Ultrastructural localization of glycoproteins in rabbit fibroblasts altered by Herpes simplex virus type 1 infection

Abstract

The periodic acid-thiocarbohydrazide (SO2)--OsO4 method was used to examine the distribution of glycoproteins in rabbit fibroblast cells infected with Herpes simplex virus type 1. In non-infected cells, a low level of staining was seen over the plasma membrane and the membranes of the Golgi apparatus. At 17 hr post-infection, the intensity of reaction was increased to include not only a relatively heavy staining of the plasma membrane, including the numerous microvilli characteristic of infected cells, and of the newly proliferated Golgi membranes, but also the envelopes of intracytoplasmic and extracellular virions. A very faint but only occasional staining also was associated with the virus-induced reduplications of the inner nuclear membrane and the envelopes of associated enveloping nucleocapsids. We suggest that such differences in the intensity of staining may be related either to the amount of glycoproteins or to the sequential maturation of the viral glycoproteins. We also observed that the structurally modified portions of the Golgi membranes at the position where intracytoplasmic naked nucleocapsids bud into the Golgi cisternae usually exhibit a more intense reaction for glycoproteins than do the adjacent portions of the Golgi membranes. This supports the evidence for an envelopment of nucleocapsids in the cytoplasm, but it does not indicate whether this event obligatorily follows or only occasionally takes the place of the envelopment of nucleocapsids at the inner nuclear membrane. In either event, the envelopes of all mature virions exhibit a prominent reaction to glycoproteins.

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“…In the current study, capsids appeared close to enlarged nuclear pores, which are dynamic cellular structures with a functional diameter of 10 nm (3), which is why, if it is to take place at all (8), transfer of capsids require unfolding of the proteins and mediated transport of much larger particles at the pore complex (3). However, reduplicated or thickened nuclear membranes housing capsids or immature viral particles were also demonstrated in accordance with earlier reports (2,(6)(7)(8)23,25). Budding viral particles were identified as early as 6 hpi, but definitive envelopment at the nuclear membranes was not encountered in the current study, which can be why only 1% of the whole cell was represented (18) and the envelopement was a very rapid event or was finished during too slow fixation of the cells.…”

“…The multistep envelopment process described is in accordance with previously reported herpes virus transport of varicella-zoster virus (55), cytomegalovirus (56), Epstein-Barr virus (57), pseudorabies (24), and HSV (2,4,6,8,23,51,58). The multistep envelopment process described is in accordance with previously reported herpes virus transport of varicella-zoster virus (55), cytomegalovirus (56), Epstein-Barr virus (57), pseudorabies (24), and HSV (2,4,6,8,23,51,58).…”

“…Then, in the terminal phase after 18 hpi, the cells are destroyed. Numerous microvilli, folded nuclei, and margination of host chromatin along the nuclear envelope are well-known characteristics of HSV-infected cells (2,(6)(7)(8)(23)(24)(25)(26). The described temporal development of viral particles is generally in agreement with previous observations (23).…”

“…Broadly, the time of appearance of gC and gD confirmed earlier studies (6,11,28), although discrepant statements exist based on the method used and the origin of the host cells (29). However, ultrastructural studies investigate only minor parts of the sample, which explains why it is difficult to find and examine the few early HSV-infected cells.…”

“…The herpes simplex virus (HSV) genome is linear double-stranded DNA of 152 kbp, which encodes at least 36 essential genes required for viral replication and 44 genes dispensable for viral growth in cell culture (2,4). The glycoproteins function as immunogenic antigens and are implicated in viral adsorption, penetration, envelopment, egress, and social behavior of infected cells (6,7). The genome encodes at least 15 membrane proteins including 11 identified glycoproteins: gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, and gM (4,5).…”