RESUMO
The CD4 glycoprotein, expressed on leukocytes belonging to subsets of T lymphocytes and to cells of monocyte/macrophage lineage, participates in the functioning of T cells and serves as a receptor for HIV-1 and HIV-2. Human eosinophils, a class of granulocytic leukocytes, have been found to express CD4. With anti-CD4 mAbs CD4 was demonstrable on eosinophils from both normal and eosinophilic donors. Eosinophils synthesized a 55-kD CD4 polypeptide immunoprecipitable with two anti-CD4 mAbs. Eosinophil CD4 bound HIV-1 gp120 as assessed by competition for anti-OKT4A, but not anti-OKT4, mAb binding. Eosinophils, normally rich in gastrointestinal and genitourinary tract tissues, increase in numbers in patients with metazoan parasitic infections. In these sites and diseases, CD4 expression by eosinophils may be pertinent to their immunologic functions and could make these cells susceptible to HIV infection.
Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Eosinófilos/metabolismo , HIV/metabolismo , Proteínas dos Retroviridae/metabolismo , Proteínas do Envelope Viral/metabolismo , Citometria de Fluxo , HumanosRESUMO
In the current study, we developed and validated a simple, rapid and safe in vivo model to test gene transfer and sensor function in vivo. Using the model, we tested the specific hypothesis that in vivo gene transfer of angiogenic factors at sites of biosensor implantation would induce neovascularization surrounding the sensor and thereby enhance biosensor function in vivo. As the in vivo site for testing of our gene transfer cell and biosensor function systems, the developing chorioallantoic membrane (CAM) of the embryo was utilized. Vascular endothelial cell growth factor (VEGF) was used as a prototype for angiogenic factor gene transfer. A helper-independent retroviral vector derived from Rous sarcoma virus (RSV), designated RCAS, was used for gene transfer of the murine VEGF (mVEGF) gene (mVEGF:RCAS) into the DF-1 chicken cell line (designated mVEGF:DF-1). Initially, the ability of VEGF:DF-1 cells to produce VEGF and RCAS viral vectors containing the mVEGF gene (mVEGF:RCAS) was validated in vitro and in vivo, as was the ability of the mVEGF:DF-1 cells to induce neovascularization in the ex ova CAM model. Using the system, we determined the ability of mVEGF:DF-1 cells to enhance acetaminophen sensor function in vivo, by inducing neovascularization at sites of sensor implantation in the ex ova CAM model. For these studies, acetaminophen sensors were placed on 8-day-old ex ova CAMs, followed by addition of media or cells (mVEGF:DF-1 cells or GFP:DF-1 cells) at the sites of biosensor implantation on the CAM. At 4 to 10 days after sensor placement, the biosensor function was determined by measuring sensor response to an intravenous injection of acetaminophen. Sensors implanted on CAMs with buffer or control cells (GFP:DF-1 cells) displayed no induced neovascularization around the sensor and had minimal/baseline sensor responses to intravenous acetaminophen injection (media, 133.33 +/- 27.64 nA; GFP:DF-1, 187.50 +/- 55.43 nA). Alternatively, the sensors implanted with mVEGF:DF-1 cells displayed massive neovascularization and equally massive sensor response to intravenous injection of acetaminophen (VEGF:DF-1, 1387.50 +/- 276.42 nA). These data clearly demonstrate that enhancing vessel density (i.e., neovascularization) around an implanted sensor dramatically enhances sensor function in vivo.
