RESUMO
A recently developed variational Bayesian analysis using pattern recognition and machine learning of single viral ribonucleoprotein (RNP) particle tracks in the cytoplasm of living cells provides a quantitative molecular explanation for active diffusion, a concept previously "explained" largely by hypothetical models based on indirect analyses such as continuum microrheology. Machine learning shows that vesicular stomatitis virus (VSV) RNP particles are temporarily confined to dynamic traps or pores made up of cytoskeletal elements. Active diffusion occurs when the particles escape from one trap to a nearby trap. In this paper, we demonstrate that actin filament disruption increased RNP mobility by increasing trap size. Inhibition of nonmuscle myosin II ATPase decreased mobility by decreasing trap size. Trap sizes were observed to fluctuate with time, dependent on nonmuscle myosin II activity. This model for active diffusion is likely to account for the dominant motion of other viral and cellular elements. IMPORTANCE RNA virus ribonucleoproteins (RNPs) are too large to freely diffuse in the host cytoplasm, yet their dominant motions consist of movements in random directions that resemble diffusion. We show that vesicular stomatitis virus (VSV) RNPs overcome limitations on diffusion in the host cytoplasm by hopping between traps formed in part by actin filaments and that these traps expand and contract by nonmuscle myosin II ATPase activity. ATP-dependent random motion of cellular particles has been termed "active diffusion." Thus, these mechanisms are applicable to active diffusion of other cellular and viral elements.
Assuntos
Citoesqueleto de Actina , Ribonucleoproteínas , Vírus da Estomatite Vesicular Indiana , Proteínas Virais , Adenosina Trifosfatases , Trifosfato de Adenosina , Animais , Teorema de Bayes , Humanos , Miosina Tipo II/metabolismo , Transporte Proteico , RNA Viral/genética , Ribonucleoproteínas/genética , Vírus da Estomatite Vesicular Indiana/genética , Proteínas Virais/genéticaRESUMO
The difficulty of glioblastoma treatment makes it a good candidate for novel therapies, such as oncolytic viruses. Vesicular stomatitis virus expressing Lassa virus glycoprotein (Lassa-VSV) showed significant promise in animal models using established glioblastoma cell lines. These experiments were to determine the susceptibility of low-passage, patient-derived cell lines to Lassa-VSV oncolysis. Four patient-derived glioblastoma cell lines were infected with Lassa-VSV that expresses green fluorescent protein (GFP) and analyzed by fluorescence microscopy, flow cytometry, and cell viability assays. Cells were also analyzed as tumorspheres containing primarily glioma stem-like cells. Three low-passage, patient-derived cells were further analyzed with RNA sequencing (RNA-seq). Individual cell lines varied somewhat in their levels of viral gene expression and time course of Lassa-VSV-induced cell death, but each was susceptible to Lassa-VSV. Brain Tumor Center of Excellence (BTCOE) 4765 cells had the highest level of expression of interferon-stimulated genes but were most susceptible to Lassa-VSV-induced cell death, indicating that more susceptible cells do not necessarily have lower interferon pathway activation. Cells cultured as tumorspheres and infected with Lassa-VSV also showed variable susceptibility to Lassa-VSV, but BTCOE 4765 cells were least susceptible. Thus, patient-derived brain tumor cells show variable responses to Lassa-VSV infection, but each of the lines was susceptible to VSV oncolysis.
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A key principle of oncolytic viral therapy is that many cancers develop defects in their antiviral responses, making them more susceptible to virus infection. However, some cancers display resistance to viral infection. Many of these resistant cancers constitutively express interferon-stimulated genes (ISGs). The goal of these experiments was to determine the role of two tumor suppressor genes, MAP3K7 and CHD1, in viral resistance and ISG expression in PC3 prostate cancer cells resistant to oncolytic vesicular stomatitis virus (VSV). MAP3K7 and CHD1 are often co-deleted in aggressive prostate cancers. Silencing expression of MAP3K7 and CHD1 in PC3 cells increased susceptibility to the matrix (M) gene mutant M51R-VSV, as shown by increased expression of viral genes, increased yield of progeny virus, and reduction of tumor growth in nude mice. Silencing MAP3K7 alone had a greater effect on virus susceptibility than did silencing CHD1. Silencing MAP3K7 and CHD1 decreased constitutive expression of ISG mRNAs and proteins, whereas silencing MAP3K7 alone decreased expression of ISG proteins, but actually increased expression of ISG mRNAs. These results suggest a role for the protein product of MAP3K7, transforming growth factor ß-activated kinase 1 (TAK1), in regulating translation of ISG mRNAs and a role of CHD1 in maintaining the transcription of ISGs.
