RESUMO
We have produced human papillomavirus type 16 E7 protein in a bacterial expression system and examined the mitogenic activity of this protein in Swiss 3T3 cells after scrape loading. The ability of E7 to induce cellular DNA synthesis in quiescent mouse fibroblasts is strongly enhanced by the presence of a single growth factor such as insulin. Although only weakly mitogenic, introduction of E7 alone resulted in the rapid induction of the transcriptionally active form of E2F, which was not enhanced further by the addition of insulin. Mutant E7 proteins defective for RB binding failed to induce the active form of E2F or act synergistically with insulin to stimulate DNA synthesis. The ability of E7 to regulate E2F may therefore be necessary, but is not sufficient, for full induction of DNA synthesis.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Mitose , Proteínas Oncogênicas Virais/farmacologia , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Sequência de Bases , Replicação do DNA , Fatores de Transcrição E2F , Técnicas In Vitro , Insulina/farmacologia , Camundongos , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/imunologia , Proteínas E7 de Papillomavirus , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1RESUMO
A monoclonal antibody defines an antigen, p68, related to hsp70, which is located in nuclei of uninfected exponential cells. Nuclear p68 is released by DNase but not RNase treatment suggesting an association with DNA. Lytic productive infection of confluent quiescent BHK 21 cells with herpes simplex virus type-2 causes p68 to accumulate in nuclei. The effect is specific for HSV-2, and does not occur in HSV-1 infected cells. Maximum nuclear accumulation of p68 requires virus DNA synthesis although a significant accumulation occurs in the absence of such synthesis. It is suggested that the nuclear accumulation of p68 is an aspect of a cellular stress response to lytic infection with HSV-2.
Assuntos
Antígenos/biossíntese , Núcleo Celular/metabolismo , Proteínas de Choque Térmico/biossíntese , Herpes Simples/metabolismo , Animais , Antígenos/genética , Linhagem Celular , Replicação do DNA , DNA Viral/biossíntese , Proteínas de Ligação a DNA/biossíntese , Escherichia coli/metabolismo , Imunofluorescência , Proteínas de Choque Térmico/genética , Técnicas Imunológicas , Simplexvirus/metabolismo , Simplexvirus/fisiologia , Replicação ViralRESUMO
Cell cultures derived from 60 different human brain tumors were screened for the presence of HSV infected cell antigens by indirect immunofluorescence using a polyclonal rabbit antiserum reacting with herpes simplex virus (HSV), 3 monoclonal antibodies recognising different HSV-specified proteins, and one monoclonal antibody T181 reacting with a DNA binding protein present in HSV-infected cells. Only one tumor (IN/157), derived from an oligodendroglioma, stained with the polyclonal antiserum. T181 but none of the other monoclonal antibodies used also specifically reacted with IN/157 cells. High levels of the T181-defined protein were detected using immunoblotting in HSV-1 infected BHK/21 cells but not in IN/157 cells. T181 may react with either an epitope shared between two different molecules in HSV-1 infected and IN/157 cells or a cell-specified polypeptide that is upregulated after HSV-1 infection.
Assuntos
Antígenos Virais/análise , Neoplasias Encefálicas/análise , Oligodendroglioma/análise , Simplexvirus/imunologia , Linhagem Celular , Epitopos/análise , Histocitoquímica , Humanos , Técnicas ImunológicasRESUMO
Monoclonal antibodies reacting with the herpes simplex virus (HSV)-encoded major DNA-binding protein defined an intracellular filamentous network. This network was associated predominantly with the infected cell nucleus and occurred in cells infected with HSV type 2. It did not co-distribute with microfilaments, microtubules or intermediate filaments, and DNA synthesis was required for its formation. We suggest explanations for the occurrence and function of this novel filamentous network structure.
