Role of simvastatin and methyl-beta-cyclodextrin [corrected] on inhibition of poliovirus infection.

Cells exposed to simvastatin or to methyl-beta-cyclodextrin show reduced poliovirus infection, without alteration in virus binding or on the kinetics of genome entry, suggesting that the steps which are altered are those post uncoating and genome entry. Reduction of infection by cyclodextrin is reversed by increasing MOI whereas that produced by simvastatin treatment is not, suggesting that the effects on infection are not due to a reduction in cholesterol. The differences in the characteristics of inhibition can be explained by the differential effects of the compounds. Cyclodextrin inhibits the store-operated calcium channels, suggesting that reduction in infection is through translational inhibition. Simvastatin produces vesicles from internal membranes which cannot sustain viral RNA synthesis, reducing infection through reduced transcription. The results indicate that the impact on viral infection by the cholesterol-modifying agents is due to the cellular changes produced rather than due to disruption of the cholesterol-rich domains.

Genome delivery and ion channel properties are altered in VP4 mutants of poliovirus.

During entry into host cells, poliovirus undergoes a receptor-mediated conformational transition to form 135S particles with irreversible exposure of VP4 capsid sequences and VP1 N termini. To understand the role of VP4 during virus entry, the fate of VP4 during infection by site-specific mutants at threonine-28 of VP4 (4028T) was compared with that of the parental Mahoney type 1 virus. Three virus mutants were studied: the entry-defective, nonviable mutant 4028T.G and the viable mutants 4028T.S and 4028T.V, in which residue threonine-28 was changed to glycine, serine, and valine, respectively. We show that mutant and wild-type (WT) VP4 proteins are localized to cellular membranes after the 135S conformational transition. Both WT and viable 4028T mutant particles interact with lipid bilayers to form ion channels, whereas the entry-defective 4028T.G particles do not. In addition, the electrical properties of the channels induced by the mutant viruses are different from each other and from those of WT Mahoney and Sabin type 3 viruses. Finally, uncoating and/or cytoplasmic delivery of the viral genome is altered in the 4028T mutants: the 4028T.G lethal mutant does not release its genome into the cytoplasm, and genome delivery is slower during infection by mutant 4028T.V 135S particles than by mutant 4028T.S or WT 135S particles. The distinctive electrical characteristics of the different 4028T mutant channels indicate that VP4 sequences might form part of the channel structure. The different entry phenotypes of these VP4 mutants suggest that the ion channels may be related to VP4's role during genome uncoating and/or delivery.

Poliovirus binding to its receptor in lipid bilayers results in particle-specific, temperature-sensitive channels.

Poliovirus (PV) infection starts with binding to its receptor (PVR), followed by a receptor-aided, temperature-sensitive conformational change of the infectious particle (sedimenting at 160S) to a particle which sediments at 135S. Reported in this communication is the successful incorporation into lipid bilayers of two forms of the receptor: the full-length human receptor and a modified clone in which the extracellular domains of the receptor were fused to a glycosylphosphatidylinositol tail. Addition of virus (160S) to receptor-containing bilayers leads to channel formation, whereas no channels were observed when the receptor-modified viral particle (135S) was added. Increasing the temperature from 21 to 31 degrees C led to a 10-fold increase in the magnitude of the single channel conductance, which can be interpreted as a conformational change in the channel structure. A mutant PV with an amino acid change in VP4 (one of the coat proteins) which is defective in genome uncoating failed to produce channels, suggesting that VP4 might be involved in the channel architecture. These studies provide the first electrophysiological characterization of the interactions between poliovirus and its receptor incorporated into a lipid bilayer membrane. Furthermore, they form the foundation for future studies aiming at defining the molecular architecture of the virus-receptor complex.

Microfabricated teflon membranes for low-noise recordings of ion channels in planar lipid bilayers.

We present a straightforward, accessible method for the fabrication of micropores with diameters from 2 to 800 micro m in films of amorphous Teflon (Teflon AF). Pores with diameters