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The DNA genome of eukaryotic cells is compacted by histone proteins within the nucleus to form chromatin. Nuclear-replicating viruses such as adenovirus have evolved mechanisms of chromatin manipulation to promote infection and subvert host defenses. Epigenetic factors may also regulate persistent adenovirus infection and reactivation in lymphoid tissues. In this review, we discuss the viral proteins E1A and protein VII that interact with and alter host chromatin, as well as E4orf3, which separates host chromatin from sites of viral replication. We also highlight recent advances in chromatin technologies that offer new insights into virus-directed chromatin manipulation. Beyond the role of chromatin in the viral replication cycle, we discuss the nature of persistent viral genomes in lymphoid tissue and cell lines, and the potential contribution of epigenetic signals in maintaining adenovirus in a quiescent state. By understanding the mechanisms through which adenovirus manipulates host chromatin, we will understand new aspects of this ubiquitous virus and shed light on previously unknown aspects of chromatin biology.
Assuntos
Infecções por Adenovirus Humanos/metabolismo , Adenovírus Humanos/patogenicidade , Cromatina/virologia , Epigênese Genética , Proteínas E1A de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/metabolismo , Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Cromatina/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Replicação ViralRESUMO
UNLABELLED: The distribution of vesicular stomatitis virus (VSV) nucleocapsids in the cytoplasm of infected cells was analyzed by scanning confocal fluorescence microscopy using a newly developed quantitative approach called the border-to-border distribution method. Nucleocapsids were located near the cell nucleus at early times postinfection (2 h) but were redistributed during infection toward the edges of the cell. This redistribution was inhibited by treatment with nocodazole, colcemid, or cytochalasin D, indicating it is dependent on both microtubules and actin filaments. The role of actin filaments in nucleocapsid mobility was also confirmed by live-cell imaging of fluorescent nucleocapsids of a virus containing P protein fused to enhanced green fluorescent protein. However, in contrast to the overall redistribution in the cytoplasm, the incorporation of nucleocapsids into virions as determined in pulse-chase experiments was dependent on the activity of actin filaments with little if any effect on inhibition of microtubule function. These results indicate that the mechanisms by which nucleocapsids are transported to the farthest reaches of the cell differ from those required for incorporation into virions. This is likely due to the ability of nucleocapsids to follow shorter paths to the plasma membrane mediated by actin filaments. IMPORTANCE: Nucleocapsids of nonsegmented negative-strand viruses like VSV are assembled in the cytoplasm during genome RNA replication and must migrate to the plasma membrane for assembly into virions. Nucleocapsids are too large to diffuse in the cytoplasm in the time required for virus assembly and must be transported by cytoskeletal elements. Previous results suggested that microtubules were responsible for migration of VSV nucleocapsids to the plasma membrane for virus assembly. Data presented here show that both microtubules and actin filaments are responsible for mobility of nucleocapsids in the cytoplasm, but that actin filaments play a larger role than microtubules in incorporation of nucleocapsids into virions.
Assuntos
Citoesqueleto de Actina/metabolismo , Citoplasma/virologia , Microtúbulos/metabolismo , Nucleocapsídeo/metabolismo , Vírus da Estomatite Vesicular Indiana/metabolismo , Montagem de Vírus , Citoesqueleto de Actina/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Núcleo Celular/virologia , Citocalasina D/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Demecolcina/farmacologia , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Microscopia Eletrônica de Varredura/métodos , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , Nucleocapsídeo/ultraestrutura , Fosfoproteínas/genética , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/genética , Proteínas Virais/efeitos dos fármacos , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/genética , Vírion/efeitos dos fármacos , Vírion/metabolismo , Montagem de Vírus/efeitos dos fármacosRESUMO
Comparing the distribution of cytoplasmic particles and organelles between different experimental conditions can be challenging due to the heterogeneous nature of cell morphologies. The border-to-border distribution method was created to enable the quantitative analysis of fluorescently labeled cytoplasmic particles and organelles of multiple cells from images obtained by confocal microscopy. The method consists of four steps: (1) imaging of fluorescently labeled cells, (2) division of the image of the cytoplasm into radial segments, (3) selection of segments of interest, and (4) population analysis of fluorescence intensities at the pixel level either as a function of distance along the selected radial segments or as a function of angle around an annulus. The method was validated using the well-characterized effect of brefeldin A (BFA) on the distribution of the vesicular stomatitis virus G protein, in which intensely labeled Golgi membranes are redistributed within the cytoplasm. Surprisingly, in untreated cells, the distribution of fluorescence in Golgi membrane-containing radial segments was similar to the distribution of fluorescence in other G protein-containing segments, indicating that the presence of Golgi membranes did not shift the distribution of G protein towards the nucleus compared to the distribution of G protein in other regions of the cell. Treatment with BFA caused only a slight shift in the distribution of the brightest G protein-containing segments which had a distribution similar to that in untreated cells. Instead, the major effect of BFA was to alter the annular distribution of G protein in the perinuclear region.
