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At least 685 records · Page 38Linked to original sources
PubMed2001

Association of bovine papillomavirus type 1 with microtubules.

Transport of BPV-1 virus from the cell membrane to the nucleus was studied in vitro in CV-1 cells. At reduced temperature (4 degrees C), BPV-1 binding to CV-1 cells was unaffected but there was no transport of virions across the cytosol. Electron microscopy showed BPV-1 virions in association with microtubules in the cytoplasm, a finding confirmed by co-immunoprecipitation of L1 protein and tubulin. Internalization of virus was unimpaired in cells treated with the microtubule-depolymerizing drug nocodazole but virions were retained in cytoplasmic vesicles and not transported to the nucleus. We conclude that a microtubule transport mechanism in CV-1 cells moves intact BPV-1 virions from the cell surface to the nuclear membrane.

Active Transport, Cell Nucleus↗
PubMed1984

Antibody-mediated infection of P388D1 cells with 17D yellow fever virus: effects of chloroquine and cytochalasin B.

Antibody-mediated 17D yellow fever virus infection of a murine macrophage-like cell line, P388D1, was not inhibited by concentrations of cytochalasin B which did inhibit phagocytosis of antibody-coated sheep red blood cells. Chloroquine did not affect antibody-mediated enhancement of viral growth but inhibited viral replication equally whether infection was carried out in the presence or absence of antibody. The results suggest that the route of internalization of virus and subsequent intracellular pathways of replication are similar for both conditions of infection.

Animals↗
PubMed1984

High-frequency deletion in recovered retrovirus vectors containing exogenous DNA with promoters.

We previously described infectious retrovirus vectors constructed from spleen necrosis virus which contain the herpes simplex virus thymidine kinase gene and the mouse alpha-globin gene (K. Shimotohno and H. M. Temin, Nature [London] 299:255-268, 1982). In the present study we report that when TK- chicken cells infected with a virus containing the mouse alpha-globin promoter and other 5' noncoding sequences in addition to the alpha-globin coding sequences were selected for thymidine kinase (TK) activity, all virus-producing TK+ cell clones shed virus with a deletion. These deletions were of different sizes and included the mouse alpha-globin coding sequences and the mouse alpha-globin transcriptional promoter. One of the deleted viruses was molecularly cloned. DNA sequencing showed that the deleted sequences are flanked by a short direct repeat. This deleted virus was also shown to have an advantage over the nondeleted parent both in multiplication and in its specific TK-transforming unit titer. In contrast to the results described above, TK+ cell clones established with viruses that contained only the coding sequences from the mouse alpha-globin gene did not delete and were stable over many cell passages. The implications of the high-frequency deletion of the viruses with internal promoters are discussed in terms of the evolution of retroviruses and the construction of retrovirus vectors.

Animals↗
PubMed1996

Replication of hepatitis A viruses with chimeric 5' nontranslated regions.

The role of the 5' nontranslated region in the replication of hepatitis A virus (HAV) was studied by analyzing the translation and replication of chimeric RNAs containing the encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES) and various lengths (237, 151, or 98 nucleotides [nt]) of the 5'-terminal HAV sequence. Translation of all chimeric RNAs, truncated to encode only capsid protein sequences, occurred with equal efficiency in rabbit reticulocyte lysates and was much enhanced over that exhibited by the HAV IRES. Transfection of FRhK-4 cells with the parental HAV RNA and with chimeric RNA generated a viable virus which was stable over continuous passage; however, more than 151 nt from the 5' terminus of HAV were required to support virus replication. Single-step growth curves of the recovered viruses from the parental RNA transfection and from transfection of RNA containing the EMCV IRES downstream of the first 237 nt of HAV demonstrated replication with similar kinetics and similar yields. When FRhK-4 cells infected with recombinant vaccinia virus producing SP6 RNA polymerase to amplify HAV RNA were transfected with plasmids coding for these viral RNAs or with subclones containing only HAV capsid coding sequences downstream of the parental or chimeric 5' nontranslated region, viral capsid antigens were synthesized from the HAV IRES with an efficiency equal to or greater than that achieved with the EMCV IRES. These data suggest that the inherent translation efficiency of the HAV IRES may not be the major limiting determinant of the slow-growth phenotype of HAV.

