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At least 109 records · Page 6Linked to original sources
PubMed1993

Increase in the basal transcriptional activity of the human foamy virus internal promoter by the homologous long terminal repeat promoter in cis.

The human foamy or spumaretrovirus HFV is a complex and exogenous retrovirus that encodes several bel genes besides the three classical retroviral genes gag, pol, and env. HFV was recently reported to contain two functionally active promoters that are both strongly trans-activated by the HFV trans-activator protein Bel 1. The occurrence of a second internal cap site underscores the complexity of the HFV genome. We have analysed whether there is interference between the HFV long terminal repeat promoter and the internal promoter located in the 3' end of env upstream of the bel genes. Recombinant clones were constructed that carry two different indicator genes, one under the control of the U3 promoter, the other under the control of the internal promoter. The portion of the basal transcriptional activity of the internal promoter that is not trans-activated by Bel 1 was increased two- to threefold in the presence of the long terminal repeat promoter. The rate of trans-activation by Bel 1 of both HFV promoters was not altered in these constructs.

Animals↗
PubMed2003

The hepatitis C virus internal ribosome entry site facilitates efficient protein synthesis in blood vessel endothelium during tumour angiogenesis.

The development of gene delivery systems for therapeutic use involves vectors (often retrovirus or adenovirus) which typically encode one target protein, but the use of internal ribosome entry sites (IRES) can confer the ability to express more than one protein from bi- or polycistronic mRNAs. IRES elements can display tissue-specific expression, so it is necessary to determine suitable IRES for specific clinical applicability. Blood vessel endothelial cells are important clinically since many different conditions involve neo-vascularisation (angiogenesis). We have demonstrated that the viral hepatitis C IRES element is a powerful mediator of protein synthesis in angiogenesis, such as found in solid tumours. Homologous recombination was used to introduce IRES-lacZ sequences into the Lmo2 gene, which is expressed in endothelial cells. beta-Galactosidase expression was determined during vascular remodelling in mouse embryos and in sprouting endothelium during growth of solid tumours, and showed that the hepatitis C IRES is used efficiently for protein synthesis in endothelial cells. This IRES element can provide the means to express two or more therapeutic genes in blood vessel endothelium in clinical conditions, such as cancer, which depend on angiogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed1993

Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment.

Adenovirus contains a heterodimeric protein complex consisting of 186 kd fiber protein that mediates high affinity virus attachment to cells and a 400 kd pentavalent subunit (penton base) that contains five Arg-Gly-Asp sequences, implying a role for integrins in adenovirus infection. We demonstrate that the vitro-nectin-binding integrins alpha v beta 3 and alpha v beta 5 promote viral infection in a novel way since antibodies against these receptors or soluble penton base block virus internalization without affecting attachment. Moreover, adenovirus binds to cultured cells lacking alpha v integrins but fail to become internalized, thus restricting infection of these cells. Transfection of alpha v(-) cells with a cDNA encoding alpha v results in the expression of integrins alpha v beta 3 and alpha v beta 5 and allows virus internalization and infection. These data indicate that adenovirus attachment and uptake into cells are separate but cooperative events that result from the interaction of distinct viral coat proteins with a receptor for attachment and alpha v integrin receptors for internalization.

Adenoviruses, Human↗
PubMed1993

Use of FITC-labeled influenza virus and flow cytometry to assess binding and internalization of virus by monocytes-macrophages and lymphocytes.

The binding of influenza virus to the surface of cells and the internalization of virus particles by all or a subset of cells are key points in the pathogenesis of viral infection. The current studies established a method for discrimination of surface-bound from internalized influenza virus. Fluorescein isothiocyanate (FITC) was attached to the viral hemagglutinin and neuroaminidase proteins; the fluorescent virus retained infectivity. A flow cytometric technique was then adapted for study of virus-cell interactions, with addition of ethidium bromide to quench green fluorescence associated with FITC-labeled virus that was cell-bound but remained external. Ethidium bromide was excluded by intact cell membranes, and internalized virions retained green fluorescence. Cells could be examined by fluorescence microscopy or flow cytometry, with flow cytometry allowing rapid, kinetic assessment of large numbers of cells and subsets of virus-exposed cells. The data showed that, whereas a majority of both monocytes-macrophages and lymphocytes bound influenza virus, a large percentage of monocytes-macrophages but only a very small percentage of lymphocytes internalized the virus. This procedure provides a simple and effective method to distinguish surface-bound from internalized influenza virus, and allows precise kinetic analyses on large numbers of cells.