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Técnicas Citológicas/métodos , Citoplasma/metabolismo , Organelas/metabolismo , Autoantígenos/metabolismo , Brefeldina A/metabolismo , Núcleo Celular/metabolismo , Fluorescência , Células HeLa , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Envelope Viral/metabolismoRESUMO
Credible but conflicting reports address the frequency of prenatal infection by species C adenovirus. This question is important because these viruses persist in lymphoid cells and suppress double-stranded DNA-break repair. Consequently, prenatal adenovirus infections may generate the aberrant clones of lymphocytes that precede development of childhood acute lymphoblastic leukemia (ALL). The present study was designed to overcome technical limitations of prior work by processing cord blood lymphocytes within a day of collection, and by analyzing sufficient numbers of lymphocytes to detect adenovirus-containing cells at the lower limits determined by our previous studies of tonsil lymphocytes. By this approach, adenoviral DNA was identified in 19 of 517 (3.7%) samples, providing definitive evidence for the occurrence of prenatal infection with species C adenoviruses in a significant fraction of neonates predominantly of African American and Hispanic ancestry. Cord blood samples were also tested for the presence of the ETV6-RUNX1 translocation, the most common genetic abnormality in childhood ALL. Using a nested PCR assay, the ETV6-RUNX1 transcript was detected in four of 196 adenovirus-negative samples and one of 14 adenovirus-positive cord blood samples. These findings indicate that this method will be suitable for determining concordance between adenovirus infection and the leukemia-associated translocations in newborns.
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Infecções por Adenoviridae/genética , Infecções por Adenoviridae/virologia , Adenoviridae/genética , DNA Viral/genética , Sangue Fetal/virologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/virologia , Adenoviridae/classificação , Adolescente , Adulto , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Feminino , Humanos , Recém-Nascido , Linfócitos/virologia , Masculino , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética , Adulto JovemRESUMO
UNLABELLED: Infection with adenovirus triggers the cellular DNA damage response, elements of which include cell death and cell cycle arrest. Early adenoviral proteins, including the E1B-55K and E4orf3 proteins, inhibit signaling in response to DNA damage. A fraction of cells infected with an adenovirus mutant unable to express the E1B-55K and E4orf3 genes appeared to arrest in a mitotic-like state. Cells infected early in G1 of the cell cycle were predisposed to arrest in this state at late times of infection. This arrested state, which displays hallmarks of mitotic catastrophe, was prevented by expression of either the E1B-55K or the E4orf3 genes. However, E1B-55K mutant virus-infected cells became trapped in a mitotic-like state in the presence of the microtubule poison colcemid, suggesting that the two viral proteins restrict entry into mitosis or facilitate exit from mitosis in order to prevent infected cells from arresting in mitosis. The E1B-55K protein appeared to prevent inappropriate entry into mitosis through its interaction with the cellular tumor suppressor protein p53. The E4orf3 protein facilitated exit from mitosis by possibly mislocalizing and functionally inactivating cyclin B1. When expressed in noninfected cells, E4orf3 overcame the mitotic arrest caused by the degradation-resistant R42A cyclin B1 variant. IMPORTANCE: Cells that are infected with adenovirus type 5 early in G1 of the cell cycle are predisposed to arrest in a mitotic-like state in a p53-dependent manner. The adenoviral E1B-55K protein prevents entry into mitosis. This newly described activity for the E1B-55K protein appears to depend on the interaction between the E1B-55K protein and the tumor suppressor p53. The adenoviral E4orf3 protein facilitates exit from mitosis, possibly by altering the intracellular distribution of cyclin B1. By preventing entry into mitosis and by promoting exit from mitosis, these adenoviral proteins act to prevent the infected cell from arresting in a mitotic-like state.
