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At least 217 records · Page 12Linked to original sources

PubMed2000

Construction of a recombinant adeno-associated virus (rAAV) vector expressing murine interleukin-12 (IL-12).

IL-12 is a heterodimeric cytokine that is known to induce tumor regression and long-term antitumor immunity. Recombinant adeno-associated virus (rAAV) vectors are advantageous for gene therapy in that they lack pathogenicity in humans, infect dividing as well as nondividing cells, and show a broad range of infectivity. We constructed an rAAV vector expressing interleukin-12 (IL-12) for cancer immunotherapy studies in a mouse model by inserting murine IL-12 (mIL-12) p35 and p40 cDNAs into the plasmid pRep4 and inserting the encephalomyocarditis virus internal ribosomal entry site between the p35 and p40 cDNAs. The mIL-12 expression cassette containing the Rous sarcoma virus promoter and a simian virus 40 polyadenylation signal was subcloned into the AAV plasmid p008Sub/NeoR, which contains two AAV inverted terminal repeat sequences and the NeoR gene driven by the thymidine kinase promoter. rAAV virions (10(4) infectious particles/ml) were generated by cotransfection of rAAV-mIL-12 and a helper plasmid (pAAV/Ad) into 293 cells previously infected with adenovirus 5. After infection of D6 fibroblasts with rAAV-mIL-12, G418-resistant clones were isolated. Each of the 1D D6 clones isolated produced up to 5.2 ng/10(6) cells/48 hours of mIL-12 as determined by enzyme-linked immunosorbent assay. Induction of interferon-gamma, enhanced lymphocyte proliferation, and cytotoxicity assays confirmed biologically functional IL-12 production by the vector. This is the first report indicating that an rAAV vector expresses mIL-12, which can be used to model the effects of mIL-12 alone and/or in combination with other antitumor agents.

Animals↗

PubMed1997

Early detection of the lytic LMP-1 protein in EBV-infected B-cells suggests its presence in the virion.

The Epstein-Barr virus (EBV)-positive B958 cell line expresses two related membrane proteins encoded by BNLF-1 open reading frames. One protein (LMP-1) has been shown to be essential for the growth transforming properties of EBV. The second protein (the lytic LMP-1) is an amino-terminally truncated form of LMP-1 whose expression is associated with induction of EBV's lytic cycle. We have investigated the expression of full-length and lytic forms of LMP-1 immediately after infection of the EBV-negative, B-lymphoma cell line BJAB. Only the lytic LMP-1 protein is present in BJAB cells early (within minutes following addition of virus) after infection with virus derived from either uninduced or tetradecanoyl phorbol acetate and sodium butyrate-induced B958 cells. Lytic LMP-1 protein levels begin to decline by 48 hr after infection, whereas levels of full-length LMP-1 increase between 24 and 48 hr after infection and then remain constant. The presence of the lytic LMP-1 protein in infected cells is independent of both protein synthesis and virus internalization. We also find the lytic LMP-1 protein in BJAB cells early after infection (within 3 hr of addition of virus) with the HH514 strain of EBV, and HH514 cells treated with tetradecanoyl phorbol acetate and sodium butyrate, express high levels of both the lytic LMP-1 and full-length LMP-1 proteins. The lytic LMP-1 protein is enriched in purified virion preparations, and immunoelectron microscopic analysis indicates that EBV virions can be specifically labeled with anti-LMP-1 antisera. Together, these results are consistent with a model in which the lytic LMP-1 protein is present in the EBV virion and is carried into the B-cell upon infection and suggest a role for this protein in early infection events and/or in EBV's lytic cycle.

Animals↗

PubMed2006

In vitro and in vivo identification of structural and sequence elements in the 5' untranslated region of Ectropis obliqua picorna-like virus required for internal initiation.