Animals↗
PubMed2001

Insertions in the gG gene of pseudorabies virus reduce expression of the upstream Us3 protein and inhibit cell-to-cell spread of virus infection.

The alphaherpesvirus Us4 gene encodes glycoprotein G (gG), which is conserved in most viruses of the alphaherpesvirus subfamily. In the swine pathogen pseudorabies virus (PRV), mutant viruses with internal deletions and insertions in the gG gene have shown no discernible phenotypes. We report that insertions in the gG locus of the attenuated PRV strain Bartha show reduced virulence in vivo and are defective in their ability to spread from cell to cell in a cell-type-specific manner. Similar insertions in the gG locus of the wild-type PRV strain Becker had no effect on the ability of virus infection to spread between cells. Insertions in the gG locus of the virulent NIA-3 strain gave results similar to those found with the Bartha strain. To examine the role of gG in cell-to-cell spread, a nonsense mutation in the gG signal sequence was constructed and crossed into the Bartha strain. This mutant, PRV157, failed to express gG yet had cell-to-cell spread properties indistinguishable from those of the parental Bartha strain. These data indicated that, while insertions in the gG locus result in decreased cell-to-cell spread, the phenotype was not due to loss of gG expression as first predicted. Analysis of gene expression upstream and downstream of gG revealed that expression of the upstream Us3 protein is reduced by insertion of lacZ or egfp at the gG locus. By contrast, expression of the gene immediately downstream of gG, Us6, which encodes glycoprotein gD, was not affected by insertions in gG. These data indicate that DNA insertions in gG have polar effects and suggest that the serine/threonine kinase encoded by the Us3 gene, and not gG, functions in the spread of viral infection between cells.

Animals↗
PubMed1993

Structural rearrangement of infecting Sindbis virions at the cell surface: mapping of newly accessible epitopes.

Sindbis virus glycoproteins E1 and E2 undergo a conformational alteration during early virus-cell interaction at the cell surface (D. Flynn, W. J. Meyer, J. M. MacKenzie, Jr., and R. E. Johnston, J. Virol. 64:3643-3653, 1990). Certain epitopes normally internal on native virus become accessible to monoclonal antibody (MAb) binding after attachment but before internalization of virus particles. These newly exposed epitopes, termed transitional epitopes, may be part of functionally important domains made accessible at the surface of the altered virus to facilitate entry into cells. Heating Sindbis virions at 51 degrees C for a short time induced a similar, although not identical, exposition of transitional epitopes on the E1 and E2 glycoproteins (W. J. Meyer, S. Gidwitz, V. K. Ayers, R. J. Schoepp, and R. E. Johnston, J. Virol. 66:3504-3513, 1992). In the current report, we have identified several of the transitional epitopes that become exposed as a consequence of early virus-cell interactions. Transitional epitope MAbs that bound to rearranged, heated virions and virus-cell complexes were used in antibody competition binding assays on heated Sindbis virions to map the spatial relationships between native, external, neutralizing antigenic sites and newly exposed transitional epitopes. Because the heated, rearranged particles retained their infectivity, MAbs that bound to transitional epitopes also were used to isolate MAb neutralization escape mutants. Sequencing the glycoprotein genes of the escape mutants identified specific E1 and E2 loci where mutation prevented MAb binding to transitional epitopes. One of the transitional epitopes identified (E2 residues 200 to 202) lies in the E2 190-216 region, which harbors two major neutralization sites, E2a and E2b, and an N-linked glycosylation site at E2 196. The glycosylation signal was eliminated by site-directed mutagenesis of a full-length cDNA clone of the Sindbis virus genome. The absence of a carbohydrate moiety did not expose the transitional epitopes mapped to this locus, suggesting that on native virions, the inaccessibility of the E2 200-202 determinant was inherent in the structure of the glycoprotein spike.

Amino Acid Sequence↗
PubMed1985

Receptor-mediated endocytosis of a ricin-colloidal gold conjugate in vero cells. Intracellular routing to vacuolar and tubulo-vesicular portions of the endosomal system.