Assuntos
Proteínas E1B de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Adenovírus Humanos/fisiologia , Interações Hospedeiro-Patógeno , Pontos de Checagem da Fase M do Ciclo Celular , Replicação Viral , HumanosRESUMO
Adenoviral infection is a major risk factor for otitis media. We hypothesized that adenovirus promotes bacterial ascension into the middle ear through the disruption of normal function in the Eustachian tubes due to inflammation-induced changes. An intranasal infection model of the chinchilla was used to test the ability of type 5 adenovirus to promote middle ear infection by Streptococcus pneumoniae. The hyperinflammatory adenovirus mutant dl327 and the nonreplicating adenovirus mutant H5wt300ΔpTP were used to test the role of inflammation and viral replication, respectively, in promotion of pneumococcal middle ear infection. Precedent infection with adenovirus resulted in a significantly greater incidence of middle ear disease by S. pneumoniae as compared to nonadenovirus infected animals. Infection with the adenovirus mutant dl327 induced a comparable degree of bacterial ascension into the middle ear as did infection with the wild-type virus. By contrast, infection with the nonreplicating adenovirus mutant H5wt300ΔpTP resulted in less extensive middle ear infection compared to the wild-type adenovirus. We conclude that viral replication is necessary for adenoviral-induced pneumococcal middle ear disease.
Assuntos
Infecções por Adenoviridae/patologia , Adenoviridae/fisiologia , Orelha Média/patologia , Otite Média/patologia , Infecções Pneumocócicas/patologia , Streptococcus pneumoniae/crescimento & desenvolvimento , Replicação Viral , Infecções por Adenoviridae/virologia , Animais , Coinfecção/microbiologia , Coinfecção/patologia , Coinfecção/virologia , Modelos Animais de Doenças , Orelha Média/microbiologia , Orelha Média/virologia , Otite Média/microbiologia , Otite Média/virologia , Infecções Pneumocócicas/microbiologia , CoelhosRESUMO
Adenovirus inundates the productively infected cell with linear, double-stranded DNA and an abundance of single-stranded DNA. The cellular response to this stimulus is antagonized by the adenoviral E1B and E4 early genes. A mutant group C adenovirus that fails to express the E1B-55K and E4orf3 genes is unable to suppress the DNA-damage response. Cells infected with this double-mutant virus display significant morphological heterogeneity at late times of infection and frequently contain fragmented nuclei. Nuclear fragmentation was due to the translocation of apoptosis inducing factor (AIF) from the mitochondria into the nucleus. The release of AIF was dependent on active poly(ADP-ribose) polymerase-1 (PARP-1), which appeared to be activated by viral DNA replication. Nuclear fragmentation did not occur in AIF-deficient cells or in cells treated with a PARP-1 inhibitor. The E1B-55K or E4orf3 proteins independently prevented nuclear fragmentation subsequent to PARP-1 activation, possibly by altering the intracellular distribution of PAR-modified proteins.
Assuntos
Proteínas E1B de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Fator de Indução de Apoptose/antagonistas & inibidores , Interações Hospedeiro-Patógeno , Proteínas Oncogênicas/metabolismo , Replicação Viral , Animais , Linhagem Celular , DNA Viral/metabolismo , Humanos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
Human adenovirus can establish latent infections in lymphoid tissues in vivo and persistent, infections in cultured lymphoid cell lines. During lytic infection, adenovirus expresses microRNAs (miRNAs) derived from the viral non-coding RNAs VAI and, especially, VAII. Here, we demonstrate that persistently adenovirus-infected human BJAB cells also produce adenovirus-derived miRNAs primarily derived from the viral VAII RNA, which contributes ~2.7% of all RNA-induced silencing complex (RISC)-associated RNAs. However, our data indicate that the 5' end of the predominant VAII-derived viral RNA, and hence its seed sequence, differs from what has been previously reported. Our data demonstrate that adenovirus expresses viral miRNAs in chronically infected lymphoid cells and raise the possibility that these may contribute to the maintenance of the latently adenovirus-infected lymphoid cells previously observed in mucosal-associated lymphoid tissues in vivo.
