Functional fluorescent protein insertions in herpes simplex virus gB report on gB conformation before and after execution of membrane fusion.
Functional fluorescent protein insertions in herpes simplex virus gB report on gB conformation before and after execution of membrane fusion.
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DOI：
10.1371/journal.ppat.1004373
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发表时间：
2014-09
期刊：
影响因子：
6.7
通讯作者：
Cohen GH
中科院分区：
文献类型：
--
作者：
Gallagher JR;Atanasiu D;Saw WT;Paradisgarten MJ;Whitbeck JC;Eisenberg RJ;Cohen GH
Entry of herpes simplex virus (HSV) into a target cell requires complex interactions and conformational changes by viral glycoproteins gD, gH/gL, and gB. During viral entry, gB transitions from a prefusion to a postfusion conformation, driving fusion of the viral envelope with the host cell membrane. While the structure of postfusion gB is known, the prefusion conformation of gB remains elusive. As the prefusion conformation of gB is a critical target for neutralizing antibodies, we set out to describe its structure by making genetic insertions of fluorescent proteins (FP) throughout the gB ectodomain. We created gB constructs with FP insertions in each of the three globular domains of gB. Among 21 FP insertion constructs, we found 8 that allowed gB to remain membrane fusion competent. Due to the size of an FP, regions in gB that tolerate FP insertion must be solvent exposed. Two FP insertion mutants were cell-surface expressed but non-functional, while FP insertions located in the crown were not surface expressed. This is the first report of placing a fluorescent protein insertion within a structural domain of a functional viral fusion protein, and our results are consistent with a model of prefusion HSV gB constructed from the prefusion VSV G crystal structure. Additionally, we found that functional FP insertions from two different structural domains could be combined to create a functional form of gB labeled with both CFP and YFP. FRET was measured with this construct, and we found that when co-expressed with gH/gL, the FRET signal from gB was significantly different from the construct containing CFP alone, as well as gB found in syncytia, indicating that this construct and others of similar design are likely to be powerful tools to monitor the conformation of gB in any model system accessible to light microscopy. 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.
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