occurred in the bands corresponding to the L and NS proteins concomitant with the appearance of several new bands of radioactivity throughout the gel. This alteration of viral proteins correlated with the loss of ability of the virus to inhibit host macromolecular synthesis. In light of these results, the role that has been ascribed to the VSV leader RNA in VSV-mediated host shut-off needs to be reevaluated.
Assuntos
Capsídeo/efeitos da radiação , RNA Polimerase Dependente de RNA , Vírus da Estomatite Vesicular Indiana/efeitos da radiação , Proteínas do Core Viral/efeitos da radiação , Proteínas Virais/efeitos da radiação , Animais , Capsídeo/fisiologia , Células L/metabolismo , Camundongos , Biossíntese de Proteínas , Raios Ultravioleta , Vírus da Estomatite Vesicular Indiana/fisiologia , Proteínas do Core Viral/fisiologia , Proteínas não Estruturais Virais , Proteínas Virais/fisiologiaRESUMO
The effect of specific photochemical and radiochemical modification of tryptophyl and cysteinyl residues of the gene 32 protein (gp 32) of bacteriophage T4 on its affinity towards single-stranded polynucleotides has been investigated. Oxidation of Cys residues of gp 32 by the free-radical anion I-.2 induces a partial loss of the protein affinity, probably by affecting the metal-binding domain which includes three of the four cysteine residues of gp 32. Ultraviolet irradiation of gp 32 in the presence of trichloroethanol results in the modification of three of its five Trp residues and total loss of the protein binding. Analysis of the relative affinity of ultraviolet-irradiated gp 32 for single-stranded polynucleotides suggest that modification of a Trp of enhanced reactivity occurs first and has no effect on the protein binding. Radiochemical modification of three Trp residues of gp 32 by (SCN)-.2 results in total loss of activity. Complexation of gp 32 with denatured DNA prior to gamma-irradiation protects two Trp residues and prevents the protein inactivation. These results suggest that at most two Trp residues are involved in stacking interactions with nucleic acid bases. However, time-resolved spectroscopic methods which allow us to monitor selectively the stacked tryptophan residues have not yielded evidence of more than a single residue undergoing such interactions.
Assuntos
Polinucleotídeos/análise , Fagos T/análise , Triptofano/análise , Proteínas Virais/análise , Sítios de Ligação/efeitos da radiação , Cisteína/análise , Desnaturação de Ácido Nucleico , Ligação Proteica , Fagos T/genética , Triptofano/efeitos da radiação , Raios Ultravioleta , Proteínas Virais/efeitos da radiaçãoAssuntos
Polinucleotídeos/efeitos da radiação , RNA Viral/efeitos da radiação , Vírus do Mosaico do Tabaco/efeitos da radiação , Raios Ultravioleta , Proteínas Virais/efeitos da radiação , Reagentes de Ligações Cruzadas , Lasers , Substâncias Macromoleculares , Polinucleotídeos/análise , RNA Viral/análise , Vírus do Mosaico do Tabaco/análise , Proteínas Virais/análiseRESUMO
Despite decades of study of the effect of near-ultraviolet radiation (NUV) on bacterial cells, insights into mechanisms of deleterious alterations and subsequent recovery are just now emerging. These insights are based on observations that 1) damage by NUV may be caused by a reactive oxygen molecule, since H2O2 may be a photoproduct of NUV; 2) some, but not all, of the effects of NUV and H2O2 are interchangeable; 3) there is an inducible regulon (oxyR) that responds to oxidative stress and is involved in protection against NUV; 4) a number of NUV-sensitive mutants are defective either in the capacity to detoxify reactive oxygen molecules or to repair DNA damage caused by NUV; and 5) recovery from NUV damage may not directly involve induction of the SOS response. Since several distinctly different photoreceptors and targets are involved, it is unknown whether NUV lethality and mutagenesis result from an accumulation of damages or whether there is a particularly critical photoeffect. To fully understand the mechanisms involved, it is important to identify the chromophore(s) of NUV, the mechanism of toxic oxygen species generation, the role of the oxidative defense regulon (oxyR), the specific lesions in the DNA, and the enzymatic events of subsequent repair.
