Entry of alphaviruses at the plasma membrane converts the viral surface proteins into an ion-permeable pore that can be detected by electrophysiological analyses of whole-cell membrane currents.
Alphaviruses are small enveloped viruses that have been used extensively as model enveloped viruses. During infection, virus particles are taken up into endosomes, where a low pH activates the viral fusion protein, E1. Fusion of the viral and the endosomal membranes releases the viral core into the cytoplasm where cores are disassembled by interaction with 60S ribosomal subunits. Recently, we have shown that in vitro this disassembly is strongly stimulated by low pH. We have proposed that after entry of the core into the cytoplasm, the viral membrane proteins that have been transferred to the endosomal membrane form an ion-permeable pore in the endosome. The resulting flow of protons from the endosome into the cytoplasm through this pore could generate a low-pH environment for core disassembly in vivo. Here we report two types of analysis aimed at the identification of such pores. First, the release of [3H]choline from the interior of liposomes was analysed in the presence of virus particles and viral proteins. Secondly, cells were infected with Sindbis or Semliki Forest alphaviruses at the plasma membrane and the possible generation of ion-permeable pores during this process was analysed by whole-cell voltage clamp analysis of the membrane current. The results obtained indicated that the proposed pores are in fact generated and allowed us to identify the formation of individual pores. Available evidence indicates that the alphavirus E1 protein probably forms these pores. Proteins homologous to the alphavirus E1 protein are present in flaviviruses and hepatitis C virus.
Wengler
Gerd
G
Institut für Virologie der Veterinärmedizin and.
Koschinski
Andreas
A
Rudolf-Buchheim-Institut für Pharmakologie, Justus-Liebig-Universität, D-35392 Giessen, Germany.
Wengler
Gisela
G
Institut für Virologie der Veterinärmedizin and.
Dreyer
Florian
F
Rudolf-Buchheim-Institut für Pharmakologie, Justus-Liebig-Universität, D-35392 Giessen, Germany.
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology / Q000821:virology*; D002463:Cell Membrane Permeability; D006224:Cricetinae; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000378:metabolism; D008561:Membrane Fusion; D008564:Membrane Potentials; D051379:Mice; D018408:Patch-Clamp Techniques; D012672:Semliki forest virus / Q000378:metabolism / Q000472:pathogenicity*; D012845:Sindbis Virus / Q000378:metabolism / Q000472:pathogenicity*; D015678:Viral Structural Proteins / Q000378:metabolism*
D008081:Liposomes; D015678:Viral Structural Proteins