Herpes simplex virus utilizes the large secretory vesicle pathway for anterograde transport of tegument and envelope proteins and for viral exocytosis from growth cones of human fetal axons.

Axonal transport of herpes simplex virus (HSV-1) is essential for viral infection and spread in the peripheral nervous system of the host. Therefore, the virus probably utilizes existing active transport and targeting mechanisms in neurons for virus assembly and spread from neurons to skin. In the present study, we used transmission immunoelectron microscopy to investigate the nature and origin of vesicles involved in the anterograde axonal transport of HSV-1 tegument and envelope proteins and of vesicles surrounding partially and fully enveloped capsids in growth cones. This study aimed to elucidate the mechanism of virus assembly and exit from axons of human fetal dorsal root ganglia neurons. We demonstrated that viral tegument and envelope proteins can travel in axons independently of viral capsids and were transported to the axon terminus in two types of transport vesicles, tubulovesicular membrane structures and large dense-cored vesicles. These vesicles and membrane carriers were derived from the trans-Golgi network (TGN) and contained key proteins, such as Rab3A, SNAP-25, GAP-43, and kinesin-1, involved in the secretory and exocytic pathways in axons. These proteins were also observed on fully and partially enveloped capsids in growth cones and on extracellular virions. Our findings provide further evidence to the subassembly model of separate transport in axons of unenveloped capsids from envelope and tegument proteins with final virus assembly occurring at the axon terminus. We postulate that HSV-1 capsids invaginate tegument- and envelope-bearing TGN-derived vesicles and utilize the large secretory vesicle pathway of exocytosis for exit from axons.

Herpes simplex virus type 1 accumulation, envelopment, and exit in growth cones and varicosities in mid-distal regions of axons.

The mechanism of anterograde transport of alphaherpesviruses in axons remains controversial. This study examined the transport, assembly, and egress of herpes simplex virus type 1 (HSV-1) in mid- and distal axons of infected explanted human fetal dorsal root ganglia using confocal microscopy and transmission electron microscopy (TEM) at 19, 24, and 48 h postinfection (p.i.). Confocal-microscopy studies showed that although capsid (VP5) and tegument (UL37) proteins were not uniformly present in axons until 24 h p.i., they colocalized with envelope (gG) proteins in axonal varicosities and in growth cones at 24 and 48 h p.i. TEM of longitudinal sections of axons in situ showed enveloped and unenveloped capsids in the axonal varicosities and growth cones, whereas in the midregion of the axons, predominantly unenveloped capsids were observed. Partially enveloped capsids, apparently budding into vesicles, were observed in axonal varicosities and growth cones, but not during viral attachment and entry into axons. Tegument proteins (VP22) were found associated with vesicles in growth cones, either alone or together with envelope (gD) proteins, by transmission immunoelectron microscopy. Extracellular virions were observed adjacent to axonal varicosities and growth cones, with some virions observed in crescent-shaped invaginations of the axonal plasma membrane, suggesting exit at these sites. These findings suggest that varicosities and growth cones are probable sites of HSV-1 envelopment of at least a proportion of virions in the mid- to distal axon. Envelopment probably occurs by budding of capsids into vesicles with associated tegument and envelope proteins. Virions appear to exit from these sites by exocytosis.