Assuntos
Bacteriófagos/efeitos da radiação , Dano ao DNA , Escherichia coli/efeitos da radiação , Raios Ultravioleta , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/efeitos da radiação , Bacteriófagos/genética , Catalase/metabolismo , Membrana Celular/efeitos da radiação , DNA Bacteriano/efeitos da radiação , DNA Viral/efeitos da radiação , Escherichia coli/genética , Peróxido de Hidrogênio/biossíntese , Mutação , Porfirinas/efeitos da radiação , Resposta SOS em Genética/efeitos da radiação , Salmonella typhimurium/genética , Salmonella typhimurium/efeitos da radiação , Proteínas Virais/efeitos da radiaçãoRESUMO
Murine splenocytes were used to study the in vitro immunosuppressive effects of UV-inactivated feline leukemia virus (FeLV-UV). FeLV-UV blocks both alloantigen (DBA/2)-induced and Con A-induced proliferation of C57BL/6 splenocytes in a dose-dependent manner. Furthermore, C57BL/6 anti-DBA/2 mixed lymphocyte cultures containing FeLV-UV fail to develop detectable DBA/2-specific cytolytic activity, although FeLV-UV has no effect on the cytolytic activity of preformed C57BL/6 anti-DBA/2 cytolytic T cells (CTL). Disruption of lymphocyte proliferation and CTL generation by FeLV-UV could not be overcome by the addition of exogenous lymphokines. These data suggest that FeLV-UV can interfere with the lymphokine reactivity of alloactivated lymphocytes. In fact, FeLV-UV blocks the lymphokine-induced proliferation of the murine IL 2-dependent cell line CTLL-20. The CTLL-20 cells were subsequently used to study the mechanism(s) by which retroviruses alter T lymphocyte function. Normally, CTLL-20 cells undergo significant proliferation when cultured in EL4 SN, an IL 2-containing culture supernatant from PMA-stimulated EL4 cells. This lymphokine-induced CTLL-20 proliferation is abrogated in a dose-dependent manner by UV-inactivated murine leukemia virus (MuLV-UV), FeLV-UV, and a purified 15,000 dalton viral protein, p15, derived from FeLV. Suppression of CTLL-20 proliferation requires only brief contact (6 hr) with FeLV-UV or with p15, but is most efficient after prolonged (24 hr) contact with these agents. Furthermore, suppression of CTLL-20 proliferation by FeLV-UV and p15 is reversible, because CTLL-20 cells which have been pretreated for 24 hr with FeLV-UV or p15 are equally as efficient at responding to EL4 SN as untreated CTLL-20. Additional studies indicate that CTLL-20 cells continue to remove IL 2 activity from EL4 SN in the presence of suppressive concentrations of FeLV-UV, and that suppressive concentrations of FeLV-UV do not remove IL 2 activity from EL4 SN. This suggests that FeLV does not block CTLL-20 proliferation by absorbing or inactivating IL 2, or by occluding IL 2 receptors, and that T lymphocytes develop an insensitivity to lymphokines after contact with FeLV-UV, which may be caused by a metabolic, rather than an immunologic, defect. Because lymphokines are requisite signals for T cell function, considerable immunosuppression would be associated with acquired lymphokine insensitivity.
Assuntos
Proteínas de Ligação a DNA , Tolerância Imunológica , Vírus da Leucemia Felina/fisiologia , Ativação Linfocitária , Linfocinas/farmacologia , Proteínas Oncogênicas de Retroviridae , Linfócitos T Citotóxicos/imunologia , Proteínas Virais/fisiologia , Animais , Linhagem Celular , Citotoxicidade Imunológica , Feminino , Imunossupressores/farmacologia , Vírus da Leucemia Felina/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas de Neoplasias/farmacologia , Proteínas Virais/efeitos da radiaçãoRESUMO
Adenovirus chromatin is constituted with three kinds of core proteins, VII, V, and mu, that are coded by the virus genome. Since a hexamer of VII contributes to formation of the nucleosome-like structure of the virion chromatin, we analyzed the interaction between DNA and VII in vitro, by the use of ultraviolet light-induced cross-linking and circular dichroism (CD) spectroscopy. It was observed that DNA and VII in a plain mixture form a structure resembling viral chromatin. The DNA in the virion core or in the simply mixed complex appears to take a tight conformation by superfolding, based on the result that the ellipticity at 275 nm of DNA was reduced to approximately 3,000 degrees, and the wave-length of the positive peak was shifted from 275 to 285 nm. The change in CD spectrum caused by interaction of VII with DNA is similar to that of a protamine rather than that of a histone mixture. The interaction of VII with DNA is preferential, and VII is capable of associating more efficiently with double stranded DNA than with single stranded. The interaction is loosened by salt (0.3 M NaCl) and tightened by magnesium ion. However, the interaction of a precursor core protein pro-VII with DNA was not as tight as that of VII and was not influenced by magnesium ion, presumably because of the existence of a hydrophobic processing sequence in the molecule.