Assuntos
Técnicas Biossensoriais , Técnicas de Transferência de Genes , Implantes Experimentais , Neovascularização Fisiológica , Fator A de Crescimento do Endotélio Vascular/genética , Acetaminofen/metabolismo , Analgésicos não Narcóticos/metabolismo , Animais , Vírus do Sarcoma Aviário/genética , Vírus do Sarcoma Aviário/metabolismo , Linhagem Celular , Tamanho Celular , Embrião de Galinha , Membranas Extraembrionárias/irrigação sanguínea , Membranas Extraembrionárias/citologia , Membranas Extraembrionárias/metabolismo , Humanos , Camundongos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
A cloned herpes simplex virus type 1 DNA polymerase gene which is biologically functional was inserted into SP6 and T7 promoter-containing vectors for in vitro transcription-translation. pol-specific RNA synthesized in vitro will direct the synthesis of a 140-kilodalton polypeptide in rabbit reticulocyte lysates. RNAs prepared from pol templates linearized at internal restriction sites specified deleted polypeptides with sizes consistent with colinearity of the pol gene and the 140-kilodalton primary translation product. The in vitro translated pol gene product was enzymatically active, with salt resistance and sensitivity to acyclovir triphosphate, similar to the enzyme activity in crude extracts of herpes simplex virus type 1-infected Vero cells. An in-frame deletion of 78 residues (amino acids residues 881 to 959) was introduced into the expression vectors to investigate the function of a region of the polypeptide (amino acids residues 881 to 895) which is conserved in nine other DNA polymerases. In a complementation assay, this mutation abolished biological activity as well as the enzymatic activity of the in vitro translated product. A BAL31 mutation deleting the upstream open reading frame of pol had no effect on biological activity in a complementation assay but was found to increase the efficiency of in vitro translation of pol RNA. Two amino-terminal deletions of 27 and 67 residues were found to greatly enhance the enzymatic activity of the in vitro translated product, while all carboxy-terminal deletions examined (the smallest being 164 residues) abolished in vitro enzymatic activity. Expression of the 67-residue amino-terminal deleted pol gene in Escherichia coli, using a bacteriophage T7-based system, resulted in accumulation of large amounts of an insoluble fusion protein. An antiserum prepared against this fusion protein precipitated the 140-kilodalton DNA polymerase from herpes simplex virus type 1-infected cell lysates.
Assuntos
DNA Polimerase I/genética , Regulação da Expressão Gênica , Biossíntese de Proteínas , Simplexvirus/genética , Animais , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Genes Virais , Teste de Complementação Genética , Mutação , Plasmídeos , Simplexvirus/enzimologia , Moldes Genéticos , Transcrição Gênica , Células VeroRESUMO
In the 5 years since its release for clinical use, acyclovir (9-[2-hydroxyethoxymethyl]guanine) has proved to be a safe and effective agent for therapy of herpes simplex and varicella-zoster infections. The drug's availability in topical, oral, and intravenous preparations has allowed its use in a range of clinical situations. Acyclovir must be phosphorylated by viral thymidine kinase in infected cells, where it then acts to inhibit viral DNA replication specifically. Epstein-Barr virus and human cytomegalovirus infections do not seem to respond to acyclovir therapy, although in-vitro effects on these viruses may be seen. Acyclovir is well absorbed and distributed, with cerebrospinal fluid levels 50% that of plasma. Clearance is almost entirely by the renal route, with a half-life of 20 hours in the anuric patient. Acyclovir has an excellent safety profile, its major adverse effect being transient serum creatinine elevations during high-dose intravenous use. Major uses include treatment of primary and recurrent genital herpes and herpes encephalitis and prophyllaxis and therapy of mucocutaneous herpes and varicella-zoster infections in immunocompromised patients. Resistance to acyclovir in herpes simplex virus is rarely encountered and does not seem to be due to long-term chronic suppressive therapy.
