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At least 253 records · Page 14Linked to original sources

PubMed1987

Radioimmunoprecipitation and Western blotting with sera of human immunodeficiency virus infected patients: a comparative study.

The sensitivity and specificity of radioimmunoprecipitation assay (RIPA) and Western blot (WB) test were compared by use of a collection of 183 sera, representing different categories of individuals, noninfected or infected with the human immunodeficiency virus (HIV). The sera were subdivided on the basis of their reactivity in at least two anti-HIV enzyme-linked immunosorbent assays (ELISA); 53 sera were negative and 61 sera were positive in both tests, whereas 69 sera showed ambiguous reactions. The reaction patterns in RIPA could be divided into 6 different groups. The same grouping could to some extent be applied to the results of WB test. RIPA provided the most efficient means for identification of the large viral envelope glycoproteins, gp120/160, whereas gp41 was detected more effectively by WB. Internal virus components reacted to a varying extent with specific antibodies in the two tests. The reaction with pol products was more pronounced with the WB tests in which extracellular material was used as antigen. In a few WB tests, however, the reaction with internal components did not reflect a prior HIV infection. No such ambiguity was observed with RIPA, reflecting the advantage of a test that uses a minimally denaturated antigen and provides appropriate conditions for identification of the large viral glycoproteins. The practical choice of confirmatory tests to be used in diagnostic laboratories requires evaluation of both the sensitivity and specificity of the tests but also their economy and convenience.

Acquired Immunodeficiency Syndrome↗

PubMed1996

A murine RNA polymerase I promoter inserted into the herpes simplex virus type 1 genome is functional during lytic, but not latent, infection.

The development of herpes simplex virus as a vector for neuronal gene delivery is dependent upon the identification and characterization of promoter elements capable of driving long-term expression during latency. The majority of RNA polymerase II (pol II) promoters studied are active during acute infection but silenced during latency. In order to investigate the potential of a murine RNA polymerase I (pol I) promoter to drive reporter gene expression during lytic and latent infection, we describe the construction and characterization of two recombinant viruses; SC16 LAT neo and SC16 US5 neo. These viruses contain a pol I-encephalomyocarditis virus internal ribosome entry site (EMCV IRES)-neomycin phosphotransferase gene (neoR) cassette inserted into the non-essential major latency associated transcript (LAT) and US5 regions respectively. Pol I promoter activity could be detected in the rodent BHK cell line, but not the primate derived Vero cell line-- consistent with the known species specificity of such promoters. This activity was specific to a virus containing an active pol I promoter. However, in situ hybridization analyses of latently infected cervical dorsal root ganglia failed to detect pol I mediated transcription of the reporter gene indicating that the murine pol I promoter is silenced following the establishment of latency. Insertion of the pol I-EMCV IRES-neoR cassette into the major LAT locus resulted in the production of a hybrid LAT transcript during latency which was translocated to the cytoplasm of latently infected neurones.

Animals↗

PubMed2001

Genetic analysis of a poliovirus/hepatitis C virus chimera: new structure for domain II of the internal ribosomal entry site of hepatitis C virus.

Internal ribosomal entry sites (IRESs) of certain plus-strand RNA viruses direct cap-independent initiation of protein synthesis both in vitro and in vivo, as can be shown with artificial dicistronic mRNAs or with chimeric viral genomes in which IRES elements were exchanged from one virus to another. Whereas IRESs of picornaviruses can be readily analyzed in the context of their cognate genome by genetics, the IRES of hepatitis C virus (HCV), a Hepacivirus belonging to Flaviviridae, cannot as yet be subjected to such analyses because of difficulties in propagating HCV in tissue culture or in experimental animals. This enigma has been overcome by constructing a poliovirus (PV) whose translation is controled by the HCV IRES. Within the PV/HCV chimera, the HCV IRES has been subjected to systematic 5' deletion analyses to yield a virus (P/H710-d40) whose replication kinetics match that of the parental poliovirus type 1 (Mahoney). Genetic analyses of the HCV IRES in P/H710-d40 have confirmed that the 5' border maps to domain II, thereby supporting the validity of the experimental approach applied here. Additional genetic experiments have provided evidence for a novel structural region within domain II. Arguments that the phenotypes observed with the mutant chimera relate solely to impaired genome replication rather than deficiencies in translation have been dispelled by constructing novel dicistronic poliovirus replicons with the gene order [PV]cloverleaf-[HCV]IRES-Deltacore-R-Luc-[PV]IRES-F-Luc-P2,3-3'NTR, which have allowed the measurement of HCV IRES-dependent translation independently from the replication of the replicon RNA.