We have prepared a conjugate (Ri-Au) of the toxic plant protein ricin and colloidal gold (particle size 5 nm) and used it for internalization studies in monolayer cultures of Vero cells. The Ri-Au conjugate was very stable, with only little release of ricin ([125I]Ri) from the gold particles within a pH range of 4.5-8.0. Within 2 h at 37 degrees C, only very little intracellular degradation of the ricin preparation ([125I]Ri-Au) occurred. The cells bound the same proportion of native ricin ([125I]Ri) and Ri-Au from the medium, and the kinetics of toxicity (decrease in cellular incorporation of [3H]leucine) of [125I]Ri and [125I]Ri-Au were also comparable. At 4 degrees C, the cell-surface binding of Ri-Au was continuous and distinct, as revealed by electron microscopy. This binding was specific, since almost no Ri-Au surface binding occurred at 4 degrees C in the presence of 0.1 M lactose or 1 mg/ml native (unlabelled) ricin. Within the first 30 min of warming prelabelled cells to 37 degrees C, the amount of surface-associated Ri-Au decreased considerably (from 150 to 60 gold particles per micron cell surface in 40 nm sections). Coated pits and vesicles were involved in the internalization of Ri-Au, and within 5-30 min at 37 degrees C Ri-Au had been delivered to vacuolar and tubulo-vesicular portions of the endosomal system, and later also to lysosomes. Analysis of very thin (ca 20 nm) serial sections revealed that most of the tubulo-vesicular elements were separate structures not connected to the membrane of the vacuolar portion. Data here presented indicate that our ricin conjugate, like many "physiological' ligands and viruses, is internalized by receptor-mediated endocytosis via the coated pit-endosomal pathway.

Animals↗
PubMed1977

Reconstitution of tobacco mosaic virus rods occurs bidirectionally from an internal initiation region: demonstration by electron microscopic serology.

RNA of a common strain of tobacco mosaic virus was sequentially reconstituted first with protein of the same strain and then with protein of a tomato strain. The reconstituted particles were treated with specific antibodies against the two strain particles and were examined electron microscopically to locate the regions of rods encapsidated with the respective proteins. The results demonstrated that reconstitution starts at an internal region of the RNA about 830 nucleotides from an end that recent studies indicate to be the 3' terminus. Evidence was obtained to show that reconstitution proceeds toward the 5' terminus of the RNA until it is fully encapsidated. Encapsidation of the 3' distal portion of the RNA apparently occurs later and in a direction opposite to that of the encapsidation of the 5' distal portion.

Binding Sites↗
PubMed1988

HIV infection does not require endocytosis of its receptor, CD4.

The T cell surface molecule CD4 interacts with class II MHC molecules on the surface of target cells as well as with the envelope glycoprotein of human immunodeficiency virus (HIV). Internalization of CD4 molecules is observed after exposure of CD4+ T cells to either phorbol esters or appropriate antigen-bearing target cells. To determine whether HIV entry proceeds via receptor-mediated endocytosis or direct viral fusion with the cell membrane, we have constructed two mutants in the cytoplasmic domain of the CD4 protein that severely impair the ability of CD4 molecules to undergo endocytosis. Quantitative infectivity studies reveal that HeLa cell lines expressing wild-type or mutant CD4 molecules are equally susceptible to HIV infection. In addition, HIV binding does not lead to CD4 endocytosis. These studies indicate that although the CD4 molecule can be internalized, HIV entry proceeds via direct fusion of the viral envelope with the cell membrane.

Acquired Immunodeficiency Syndrome↗
PubMed1984

Uptake and transport of herpes simplex virus in neurites of rat dorsal root ganglia cells in culture.

Attachment and neuritic transport of herpes simplex virus (HSV) type 1 (McIntyre) were studied in a cell culture system with dissociated cells of rat dorsal root ganglia. The two-chamber cell culture system containing a diffusion barrier penetrated by neurites of cultured sensory neurons permitted infection of neurites extending outside the diffusion barrier without exposure of the neuronal cell soma. HSV adsorbed to neuritic extensions and reached the neuronal soma within 1.5 h post-inoculation. Neuritic uptake and transport of HSV were inhibited in the presence of cytochalasin B. Internalization of virus in neurites was preceded by attachment of virus to the neurite plasma membrane. Neurites transported viral nucleocapsids (NC) through the diffusion barrier of the cultures. Destruction of the neuritic extensions before or shortly after peripheral virus inoculation blocked spread of infection to the cell soma. No infection was established when neuritic extensions were exposed to viral NC and NC were then not observed inside the neurite plasma membrane. Virus produced in neurons, when HSV was inoculated into the inner culture chamber containing the neuronal cell bodies, was transported as enveloped virus in cytoplasmic vesicles from the neuronal cell body towards the periphery. Schwann cells were infected by viropexis. Shortly after infection virions were observed in vacuoles of the cytoplasm.

