Heinz Zeichhardt

Collagen degradation in a murine myocarditis model: relevance of matrix metalloproteinase in association with inflammatory induction

OBJECTIVE Myocardial collagen degradation is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase (TIMPs). The possible relevance of MMPs in association with the inflammatory induction was investigated in a murine coxsackievirus B3 myocarditis model. METHODS Hearts from viral infected and sham-infected BALB/c mice were analyzed by semi-quantitative RT-PCR, picrosirius red staining, Western blot analysis, and immunohistochemistry. RESULTS In viral infected mice, both mRNA and protein abundance for collagen type I remained unaltered. In addition, picrosirius red staining showed the unchanged total collagen content. However, degraded soluble fraction of collagen type I protein was increased. Moreover, the mRNA abundance for MMP-3 and MMP-9 was upregulated, whereas the mRNAs for TIMP-1 and TIMP-4 were downregulated, respectively. The upregulation of MMP-3/MMP-9 and downregulation of TIMP-4 were confirmed at the protein level, and were associated with significantly increased mRNA levels of interleukin 1beta, tumor necrosis factor-alpha, transforming growth factor-beta1 and interleukin-4. CONCLUSION The increment of MMPs in the absence of counterbalance by TIMPs may lead to a functional defect of the myocardial collagen network by posttranslational mechanisms. This may contribute significantly to the development of left ventricular dysfunction in murine viral myocarditis. The inflammatory response with induction of cytokines may mediate the dysregulation of the myocardial MMP/TIMP systems.

Entry of Poliovirus Type 1 and Mouse Elberfeld (ME) Virus into HEp-2 Cells: Receptor-mediated Endocytosis and Endosomal or Lysosomal Uncoating

Poliovirus type 1 appeared from electron microscope studies to enter HEp-2 cells by receptor-mediated endocytosis. On adsorption the virus was evenly distributed over the cell surface, with some preference for the microvilli and their bases. Invagination of the cell surface membrane with the attached virus commenced at coated pits and led to the formation of virus-containing coated vesicles in the cytoplasm. These coated vesicles fused with intracellular vesicles to form endosomes. When cells infected with poliovirus or Mouse Elberfeld virus were treated with the weak bases chloroquine, NH4Cl or the ionophore monensin to raise the intraendosomal and intralysosomal pH above 6, virus-directed macromolecular synthesis and production of progeny were prevented. These results suggest that the virus genomes are released to the cytoplasm via endosomes and/or lysosomes by a pH-dependent process.

Inhibition of poliovirus uncoating by disoxaril (WIN 51711)

Disoxaril [WIN 51711, 5-[7-[4(4,5-dihydro-2-oxazolyl)phenoxy]heptyl]-3- methylisoxazole] inhibits the replication of polioviruses types 1 and 2 in HeLa cells by stabilizing the virus capsid, which results in the inhibition of the pH-dependent viral uncoating in endosomes and/or lysosomes. As shown by electron microscopy the virus entered into the cell by receptor-mediated endocytosis via coated pits and coated vesicles into endosomes irrespective of the presence or absence of the compound. Measurements of viral RNA synthesis showed that disoxaril completely inhibited the arrival of viral RNA in the cytoplasm for new RNA synthesis only when the inocula were preincubated with disoxaril for 15 min at 37 degrees at 0.3 microgram disoxaril/ml for poliovirus type 1 and 0.03 microgram disoxaril/ml for poliovirus type 2. Simultaneous addition of the compound and virus resulted in reduced inhibition of viral RNA synthesis. The inhibitory effect of the compound could be partially reversed up to 25 min p.i. if the compound was eluted from the cells.