Ectropis obliqua picorna-like virus (EoPV) is a newly described insect virus that is classified as a putative member of the genus Iflavirus. The virus possesses a large, positive-sense RNA genome encoding a single polyprotein that shares physicochemical properties with those of members of the family Picornaviridae. The 5' untranslated region (5' UTR) plays an important role in picornavirus translation initiation, as it contains an internal ribosome entry site (IRES) that mediates cap-independent translation. To investigate translation in EoPV, an extensive range of mutations were engineered within the 5' UTR and the effects of these changes were examined in vitro and in vivo by using a bicistronic construct. Results showed that deletions within the first 63 nt had little impact on IRES activity, whilst core IRES function was contained within stem-loops C and D, as their removal abrogated IRES activity significantly. In contrast to these findings, removal of stem-loop G containing two cryptic AUGs caused a remarkable increase in IRES activity, which was further investigated by site-directed mutagenesis at these two positions. It was also confirmed that initiation of protein synthesis occurs at AUG6 (position 391-394) and not at the AUG immediately downstream of the polypyrimidine tract. Mutation of the polypyrimidine tract (CCTTTC) had a slight effect on EoPV IRES activity. Furthermore, mutations of the RAAA motif led to a decrease in IRES activity of approximately 40 % in vitro, but these results were not supported by in vivo experiments. In conclusion, this study reveals that the EoPV IRES element is unique, although it has features in common with the type II IRESs.

5' Untranslated Regions↗

PubMed2001

Multigene lentiviral vectors based on differential splicing and translational control.

Lentiviral vectors, so far, have been optimized for the expression of a single open reading frame. Certain practical applications of gene therapy will, however, require expression of multiple genes. The goal of this study was to explore the feasibility of directing expression of two marker genes from a lentiviral vector. We designed two types of multigene lentiviral vectors. First, we used a strategy based on the natural splicing signals of HIV-1, by which multiple mRNAs are generated from a single transcriptional unit. A second strategy was construction of a polycistronic mRNA using a translational cis-acting element, the encephalomyocarditis virus internal ribosome entry site (IRES). Our studies show that the inclusion of multiple genes in lentiviral vectors does not result in reduction in virus titers or in the loss of ability to infect nondividing cells. We introduced mutations in tat and/or rev to test whether splicing modulates the relative levels of expression of reporter genes. We also developed a truncated version of tat, which is devoid of the apoptosis-associated domain. Inclusion of this tat mutant in a lentiviral vector resulted in the generation of virus with titers similar to those of lentivirus vectors expressing wild-type tat.

Alternative Splicing↗

PubMed2005

Sindbis virus translation is inhibited by a PKR/RNase L-independent effector induced by alpha/beta interferon priming of dendritic cells.

The tropism of Sindbis virus (SB) for cells of the dendritic cell (DC) lineage and the virulence of SB in vivo are largely determined by the efficacy of alpha/beta interferon (IFN-alpha/beta)-mediated antiviral responses. These responses are essentially intact in the absence of PKR and/or RNase L (K. D. Ryman, L. J. White, R. E. Johnston, and W. B. Klimstra, Viral Immunol. 15:53-76, 2002). In the present studies, we investigated the nature of antiviral effects and identity of antiviral effectors primed by IFN-alpha/beta treatment of bone marrow-derived DCs (BMDCs) generated from mice deficient in PKR and RNase L (TD). IFN-alpha/beta priming exerted significant antiviral activity at very early stages of SB replication and most likely inhibited the initial translation of infecting genomes. The early effect targeted cap-dependent translation as protein synthesis from an SB-like and a simple RNA were inhibited by interferon treatment, but an encephalomyocarditis virus internal ribosome entry site-driven element exhibited no inhibition. Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 was defective after virus infection of TD cells, suggesting other mechanisms of translation inhibition. To identify components of these alternative antiviral pathway(s), we have compared global gene regulation in BMDCs derived from normal 129 Sv/Ev, IFNAR1-/-, and TD mice following infection with SB or treatment with IFN-alpha/beta. Candidate effectors of alternative antiviral pathways were those genes induced by virus infection or IFN-alpha/beta treatment in 129 Sv/Ev and TD-derived BMDC but not in virus-infected or IFN-alpha/beta-treated IFNAR1-/- cells. Statistical analyses of gene array data identified 44 genes that met these criteria which are discussed.

ATP-Binding Cassette Transporters↗

PubMed1996

Adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells.