Animals↗
PubMed2000

Cloning and characterization of a retroviral plasmid, pCC1, for combination suicide gene therapy.

Introduction of the herpes simplex virus thymidine kinase (HSV-TK) gene into mammalian cells confers specific sensitivity to killing by the anti-herpes drug ganciclovir (GCV). This gene has therefore been used in a number of cancer gene therapy protocols as a therapeutic gene. However, the therapeutic efficacy of HSV-TK/GCV in cancer gene therapy experiments can be augmented by additional therapeutic genes. We have cloned a retroviral plasmid, pCC1, containing a fusion gene of HSV-TK and Sh-ble driven by an internal simian virus 40 early promoter. This gene encodes a fusion protein that confers GCV sensitivity and Zeocin resistance when introduced into mammalian cells. A multiple cloning site (MCS) allows the introduction of a second therapeutic gene under the transcriptional control of the Moloney murine leukemia virus 5' long terminal repeat. We have generated packaging cell lines electroporated with pCC1 or pCC1 rtIL-2 S (rat interleukin-2 gene cloned in the sense direction in the MCS), the supernatants of which transfer GCV sensitivity only, or both GCV sensitivity and rtIL-2 production, respectively to rat ovarian cancer cells. This plasmid may be useful for the study of combination suicide gene therapy strategies.

Animals↗
PubMed2004

Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells.

The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host.

Cell Adhesion Molecules↗
PubMed1992

Defective interfering L RNA segments of tomato spotted wilt virus retain both virus genome termini and have extensive internal deletions.

Defective interfering (DI) RNA molecules derived from the genomic L RNA segment of tomato spotted wilt virus (TSWV) were generated during sequential passage of the virus at high multiplicity. Characterization of DI RNAs from four distinct isolates by Northern blot analysis and sequence determination revealed that both the 5' and 3' genomic termini were retained in these molecules. Each DI RNA contained a single internal deletion of approximately 60% to 80% of the L RNA segment. All DI RNAs studied maintain an open reading frame (ORF) which suggests that these defective molecules should be translatable by ribosomes. Detection of only defective molecules with ORFs indicates either that association with ribosomes or translation is a prerequisite for the selection and maintenance of replicating DI RNAs, or that the truncated proteins produced play a role in their selection or replication. Analysis of the junction sites in the DI RNAs showed that short nucleotide sequences are repeated, one at the release and another at the reinitiation point on the L RNA. One of these is lost during the generation of the DI molecules. The presence of repeated sequences at the junction sites seems to be unique for tospovirus DI L RNAs; they have not been described for other DI systems of either positive- or negative-strand RNA viruses. A model for TSWV DI RNA generation is proposed in which the viral polymerase can 'jump' across the internal sequences from one secondary structure to another containing the repeated sequences, during the replication of the viral complementary L RNA segment.

Base Sequence↗
PubMedDate not supplied

[Serological markers of hepatitis B virus in hospitalized patients (Gastroenterology and Internal Medicine Service of the University Hospital Center of Brazzaville - Congo )].

The prevalence of serum markers of the hepatitis B virus was studied in 139 patients, 88 men and 51 women, at the Gastroenterology and Internal Medicine Department at the University Hospital in Brazzaville (Congo). The findings show that 125 individuals (89%), 79 men and 46 women, show signs of infection. Only 14 patients, 9 men and 5 women, show no hepatitis B virus markers. 64 individuals (46%) are carriers of Ag HBS, and among these, 23 (35.9%) are carriers of Ag HBe. Ac anti HBC was found 116 times (83.4%): 12 times by itself, and 16 times in association with Ac anti HBS. 43 individuals (30.9 %) are carriers of Ac anti HBS. Such high frequency of Ac anti HBS, whether or not accompanied by Ac anti HBC, argues in favor of the age of the infection. The study points out the high frequency of hepatitis B virus markers (89.8 %) compared with blood donors (7 to 9 %). This should incite government officials to set up some preventive procedures.

Adolescent↗
PubMed1996

Vaccinia virus glycoprotein A34R is required for infectivity of extracellular enveloped virus.