Base Sequence↗

PubMed2006

The degree of apoptosis as an immunostimulant for a DNA vaccine against HIV-1 infection.

To regulate the expression of the apoptotic gene, we constructed bicistronic DNA vaccines that encode for HIV env and caspase-3 mutant (casp 3m) that are expressed via the encephalomyocarditis virus internal ribosomal entry site (IRES) or cytomegalovirus (CMV) promoter-dependent translations. While IRES-casp 3m induced weak apoptosis and caused little reduction in antigen expression, CMV-casp 3m elicited strong apoptosis and led to a marked decrease in the antigen expression. Therefore, IRES-casp 3m augmented HIV-specific immune responses, and IRES-casp 3m induced significant protection against the vaccinia-HIV chimeric virus. These results suggest that the appropriate level of apoptosis is important for DNA vaccine development.

AIDS Vaccines↗

PubMed1996

Open reading frame 5 of porcine reproductive and respiratory syndrome virus as a cause of virus-induced apoptosis.

The gene product of open reading frame 5 (p25) of porcine reproductive and respiratory syndrome (PRRS) virus has been expressed by coinfection of culture cells with vaccinia virus expressing the T7 RNA polymerase and a recombinant vaccinia virus encoding the open reading frame 5 gene under the T7 promoter and the encephalomyocarditis virus internal ribosome entry site. In spite of the reported efficiency of the expression system, very poor accumulation of p25 protein was observed and a strong cytotoxicity was produced in the doubly infected cells. This cell toxicity was shown to occur by induction of apoptosis, as indicated by nucleosome ladder formation, chromatin condensation, and rRNA degradation. Apoptosis induction was also observed after infection of cultured cells with an adapted PRRS virus strain and after infection of swine macrophage cells with a PRRS virus field strain. Contrary to the observations made for other cases of virus-induced apoptosis, we could not prevent p25 protein-induced apoptosis by using a cell line permanently expressing Bcl-2 protein.

Animals↗

PubMed1986

Recognition of influenza A virus nucleoprotein by human cytotoxic T lymphocytes.

A recombinant vaccinia virus (NP-VAC) containing cDNA corresponding to segment 5, the nucleoprotein (NP) gene of influenza A/PR/8/34 virus was used to examine the specificity of human influenza virus immune cytotoxic T lymphocytes (CTL). Effector cell preparations from two donors recognized autologous lymphocytes that had been infected with NP-VAC. Lysis was specific because cells infected with vaccinia virus were not killed and recognition was HLA-restricted. In one donor, the influenza virus-specific CTL response changed with time so that his effector cells no longer recognized autologous lymphocytes infected with NP-VAC. However, a component that was NP-specific remained because these CTL lysed the more sensitive autologous B lymphoblastoid cells that had been infected with NP-VAC. In four other donors, no NP-specific CTL response could be detected using autologous lymphocyte targets. Thus NP, an internal virus protein, is one antigen that is recognized by human influenza A virus-specific CTL, but it is likely that other individual virus components contribute to the total CTL response.

Cytotoxicity, Immunologic↗

PubMed2001

Variations in disparate regions of the murine coronavirus spike protein impact the initiation of membrane fusion.

The prototype JHM strain of murine hepatitis virus (MHV) is an enveloped, RNA-containing coronavirus that has been selected in vivo for extreme neurovirulence. This virus encodes spike (S) glycoproteins that are extraordinarily effective mediators of intercellular membrane fusion, unique in their ability to initiate fusion even without prior interaction with the primary MHV receptor, a murine carcinoembryonic antigen-related cell adhesion molecule (CEACAM). In considering the possible role of this hyperactive membrane fusion activity in neurovirulence, we discovered that the growth of JHM in tissue culture selected for variants that had lost murine CEACAM-independent fusion activity. Among the collection of variants, mutations were identified in regions encoding both the receptor-binding (S1) and fusion-inducing (S2) subunits of the spike protein. Each mutation was separately introduced into cDNA encoding the prototype JHM spike, and the set of cDNAs was expressed using vaccinia virus vectors. The variant spikes were similar to that of JHM in their assembly into oligomers, their proteolysis into S1 and S2 cleavage products, their transport to cell surfaces, and their affinity for a soluble form of murine CEACAM. However, these tissue culture-adapted spikes were significantly stabilized as S1-S2 heteromers, and their entirely CEACAM-dependent fusion activity was delayed or reduced relative to prototype JHM spikes. The mutations that we have identified therefore point to regions of the S protein that specifically regulate the membrane fusion reaction. We suggest that cultured cells, unlike certain in vivo environments, select for S proteins with delayed, CEACAM-dependent fusion activities that may increase the likelihood of virus internalization prior to the irreversible uncoating process.