A major impediment to the effective use of adenovirus vectors for gene therapy is a lack of knowledge of how these vectors interact with diverse cell types in vivo. Adenovirus attachment to most human cell types is mediated by the fiber protein, which binds to an as yet unidentified cell receptor. In contrast to this, we report that adenovirus type 2 (Ad2) attachment to hematopoietic cells is facilitated by interaction of the penton base protein with members of the beta2 integrin family. Adenovirus particles were capable of binding to human monocytic cells, which lack fiber receptors, and virus binding could be blocked by a soluble penton base or by a function-blocking monoclonal antibody to integrin alphaMbeta2. To confirm the role of alphaMbeta2 integrins in Ad2 binding to hematopoietic cells, we analyzed virus attachment and gene delivery to CHO cells expressing recombinant beta2 integrins. alphaMbeta2-expressing CHO cells supported 3- to 5-fold-higher levels of Ad2 binding and 5- to 10-fold-larger amounts of gene delivery than did nontransfected CHO cells, indicating that alphaMbeta2 facilitates adenovirus attachment to and infection of hematopoietic cells. While beta2 integrins promote Ad2 attachment to hematopoietic cells, further studies demonstrated that alphav integrins were required for the next step in infection, virus internalization into cell endosomes. These studies reveal a novel pathway of Ad2 infection of hematopoietic cells mediated by distinct integrins which facilitate separate events in virus entry. They also suggest a possible strategy for selective adenovirus-mediated gene delivery to hematopoietic cells.

Adenoviruses, Human↗

PubMed1997

International HIV and AIDS prevention: Japan/United States collaboration.

As the epicenter of the HIV/AIDS pandemic shifts from Africa to Asia, Japan is becoming ever more aware of the importance of containing and preventing spread of the virus. International collaboration, particularly with the United States, is a logical approach because it allows utilization of expertise from countries in other stages of the pandemic, can prevent duplication of efforts, and complements efforts of the other countries. Further, both Japan and the United States can use their combined influence and prestige to encourage cooperation among all nations. In 1994, Japan established the Global Issues Initiative to extend cooperation to developing countries in the areas of population and AIDS control. It has disbursed more than $460 million (U.S.$) to promote active cooperation and stimulate international attention to the importance of addressing these health issues. Japan has established four main programs for international collaboration for control of HIV and AIDS, three operated by ministries and one by a Japanese nongovernmental organization. Japanese/United States collaboration is developing through the United States/Japan Cooperative Medical Sciences Program, the Common Agenda for Cooperation in Global Perspective, the Paris Summit, and the United Nations Joint Programme on AIDS. It is critical that Japan and the United States, as the two largest donors to international development, demonstrate, through their collaboration, ways to maximize the use of limited resources, reduce duplication, and promote sustainable development programs in which HIV prevention and AIDS care programs are systemically integrated.

Acquired Immunodeficiency Syndrome↗

PubMed1987

Fusion of influenza virus in an intracellular acidic compartment measured by fluorescence dequenching.

The fusion of influenza virus with cultured cells has been investigated. The virus was labelled with the fluorescent probe octadecyl rhodamine B and fusion was monitored as fluorescence dequenching due to dilution of the probe from the viral into a cellular target membrane. Fusion with the plasma membrane does not occur, unless the extracellular pH is temporarily lowered. At neutral pH fusion occurs only after a lag phase of 10-15 min, the time required for virus internalization, and the reaction is inhibited by NH4Cl, indicating that it takes place in an intracellular acidic compartment, most likely the endosome. This suggests that influenza virus infects cells via the endocytic pathway.

Ammonium Chloride↗

PubMed1995

Upregulation of integrins alpha v beta 3 and alpha v beta 5 on human monocytes and T lymphocytes facilitates adenovirus-mediated gene delivery.

