Early Events in Herpes Simplex Virus Infection: a Radioautographic Study

Abstract

The early events in herpes simplex virus infection were studied by means of radioautography. The virus was rapidly taken up by the host cells and uncoated. Viral deoxyribonucleic acid (DNA) reached the nuclear sites of replication in 15 to 30 min after infection. The viral DNA occasionally associated with chromosomes or condensed chromatin but was more frequently found to be randomly distributed. Viral progeny appeared 3 hr after infection. These particles did not show any particular spatial relationship to the parental DNA. The morphological latent period lasted 2.5 hr.Morphological studies on the entry of herpes simplex virus (HSV) into tissue culture cells and subsequent early intracellular events have been described (3-5, 7, 8, 10). During this early phase of the infection process, the virus can be followed until the genome is uncoated. The actual transport of the genome to the nucleus, the site of virus replication, cannot be seen. The present study therefore was undertaken to determine the path of the viral deoxyribonucleic acid (DNA) from cell entry to appearance of viral progeny in the nucleus by means of radioautography. This permitted the determination of the length of time required for the viral DNA to reach the nucleus, the intranuclear sites of replication, and spatial relationships of viral progeny to the parental DNA. MATERIALS AND METHODSTissue culture. A BHK-21 line of Syrian hamster kidney cells was grown as monolayers in Blake bottles in a previously described medium (9), which consisted of a mixture of basal medium Eagle (2X) in Hanks balanced salt solution, 10% fetal calf serum, and 10% Tryptose phosphate broth.Virus. The H strain of HSV was received through the courtesy of T. Tokumaru. The virus had been propagated in primary rabbit kidney cells. Before use in these experiments, it was passed five times in BHK-21 cells.Infectivity assay. Plastic petri dishes (60 mm) were seeded with BHK-21 cells in the above medium. They were incubated at 37 C in a 5% CO2 atmosphere. Confluent monolayers were apparent after 24 hr. At this time, the medium was drained and the cells were inoculated with 0.2 ml of the various dilutions of virus.After an adsorption period of 1 hr at 37 C, with occasional agitation, the infected monolayers of cells were overlaid with 5 ml of medium containing 0.5% Difco agar. Cultures were incubated for 72 hr in CO2 atmosphere. After incubation, the overlay medium was removed, and the cells were fixed in 10% neutral Formalin and stained with Leishman's stain (9). The apparent plaques were enumerated and titers were expressed as number of plaque forming units (PFU) per ml.Preparation of labeled virus. Monolayer cultures of 108 BHK-21 cells were infected with HSV at an input multiplicity of 10. At the time of infection, 25 uc/ml of 3H-thymidine per ml (Schwarz BioResearch Inc.), with a specific activity of 6.0 c/mmole, was added to the virus-containing medium. The cultures were incubated at 37 C for 18 to 20 hr when cytopathic effect was evident. The medium was discarded and the ce...

Early events in cell-animal virus interactions.

“…With herpes simplex virus, doubly-labeled inoculum was similarly employed to study uncoating. Occurrence of a time-related transfer into the nucleus of only the DNA and retention of the bulk of the protein in the cytoplasm could be demonstrated (80,95). Intracytoplasmic separation of DNA cores is described in an electron microscopy study of the same agent (136).…”

“…Several studies cited previously (47) and published more recently show evidence for rapid clearance from the surface of attached inoculum particles (1,51,67,95,136,140). Some workers report either exclusive or almost exclusive transfer from the surface by viropexis of both enveloped virions and the nucleoprotein cores (1,51,95). Others propose that transfer of cores into the cytoplasm may occur as a consequence of either the digestion or fusion of both the virus envelope and contiguous plasma membrane or by phagocytosis (136,140).…”

Similarities and Differences in the Development of Laboratory Strains and Freshly Isolated Strains of Herpes Simplex Virus in HEp-2 Cells: Electron Microscopy

“…A recent survey of the multiplication of herpesviruses (28) revealed considerable differences in the results of electron microscopic studies of various phases of herpesvirus development. Thus, differences have been reported in the mode of entry of herpesviruses (6,16,23), in the site of envelopment of herpesviruses (8,11,22,33,35), and in the mode of egress of the virus from infected cells (22,33). The differences in the results obtained in these studies are in some instances considerably greater than would be expected from mere variation in techniques or in interpretation of data.…”

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

“…They are prominent immunogenic antigens and are therefore, targets for the immune system. In addition, some of them are implicated in the fusion of the virion envelope to the membrane of the host cell at the beginning of the infectious process [8,13,19,21,27], and others are involved in the modification of the social behavior of infected cells [17,18].…”