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At least 955 records · Page 53Linked to original sources
PubMed1979

Protein synthesized early after infection is linked to the termini of adenovirus type 2 DNA synthesized in vivo and in vitro.

The human adenovirus DNA genome contains a protein (CBP, or covalently bound protein) linked to each 5' terminus. To assess whether CBP is synthesized early, infected cells were incubated with hydroxyurea from 1 to 18 h postinfection, the hydroxyurea was removed, cycloheximide was added, and viral DNA was labeled with [3H]thymidine from 18 to 23 h postinfection. Removal of hydroxyurea at 18 h postinfection permits the synthesis of viral DNA, whereas cycloheximide maintains the block in late viral protein synthesis. Three lines of evidence are presented to show that viral 3H-labeled DNA prepared by this procedure was linked to CBP: (I) the DNA sedimented more rapidly than protein-free DNA (i.e., protinase treated) in neutral sucrose gradients containing guanidine hydrochloride; (ii) the DNA banded at a lower density than protein-free DNA in CsCl gradients containing guanidine hydrochloride; and (iii) neither the 3H-labeled DNA nor the end fragments produced by EcoRI digestion entered a 1.4% agarose gel during electrophoresis. These experiments are strong evidence that CBP is not a product of a late viral gene and is therefore the product of either an early viral gene or a cell gene. Experiments were performed to test whether CBP is attached to viral DNA synthesized in vitro by a soluble complex that synthesizes exclusively viral DNA as completed viral genomes in vitro. In vitro-labeled DNA was analyzed by velocity sedimentation, equilibrium sedimentation, and agarose gel electrophoresis as described above. Our results indicate that the majority of in vitro-synthesized DNA molecules were attached to CBP. These results, which indicate that CBP is synthesized early after infection and is attached to viral DNA labeled in vitro by a soluble replication complex, are consistent with the idea that CBP may play a role in viral DNA replication.

Adenoviruses, Human↗
PubMed2004

Binding of fluorescent dye to genomic RNA inside intact human rhinovirus after viral capsid penetration investigated by capillary electrophoresis.

RiboGreen is used for concentration measurements of RNA. Upon binding to the RNA, an approximately 1000-fold increase in sensitivity in comparison with the UV absorbance of the free polynucleotide is observed. In the present work, we demonstrate that this dye can penetrate in a time- and temperature-dependent manner the intact viral capsids of human rhinovirus serotypes 2 and 14, where it forms a fluorescent complex with the viral RNA. Capillary electrophoresis with laser-induced fluorescence detection of virus incubated with RiboGreen shows that the electrophoretic mobility of the viruses remained unchanged upon dye-binding. As shown for human rhinovirus serotype 2, its native conformation was conserved, since it still bound a recombinant soluble receptor fragment derived from the very low density lipoprotein receptor. The labeled RNA was released by heat-induced uncoating of the virus, and the RNA-dye complex could be directly detected if degradation was prevented with an RNase inhibitor. This in vitro labeling of viral RNA encased within a protein shell demonstrates the virion's dynamic nature that temporarily allows access of a low-molecular-mass compound to the otherwise protected RNA. It might be of great value for experiments requiring fluorescent viral particles with an unmodified surface, such as investigations of endocytosis and viral uncoating on the single molecule level.

Capsid Proteins↗
PubMed1983

Association between transplantation antigens and a viral membrane protein synthesized from a mammalian expression vector.

Cells infected or transformed by adenovirus-2 express complexes of the viral E19 protein and class I transplantation antigens. To eliminate the complication of the numerous metabolic changes in adenovirus-infected and -transformed cells, we detached the E19 gene from its viral background by constructing transient expression vectors where the E19 coding sequence is flanked by the SV40 early promoter and the 3' region of the rabbit beta-globulin gene. E19 production was assayed 2 days after transfection of monkey COS or human HeLa cells with vector DNA. Efficient E19 gene expression depends on the presence of an intron (the second beta-globin intron), and the E19 protein is properly processed and anchored in the plasma membrane of transfected cells. The vector-synthesized E19 protein is also associated with class I transplantation antigens in human cells, since a fraction of it is coprecipitated both with antiserum against HLA antigens and with monoclonal antibodies against beta 2-microglobulin.

Adenoviridae↗
PubMed1990

Different type of hepatitis B virus (HBV) DNA integrants that may reflect the integration process.