Entry of human adenovirus into host cells involves interaction of virus particles with two distinct receptors. The initial binding event is mediated by the fiber protein, while subsequent interaction of the penton base protein with alpha v integrins promotes virus internalization and/or penetration. Although these interactions in epithelial and endothelial cells have been well characterized, relatively little is known as to whether these events occur during virus infection of human peripheral blood mononuclear cells. We demonstrate that freshly isolated peripheral blood monocytes and T lymphocytes express very small amounts of alpha v integrins and also are resistant to adenovirus infection. Exposure of monocytes to hematopoietic growth factors granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor induced expression of cell surface alpha v integrins, promoted the binding of penton base protein, and also rendered these cells susceptible to adenovirus-mediated gene delivery. Stimulation of T cells with a mitogen, phytohemagglutinin, or a cell-activating agent, phorbol myristate acetate, induced expression of alpha v integrins and also enhanced adenovirus-mediated gene delivery. These studies further indicate that alpha v integrins play a crucial role in adenovirus infection and also provide a useful strategy for enhancing adenovirus-mediated gene delivery into human peripheral blood mononuclear cells.

Adenoviruses, Human↗

PubMed1985

Attenuation of murine coronavirus infection by ammonium chloride.

Ammonium chloride at a concentration of 20 mM delayed by 4-5 hr the production of virus progeny in mouse L-2 cells infected at high multiplicity with mouse hepatitis virus (MHV). This delay was seen in the production of both intracellular and extracellular virus. However, the final titers were similar to those produced by MHV-infected cells maintained in normal medium. The manifestation of virus-induced cell fusion was similarly found to be delayed, but not otherwise decreased in severity, when ammonium chloride was present in the culture medium. Ammonium chloride caused similar delays in production of virus-specific, positive-sense RNAs and of viral polypeptides. The relative proportions and apparent molecular weights of viral RNAs and polypeptides were similar to those found in MHV-infected cells cultured in normal medium. In vitro translation of endogenously produced viral RNAs in cell extracts, prepared from MHV-infected cells, was not inhibited by ammonium chloride. Thus, ammonium chloride has no specific, inhibitory effect on viral protein synthesis. Ammonium chloride did not reduce the number of virus-infected cells in culture, as monitored by infectious center assay. Analysis of early events in MHV infection showed that ammonium chloride did not affect adsorption or internalization of MHV by L-2 cells. However, the subsequent eclipse phase, as monitored by decline in infectivity of internalized virus inoculum proceeded less efficiently in the presence of ammonium chloride. On the basis of the known inhibitory effects of ammonium chloride on lysosomal/endosomal functions, the results suggest an endosomal mechanism of MHV uncoating. Thus the primary effect of ammonium chloride on MHV infection of L-2 cells is to attenuate virus uncoating, thereby chronologically displacing all subsequent virus-encoded functions.

Ammonium Chloride↗

PubMed1983

The estimation of host antigen in experimental and commercial influenza subunit preparations by delayed-type hypersensitivity reaction.

A delayed-type hypersensitivity (DTH) test has been used to detect the presence of host-specific antigen(s) in highly-purified egg-grown influenza virus and virus components. Cyclophosphamide-treated mice, primed with an extract from allantoic fluid, showed DTH reactions when challenged with whole virus, spikeless virus particles or polymer-type subunits. All commercial egg-grown influenza vaccines showed the presence of host antigen, whereas lipid-free derivatives such as internal virus protein complexes and monomer HA preparations lacked the activity. These findings are discussed in relation to vaccine application.

Animals↗

PubMed2000

The first step of adenovirus type 2 disassembly occurs at the cell surface, independently of endocytosis and escape to the cytosol.

Disassembly is a key event of virus entry into cells. Here, we have investigated cellular requirements for the first step of adenovirus type 2 (Ad2) disassembly, the release of the fibers. Although fiber release coincides temporally with virus uptake, fiber release is not required for Ad2 endocytosis. It is, however, inhibited by actin-disrupting agents or soluble RGD peptides, which interfere with integrin-dependent endocytosis of Ad2. Fiber release occurs at the cell surface. Actin stabilization with jasplakinolide blocks Ad2 entry at extended cell surface invaginations and efficiently promotes fiber release, indicating that fiber release and virus endocytosis are independent events. Fiber release is not sufficient for Ad2 escape from endosomes, since inhibition of protein kinase C (PKC) prevents Ad2 escape from endosomes but does not affect virus internalization or fiber release. PKC-inhibited cells accumulate Ad2 in small vesicles near the cell periphery, indicating that PKC is also required for membrane trafficking of virus. Taken together, our data show that fiber release from incoming Ad2 requires integrins and filamentous actin. Together with correct subcellular transport of Ad2-containing endosomes, fiber release is essential for efficient delivery of virus to the cytosol. We speculate that fiber release at the surface might extend the host range of Ad2 since it is associated with the separation of a small fraction of incoming virus from the target cells.