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Adenovírus Humanos/fisiologia , Linfócitos/virologia , MicroRNAs/biossíntese , MicroRNAs/genética , RNA Viral/genética , RNA Viral/metabolismo , Latência Viral , Adenovírus Humanos/genética , Linhagem Celular , Inativação Gênica , Humanos , Modelos Moleculares , Conformação de Ácido NucleicoRESUMO
Individuals with HIV infection and two apolipoprotein L1 gene (APOL1) risk variants frequently develop nephropathy. Here we tested whether non-HIV viral infections influence nephropathy risk via interactions with APOL1 by assessing APOL1 genotypes and presence of urine JC and BK polyoma virus and plasma HHV6 and CMV by quantitative polymerase chain reaction. We analyzed 300 samples from unrelated and related first-degree relatives of African Americans with nondiabetic nephropathy using linear and nonlinear mixed models to account for familial relationships. The four groups evaluated were APOL1 zero/one versus two risk alleles, with or without nephropathy. Urine JCV and BKV were detected in 90 and 29 patients, respectively, whereas HHV6 and CMV were rare. Adjusting for family age at nephropathy, gender, and ancestry, presence of JCV genomic DNA in urine and APOL1 risk alleles were significantly negatively associated with elevated serum cystatin C, albuminuria (albumin-to-creatinine ratio over 30 mg/g), and kidney disease defined as an eGFR under 60 ml/min per 1.73 m(2) and/or albuminuria in an additive (APOL1 plus JCV) model. BK viruria was not associated with kidney disease. Thus, African Americans at increased risk for APOL1-associated nephropathy (two APOL1 risk variants) with JC viruria had a lower prevalence of kidney disease, suggesting that JCV interaction with APOL1 genotype may influence kidney disease risk.
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Apolipoproteínas/genética , Negro ou Afro-Americano/genética , Vírus JC/isolamento & purificação , Nefropatias/genética , Nefropatias/virologia , Lipoproteínas HDL/genética , Infecções por Polyomavirus/virologia , Infecções Tumorais por Vírus/virologia , Adulto , Idoso , Albuminúria/etnologia , Albuminúria/genética , Albuminúria/virologia , Apolipoproteína L1 , Distribuição de Qui-Quadrado , Cistatina C/sangue , DNA Viral/urina , Feminino , Interação Gene-Ambiente , Predisposição Genética para Doença , Taxa de Filtração Glomerular , Humanos , Vírus JC/genética , Nefropatias/sangue , Nefropatias/etnologia , Nefropatias/fisiopatologia , Nefropatias/prevenção & controle , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Dinâmica não Linear , North Carolina/epidemiologia , Fenótipo , Infecções por Polyomavirus/etnologia , Prevalência , Fatores de Risco , Infecções Tumorais por Vírus/etnologiaRESUMO
BACKGROUND: This study evaluated the properties of scaffold derived from freeze-dried human Achilles tendon allograft for use in anterior cruciate ligament (ACL) reconstruction. Our hypothesis was that such an allograft could be processed using a method to remove cellular and infectious material, producing a cytocompatible, architecturally modified scaffold possessing tensile properties suitable for ACL reconstruction. METHODS: Fifty-two allografts were provided by a tissue bank. Twenty-one were used as controls to assess cellularity, DNA content, microarchitecture, porosity, cytocompatibility, and tensile properties in vitro (n = 13) and in vivo (n = 8). Thirty-one were processed to produce scaffolds that were similarly assessed for these properties in vitro (n = 23) and in vivo (n = 8). The elimination of added enveloped and nonenveloped viruses was also determined in vitro after each processing step. RESULTS: A subjective decrease in cellularity and a significant decrease in DNA content were observed in the scaffolds compared with the allografts from which they had been derived. The porosity was increased significantly, and the scaffolds were cytocompatible in vitro. Processing resulted in significantly increased elongation of the scaffolds (138% of the elongation of the unprocessed allograft) during tensile testing. No other significant differences in tensile properties were observed in vitro or in vivo. The number of infiltrating host cells and the depth to which those cells infiltrated were significantly greater in the scaffolds. No enveloped viruses and only two of 10(8) nonenveloped viruses were detected in the scaffolds after processing, corresponding to a sterility assurance level of 0.2 × 10(-7). CONCLUSIONS: Allografts were processed using a method that removed cellular and infectious material to produce a decellularized, cytocompatible, architecturally modified scaffold with tensile properties that differed minimally from those of human allograft tissue both in vitro and in vivo. The scaffold production process also resulted in an increase in porosity that led to increased cell infiltration in vivo.
