Karl-Otto Habermehl

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.

Comparison of heparin-sensitive attachment of pseudorabies virus (PRV) and herpes simplex virus type 1 and identification of heparin-binding PRV glycoproteins

To determine whether heparan sulphate residues on the cellular surface could serve as an attachment receptor for pseudorabies virus (PRV), the effect of heparin on PRV in plaque reduction and adsorption tests was investigated. PRV was significantly less sensitive to heparin than was herpes simplex virus type 1 (HSV-1). At concentrations of 500 micrograms/ml heparin the number of plaques formed by PRV was reduced to 7% of the untreated control whereas the number of plaques formed by HSV-1 was reduced to below 0.1%. Adsorption of PRV to host cells was also less sensitive to heparin treatment than was adsorption of HSV-1. Experiments concerning the binding sites of PRV showed that heparin binds to the disulphide-linked glycoprotein complex gII (PRV gB), gIII (PRV gC) and probably gV.

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.

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.

Specific cross-linking of capsid proteins to virus RNA by ultraviolet irradiation of poliovirus.

Summary Poliovirus was irradiated with u.v. light under conditions causing approx. 5% cross-linking of capsid protein to virus RNA. Cross-linked RNA-protein complexes, freed from unbound protein, were treated with nuclease, and then analysed on SDS-polyacrylamide gels. The smallest capsid polypeptide VP4 was found to be associated with the RNA to the greatest degree, followed by VP2 and VP1, while VP3 was attached only in trace amounts. Low radiation doses, which produced cross-linking of RNA to protein, did not cause breakdown of the virus particles or conformational changes of the capsid as examined physically and serologically. However, higher doses caused structural alterations of the virus capsid.

A peptide-model for the heparin-binding property of pseudorabies virus glycoprotein III

The pseudorabies virus glycoprotein III (PrV-gIII) has been identified previously as the major viral component binding to a heparin-like receptor on the surface of target cells. The amino acid sequence of gIII contains three regions corresponding to consensus sequences for heparin binding. A synthetic peptide corresponding to amino acids 134 to 141 of PrV-gIII bound heparin in a dot blot assay. In contrast, a synthetic peptide derived from amino acids 290–299 of PrV-gIII did not bind heparin. We therefore conclude that the region containing amino acid 134–141 is involved in binding to the heparin-like cellular receptor.

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.

Differences in the morphology of herpes simplex virus infected cells: I. Comparative scanning and transmission electron microscopic studies on HSV-1 infected HEp-2 and chick embryo fibroblast cells.

Infection with herpes simplex virus type 1 (HSV-1) induces different morphological changes in different cell lines. This is demonstrated by comparative scanning (SEM and transmission (TEM) electron microscopic investigations of cell cultures prepared under identical conditions. SEM of HSV-1 infected HEp-2 cells reveals a slightly altered cell surface: only the number of the microvilli is reduced. Large amounts of released virions are detectable adhering to the outer plasma membrane. Ultra-thin sections show typical virus maturation steps in the nuclei (formation of nucleocapsids and virus budding from the inner lamella of the nuclear membrane) and in the cytoplasm (egress of enveloped nucleocapsids through membranous structures). HSV-infected primary chick embryo fibroblast (CEF) cells are characterized by crumpled and rough surfaces without virus particles adhering to the membrane. Ultra-thin sections exhibit atypical virus maturation with many unenveloped nucleocapsids within the cytoplasm. The distribution of HSV-induced antigen(s) on the surface of the infected cells is identical in the two cell systems as determined by the peroxidase labelling technique. The c.p.e. (as seen by phase contrast light microscopy) is similar in both HEp-2 and CEF cells: both fusion and rounding up is induced in the infected cells.