Direct Transfer of Viral and Cellular Proteins from Varicella-Zoster Virus-Infected Non-Neuronal Cells to Human Axons

Abstract

Varicella Zoster Virus (VZV), the alphaherpesvirus that causes varicella upon primary infection and Herpes zoster (shingles) following reactivation in latently infected neurons, is known to be fusogenic. It forms polynuclear syncytia in culture, in varicella skin lesions and in infected fetal human ganglia xenografted to mice. After axonal infection using VZV expressing green fluorescent protein (GFP) in compartmentalized microfluidic cultures there is diffuse filling of axons with GFP as well as punctate fluorescence corresponding to capsids. Use of viruses with fluorescent fusions to VZV proteins reveals that both proteins encoded by VZV genes and those of the infecting cell are transferred in bulk from infecting non-neuronal cells to axons. Similar transfer of protein to axons was observed following cell associated HSV1 infection. Fluorescence recovery after photobleaching (FRAP) experiments provide evidence that this transfer is by diffusion of proteins from the infecting cells into axons. Time-lapse movies and immunocytochemical experiments in co-cultures demonstrate that non-neuronal cells fuse with neuronal somata and proteins from both cell types are present in the syncytia formed. The fusogenic nature of VZV therefore may enable not only conventional entry of virions and capsids into axonal endings in the skin by classical entry mechanisms, but also by cytoplasmic fusion that permits viral protein transfer to neurons in bulk.

Function and regulation of the fusogen EFF-1 during axonal repair

“…It has been noted that EFF-1 and AFF-1 are sometimes expressed simultaneously in adjacent cells, and that this alternate expression could help to specify fusion partners 40,41 . In the pharynx, two neighboring cells undergo developmental self-fusion to form unicellular tubes.…”

“…EFF-1 must be inserted into both membranes to mediate fusion; current models suggest that its mechanism of action involves the formation of trimers in trans across apposing membranes 28,31 . Its activity is known to be precisely regulated in development, ensuring it mediates fusion at the correct place and time 23, .AFF-1 functions similarly to EFF-1 by mediating specific membrane fusion events during C.elegans development [39][40][41][42] . The mechanism through which it mediates fusion has not yet been determined, but similarly to EFF-1, it requires insertion into both membranes 26 .…”

“…Active signaling from this fragment has been shown to be important for the efficiency of axonal fusion 8 . Molecules acting on the distal fragment are also likely to play a crucial role in determining whether fusion occurs, or regeneration is instead attempted through 40,42 ; this is in addition to the self-fusion of the PLM and PVD neurons described in Section 1.2. …”

“…In the pharynx, two neighboring cells undergo developmental self-fusion to form unicellular tubes. One expresses EFF-1 for this purpose, whereas the other expresses AFF-1, potentially preventing cross-fusion between the two cells 40 . In contrast, as mentioned in Section 1.2, EFF-1 and AFF-1 appear to be co-expressed during PVD dendritic repair and act cooperatively, albeit for mediating different forms of membrane sculpting.…”

“…elegans development [39][40][41][42] . The mechanism through which it mediates fusion has not yet been determined, but similarly to EFF-1, it requires insertion into both membranes 26 .…”