Animals↗

PubMed1988

Effect of ammonium chloride on the multiplication of infectious pancreatic necrosis virus.

The multiplication of IPNV in CHSE cells is inhibited by ammonium chloride. This inhibition is complete if NH4Cl is added early in the infective cycle. Immunofluorescence analysis shows that NH4Cl reduces the number of virus producing cells and thus suggests it may act by inhibiting virus internalization. In addition, NH4Cl can produce partial inhibition of IPNV multiplication if added later in the replicative cycle. Analysis of 3H-uridine incorporation into viral RNA shows that this later effect could be an inhibition of viral RNA synthesis.

Ammonium Chloride↗

PubMed1998

Gene transfer using a disabled herpes virus vector containing the EMCV IRES allows multiple gene expression in vitro and in vivo.

The design of recombinant HSV-1 vectors for delivery of transgenes to the central nervous system is undergoing constant development. Problems associated with the construction and use of such vectors include the requirement for detection of recombinant versus nonrecombinant virus in vitro and also the identification of transduced cells in vivo. This could be overcome by the insertion of reporter genes such as lacZ or green fluorescent protein (GFP) under a separate promoter to the transgene to be expressed. In this case, however, reporter gene expression does not necessarily confirm transgene expression as a separate RNA must be produced. This study reports the use of an encephalomyocarditis virus internal ribosome entry site (IRES) to enable the translation of two reporter genes from a single mRNA transcript driven by the same promoter within a disabled HSV vector, and discusses the potential advantages of this approach.

Animals↗

PubMed1997

Expression of a recombinant monoclonal antibody from a bicistronic mRNA.

The variable regions of the murine monoclonal antibody A1, which effectively neutralizes the infection of susceptible cells by the murine hepatitis virus strain JHM, were cloned, sequenced, and expressed in mammalian cells as a functional recombinant antibody. To accomplish the concurrent synthesis of both antibody chains, the light- and heavy-chain-coding regions were inserted into a bicistronic expression cassette based on the encephalomyocarditis virus internal ribosomal entry site. The strategy of combining both coding regions in one bicistronic mRNA allows for the rapid isolation of cell clones expressing high levels of recombinant antibody.

Amino Acid Sequence↗

PubMed2005

A hydrophobic domain in the large envelope protein is essential for fusion of duck hepatitis B virus at the late endosome.

The duck hepatitis B virus (DHBV) envelope is comprised of two transmembrane (TM) proteins, the large (L) and the small (S), that assemble into virions and subviral particles. Secondary-structure predictions indicate that L and S have three alpha-helical, membrane-spanning domains, with TM1 predicted to act as the fusion peptide following endocytosis of DHBV into the hepatocyte. We used bafilomycin A1 during infection of primary duck hepatocytes to show that DHBV must be trafficked from the early to the late endosome for fusion to occur. Alanine substitution mutations in TM1 of L and S, which lowered TM1 hydrophobicity, were used to examine the role of TM1 in infectivity. The high hydrophobicity of the TM1 domain of L, but not of S, was shown to be essential for virus infection at a step downstream of receptor binding and virus internalization. Using wild-type and mutant synthetic peptides, we demonstrate that the hydrophobicity of this domain is required for the aggregation and the lipid mixing of phospholipid vesicles, supporting the role of TM1 as the fusion peptide. While lipid mixing occurred at pH 7, the kinetics of insertion of the fusion peptide was increased at pH 5, consistent with the location of DHBV in the late-endosome compartment and previous studies of the nonessential role of low pH for infectivity. Exchange of the TM1 of DHBV with that of hepatitis B virus yielded functional, infectious DHBV particles, suggesting that TM1 of all of the hepadnaviruses act similarly in the fusion mechanism.