Through analyses of HBV DNA integratns in human cellular DNA, we identified three different integrant types, each of which may reflect the process of primary integrant formation by the viral DNA. The first type, which we call "simple type" consists of integrants found in some hepatocellular carcinomas (HCC's). The structure of the viral genome is simple, and part of it is deleted. The viral cohesive end sequence appears at one of the viral-cellular DNA junctions, and integration has elicited a microdeletion in the target cellular DNA sequence. This structure suggests viral DNA replication intermediates as substrates for integration. Judging from its frequency in HCC, this type may represent the most preferred one, if not all, among the primary integration products. The second type, which we call "complex type" is essentially the same as the first type, except tht the viral genome structure is complex. We considered the possibility that they may have been produced via the same process, using preformed complex viral genomes such as "novel form DNA's" (Rogler and Summers, 1982) as substrates. In cultured fetal hepatocytes, integration of HBV DNA can occur only a few days after infection. Among such integrants, we found a third type integrant, having a simple viral genome, but having a larger cellular DNA deletion. We propose that different forms of viral DNA may be used as substrates in the integration process, and the process is characterized by its eliciting of deletions of different size in the target cellular DNA. The most preferred substrate may be the one producing the simple type integrants, and the most frequently occurring deletion in the target DNA may be the microdeletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1977

Simplified radioimmunoassay for viral antigens: use of Staphylococcus aureus as an adsorbent for antigen-antibody complexes.

In a rapid method for the radioimmunoassay (RIA) of viral antigens, Staphylococcus aureus was used as the adsorbent for antigen-antibody complexes ("protein A" molecules on the cell walls of certain strains of staphylococci have a strong affinity toward IgG molecules). The results showed that this method could be used instead of the double-antibody technique, with the same or probably higher sensitivity in precipitation as well as competition RIA's.

Adsorption↗
PubMed1999

Genome activation in alfamo- and ilarviruses.

Alfamo- and ilarviruses are characterized by the deficiency of their genomes (three messenger-sense RNAs) to start an infection cycle. The RNAs are in capsids built from a single species of protein of about 24 kD. A few dimers of this coat protein per RNA molecule are sufficient to activate the genome. Since the first description of genome activation [Bol JF, van Vloten-Doting L, Jaspars EMJ (1971) Virology 46: 73-85] three models have been proposed concerning its mechanism: the protection, the replicase and the messenger release hypotheses. The first two models make use of the fact that in these genera of RNA viruses the 3' termini of the RNAs bind the coat protein very strongly. The resulting structure would provide protection against 3'- to 5' exoribonucleases, or would permit correct initiation of minus-strand synthesis, respectively. However, naked inoculated RNAs of alfalfa mosaic virus appear to be quite stable in the cell, and in vitro the coat protein is inhibiting rather than stimulating initiation of minus-strand synthesis. The messenger release hypothesis states that the coat protein is needed for the release of viral messenger RNAs from membranous replication complexes throughout the whole viral replication cycle. This is supported by in vivo and in vitro observations, but as yet a detailed molecular mechanism is difficult to give.

Alfamovirus↗
PubMed2005

Retroviral DNA integration--mechanism and consequences.

Integration of retroviral cDNA into the host cell chromosome is an essential step in its replication. This process is catalyzed by the retroviral integrase protein, which is conserved among retroviruses and retrotransposons. Integrase binds viral and host DNA in a complex, called the preintegration complex (PIC), with other viral and cellular proteins. While the PIC is capable of directing integration of the viral DNA into any chromosomal location, different retroviruses have clear preferences for integration in or near particular chromosomal features. The determinants of integration site selection are under investigation but may include retrovirus-specific interactions between integrase and tethering factors bound to the host cell chromosomes. Research into the mechanisms of retroviral integration site selection has shed light on the phenomena of insertional mutagenesis and viral latency.

DNA↗
PubMed1981

The release of N-acetyl- and N-glycolloyl-neuraminic acid from soluble complex carbohydrates and erythrocytes by bacterial, viral and mammalian sialidases.