Assuntos
Tendão do Calcâneo/diagnóstico por imagem , Tendão do Calcâneo/transplante , Alicerces Teciduais , Transplante Homólogo/métodos , Tendão do Calcâneo/citologia , Ligamento Cruzado Anterior/citologia , Ligamento Cruzado Anterior/cirurgia , Ligamento Cruzado Anterior/ultraestrutura , Materiais Biocompatíveis , DNA/ultraestrutura , Liofilização , Humanos , Técnicas In Vitro , RNA/ultraestrutura , Resistência à Tração , Alicerces Teciduais/virologia , UltrassonografiaRESUMO
Vesicular stomatitis virus (VSV) is a promising oncolytic agent against a variety of cancers. However, it has never been tested in any pancreatic cancer model. Pancreatic ductal adenocarcinoma (PDA) is the most common and aggressive form of pancreatic cancer. In this study, the oncolytic potentials of several VSV variants were analyzed in a panel of 13 clinically relevant human PDA cell lines and compared to conditionally replicative adenoviruses (CRAds), Sendai virus and respiratory syncytial virus. VSV variants showed oncolytic abilities superior to those of other viruses, and some cell lines that exhibited resistance to other viruses were successfully killed by VSV. However, PDA cells were highly heterogeneous in their susceptibility to virus-induced oncolysis, and several cell lines were resistant to all tested viruses. Resistant cells showed low levels of very early VSV RNA synthesis, indicating possible defects at initial stages of infection. In addition, unlike permissive PDA cell lines, most of the resistant cell lines were able to both produce and respond to interferon, suggesting that intact type I interferon responses contributed to their resistance phenotype. Four cell lines that varied in their permissiveness to VSV-ΔM51 and CRAd dl1520 were tested in mice, and the in vivo results closely mimicked those in vitro. While our results demonstrate that VSV is a promising oncolytic agent against PDA, further studies are needed to better understand the molecular mechanisms of resistance of some PDAs to oncolytic virotherapy.
Assuntos
Carcinoma Ductal Pancreático/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/fisiologia , Neoplasias Pancreáticas/terapia , Vírus da Estomatite Vesicular Indiana/fisiologia , Animais , Carcinoma Ductal Pancreático/imunologia , Linhagem Celular Tumoral , Humanos , Interferon Tipo I/imunologia , Masculino , Camundongos , Camundongos Nus , Terapia Viral Oncolítica/instrumentação , Vírus Oncolíticos/genética , Neoplasias Pancreáticas/imunologia , Vírus da Estomatite Vesicular Indiana/genéticaRESUMO
Species C adenovirus establishes a latent infection in lymphocytes of the tonsils and adenoids. To understand how this lytic virus is maintained in these cells, four human lymphocytic cell lines that support the entire virus life cycle were examined. The T-cell line Jurkat ceased proliferation and died shortly after virus infection. BJAB, Ramos (B cells), and KE37 (T cells) continued to divide at nearly normal rates while replicating the virus genome. Viral genome numbers peaked and then declined in BJAB cells below one genome per cell at 130 to 150 days postinfection. Ramos and KE37 cells maintained the virus genome at over 100 copies per cell over a comparable period of time. BJAB cells maintained the viral DNA as a monomeric episome. All three persistently infected cells lost expression of the cell surface coxsackie and adenovirus receptor (CAR) within 24 h postinfection, and CAR expression remained low for at least 340 days postinfection. CAR loss proceeded via a two-stage process. First, an initial loss of cell surface staining for CAR required virus late gene expression and a CAR-binding fiber protein even while CAR protein and mRNA levels remained high. Second, CAR mRNA disappeared at around 30 days postinfection and remained low even after virus DNA was lost from the cells. At late times postinfection (day 180), BJAB cells could not be reinfected with adenovirus, even when CAR was reintroduced to the cells via retroviral transduction, suggesting that the expression of multiple genes had been stably altered in these cells following infection.