Assuntos
Aciclovir , Aciclovir/efeitos adversos , Aciclovir/análogos & derivados , Aciclovir/farmacocinética , Aciclovir/farmacologia , Aciclovir/uso terapêutico , Varicela/tratamento farmacológico , Interações Medicamentosas , Resistência Microbiana a Medicamentos , Ganciclovir , Herpes Simples/tratamento farmacológico , Herpes Zoster/tratamento farmacológico , Herpesviridae/efeitos dos fármacos , Humanos , Complicações Pós-Operatórias/prevenção & controle , TransplanteRESUMO
Expression of the Epstein-Barr virus (EBV) DNA polymerase (EBVpol) open reading frame (BALF5) by in vitro transcription-translation yielded a 116-kDa primary translation product. Enzymatic DNA polymerase activity of the in vitro translated polypeptide required the presence of the 47-kDa BMRF1 (EA-D) gene product. Antiserum raised to the BALF5 gene product expressed in Escherichia coli specifically precipitated a 116-kDa polypeptide in extracts of latently infected lymphoblastoid cells induced for EBV replication. Immunofluorescence microscopy revealed colocalization of the EBVpol and EA-D(BMRF1) to discrete foci within the nuclei of induced cells; however, the blockade of viral DNA synthesis resulted in diffuse nuclear staining patterns for both antigens. Bromodeoxyuridine staining of these discrete foci colocalizing with EBVpol suggests that they are sites of early viral DNA synthesis. These observations suggest that EA-D(BMRF1) may be an accessory protein of the EBV DNA polymerase which colocalizes in vivo with EBVpol to sites of viral DNA replication and cooperates in vitro to form an active EBVpol holoenzyme.
Assuntos
Antígenos Virais/metabolismo , Núcleo Celular/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , Herpesvirus Humano 4/genética , Fosfoproteínas/metabolismo , Simplexvirus/genética , Replicação Viral , Antígenos Virais/análise , Linfoma de Burkitt , Linhagem Celular , DNA Viral/genética , Escherichia coli/genética , Imunofluorescência , Herpesvirus Humano 4/enzimologia , Herpesvirus Humano 4/fisiologia , Humanos , Fases de Leitura Aberta , Fosfoproteínas/análise , Plasmídeos , Biossíntese de Proteínas , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
Seven point mutations were introduced into region I of the herpes simplex virus type 1 DNA polymerase, which is most highly conserved among DNA polymerases and has no drug sensitivity markers mapped to it. The functional consequences of these mutations were studied in an in vitro transcription-translation system in which T7 transcripts of cloned polymerase genes were used to generate enzymatically active polypeptides in reticulocyte lysate. Analysis of labeled polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis failed to show any alterations of stability caused by these mutations. The mutations G885R, D886N, T887K, D888A, and G896V lacked polymerase activity and failed to be stimulated by cotranslation of the herpes simplex virus 65-kilodalton DNA-binding protein, whereas R881G and S889A retained both polymerase activity and the capacity to be stimulated by the 65-kilodalton DNA-binding protein.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Genes Virais , Mutação , Simplexvirus/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Polimerase Dirigida por DNA/metabolismo , Cinética , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Plasmídeos , Biossíntese de Proteínas , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Simplexvirus/enzimologia , Fagos T/genéticaRESUMO
Most point substitutions in the highly-conserved 885-GDTDS motif of the HSV-1 DNA polymerase inactivate polymerase elongation activity. However, in an assay system based on expression by in vitro transcription-translation, the mutant GDTDA (S889A) possessed wild-type elongation activity which was highly resistant to phosphonoacetic acid and acyclovir triphosphate, but retained sensitivity to aphidicolin.