Internalization of human rhinovirus 14 into HeLa and ICAM-1-transfected BHK cells

Abstract Virus adsorption and uptake of human rhinovirus 14 (HRV14) were studied with HeLa cells and baby hamster kidney (BHK) cells which were transfected with the HRV14 receptor intercellular adhesion molecule-1 (ICAM-1). Transmission electron microscopy of HeLa cells revealed that HRV14 was internalized via clathrin-coated pits and -coated vesicles. A minority of virus particles also used uncoated vesicles for entry. The internalization showed the characteristics of receptor-mediated endocytosis. Presence of the carboxylic ionophore monensin inhibited viral uncoating, indicating a pH-dependent entry mechanism. The expression of ICAM-1 on the surface of the ICAM-1 transfected baby hamster kidney cells (BHK-ICAM cells) allowed extensive virus adsorption and internalization through membrane channels. Virus particles were lined up in these channels like pearls on a string, but did not induce a productive infection. Although ICAM-1 was expressed to the same degree on BHK-ICAM and HeLa cells, HRV14 induced neither viral protein and RNA syntheses nor infectious virus progeny in BHK-ICAM cells. ICAM-1 on the transfected BHK cells was a functional active receptor as it rendered these cells permissive to coxsackievirus A21. These results suggest that HRV14 uptake into BHK-ICAM cells is blocked directly in or shortly after its final step of internalization, the uncoating. Our findings underline that the receptor ICAM-1 determines virus uptake into cells, however, is not sufficient to confer susceptibility of BHK cells to HRV14 infection.

Detection of antibodies against viral capsid proteins of human herpesvirus 8 in AIDS-associated Kaposi's sarcoma

Abstract Sequences of a new herpesvirus with homology to gammaherpesvirinae were recently identified in AIDS-associated Kaposi’s sarcoma (KS). Subsequently this novel virus, called KS-associated virus (KSHV) or human herpesvirus (HHV) 8 was detected in classical KS and AIDS-associated body cavity based lymphomas by polymerase chain reaction. In this report major and minor capsid proteins of HHV-8 were molecularly cloned and produced as recombinant proteins in Escherichia coli. Sera from 69 HIV-1 infected patients with KS, 30 HIV-1 infected patients without KS and 106 control individuals were tested by enzyme-linked immunosorbent assay for anti-HHV-8 capsid IgM and IgG antibodies. Sera from four patients were tested over periods ranging from 18 months to 6 years. IgG antibodies directed against HHV-8 capsid antigens were detected in patients with AIDS-associated KS and in some AIDS patients without KS. Seroconversion with IgM and IgG antibodies directed against HHV-8 capsid proteins occurred more than 1 year prior to diagnosis of KS. In a considerable portion of KS patients no IgM or IgG antibodies against HHV-8 capsid proteins were detected. In these patients there was an inverse relationship between antibodies against HHV-8orf26 and the CD4/CD8 ratio, suggesting that the inconsistency of anti-HHV-8orf26 antibodies is due at least partly to an impaired immune response. No reactivity against HHV-8 capsid antigens was detected in the vast majority of sera from HIV-negative control individuals. Our findings indicate that a specific humoral immune response against capsid proteins is raised in HHV-8 infected individuals, and that anti-capsid antibodies can be used to diagnose HHV-8 infection. The correlation between occurrence of anti-HHV-8 antibodies and KS supports the hypothesis of a causative role of HHV-8.

Recovery of structurally intact and infectious poliovirus type 1 from HeLa cells during receptor-mediated endocytosis

Poliovirus type 1 enters HeLa cells by receptor-mediated endocytosis as an intact virus. Up to 30 min after adsorption complete virus particles still containing VP4 and sedimenting with 156 S could be recovered from the cells. These virus particles were N-antigenic and infectious. Thirty minutes after adsorption the recovery of intact and infectious virus decreased. This decrease presumably reflects viral uncoating in the acidic endosomes and/or lysosomes because virus particles could be localized in endosomes at this time. The direct involvement of clathrin-coated structures in the endocytosis of poliovirus has been deduced from the enclosure of poliovirus in coated vesicles at 10 min after adsorption. At this time intact and infectious virus could be recovered only after the coated vesicles were disrupted by treatment with 0.5 M Tris at pH 7.0.

