Effects of Photobleaching on Membrane Fusion and Infectivity of Dye Labeled Sindbis Virus

Sindbis virus;fluorescence dequenching assay;membrance fusion;photoinactivation;R18 fluorescence dye

Thongthai, Wor ; Karen Daniels, Committee Member,Robert Riehn, Committee Member,Keith Weninger, Committee Chair,Thongthai, Wor ; Karen Daniels ; Committee Member ; Robert Riehn ; Committee Member ; Keith Weninger ; Committee Chair

University:North Carolina State University

关键词: Sindbis virus;    fluorescence dequenching assay;    membrance fusion;    photoinactivation;    R18 fluorescence dye;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/37/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

Membrane fusion has long been accepted as a common cell entry mechanism for enveloped viruses.We have observed membrane fusion between Sindbis virus (SIN) and liposomes formed from purified lipids using a fluorescence dequenching assay.We verified that labeling Sindbis virus with R18, DiI, and DiD fluorescent dyes did not decrease its infectivity as determined by plaque assays.We found that infectivity was eliminated after extensive photobleaching of R18-labeled Sindbis virus, and in contrast, DiI and DiD labeled Sindbis both maintain infectivity following bleaching.Even though photobleached R18-labeled Sindbis was not infectious, we verified that it is still fully competent for low pH triggered membrane fusion using a liposome assay where fusion was indicated by the dequenching of a second membrane dye incorporated into the liposome.We attribute the inactivation of Sindbis virus to the propensity of R18 to translocate to the inner leaflet of the viral membrane, thus exposing the viral genome to the byproducts of photobleaching.Possible mechanisms of photoinactivation are discussed.