Adult↗
PubMed1985

Endocytosis in Saccharomyces cerevisiae: internalization of enveloped viruses into spheroplasts.

When vesicular stomatitis virus was incubated with Saccharomyces cerevisiae spheroplasts at 37 degrees C, part of the virus was internalized by the spheroplasts as shown by the following criteria. (i) The spheroplast-associated virus was protected from proteinase K digestion, which releases surface-bound virus by degrading the envelope glycoproteins. (ii) The spheroplast-associated virus was resistant to mild Triton X-100 treatment, which readily solubilizes the virus. The same results were obtained with Semliki Forest virus. Internalization of the two viruses followed linear kinetics up to 90 min at 37 degrees C. Internalization was concentration- and temperature-dependent. At 11 degrees C no uptake could be detected for at least 2 h. Homogenization and organelle fractionation protocols were designed for the S. cerevisiae spheroplasts to study the compartments into which the virions were internalized. Three compartments containing both marker viruses could be separated in density gradients. One coincided with vacuole markers, one banded at a slightly higher and one at a similar density to the plasma membrane markers. Thus, S. cerevisiae spheroplasts appear to have the capability of endocytosing particulate markers like viruses. The companion paper describes internalization of two soluble macromolecules, alpha-amylase and fluorescent dextran, into intact cells.

Cell Fractionation↗
PubMed1979

Amino-terminal sequence of bovine leukemia virus major internal protein: homology with mammalian type C virus p30 structural proteins.

The amino acid composition, the COOH-terminal amino acid, and the NH2-terminal amino acid sequence of the first 55 residues of the major internal structural protein, p24, of bovine leukemia virus (BLV) were determined. The compositional data and the results of end-group analysis revealed that, although BLV p24 is chemically distinct, it more closely resembles the p30 structural proteins than the other gag gene products of mammalian retroviruses. It was found that BLV p24 shares the common NH2-terminal proline and COOH-terminal leucine but lacks the common prolylleucylarginine tripeptide and the larger conserved region found near the NH2 terminus of all mammalian type C viral p30s. Alignment of the amino acid sequence of BLV p24 with the previously determined sequence of feline leukemia virus p27 revealed a statistically significant sequence homology. A more distant relationship was found between BLV p24 and other mammalian p30s. The finding of a definite sequence homology between BLV p24 and mammalian type C virus p30s clearly establishes the origin of these contemporary viral proteins from common progenitor genes.

Amino Acid Sequence↗
PubMed2001

Mechanisms of integrin-mediated virus attachment and internalization process.

Viruses that propagate within vertebrate hosts have adapted many strategies to infect host cells. One of the first steps in a viral infection is the binding of the virus to cell surface molecules. This interaction between a virus and its receptors plays a key role in the multiplication cycle. Entry of viruses into cells is a complex, multistep process, and for several viruses, cell attachment and internalization are distinct steps. A number of virus receptors have been identified; a common family of viral receptors is the integrin family. Integrins are a widely expressed family of cell adhesion receptors, by which cells attach to extracellular matrices; they also mediate important cell-cell adhesion events. Integrins are involved in a number of tissue remodeling events, including embryogenesis, angiogenesis, wound repair, and bone resorption. In addition, several integrins are used by manyviruses in theirinfectious cycle. Virus-integrin interactions maybe more complex than previously thought because several viruses can interact with unique integrin regions or can activate distinct signaling pathways. This article will discuss the strategies devised by many viruses in their integrin-mediated attachment or cell entry.

Adenoviridae↗
PubMed2004

Differential effects on the hepatitis C virus (HCV) internal ribosome entry site by vitamin B12 and the HCV core protein.