Animals↗

PubMed1999

General and specific immunosuppression caused by antiviral T-cell responses.

Immunosuppression caused by the non-cytopathic lymphocytic choriomeningitis virus (LCMV) (an RNA virus) is mediated by antiviral cytotoxic T cells that destroy LCMV-infected cells, also of the immune system. While this immunopathological destruction of antigen-presenting cells, macrophages and follicular dendritic cells and of some CD4+ T cells causes general immunosuppression and impairs immune response to third party antigens, it also enhances exhaustion/deletion of LCMV-specific CD8+ T-cell responses. LCMV seems in addition to infect neutralizing antibody-producing B cells via the specific receptor; immunopathological LCMV specific CD8+ T-cell-mediated elimination of these infected B cells (but not of uninfected internal virus antigen-specific B cells) causes a highly specific immunosuppression that delays neutralizing antibody responses and thereby enhances virus persistence. Both generalized and specific immunosuppression by CD8+ T-cell-mediated immunopathology may be involved in human infections with HIV, hepatitis B virus or hepatitis C virus.

Animals↗

PubMed2005

The polypyrimidine tract-binding protein (PTB) is required for efficient replication of hepatitis C virus (HCV) RNA.

Our earlier work found that the polypyrimidine tract-binding protein (PTB) specifically interacts with the poly(U/C) tract of the hepatitis C virus (HCV) 3' untranslated region (3'UTR) [Luo, G., 1999. Cellular proteins bind to the poly(U) tract of the 3' untranslated region of hepatitis C virus RNA genome. Virology 256, 105-118]. We report here that the phosphorylated form of PTB is associated with the membrane-bound HCV replication complex. To determine whether the PTB is required for HCV RNA replication, synthetic small interfering RNAs (siRNAs) were used to specifically knockdown PTB expression in the HCV replicon-harboring Huh7 cells. The level of PTB expression was efficiently reduced by PTB-specific siRNAs. Consequently, the levels of HCV proteins as well as HCV RNA were proportionally decreased by increasing concentrations of PTB siRNA. However, the translation mediated by the encephamyocarditis virus internal ribosome entry site was unaffected, suggesting that the inhibition of HCV RNA replication by PTB siRNA was not due to its effect on the EMCV IRES-mediated translation. Collectively, these findings demonstrate that PTB is required for efficient HCV RNA replication in the cell.

Cell Line↗

PubMed2006

Identification of a structural motif crucial for infectivity of hepatitis B viruses.

Infectious entry of hepatitis B viruses (HBV) has nonconventional facets. Here we analyzed whether a cell-permeable peptide [translocation motif (TLM)] identified within the surface protein of human HBV is a general feature of all hepadnaviruses and plays a role in the viral life cycle. Surface proteins of all hepadnaviruses contain conserved functional TLMs. Genetic inactivation of the duck HBV TLMs does not interfere with viral morphogenesis; however, these mutants are noninfectious. TLM mutant viruses bind to cells and are taken up into the endosomal compartment, but they cannot escape from endosomes. Processing of surface protein by endosomal proteases induces their exposure on the virus surface. This unmasking of TLMs mediates translocation of viral particles across the endosomal membrane into the cytosol, a prerequisite for productive infection. The ability of unmasked TLMs to translocate processed HBV particles across cellular membranes was shown by confocal immunofluorescence microscopy and by infection of nonpermissive cell lines with HBV processed in vitro with endosomal lysate. Based on these data, we propose an infectious entry mechanism unique for hepadnaviruses that involves virus internalization by receptor-mediated endocytosis followed by processing of surface protein in endosomes. This processing activates the function of TLMs that are essential for viral particle translocation through the endosomal membrane into the cytosol and productive infection.

Amino Acid Motifs↗

PubMed1999

Antiherpetic activity and mode of action of natural carrageenans of diverse structural types.