Assuntos
Antivirais/farmacologia , Sequência Conservada , DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases/genética , Proteínas Virais , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Viral , DNA Polimerase Dirigida por DNA/metabolismo , Resistência Microbiana a Medicamentos/genética , Eletroforese em Gel de Poliacrilamida , Exodesoxirribonucleases/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação PuntualRESUMO
The Epstein-Barr virus (EBV) BMRF1 gene product is necessary for DNA polymerase catalytic subunit (BALF5) activity in 100 mM ammonium sulfate. To map regions of BMRF1 necessary for polymerase accessory function, linker insertion and deletion mutant BMRF1 polypeptides were expressed by in vitro transcription-translation and assayed for DNA polymerase elongation activity and binding to double-stranded DNA (dsDNA)-cellulose. Amino-terminal deletions up to residue 303 were defective for stimulation of elongation. Deletions between residues 44 and 194 and residues 238 and 303 abolished binding to dsDNA-cellulose. The region from residues 194 to 238, therefore, is necessary for stimulation of BALF5 elongation but dispensable for dsDNA-cellulose binding. Deletion analysis also localized reactive epitopes of two neutralizing monoclonal antibodies to BMRF1 to a carboxy-terminal region which is dispensable for activity. These data suggest that a bipartite DNA-binding region is an essential component of the DNA polymerase accessory function and that the two noncontiguous regions are separated by a region (residues 194 to 217) which is essential for stimulation; therefore, it may interact with the BALF5 catalytic subunit of EBV DNA polymerase. Both EBV BMRF1 and herpes simplex virus UL42 gene products are DNA polymerase accessory proteins which bind dsDNA and increase the processivity of their corresponding catalytic components. Outstanding similarities between their primary amino acid sequences are not evident. However, it appears that their structural organizations are similar.
Assuntos
Antígenos Virais/genética , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Herpesvirus Humano 4/genética , Proteínas Virais , Anticorpos Monoclonais , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Sequência de Bases , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mapeamento de Epitopos , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/química , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
Quantitation of HIV-1 in blood is now widely used by clinicians to manage antiviral therapy. Current methods to detect viral RNA are expensive, have slow turnaround times, and do not directly quantitate infectious particles. Indicator cell assay (ICA) methods for titering HIV-1 rely on the activation of HIV-1 long terminal repeat (LTR)-driven expression of a reporter gene by the viral tat gene product, which is expressed early in the course of infection. The Aequorea victoriana green fluorescent protein (GFP) has proven to be a useful reporter gene for detecting tat-mediated HIV-LTR activation. A general approach to developing a clinically useful ICA required a method of introducing the LTR-GFP expression cassette into various HIV1-infectable cell lines. The LTR-GFP expression cassette was inserted into the LXSN retrovector in a reverse orientation with respect to transcription from the 5' LTR. In cells transduced by the RH5 retrovector, GFP expression was tightly dependent on expression of HIV-1 tat. The PM1 human T-cell line was transduced with RH5 and was further engineered to express the CCR5 HIV-1 CD4 coreceptor constitutively. The resulting cell line, D5-R5, was susceptible to infection by primary HIV-1 strains, macrophage-tropic (M-tropic) and T-cell tropic (T-tropic) laboratory strains, and syncytium-inducing (SI) and and non-SI (NSI) variants. Four days after HIV-1 infection of the indicator cells, GFP expression was detected and quantitated by fluorescence activated cell sorter (FACS), without any false-positive signals. This GFP-based ICA method is of potential use in clinical management of HIV-1, especially in the detection and recovery of drug-resistant virus and the direct determination of antiviral drug sensitivities.
Assuntos
HIV-1/isolamento & purificação , Indicadores e Reagentes , Proteínas Luminescentes , Macrófagos/virologia , Linfócitos T/virologia , Linhagem Celular , Separação Celular , Citometria de Fluxo , Proteínas de Fluorescência Verde , Repetição Terminal Longa de HIV , HIV-1/patogenicidade , Células HeLa , Humanos , Microscopia de Fluorescência , TransfecçãoRESUMO
Several systems for the detection of HIV-1 have been described in which HIV-1-susceptible cells contain a reporter gene (chloramphenicol acetyltransferase, beta-galactosidase, or alkaline phosphatase) under the control of the HIV-1 long terminal repeat (LTR). Upon infection by HIV-1, the expression of the viral tat product increases transcription from the HIV-1 LTR promoter, leading to high-level expression of the reporter gene product. Previously described reporter systems require processing of the cells by lysis, fixation, or other steps following infection to detect the reporter gene product. In the present study, the Aequorea green fluorescent protein S65T variant (GFP-S65T) was used in a reporter system for detecting HIV-1. HeLa-CD4 cells transfected with the plasmid pRH1, which encodes GFP-S65T under the control of the HIV-1 LTR promoter, and either co-transfected with a plasmid encoding the HIV-1 tat product or superinfected with HIV-1, expressed high levels of GFP-S65T, which was readily detected by fluorescence microscopy and fluorescence-activated cell-sorting analysis. The advantages of this system include its simplicity, sensitivity, and ability to detect and sort live HIV-1-infected cells using readily available instruments. The construction of cell lines stably transfected with pRH1 will provide a tool for titering HIV-1 and sorting HIV-1-infected cells.