To investigate the role of the hepatitis C virus internal ribosome entry site (HCV IRES) domain IV in translation initiation and regulation, two chimeric IRES elements were constructed to contain the reciprocal domain IV in the otherwise HCV and classical swine fever virus IRES elements. This permitted an examination of the role of domain IV in the control of HCV translation. A specific inhibitor of the HCV IRES, vitamin B(12), was shown to inhibit translation directed by all IRES elements which contained domain IV from the HCV and the GB virus B IRES elements, whereas the HCV core protein could only suppress translation from the wild-type HCV IRES. Thus, the mechanisms of translation inhibition by vitamin B(12) and the core protein differ, and they target different regions of the IRES.

Hepacivirus↗
PubMed2000

Mutational analysis of the GB virus B internal ribosome entry site.

GB virus B (GBV-B) is a recently discovered hepatotropic flavivirus that is distantly related to hepatitis C virus (HCV). We show here that translation of its polyprotein is initiated by internal entry of ribosomes on GBV-B RNA. We analyzed the translational activity of dicistronic RNA transcripts containing wild-type or mutated 5' nontranslated GBV-B RNA (5'NTR) segments, placed between the coding sequences of two reporter proteins, in vitro in rabbit reticulocyte lysate and in vivo in transfected BT7-H cells. We related these results to a previously proposed model of the secondary structure of the GBV-B 5'NTR (M. Honda, et al. RNA 2:955-968, 1996). We identified an internal ribosome entry site (IRES) bounded at its 5' end by structural domain II, a location analogous to the 5' limit of the IRES in both the HCV and pestivirus 5'NTRs. Mutational analysis confirmed the structure proposed for domain II of GBV-B RNA, and demonstrated that optimal IRES-mediated translation is dependent on each of the putative RNA hairpins in this domain, including two stem-loops not present in the HCV or pestivirus structures. IRES activity was also absolutely dependent on (i) phylogenetically conserved, adenosine-containing bulge loops in domain III and (ii) the primary nucleotide sequence of stem-loop IIIe. IRES-directed translation was inhibited by a series of point mutations predicted to stabilize stem-loop IV, which contains the initiator AUG codon in its loop segment. A reporter gene was translated most efficiently when fused directly to the initiator AUG codon, with no intervening downstream GBV-B sequence. This finding indicates that the 3' limit of the GBV-B IRES is at the initiator AUG and that it does not require downstream polyprotein-coding sequence as suggested for the HCV IRES. These results show that the GBV-B IRES, while sharing a common general structure, differs both structurally and functionally from other flavivirus IRES elements.

5' Untranslated Regions↗
PubMed2005

Replacement of primary chicken embryonic fibroblasts (CEF) by the DF-1 cell line for detection of avian leucosis viruses.

International regulations prescribe that the absence of avian leucosis viruses (ALV) in avian live virus vaccines has to be demonstrated. Primary chicken embryo fibroblasts (CEF) from special SPF chicken lines are normally used for detection of ALV. The suitability of the DF-1 cell line for ALV-detection, as alternative for primary CEF, was studied in three types of experiments: (1) in titration experiments without cell passage, (2) in experiments with passages in cell cultures according to European Pharmacopoeia requirements, and (3) in experiments with commercial live avian vaccines that had been spiked with known amounts of ALV. In all tests the sensitivity of ALV-A and ALV-J detections on DF-1 cells was at least as high as on primary CEF. The sensitivity of ALV-B detection was always superior when DF-1 cells were used. ALV were detected earlier in all comparative tests when DF-1 cells were used. ALV-A, ALV-B and ALV-J all induced CPE on DF-1 cells, whereas no clear CPE was seen on CEF-cells. For reasons of sensitivity, standardisation as well as reduction of animal use, the data support the use of DF-1 cells to monitor absence of ALV in vaccine virus seed lots or finished products.

Animals↗
PubMed1993

Interaction of polypyrimidine tract binding protein with the encephalomyocarditis virus mRNA internal ribosomal entry site.