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Infecções por Adenoviridae/virologia , Adenoviridae/fisiologia , Linfócitos/virologia , Latência Viral , Adenoviridae/genética , Linhagem Celular , Genoma Viral , Humanos , Modelos BiológicosRESUMO
During a productive infection, species C adenovirus reprograms the host cell to promote viral translation at the expense of cellular translation. The E1B 55-kilodalton (E1B-55K) and E4 open reading frame 6 (E4orf6) proteins are important in this control of gene expression. As part of a ubiquitin-protein ligase, these viral proteins stimulate viral mRNA export, inhibit cellular mRNA export, promote viral gene expression, and direct the degradation of certain host proteins. We report here that the E1B-55K and E4orf6 proteins limited phosphorylation of eIF2alpha and the activation of the eIF2alpha kinase PKR. Phospho-eIF2alpha levels were observed to rise and fall at least twice during infection. The E1B-55K and E4orf6 proteins prevented a third increase at late times of infection. PKR appeared to phosphorylate eIF2alpha only in the absence of E1B-55K/E4orf6 function. PKR activation and eIF2alpha phosphorylation was unrelated to the cytoplasmic levels of the adenovirus inhibitor of PKR, VA-I RNA. Nonetheless, expression of a PKR inhibitor, the reovirus double-stranded RNA-binding protein sigma 3, prevented PKR activation and eIF2alpha phosphorylation. The sigma 3 protein largely corrected the defect in viral late protein synthesis associated with the E1B-55K and E4orf6 mutant viruses without affecting cytoplasmic levels of the late viral mRNA. The ubiquitin-protein ligase activity associated with the E1B-55K/E4orf6 complex was necessary to prevent activation of PKR and phosphorylation of eIF2alpha. These findings reveal a new contribution of the E1B-55K/E4orf6 complex to viral late protein synthesis and the existence of multiple layers of regulation imposed on eIF2alpha phosphorylation and PKR activation in adenovirus-infected cells.
Assuntos
Infecções por Adenoviridae/metabolismo , Adenoviridae/genética , Proteínas E1B de Adenovirus/fisiologia , Fator de Iniciação 2 em Eucariotos/genética , Fases de Leitura Aberta , Adenoviridae/metabolismo , Proteínas E1B de Adenovirus/genética , Citoplasma/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Mutação , Fosforilação , Reoviridae/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares , eIF-2 Quinase/metabolismoRESUMO
Adenovirus infection activates cellular DNA damage response and repair pathways. Viral proteins that are synthesized before viral DNA replication prevent recognition of viral genomes as a substrate for DNA repair by targeting members of the sensor complex composed of Mre11/Rad50/NBS1 for degradation and relocalization, as well as targeting the effector protein DNA ligase IV. Despite inactivation of these cellular sensor and effector proteins, infection results in high levels of histone 2AX phosphorylation, or gammaH2AX. Although phosphorylated H2AX is a characteristic marker of double-stranded DNA breaks, this modification was widely distributed throughout the nucleus of infected cells and was coincident with the bulk of cellular DNA. H2AX phosphorylation occurred after the onset of viral DNA replication and after the degradation of Mre11. Experiments with inhibitors of the serine-threonine kinases ataxia telangiectasia mutated (ATM), AT- and Rad3-related (ATR), and DNA protein kinase (DNA-PK), the kinases responsible for H2AX phosphorylation, indicate that H2AX may be phosphorylated by ATR during a wild-type adenovirus infection, with some contribution from ATM and DNA-PK. Viral DNA replication appears to be the stimulus for this phosphorylation event, since infection with a nonreplicating virus did not elicit phosphorylation of H2AX. Infected cells also responded to high levels of input viral DNA by localized phosphorylation of H2AX. These results are consistent with a model in which adenovirus-infected cells sense and respond to both incoming viral DNA and viral DNA replication.