Neutralization of poliovirus by polyclonal antibodies requires binding of a single IgG molecule per virion

SummaryNeutralization of poliovirus type 1 was studied using radioactively labelled polyclonal IgG. With nonsaturating antibody concentrations various virus-antibody complexes were produced which were isolated by sucrose gradient centrifugation and identified by electron microscopy as virus monomers, dimers, trimers, tetramers and pentamers. The neutralization rate (n. r.) of each of the virus-antibody complexes relative to non-neutralized virus and the stoichiometry have been estimated. The monomer fraction showed that about every fifth virion was associated with one IgG molecule and neutralized. The antibody was bivalently attached. The majority of virus particles formed aggregates of different sizes, which were cross-linked by antibodies. The following neutralization rates and ratios of IgG to virus (IgG/V) were determined for the oligomers: dimers, 59.2 per cent n. r. and 0.55 IgG/V; trimers, 67.3 per cent n. r. and 0.66 IgG/V; tetramers, 79.0 per cent n. r. and 0.75 IgG/V; pentamers, 86.3 per cent n. r. and 0.98 IgG/V.Two different mechanisms of neutralization are proposed: i) an antibody-mediated mechanism specifically inhibits infectivity of the monomer virus-antibody complexes and ii) reduction of infectivity of oligomer virus-antibody complexes is caused simply by reduction of the actual number of infectious units.Immunoprecipitation of the denatured capsid proteins showed that only VP1 was recognized by the polyclonal IgGs.

EFFECT OF THE QUASSINOIDS GLAUCARUBOLONE AND SIMALIKALACTONE D ON GROWTH OF CELLS PERMANENTLY INFECTED WITH FELINE AND HUMAN IMMUNODEFICIENCY VIRUSES AND ON VIRAL INFECTIONS

Growth of Crandall feline kidney cells permanently infected with feline immunodeficiency virus was inhibited by the anti-cancer quassinoid glaucarubolone whereas growth of uninfected cells was not inhibited. Similar results were obtained for human MOLT-4 cells infected with HIV-1. The results suggest that cell lines permanently infected with either the feline or the human lentivirus exhibit growth response characteristics to the quassinoids in common with other cell lines malignantly transformed. In addition the quassinoids may delay viral infection suggesting some commonality between the mechanism responsible for inhibition of the growth of the transformed phenotype and viral infection.

Dense Particles and Slow Sedimenting Particles Produced by Ultraviolet Irradiation of Poliovirus

Low doses of u.v. radiation rapidly inactivate poliovirus, and the virus is progressively converted into dense particles (DPs) of buoyant density 1.44 g/ml in CsCl. The DPs are structurally and antigenically related to standard virus (N-antigen), i.e. they are indistinguishable from virus in their RNA and protein content and in their sedimentation properties. Furthermore, there is no difference in reactivity of the structural proteins of virus and DPs with the monofunctional reagent [3H]N-succinimidyl propionate (3H-NSP). However, DPs differ from virus in that their capsids are permeable to several ions, and they can be degraded by RNase and protease. Increasing the radiation dose causes a successive transformation of DPs into 105S slow-sedimenting particles (SSPs). The SSPs are antigenically related to 76S artificial empty capsids (AECs) or H-antigen, but they differ physically and structurally from them. The SSPs have a higher S value than AECs and contain all the capsid proteins, including VP4, and the RNA, both of these macromolecules being absent from AECs. It is concluded, therefore, that transformation from N- to H-antigenicity by u.v. radiation does not require release of RNA and VP4. Conversion of virus particles to SSPs correlates with altered reactivity of VP2 and to a lesser extent VP1 and VP3, with the monofunctional reagent 3H-NSP.

Glycoprotein B (gB) of pseudorabies virus interacts specifically with the glycosaminoglycan heparin

We have previously shown that the pseudorabies virus (PrV) glycoproteins gB and gC (former PrV-gII and PrV-gIII) exhibit heparin-binding properties. While PrV-gC functions as the major adsorption protein, the biological role of the heparin-binding properties of PrV-gB are not understood. We used a gC-deleted PrV-mutant, PrV (dlg92/dltk), to analyse the heparin-binding properties of PrV-gB and the biological role of the PrV-gB-protein in adsorption. PrV-gB was the only glycoprotein of this vaccine strain binding to immobilised heparin in in vitro assays. Presence of the gC-protein was not necessary for the interaction of gB with heparin. Soluble heparin also interfered with adsorption of this mutant virus to a similar extent as it blocked adsorption of wild-type PrV (Ka), but it had only a minor inhibitory effect on infectivity of the mutant strain. These results show that PrV-gB interacts specifically with immobilized heparin and heparin-like structures on the cell surface, but this interaction is not required for a productive infection.