A series of substrates, sialyl(2 leads to 6)GalNAc and ganglioside GM3, containing either N-acetylneuraminic acid (AcNeu) or N-glycolloylneuraminic acid (GcNeu), has been prepared. The trisaccharide GcNeu(2 leads to 3)lactose was preapred by ozonolysis of GcNeu-GM3, and the disaccharides AcNeu(2 leads to 6)GalNAc and GcNeu(2 leads to 6)GalNAc were isolated from bovine submandibular-gland mucin by alkali elimination. Sialidases from Newcastle-disease virus, fowl-plague virus, influenza virus A2, Clostridium perfringens, Vibrio cholerae, Arthrobacter ureafaciens and human liver lysosomes were studied with the above substrates and all showed poorer cleavage of GcNeu-containing substrates when compared with the corresponding AcNeu-containing compounds. This was reflected in the Km and Vmax. values of these sialidases. Differences between viral and bacterial sialidases could be detected on the basis of their kinetic constants and time curves of sialic acid release. Preferred release of AcNeu relative to GcNeu was also observed with bovine submandibular gland mucin and a mixture of human and porcine erythrocytes, macromolecular substrates containing both AcNeu and GcNeu. The significance of differential cleavage of AcNeu and GcNeu by sialidases is considered together with examples of the role of GcNeu in physiologicaL systems.

Animals↗
PubMed1994

Somatic gene therapy for cystic fibrosis.

There is considerable potential to ameliorate the pulmonary disease in cystic fibrosis (CF) using somatic gene therapy. Even low levels of expression of the gene in airways epithelium may be beneficial. Adenoviral vectors, DNA-liposome complexes, adeno-associated viral vectors and DNA-ligand complexes have been used effectively in vitro and have been tested in animals to varying extent. Adenoviral vectors and DNA-liposome complexes are being used to deliver the CF gene to patient airways in phase I clinical trials. Transient correction of the electrophysiological defect in human CF nasal epithelium has been achieved. Major goals are (i) to demonstrate that expression of the CF gene in airways epithelium will ameliorate lung disease in CF patients, and (ii) to achieve long-term expression of the introduced gene either through a single delivery with persistent expression or through the ability to use a delivery system repetitively with safety and efficacy.

Cystic Fibrosis↗
PubMed1995

Antiviral protective immunity induced by major histocompatibility complex class I molecule-restricted viral T-lymphocyte epitopes inserted in various positions in immunologically self and nonself proteins.

Injection into mice of chimeric proteins consisting of a portion of either the simian virus 40 large tumor antigen or nonstructural protein 1 of influenza A virus or of the murine tumor suppressor p53 on one hand and T-cell epitopes of lymphocytic choriomeningitis virus on the other resulted in antiviral protective immunity, which was independent of the epitopes' position in the protein and the same whether the latter was immunologically nonself or self. Mice of different haplotypes were protected when the corresponding class I molecule-restricted epitopes had been inserted close to each other in one carrier protein.

Amino Acid Sequence↗
PubMed1997

Genetic dissection of interaction between poliovirus 3D polymerase and viral protein 3AB.

Poliovirus RNA-dependent RNA polymerase 3D and viral protein 3AB are both thought to be required for the initiation of RNA synthesis. These two proteins physically associate with each other and with viral RNA replication complexes found on virus-induced membranes in infected cells. An understanding of the interface between 3D and 3AB would provide a first step in visualizing the architecture of the multiprotein complex that is assembled during poliovirus infection to replicate and package the viral RNA genome. The identification of mutations in 3D that diminish 3D-3AB interactions without affecting other functions of 3D polymerase is needed to study the function of the 3D-3AB interaction in infected cells. We describe the use of the yeast two-hybrid system to isolate and characterize mutations in 3D polymerase that cause it to interact less efficiently with 3AB than wild-type polymerase. One mutation, a substitution of leucine for valine at position 391 (V391L), resulted in a 3AB-specific interaction defect in the two-hybrid system, causing a reduction in the interaction of 3D polymerase with 3AB but not with another viral protein or a host protein tested. In vitro, purified 3D-V391L polymerase bound to membrane-associated 3AB with reduced affinity. Poliovirus that contained the 3D-V391L mutation was temperature sensitive, displaying a pronounced conditional defect in RNA synthesis. We conclude that interaction between 3AB and 3D or 3D-containing polypeptides plays a role in RNA synthesis during poliovirus infection.

Alleles↗
PubMed2002

An efficient and versatile mammalian viral vector system for major histocompatibility complex class I/peptide complexes.

