Functional Fluorescent Protein Insertions in Herpes Simplex Virus gB Report on gB Conformation before and after Execution of Membrane Fusion


Viral fusion proteins undergo complicated conformational changes in order to fuse viral and host membranes during viral entry. Conformational changes between prefusion and postfusion states also allow the virus to hide critical regions of the fusion machinery from the immune system. The structure of herpes simplex virus fusion protein gB is known only in its postfusion state, while the prefusion structure is unknown. To study the prefusion state, we created fluorescent protein (FP) insertions within gB and tested them for fusion activity. Due to the size of the fluorescent protein insertion, regions in gB that tolerate this insertion must be solvent exposed, thereby describing structural features of the prefusion structure. We created functional gB constructs with FP insertions in two of the three globular domains of gB, while non-functional insertions in the third domain suggested that it may be buried in the prefusion structure. Additionally, we created a dual-labeled FP gB construct which we found to report on the conformation of gB before and after fusion. Using this dual-labeled gB construct, we have demonstrated how fluorescence-based methods can be used to directly study dynamics of viral fusion proteins in living cells.


Vyšlo v časopise: Functional Fluorescent Protein Insertions in Herpes Simplex Virus gB Report on gB Conformation before and after Execution of Membrane Fusion. PLoS Pathog 10(9): e32767. doi:10.1371/journal.ppat.1004373
Kategorie: Research Article
prolekare.web.journal.doi_sk: 10.1371/journal.ppat.1004373

Souhrn

Viral fusion proteins undergo complicated conformational changes in order to fuse viral and host membranes during viral entry. Conformational changes between prefusion and postfusion states also allow the virus to hide critical regions of the fusion machinery from the immune system. The structure of herpes simplex virus fusion protein gB is known only in its postfusion state, while the prefusion structure is unknown. To study the prefusion state, we created fluorescent protein (FP) insertions within gB and tested them for fusion activity. Due to the size of the fluorescent protein insertion, regions in gB that tolerate this insertion must be solvent exposed, thereby describing structural features of the prefusion structure. We created functional gB constructs with FP insertions in two of the three globular domains of gB, while non-functional insertions in the third domain suggested that it may be buried in the prefusion structure. Additionally, we created a dual-labeled FP gB construct which we found to report on the conformation of gB before and after fusion. Using this dual-labeled gB construct, we have demonstrated how fluorescence-based methods can be used to directly study dynamics of viral fusion proteins in living cells.


Zdroje

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