Assuntos
Adenovírus Humanos/genética , Cromatina/metabolismo , DNA Viral/metabolismo , Desoxirribonucleoproteínas/metabolismo , Raios Ultravioleta , Proteínas Virais/metabolismo , Carcinoma , Linhagem Celular , Cromatina/efeitos da radiação , Cromatina/ultraestrutura , Dicroísmo Circular , DNA Viral/efeitos da radiação , Humanos , Cinética , Neoplasias Bucais , Conformação de Ácido Nucleico , Conformação Proteica , Proteínas do Core Viral , Proteínas Virais/efeitos da radiaçãoRESUMO
The rates of inactivation of synthesis of individual virus-specified proteins by ultraviolet radiation provided an estimate of the target sizes of individual viral genes. In a control experiment with Semliki Forest virus, the genes for the structural proteins mapped in the known sequence 5' C-PE2-E1 3', and in accordance with initiation of translation from a single site on 26-S mRNA. Under the same conditions, the inactivation of synthesis of seven Kunjin virus-specified proteins also followed first-order kinetics, but the largest target size was equivalent to only about half the length of the genome. No gene sequence could be deduced using the premise that translation was initiated at a single site. However, genes for the major nonstructural proteins could be mapped in the order 5' P98-P71-P10 3' from a postulated ribosomal attachment site about midway along the RNA. The genes for the structural proteins C and E could then be mapped in a preceding sequence 5'...C-E-GP19-P21 3', with a vacant upstream region sufficient to code for at least one of two flavivirus genes not expressed in these experiments. The implications of the two postulated translational units are discussed in relation to previous data on translation strategy of flaviviruses.
Assuntos
Flavivirus/genética , Biossíntese de Proteínas , Animais , Sequência de Bases , Peso Molecular , Ribossomos/metabolismo , Raios Ultravioleta , Proteínas Virais/biossíntese , Proteínas Virais/efeitos da radiação , Proteínas Estruturais ViraisRESUMO
RNA and protein interaction in the structure of influenza virus ribonucleoprotein (RNP) was studied by ultraviolet (UV) irradiation. After UV-irradiation of virion RNP for 1 hour only 6% of 3H-uridine-labeled RNA was found to go into the aqueous phase upon phenol-detergent extraction. Pretreatment of RNP with small doses of pancreatic RNase before RNA extraction slightly increased (up to 18%) the amount of RNA going into the aqueous phase. About 90% RNA was found after extraction in the aqueous phase in the nonirradiated material. As a result of UV-irradiation of RNP, RNA in RNP became more resistant to RNase: the residual acid-insoluble radioactivity was 21% whereas with nonirradiated RNP it was 3.2%. The results of the analysis of RNP labeled for protein in polyacrylamide gel and SDS-sucrose gradient after UV-irradiation and ribonuclease treatment indicate the formation of UV-induced linkages between RNA and NP protein.
Assuntos
Vírus da Influenza A/efeitos da radiação , Nucleoproteínas/efeitos da radiação , RNA Viral/efeitos da radiação , Ribonucleoproteínas/efeitos da radiação , Raios Ultravioleta , Proteínas Virais/efeitos da radiação , Animais , Embrião de Galinha , Interações Medicamentosas/efeitos da radiação , Eletroforese em Gel de Poliacrilamida , Vírus da Influenza A/análise , RNA Viral/análise , Ribonucleoproteínas/análise , Proteínas Virais/análiseRESUMO
Ultraviolet (UV) irradiation of type I poliovirus resulted in a modified (M) particle that had lost infectivity, lacked ability to adsorb to HeLa cells, lacked VP4, and reduced in S value. Additional irradiation resulted in the loss of VP2, further reduction in S value, and permeability of the capsid to RNAse, This particle (C) as well as M contain the genome. Acid pH (5.5-65) and sulfhydryl-reducing substances (dithiothreitol. reduced glutathione, and L-cysteine) inhibited UV-induced modification of the capsid. UV irradiation at alkaline pH (7.5-8.5) resulted in more extensive modification of the capsid than irradiation at neutral pH. Ionic compounds were found to inhibit the modifying reaction.