Actins↗

PubMed1994

Membrane fusion as a determinant of the infectibility of cells by vesicular stomatitis virus.

When rat embryo fibroblasts (REF) and BHK21 cells were treated with VSV at the same m.o.i., 8- to 10-fold fewer REF were infected than BHK21 cells; REF also showed a similar decrease in the production of progeny virions per cell. When other aspects of the infectious cycle were studied in the two cells, it was found that the time course of virus production was similar in BHK21 cells and REF, virus grown in REF showed the same infectivity in both cell types as virus grown in BHK21 cells, and all REF in a culture could eventually be infected. There was virtually no difference in the binding of VSV to the cells, and REF actually internalized virus at a slightly greater rate than BHK21 cells. Direct fusion of VSV with the plasma membrane of the two cell types resulted in infection of the cells, but did not abolish the difference in infectibility. When this fusion was quantitated using 125I-VSV, BHK21 cells fused 6-fold more virus than REF, a difference that accounts for most of the difference in the infectibility of the two cell types. The results indicate that constituents of the host cell plasma membrane modulate the fusion of VSV.

Animals↗

PubMed1996

A variant of MDCK cell line which restricted growth of influenza viruses mainly through suppression of viral primary transcription.

By serial subculture of MDCK cells which survived high multiplicity infections with AWBY-140, a weakly cytolytic mutant of influenza virus A/WSN (H1N1), we established a variant cell line (MDCK-L cells) that was uniquely resistant to infection with influenza A and B viruses, yielding 3 to 4 orders lower amount of progeny virus compared with MDCK cells. Competitive polymerase chain reaction revealed that the amount of primary transcript produced in MDCK-L cells infected with 10 PFU/cell of influenza virus A/Aichi was suppressed to 1/100 of that in MDCK cells similarly infected, although the amount of virus adsorbed to MDCK-L cells was 1/4 of MDCK cells. Even when MDCLK-L cells were infected with 40 PFU/cell of Aichi to overcome the lower amount of internalized virus in those cells, the results were the same. The synthesis of v-, c- and mRNAs, as well as proteins of infected A/Aichi was below detectable level in MDCK-L cells, in contrast with MDCK cells, where they were clearly demonstrable by ribonuclease protection assay or polyacrylamide gel electrophoresis.

Animals↗

PubMed1993

The integrin VLA-2 binds echovirus 1 and extracellular matrix ligands by different mechanisms.

The integrin VLA-2 mediates cell adhesion to collagen and laminin and also functions as a virus receptor, mediating cell surface attachment and infection by a human pathogen, echovirus 1. To determine whether extracellular matrix proteins and virus interact with VLA-2 in the same manner, we carried out a detailed comparison of these two functions and found that they differed markedly in six different respects. In contrast to the ECM/VLA-2 interaction, echovirus 1 binding did not discriminate between functional forms of VLA-2, showed a different pattern of inhibition by anti-beta1 and -alpha 2 antibodies, was not stimulated by phorbol esters, was not activated by beta 1 antibodies that stimulate ECM binding, was not inhibited by any particular divalent cation, and most notably was not inhibited by EDTA. These striking differences were found both with intact cells expressing VLA-2 and with solubilized VLA-2, suggesting that VLA-2 interacts with these different ligands by markedly different mechanisms, and probably at different functional sites. In addition, alterations in the alpha 2 cytoplasmic domain that had marked effects on cellular responses to collagen and laminin had no effect on virus internalization and cell killing. Thus VLA-2-mediated events that occur after receptor occupancy by extracellular matrix proteins also appear to be distinct from those that occur after receptor interaction with virus.

Cell Adhesion↗

PubMed1998

Virus-specific MHC-class II-restricted TCR-transgenic mice: effects on humoral and cellular immune responses after viral infection.

