Role of plasma membrane lipid microdomains in respiratory syncytial virus filament formation.
The fusion protein (F) of respiratory syncytial virus (RSV) is the envelope glycoprotein responsible for the characteristic cytopathology of syncytium formation. RSV has been shown to bud from selective areas of the plasma membrane as pleomorphic virions, including both filamentous and round particles. With immunofluorescent microscopy, we demonstrated evidence of RSV filaments incorporating the fusion protein F and colocalizing with a lipid microdomain-specific fluorescent dye, 1,1-dihexadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate. Western blot analysis of Triton X-100 cold-extracted membrane fractions confirmed the presence of RSV proteins within the lipid microdomains. RSV proteins also colocalized with cellular proteins associated with lipid microdomains, caveolin-1, and CD44, as well as with RhoA, a small GTPase. ADP-ribosylation of RhoA by Clostridium botulinum exotoxin inactivated RhoA signaling and resulted in the absence of RSV-induced syncytia despite no significant change in viral titer. We demonstrated an overall decrease in both the number and length of the viral filaments and a shift in the localization of F to nonlipid microdomain regions of the membrane in the presence of C3 toxin. This suggests that the selective incorporation of RSV proteins into lipid microdomains during virus assembly may lead to critical interactions of F with cellular proteins, resulting in microvillus projections necessary for the formation of filamentous virus particles and syncytium formation. Thus, manipulation of membrane lipid microdomains may lead to alterations in the production of viral filaments and RSV pathogenesis and provide a new pharmacologic target for RSV therapy.
McCurdy
Lewis H
LH
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-3017, USA. bgraham@nih.gov
Graham
Barney S
BS
D036002:ADP Ribose Transferases / Q000494:pharmacology; D001905:Botulinum Toxins / Q000494:pharmacology; D051242:Caveolin 1; D022461:Caveolins / Q000032:analysis; D002460:Cell Line; D003505:Cyclodextrins / Q000494:pharmacology; D006801:Humans; D018960:Hyaluronan Receptors / Q000032:analysis; D021962:Membrane Microdomains / Q000502:physiology*; D012136:Respiratory Syncytial Viruses / Q000502:physiology*; D014760:Viral Fusion Proteins / Q000502:physiology*; D019065:Virus Assembly; D020742:rhoA GTP-Binding Protein / Q000032:analysis
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D003505:Cyclodextrins; D018960:Hyaluronan Receptors; D014760:Viral Fusion Proteins; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D020742:rhoA GTP-Binding Protein