Translation of encephalomyocarditis virus (EMCV) mRNA occurs in a cap-independent manner, requiring instead a cis-acting element termed the internal ribosomal entry site (IRES). Binding of a 57-kDa ribosome-associated protein (p57) to the EMCV IRES has been found to correlate with cap-independent translation. p57 has recently been reported to be very similar, if not identical, to the polypyrimidine tract binding protein (pPTB), a spliceosome-associated factor possibly involved in U2 snRNP/pre-mRNA complex formation of 3'-splice-site recognition. The interaction between purified pPTB and the EMCV IRES was characterized in this study using nitrocellulose filter binding and UV cross-linking assays. pPTB bound the EMCV IRES with high affinity (Kd = 40 nM at 25 degrees C, pH 5.5, 80 mM ionic strength). pPTB also bound strongly to RNA fragments containing either the 5'-end, 3'-end, or an internal stem-loop of the IRES. The binding properties of 16 RNA variants derived from the IRES revealed however that purified pPTB bound with less specificity than pPTB in a mixture of cytoplasmic HeLa cell polypeptides. The addition of HeLa extract to purified pPTB increased the binding specificity, suggesting that factors within the extract alter the binding specificity of pPTB. The binding of pPTB to the full-length IRES and three IRES-derived fragments was studied in detail. Complex formation was optimal at low pH and was driven entirely by entropy. As many as four ion pairs are formed upon binding, with electrostatic interactions accounting for approximately 35% of the total free energy of complex formation.

Base Sequence↗
PubMedDate not supplied

Efficacy of recombinant fowl poxvirus vaccine in protecting chickens against a highly pathogenic Mexican-origin H5N2 avian influenza virus.

Internationally and nationally, governments and the poultry industries have used various strategies to control avian influenza (AI), ranging from a minimum of living with mildly pathogenic AI virus (AIV) infections to the other extreme of implementing a total quarantine-slaughter approach for eradication of highly pathogenic (HP) forms of the disease. However, recent economic considerations in various countries have prompted a broader reevaluation of vaccination as one of several tools to be used in AI control programs, including H5 and H7 HP AI. In the current study, 1-day-old chickens were immunized with a recombinant fowl poxvirus vaccine containing a hemagglutinin gene insert (Vector-HA) from an H5 AIV. Vector-HA- and negative control (vector-control)-vaccinated chicks were challenged with a HP H5N2 AIV isolated from chickens in Mexico. All immunized chickens were antibody negative on the agar gel precipitin test, indicating that vaccination would not interfere with routine AI serologic surveillance programs in the United States. However, in the hemagglutinin-inhibition test, a few immunized chickens (8%) had low serologic titers. Protection against illness (90-100%) and death (90-100%) was provided by the vector-HA vaccine from 3 wk of age to the end of the 20-wk study. The number of chickens shedding the challenge AIV from their enteric tracts was significantly reduced (50-75%) and the quantity of challenge AIV shed from respiratory and enteric tracts was significantly reduced (10(1)-10(2.1) mean embryo lethal dose/ml) in most vector-HA vaccine groups when compared with vector-control groups. Furthermore, vector-HA vaccination reduced in contact transmission of HP AI challenge virus to both vector-HA- and vector-control-vaccinated chickens. These findings indicate the recombinant fowl poxvirus vaccine can be a useful tool in an AI control program by preventing illness and death in chickens and reducing intestinal and respiratory shedding of H5 AIV. However, for an AI control program to be successful, enhanced biosecurity and surveillance must be practiced, and the vaccine's use must be controlled by an industry and/or government task force.

Animals↗
PubMed1988

The expression of novel antigens from the Epstein-Barr virus large internal repeat.

A large Epstein-Barr virus (EBV) B95-8 genomic bank has been prepared in an Escherichia coli expression vector and screened with a pool of sera from human infectious mononucleosis patients. Four immunopositive clones which also contained sequences from the viral large internal repeat were selected. DNA sequence analysis has located them on the repeat sequence and shown that they come from three potential open reading frames and that two of them consist of overlapping reading frames. This must imply extensive intron/exon splicing or that the repeat itself encodes several different proteins. The four expressed epitopes were shown to be present simultaneously in independent cases of infectious mononucleosis. These have not been previously described and based on the experimental design, they must reflect the situation in vivo.

Amino Acid Sequence↗