The vaccinia virus strain Western Reserve (WR) A34R gene encodes a C-type lectin-like glycoprotein, gp22-24, that is present in the outer membrane of extracellular enveloped virus (EEV) with type II membrane topology (S.A. Duncan and G.L. Smith, J. Virol. 66:1610-1621, 1992). Here we that a WR A34R deletion mutant (WR delta A34R) released 19- to 24-fold more EEV from infected cells than did WR virus, but the specific infectivity of the released virions was reduced 5- to 6-fold. Rupture of the WR delta A34R EEV outer envelope by freeze-thawing increased virus infectivity by five- to sixfold, because of the release of infectious intracellular mature virus. All other known EEV-specific proteins are incorporated into WR delta A34R EEV, and thus the loss of gp22-24 is solely responsible for the reduction of EEV specific infectivity. The WR delta A34R virus is highly attenuated in vivo compared with WR or a revertant virus in which the A34R gene was reinserted into WR delta A34R. This attenuation is consistent with the known important role of EEV in virus dissemination and virulence. Vaccinia virus strain International Health Department-J (IHD-J) produces large amounts of EEV and forms comets because of an amino acid substitution within the A34R protein (R. Blasco, R. Sisler, and B. Moss, J. Virol. 67:3319-3325, 1993), but despite this, IHD-J EEV has a specific infectivity equivalent to that of WR EEV. Substitution of the IHD-J A34R gene into the WR strain induced comet formation and greater release of EEV, while coexpression of both genes did not; hence, the WR phenotype is dominant. All orthopoxviruses tested express the A34R protein, but most viruses, including variola virus, have the WR rather than the IHD-J A34R genotype. The A34R protein affects plaque formation, EEV release, EEV infectivity, and virus virulence.

Animals↗
PubMed1997

EBV binds to lymphocytes of transgenic mice that express the human CR2 gene.

Epstein Barr virus (EBV) is unable to bind to or infect normal mouse lymphocytes. A construct containing the human complement receptor type 2 (CR2) gene, the receptor for EBV, was placed under the control of the IgH/c-fos enhancer/promoter and microinjected into single cell embryos. A total of five transgenic mouse lines were established and four expressed hCR2 mRNA. Flow cytometry and immunostaining revealed that approximately 15-30% of the lymphocytes from the thymus, spleen and lymph nodes expressed hCR2 protein on their surface and bound EBV. Despite this binding, less than 1% of the cells showed evidence that the virus was internalized or replicated. Transgenic mouse lymphocytes, expressing hCR2, could not be immortalized with EBV. It is concluded that the simple expression of hCR2 receptor on mouse lymphocytes is not sufficient for efficient infection.

Animals↗
PubMed2006

Inhibition of TNF receptor 1 internalization by adenovirus 14.7K as a novel immune escape mechanism.

The adenoviral protein E3-14.7K (14.7K) is an inhibitor of TNF-induced apoptosis, but the molecular mechanism underlying this protective effect has not yet been explained exhaustively. TNF-mediated apoptosis is initiated by ligand-induced recruitment of TNF receptor-associated death domain (TRADD), Fas-associated death domain (FADD), and caspase-8 to the death domain of TNF receptor 1 (TNFR1), thereby establishing the death-inducing signaling complex (DISC). Here we report that adenovirus 14.7K protein inhibits ligand-induced TNFR1 internalization. Analysis of purified magnetically labeled TNFR1 complexes from murine and human cells stably transduced with 14.7K revealed that prevention of TNFR1 internalization resulted in inhibition of DISC formation. In contrast, 14.7K did not affect TNF-induced NF-kappaB activation via recruitment of receptor-interacting protein 1 (RIP-1) and TNF receptor-associated factor 2 (TRAF-2). Inhibition of endocytosis by 14.7K was effected by failure of coordinated temporal and spatial assembly of essential components of the endocytic machinery such as Rab5 and dynamin 2 at the site of the activated TNFR1. Furthermore, we found that the same TNF defense mechanisms were instrumental in protecting wild-type adenovirus-infected human cells expressing 14.7K. This study describes a new molecular mechanism implemented by a virus to escape immunosurveillance by selectively targeting TNFR1 endocytosis to prevent TNF-induced DISC formation.

Adenoviridae↗