The lambda-carrageenan 1T1, the kappa/iota-carrageenan 1C1 and the mu/nu-type 1C3, isolated from the red seaweed Gigartina skottsbergii, proved to be potent and selective inhibitors of herpes simplex virus (HSV) types 1 and 2. The antiviral IC50 values determined by virus yield inhibition assay in different cell lines ranged from 0.4 to 3.3 microg/ml, and no cytotoxic effects, measured by trypan blue exclusion on stationary or proliferating cells, tetrazolium salt method or cell protein synthesis, were observed. Time of addition and attachment studies suggested that the main target for antiviral action of the three carrageenans was virus adsorption, whereas no effect on virus internalization, or early or late protein synthesis was detected. However, the lambda-carrageenan 1T1 was still significantly inhibitory when added any time after adsorption. The pretreatment of virions with the carrageenans showed that 1C1 and 1C3 lacked direct inactivating effect at concentrations near the antiviral IC50 but 1T1 exerted virucidal action. The cyclization of 1T1 to afford the derivative 1T1T1 maintained the antiviral activity but eliminated the virucidal properties. Thus, the structure of 1T1 seems to be responsible for its differential behavior from 1C1 and 1C3, probably allowing a more stable binding to HSV, leading to virion inactivation. In contrast, 1C1 and 1C3 fail to bind with high affinity to virus alone, but are able to interfere with the interaction between HSV particles and the cell.

Adsorption↗

PubMed1994

Integrin alpha v beta 5 selectively promotes adenovirus mediated cell membrane permeabilization.

Human adenovirus type 2 (Ad2) enters host cells by receptor-mediated endocytosis, an event mediated by the virus penton base binding to cell surface integrins alpha v beta 3 and alpha v beta 5. While both alpha v integrins promote virus internalization, alpha v beta 5 is involved in the subsequent event of membrane permeabilization. Cells transfected with the beta 5 or beta 3 subunit, expressing either alpha v beta 5 and alpha v beta 3, respectively, were capable of supporting Ad2 infection to varying degrees. In this case, cells expressing alpha v beta 5 were significantly more susceptible to Ad2-induced membrane permeabilization, as well as to Ad2 infection, than cells expressing alpha v beta 3. Adenovirus-mediated gene delivery was also more efficient in cells expressing alpha v beta 5. These results suggest that the interaction of alpha v beta 5 with Ad2 penton base facilitates the subsequent step of virus penetration into the cell. These studies provide evidence for the involvement of a cellular receptor in virus-mediated membrane permeabilization and suggest a novel biological role for integrin alpha v beta 5 in the infectious pathway of a human adenovirus.

Adenoviruses, Human↗

PubMedDate not supplied

[Study of Sendai virus proteins: the proteolytic activity in the composition of the viral particles].

Disruption of virions with non-ionic detergent induced cleavage of virus proteins in purified preparations of Sendai virus. The proteolytic activity was detected in fraction of both the soluble proteins and subvirus particles. In the fraction of soluble proteins which initially had contained glycoproteins of the viral external envelope--HN and F--, both proteins were cleaved at a different relative rate. In parallel with polypeptide cleavage, the hemagglutinating and neuraminidase activity of the analysed specimens decreased. In the fraction of subvirus particles, sequential cleavage of inner virion proteins, P, m1, M, and NP, was observed, and also the formation of certain classes of intermediate products which had two typical features; (1) the products retained their connection with subvirus particles, and (2) the products of hydrolysis of the major structural proteins of the internal virus ribonucleoprotein, --P and NP--, had the electrophoretic motility similar to that of minor components, m2--m5. These observations demonstrate the principal possibility of the origin of some minor Sendai virus proteins as a result of degradation of major virus proteins.

Electrophoresis, Polyacrylamide Gel↗

PubMed2000

Design and intracellular activity of a human single-chain antibody to human immunodeficiency virus type 1 conserved gp41 epitope.

A human lymphoid cell line (F172-D8) excreting a human immunodeficiency virus type 1 (HIV-1) anti-gp41 monoclonal antibody was used to construct a plasmid containing the cDNA of the single-chain variable fragment (scFvD8) corresponding to this antibody. A stable human osteosarcoma cell line was obtained which expressed the scFvD8 protein in the cytoplasm. Whereas a cell line transfected with a control construct (pCI-neo) was readily and productively infected with laboratory (Ba-L) or primary HIV-1 isolates, the scFvD8 cell line did not support productive infection. Binding of the virus, internalization, and reverse transcription were not altered by scFvD8 expression, but gp160 expression was dramatically reduced. These data suggest that cytoplasmic expression of this artificial single-chain antibody can interfere with gp160 expression, thereby reducing the production of mature viral envelope proteins.

Cell Line↗