Assuntos
DNA Viral/genética , Genes Reporter/genética , Infecções por HIV/diagnóstico , HIV-1/genética , Proteínas Luminescentes , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Citometria de Fluxo , Genes tat/genética , Proteínas de Fluorescência Verde , Repetição Terminal Longa de HIV/genética , HIV-1/isolamento & purificação , Células HeLa/virologia , Humanos , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Microscopia de FluorescênciaRESUMO
The 65-kilodalton DNA-binding protein (65KDBP) of herpes simplex virus type 1 (HSV-1), the product of the UL42 gene, is required for DNA replication both in vitro and in vivo, yet its actual function is unknown. By two independent methods, it was shown that the 65KDBP stimulates the activity of the HSV-1-encoded DNA polymerase (Pol). When Pol, purified from HSV-1-infected cells, was separated from the 65KDBP, much of its activity was lost. However, addition of the 65KDBP, purified from infected cells, stimulated the activity of Pol 4- to 10-fold. The ability of a monoclonal antibody to the 65KDBP to remove the Pol-stimulating activity from preparations of the 65KDBP confirmed that the activity was not due to a trace contaminant. Furthermore, the 65KDBP did not stimulate the activity of other DNA polymerases derived from T4, T7, or Escherichia coli. The 65KDBP gene transcribed in vitro from cloned DNA and translated in vitro in rabbit reticulocyte lysates also was capable of stimulating the product of the pol gene when the RNAs were cotranslated. The product of a mutant 65KDBP gene missing the carboxy-terminal 28 amino acids exhibited wild-type levels of Pol stimulation, while the products of two large deletion mutants of the gene could not stimulate Pol activity. These experiments suggest that the 65KDBP may be an accessory protein for the HSV-1 Pol.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Genes Virais , Simplexvirus/enzimologia , Proteínas Estruturais Virais/genética , Animais , DNA Polimerase Dirigida por DNA/genética , Cinética , Peso Molecular , Mutação , Plasmídeos , Biossíntese de Proteínas , Simplexvirus/genética , Transcrição Gênica , Células VeroRESUMO
We have constructed a transformation-defective polyoma virus mutant (Py 1387-T) that directs the synthesis of a normal small tumor antigen, a functional large tumor antigen, and a truncated (51,000-dalton) middle-sized tumor (mT) antigen that lacks 37 amino acids at its COOH terminus. The shortened mT polypeptide is missing the hydrophobic "tail" thought to be responsible for the anchorage of this protein into the plasma membrane and is in fact in cytosol fractions. This truncated mT polypeptide is inactive in an in vitro protein kinase assay and is altered in its phosphorylation in vivo. Mutant 1387-T differs from wild-type virus in having a T.A base pair instead of a C.G base at nucleotide position 1387. This change was introduced into viral DNA by using a synthetic undecanucleotide as a specific mutagen. Wild-type polyoma DNA was rendered single stranded by molecular cloning into coliphage M13. The oligonucleotide, which hybridizes with a mismatch at the site to be altered, was used to prime the synthesis of double-stranded closed circular DNA. Progeny recombinant phage were screened by DNA sequence analysis for the desired base change. The polyoma mutant was reconstructed from recombinant phage replicative form DNA molecules containing the mutation.