Assuntos
Adenovírus Humanos/fisiologia , Replicação do DNA , DNA Viral/biossíntese , Histonas/metabolismo , Replicação Viral , Infecções por Adenovirus Humanos/virologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Proteína Homóloga a MRE11 , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Clinical trials have shown oncolytic adenoviruses to be tumor selective with minimal toxicity toward normal tissue. The virus ONYX-015, in which the gene encoding the early region 1B 55-kDa (E1B-55K) protein is deleted, has been most effective when used in combination with either chemotherapy or radiation therapy. Therefore, improving the oncolytic nature of tumor-selective adenoviruses remains an important objective for improving this form of cancer therapy. Cells infected during the G(1) phase of the cell cycle with the E1B-55K deletion mutant virus exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less efficiently killed than cells infected during the S phase. Here we demonstrate that the G(1) restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1). E4orf1 has been reported to signal through the phosphatidylinositol 3'-kinase pathway leading to the activation of Akt, mTOR, and p70 S6K. Evidence presented here shows that E4orf1 may also induce the phosphorylation of Akt and p70 S6K in a manner that depends on Rac1 and its guanine nucleotide exchange factor Tiam1. Accordingly, agents that have been reported to disrupt the Tiam1-Rac1 interaction or to prevent phosphorylation of the ribosomal S6 kinase partially alleviated the E4orf1 restriction to late viral protein synthesis and enhanced tumor cell killing by the E1B-55K mutant virus. These results demonstrate that E4orf1 limits the oncolytic nature of a conditionally replicating adenovirus such as ONYX-015. The therapeutic value of similar oncolytic adenoviruses may be improved by abrogating E4orf1 function.
Assuntos
Adenoviridae/crescimento & desenvolvimento , Deleção de Genes , Proteínas Oncogênicas Virais/metabolismo , Vírus Oncolíticos/crescimento & desenvolvimento , Adenoviridae/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Proteína Oncogênica v-akt/metabolismo , Proteínas Oncogênicas Virais/genética , Vírus Oncolíticos/genética , Fosforilação , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Vacinas Virais , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The localization of the adenovirus E1B-55K-E4orf6 protein complex is critical for its function. Prior studies demonstrated that E4orf6 directs the nuclear localization of E1B-55K in human cells and in rodent cells that contain part of human chromosome 21. We show here that the relevant activity on chromosome 21 maps to RUNX1. RUNX1 proteins are transcription factors that serve as scaffolds for the assembly of proteins that regulate transcription and RNA processing. After transfection, the RUNX1a, RUNX1b, and RUNX1-DeltaN variants allowed E4orf6-directed E1B-55K nuclear localization. The failure of RUNX1c to allow nuclear colocalization was relieved by the deletion of amino-terminal residues of this protein. In the adenovirus-infected mouse cell, RUNX1 proteins were localized to discrete structures about the periphery of viral replication centers. These sites are enriched in viral RNA and RNA-processing factors. RUNX1b and RUNX1a proteins displaced E4orf6 from these sites. The association of E1B-55K at viral replication centers was enhanced by the RUNX1a and RUNX1b proteins, but only in the absence of E4orf6. In the presence of E4orf6, E1B-55K occurred in a perinuclear cytoplasmic body resembling the aggresome and was excluded from the nucleus of the infected mouse cell. We interpret these findings to mean that a dynamic relationship exists between the E4orf6, E1B-55K, and RUNX1 proteins. In cooperation with E4orf6, RUNX1 proteins are able to modulate the localization of E1B-55K and even remodel virus-specific structures that form at late times of infection. Subsequent studies will need to determine a functional consequence of the interaction between E4orf6, E1B-55K, and RUNX1.
Assuntos
Proteínas E1B de Adenovirus/fisiologia , Proteínas E4 de Adenovirus/metabolismo , Cromossomos Humanos Par 21/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , DNA Viral/biossíntese , RNA Viral/biossíntese , Replicação Viral/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Linhagem Celular Tumoral , Mapeamento Cromossômico , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Técnica Indireta de Fluorescência para Anticorpo , Componentes do Gene , Humanos , Camundongos , Replicação Viral/genéticaRESUMO
Adenoviruses (Ads) are small DNA tumor viruses that have played a pivotal role in understanding eukaryotic cell biology and viral oncogenesis. Among other cellular pathways, Ad usurps cell cycle progression following infection. Likewise, progression of the viral infection is influenced by the host cell cycle. We describe here methods developed for synchronizing dividing cell populations and for analysis of cell cycle synchrony by flow cytometry. Furthermore, three methods used to evaluate the outcome of Ad infection in synchronized cell populations are described. These include two assays for infectious centers and an assay for analyzing production of progeny virus by transmission electron microscopy. These methods have been used to demonstrate that Ads that fail to direct synthesis of the E1B 55-kDa or E4orf6 proteins replicate most effectively upon infecting cells in S phase.