A transgenic mouse expressing MHC class II-restricted TCR with specificity for a lymphocytic choriomeningitis virus (LCMV) glycoprotein-derived T helper cell epitope was developed to study the role of LCMV-specific CD4+ T cells in virus infection in vivo. The majority of CD4+ T cells in TCR transgenic mice expressed the transgenic receptor, and LCMV glycoprotein-specific TCR transgenic CD4+ T cells efficiently mediated help for the production of LCMV glycoprotein-specific isotype-switched antibodies. In contrast, LCMV glycoprotein-specific TCR transgenic mice exhibited a drastically reduced ability to provide help for the generation of antibody responses specific for the virus-internal nucleoprotein, indicating that intramolecular/intrastructural help is limited to antigens that are accessible to B cells on the viral surface. Antiviral cellular immunity was studied with noncytopathic LCMV and recombinant cytopathic vaccinia virus expressing the LCMV glycoprotein. TCR transgenic mice failed to efficiently control LCMV infection, demonstrating that functional LCMV-specific CD4+ T cells--even if activated and present at extremely high frequencies--cannot directly mediate protective immunity against LCMV. Despite the fact that LCMV-primed CD4+ T cells from TCR transgenic mice as well as from control mice showed low MHC class II-restricted cytotoxic activity in vivo, this did not correlate with protection against LCMV replication in vivo. In contrast, CD4+ T cells from TCR-transgenic mice mediated efficient protection against infection with recombinant vaccinia virus. These results further support the need for different immune effector functions for protective immunity against different viral infections.

Adoptive Transfer↗

PubMed2004

Itinerary of hepatitis B viruses: delineation of restriction points critical for infectious entry.

Little is known about cellular determinants essential for human hepatitis B virus infection. Using the duck hepatitis B virus as a model, we first established a sensitive binding assay for both virions and subviral particles and subsequently elucidated the characteristics of the early viral entry steps. The infection itinerary was found to initiate with the attachment of viral particles to a low number of binding sites on hepatocytes (about 10(4) per cell). Virus internalization was fully accomplished in less than 3 h but was then followed by a period of unprecedented length, about 14 h, until completion of nuclear import of the viral genome. Steps subsequent to virus entry depended on both intact microtubules and their dynamic turnover but not on actin cytoskeleton. Notably, cytoplasmic trafficking of viral particles and emergence of nuclear covalently closed circular DNA requires microtubules during entry only at and for specific time periods. Taken together, these data disclose for the first time a series of steps and their kinetics that are essential for the entry of hepatitis B viruses into hepatocytes and are different from those of any other virus reported so far.

Actins↗

PubMed2005

Murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release.

Thorp and Gallagher first reported that depletion of cholesterol inhibited virus entry and cell-cell fusion of mouse hepatitis virus (MHV), suggesting the importance of lipid rafts in MHV replication (E. B. Thorp and T. M. Gallagher, J. Virol. 78:2682-2692, 2004). However, the MHV receptor is not present in lipid rafts, and anchoring of the MHV receptor to lipid rafts did not enhance MHV infection; thus, the mechanism of lipid rafts involvement is not clear. In this study, we defined the mechanism and extent of lipid raft involvement in MHV replication. We showed that cholesterol depletion by methyl beta-cyclodextrin or filipin did not affect virus binding but reduced virus entry. Furthermore, MHV spike protein bound to nonraftraft membrane at 4 degrees C but shifted to lipid rafts at 37 degrees C, indicating a redistribution of membrane following virus binding. Thus, the lipid raft involvement in MHV entry occurs at a step following virus binding. We also found that the viral spike protein in the plasma membrane of the infected cells was associated with lipid rafts, whereas that in the Golgi membrane, where MHV matures, was not. Moreover, the buoyant density of the virion was not changed when MHV was produced from the cholesterol-depleted cells, suggesting that MHV does not incorporate lipid rafts into the virion. These results indicate that MHV release does not involve lipid rafts. However, MHV spike protein has an inherent ability to associate with lipid rafts. Correspondingly, cell-cell fusion induced by MHV was retarded by cholesterol depletion, consistent with the association of the spike protein with lipid rafts in the plasma membrane. These findings suggest that MHV entry requires specific interactions between the spike protein and lipid rafts, probably during the virus internalization step.

Animals↗