Viroporinas de virus animales con genoma RNA

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 13-02-2006Early permeabiiization induced by Sindbis virus particles in BHK cells efficiently pmmotes the co-intemalization of macmmolecules such as alpha-sarcin. By contmt, during late membrane permeabilization, associated with synthesis of virus stnictural pmtehs, only small campounds enter into cells. Vimporins are a new family of vid pmteins that enhance membrane penneability. They are small and very hydrophobic integral membrane pmteins, generally dispensable in virus replication though they play an important mle in virion maturation and budding pmcesses. Most of them have the ability to form ion channels in artificial lipid bilayers. 6K from alphavinrses, M2 from influenza A, 2B from picomavimses, SH h m syncitial respiratory virus, Vpu from human immunodefiency v h m e 1 (HIV-1) and p10 from avian reovirus, among others, have been included in this group. 6K pmtein is a major determinant of late membrane leakiness dwing infection. lnterfacial 6K regions, which are important for membrane permeabilization and v i d budding, have been characterized by expressing 6K variants in bacteria and mammalian ceiis. The permeabilization region of poliovims 2B vimporin was deíined by using synthetic peptides derived from 2B sequence. The peptide P3-25 that corresponds with amphipathic a-helix of 2B, permeabilized both liposomes and mammalian cells. Momver, this peptide penetrates into cells and is located in mitochondna. 2B pmtein expression in BHK cells altered mitochondria panern, inducing a perinuclear redistribution of these organelles. 'Ihis phenomenon wrrelates with the capacity of 2B to induce apoptosis. Synthesis of E protein h m mouse hepatitis virus (MHV-A59) and p7 pmtein from hepatitis C virus (HCV) in bacteria pmvoked membrane permeabilization, as well as gmwih arrest and cellular lysis, in a way similar to that found with the 6K vimporin from alphavims. iu BHK cells, expression of E, p7 and NS4A pmtein from HCV, also enhanced membrane permeabilization, though to different extents. Brefeldin A, an antibiotic that blocks the exocytic transport, powerfully blocked late membrane permeabilization induced both by SV infection and 6K pmtein as well as v i r o p o ~ac tivity of E pmtein. Hence, we believe tha! E and p7 pmteins can be considered new members of the vimporin familiy. The permeabilization capacity of several viroporins was comparatively analyzed and a classification was pmposed. Al1 viroporins studied in this work were able to induce apoptosis when expressed in BHK cells