Assuntos
Antígenos Virais/fisiologia , Transformação Celular Viral , Polyomavirus , Proteínas Quinases/metabolismo , Animais , Antígenos Virais de Tumores , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma , DNA Recombinante , Camundongos , Mutação , Fosfotirosina , Polyomavirus/imunologia , Relação Estrutura-Atividade , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Using indirect immunofluorescence, well-characterized monoclonal and polyclonal antibodies, and temperature-sensitive (ts) mutants of herpes simplex virus type 1, we demonstrated that the 65-kilodalton DNA-binding protein (65KDBP), the major DNA-binding protein (infected cell polypeptide 8 [ICP8]), and the viral DNA polymerase (Pol) colocalize to replication compartments in the nuclei of infected cells under conditions which permit viral DNA synthesis. When viral DNA synthesis was blocked by incubation of the wild-type virus with phosphonoacetic acid, the 65KDBP, Pol, and ICP8 failed to localize to replication compartments. Instead, ICP8 accumulated nearly exclusively to prereplication sites, while the 65KDBP was only diffusely localized within the nuclei. Although some of the Pol accumulated in prereplication sites occupied by ICP8 in the presence of phosphonoacetic acid, a significant amount of Pol also was distributed throughout the nuclei. Examination by double-labeling immunofluorescence of DNA- ts mutant virus-infected cells revealed that the 65KDBP also did not colocalize with ICP8 to prereplication sites at temperatures nonpermissive for virus replication. These results are in disagreement with the hypothesis that ICP8 is the major organizational protein responsible for attracting other replication protein to prereplication sites in preparation for viral DNA synthesis (A. de Bruyn Kops and D. M. Knipe, Cell 55:857-868, 1988), and they suggest that other viral proteins, perhaps in addition to ICP8, or replication fork progression per se are required to organize the 65KDBP.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/biossíntese , DNA Polimerase Dirigida por DNA/biossíntese , Simplexvirus/metabolismo , Animais , Anticorpos Monoclonais , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/análise , DNA Polimerase Dirigida por DNA/análise , Imunofluorescência , Peso Molecular , Mutação , Ácido Fosfonoacéticos/farmacologia , Simplexvirus/efeitos dos fármacos , Células VeroRESUMO
The herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene has been cloned into an Escherichia coli-yeast shuttle vector fused to the galactokinase gene (GAL-1) promoter. Genes controlled by the GAL-1 promoter are induced by galactose, uninduced by raffinose, and repressed by glucose. Cell extracts from a strain of Saccharomyces cerevisiae harboring this vector (Y-MH202, expresser cells) grown in the presence of galactose and assayed in high salt (100 mM ammonium sulfate) contained a novel DNA polymerase activity. No significant high-salt DNA polymerase activity was detected in extracts from expresser cells grown in the presence of raffinose or in extracts from control cells containing the E. coli-yeast shuttle vector without the HSV-1 DNA polymerase gene grown in the presence of raffinose of galactose. Immunoblot analysis of the cell extracts by using a polyclonal rabbit antiserum prepared against a highly purified HSV-1 DNA polymerase preparation revealed the specific induction of the HSV-1 approximately 140-kilodalton DNA polymerase polypeptide in expresser cells grown in galactose. Extracts from the same cells grown in raffinose or control cells grown in either raffinose or galactose did not contain this immunoreactive polypeptide. The high-salt DNA polymerase activity in the extracts from expresser cells grown in galactose was inhibited greater than 90% by either acyclovir triphosphate or aphidicolin, as expected for HSV-1 DNA polymerase. In addition, the high-salt polymerase enzyme activity could be depleted from extracts by immunoprecipitation by using purified immunoglobulin G from this same polyclonal rabbit antiserum. These results demonstrate the successful expression of functional HSV-1 DNA polymerase enzyme in S. cerevisiae.