Assuntos
Proteínas de Ligação a DNA/análise , Fibroblastos/ultraestrutura , Simplexvirus/análise , Proteínas Virais/análise , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Núcleo Celular/ultraestrutura , Cricetinae , Citoesqueleto/ultraestrutura , Replicação do DNA , DNA Viral/biossíntese , Proteínas de Ligação a DNA/imunologia , Fibroblastos/microbiologia , Imunofluorescência , Rim , Mesocricetus , Simplexvirus/imunologia , Proteínas Virais/imunologia , Replicação ViralRESUMO
Herpes simplex virus-1 (HSV-1) infection of cultured human neural cells causes the accumulation of a host cell-encoded nuclear protein identified as a 57,000 mol.wt. stress protein by monoclonal antibody TI56. This protein is cell cycle-related in fibroblast, may mediate host cell control during HSV infection and could play a role in the regulation of HSV latency.
Assuntos
Proteínas de Choque Térmico/metabolismo , Herpes Simples/metabolismo , Anticorpos Monoclonais , Feto , Fibroblastos , Imunofluorescência , Gânglios Espinais , Humanos , Medula EspinalRESUMO
Antigenic determinants common to distinct proteins may be unambiguously identified by the use of monoclonal antibodies. Some monoclonal antibodies to mammalian neurofilaments have recently been shown to cross-react with the neurofibrillary tangles found at high density in the brains of senile dements with Alzheimers disease (SDAT). Here, we show that these antibodies also cross-react with chromatin proteins, including the linker histones H1 and H1(0). Elevated levels of histone H1(0) have also been reported in SDAT brains.
Assuntos
Cromatina/análise , Citoesqueleto/imunologia , Epitopos/análise , Doença de Alzheimer/patologia , Animais , Anticorpos Monoclonais , Encéfalo/patologia , Reações Cruzadas , Imunofluorescência , Histonas/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
A monoclonal antibody reacts with a polypeptide of 68 000 mol. wt. (p68) that accumulates to high levels during heat shock. The intracellular distribution of this antigen in normal and heat-shocked cells has been studied. It is a major component of non-stressed cells, where it is located predominantly in the cytoplasm, but also occurs in the nucleus. The nuclear accumulation is growth regulated, in that exponentially growing cells have strong nuclear immunofluorescence and confluent cells little. It is concentrated at the leading edge of motile fibroblasts and co-distributes with actin-containing microfilaments. Heat shock causes cytoplasmic and nuclear accumulation and there is new deposition in the periphery of cells. In normal cells the antigen in the nucleus is located in the nuclear lamina and matrix which increases during heat shock. The distribution of this molecule and the structures with which it interacts suggests that it is important in mediating the effects of heat shock.
Assuntos
Anticorpos Monoclonais/imunologia , Núcleo Celular/análise , Proteínas de Choque Térmico/análise , Animais , Antígenos/análise , Células Cultivadas , Cricetinae , Citoesqueleto/análise , Embrião de Mamíferos , Fibroblastos/análise , Proteínas de Choque Térmico/imunologia , Proteínas de Filamentos Intermediários/análise , Mesocricetus , Camundongos , Microtúbulos/análise , RatosAssuntos
Sequência de Bases , Regulação da Expressão Gênica , Neoplasias/genética , Humanos , OncogenesRESUMO
A monoclonal antibody, produced from mice immunized with a herpes simplex virus (HSV)-infected cell extract, reacts with a molecule which is present in uninfected cells and which accumulates in large amounts during HSV 2 infection. In uninfected cells this molecule is growth regulated, in that exponentially growing cells have intense nuclear immunofluorescence, whereas confluent quiescent cells have little. It has a mol. wt. of 57 000 (p57) in exponential cells, and one of 61 000 (p61) in quiescent cells. In HSV 2-infected cells, p57 accumulates and nuclear and cytoplasmic immunofluorescence increases. In uninfected cells, p57 also accumulates during heat-shock treatment, and this is associated with a new immunofluorescence throughout the cytoplasm. We suggest that HSV 2 infection induces a cellular stress response which is involved in the shut-off of host cell polypeptide synthesis.