We report a Sendai virus (SeV) vector system for expression of major histocompatibility complex (MHC) class I/peptide complexes. We cloned the extracellular domain of a human MHC class I heavy chain, HLA-A*2402, and human beta-2 microglobulin (beta2m) fused with HLA-A*2402-restricted human immunodeficiency virus type 1 (HIV-1) cytotoxic T-lymphocyte (CTL) epitopes (e-beta2m) in separate SeV vectors. When we coinfected nonhuman mammalian cells with the SeVs, naturally folded human MHC class I/peptide complexes were secreted in the culture supernatants. Biotin binding peptide sequences on the C terminus of the heavy chain were used to tetramerize the complexes. These tetramers made in the SeV system recognized specific CD8-positive T cells in peripheral blood mononuclear cells of HIV-1-positive patients with a specificity and sensitivity similar to those of MHC class I tetramers made in an Escherichia coli system. Solo infection of e-beta2m/SeV produced soluble e-beta2m in the culture supernatant, and cells pulsed with the soluble protein were recognized by specific CTLs. Furthermore, when cells were infected with e-beta2m/SeV, these cells were recognized by the specific CTLs more efficiently than the protein pulse per se. SeV is nonpathogenic for humans, can transduce foreign genes into nondividing cells, and may be useful for immunotherapy to enhance antigen-specific immune responses. Our system can be used not only to detect but also to stimulate antigen-specific cellular immune responses.

Animals↗
PubMed2001

Diverse repertoire of the MHC class II-peptide complexes is required for presentation of viral superantigens.

Among other features, peptides affect MHC class II molecules, causing changes in the binding of bacterial superantigens (b-Sag). Whether peptides can alter binding of viral superantigens (v-Sag) to MHC class II was not known. Here we addressed the question of whether mutations limiting the diversity of peptides bound by the MHC class II molecules influenced the presentation of v-Sag and, subsequently, the life cycle of the mouse mammary tumor virus (MMTV). T cells reactive to v-Sag were found in mice lacking DM molecules as well as in A(b)Ep-transgenic mice in which MHC class II binding grooves were predominantly occupied by an invariant chain fragment or Ealpha(52-68) peptide, respectively. APCs from the mutant mice failed to present v-Sag, as determined by the lack of Sag-specific T cell activation, Sag-induced T cell deletion, and by the aborted MMTV infection. In contrast, mice that express I-A(b) with a variety of bound peptides presented v-Sag and were susceptible to MMTV infection. Comparison of v-Sag and b-Sag presentation by the same mutant cells suggested that presentation of v-Sag had requirements similar to that for presentation of toxic shock syndrome toxin-1. Thus, MHC class II peptide repertoire is critical for recognition of v-Sag by the T cells and affects the outcome of infection with a retrovirus.

Animals↗
PubMed1990

Effect of cellular density and viral oncogenes on the major histocompatibility complex class I antigen response to gamma-interferon in BALB-c/3T3 cells.

Cellular density in culture has profound effects on major histocompatibility complex (MHC) class I antigen expression in BALB/c-3T3 cells. Cells which have been confluent for greater than 24 h demonstrate a 2- to 6-fold increase in MHC class I antigen expression compared to subconfluent cells. These density-associated changes in MHC class I antigen expression occur both in untransformed and in v-mos or v-rasKi-transformed cells. The density-associated increases are specific for MHC class I antigens and do not occur with the cytoskeletal antigen actin. Transformation of the BALB/c-3T3 cells by either v-rasKi or v-mos has little or no direct effect on MHC class I expression under standard culture conditions. However, both oncogenes can indirectly alter the enhancement of MHC class I antigen expression in response to gamma-interferon. Incubation of untransformed BALB/c-3T3 cells with gamma-interferon leads to greater relative and absolute increases in MHC class I antigen expression in confluent cells than it does in subconfluent cells. In contrast, in v-rasKi- and v-mos-transformed cells, the subconfluent cells have a greater increase in MHC class I antigen expression in response to gamma-interferon than the cells which have exceeded monolayer confluence. The dense v-rasKi- and v-mos-transformed BALB/c-3T3 cell cultures are able to deplete their medium of the exogenous gamma-interferon, and this depletion of gamma-interferon causes the increase in the MHC class I antigen expression to be less sustained with lower peak expressions than the expression found in subconfluent cells. Supplementation with additional gamma-interferon can restore the full enhancement of MHC class I antigen in the transformed BALB/c-3T3 cells. The v-mos- and v-rasKi-transformed cells are more likely to deplete their medium of exogenous gamma-interferon because these cells can exceed monolayer confluence and thus achieve 10-fold higher densities than the untransformed BALB/c-3T3 cells. At high cellular densities, untransformed cells can partially deplete their medium of exogenous gamma-interferon, but this phenomenon is generally less pronounced than in the transformed cells.

Animals↗