The Herpesviruses — A Biochemical Definition of the Group

Roizman, Bernard

doi:10.1007/978-3-642-46166-8_1

Cited by 64 publications

(36 citation statements)

References 244 publications

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“…Furthermore, the spread of incorporation at later times appeared to be due to extension and fusion of existing labelling sites rather than to a generalized spread which might be expected if DNA had moved rapidly away from its initial location, before or during synthesis. The integrity of the regions of incorporation is unlikely to be due to localization of virus DNA synthesis within a discrete physical entity, as neither this study nor that of Roizman (1969) revealed any association between HSV DNA synthesis and a recognizable morphological structure. Although Hay & Hay (1981) reported HSV DNA synthesis in isolated chromatin preparations, the present and earlier studies have shown no preferential association between replicating HSV DNA and chromatin.…”

contrasting

confidence: 74%

“…Light microscopic autoradiography of cells labelled from 23 to 24 h post-infection showed that the entire nuclear volume was involved, with the exception of the marginated chromatin (Munk & Sauer, 1964). Roizman (1969) obtained similar results from electron microscopic autoradiographic studies on cells labelled for 15 min at 4 h post-infection, and showed that the incorporated label was not associated with any identifiable sub-nuclear structure. More recently, Luetzeler & Heine (1978) have studied the morphological changes that occur following infection with HSV-1.…”

supporting

confidence: 53%

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Intranuclear Distribution of Herpes Simplex Virus Type 2 DNA Synthesis: Examination by Light and Electron Microscopy

Rixon

Atkinson

Hay

1983

Journal of General Virology

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SUMMARYUptake of [3H]thymidine by serum-starved baby hamster kidney cells infected with herpes simplex virus type 2 was investigated by light microscopic and electron microscopic autoradiography. The distribution of incorporated label altered throughout the period of viral DNA synthesis. It was restricted initially to a few well-defined sites within the nucleus which increased in size and spread as infection proceeded until the entire nucleus was involved. The label was not associated with any identifiable subnuclear structure although there was a high degree of association with the nuclear membrane at early times post-infection.Herpes simplex virus (HSV) DNA synthesis takes place entirely within the nucleus of infected cells. Light microscopic autoradiography of cells labelled from 23 to 24 h post-infection showed that the entire nuclear volume was involved, with the exception of the marginated chromatin (Munk & Sauer, 1964). Roizman (1969) obtained similar results from electron microscopic autoradiographic studies on cells labelled for 15 min at 4 h post-infection, and showed that the incorporated label was not associated with any identifiable sub-nuclear structure. More recently, Luetzeler & Heine (1978) have studied the morphological changes that occur following infection with HSV-1. At very late times (24 to 72 h post-infection) they observed that labelled virus DNA was strongly associated with bundles of tightly packed filaments within the nucleus of infected cells.Autoradiographic examination of labelled HSV DNA in infected cells is greatly simplified if the background of host cell DNA synthesis can be removed or diminished. Serum starvation of baby hamster kidney (BHK-21/C 13) cells over a 7 day period reduces the level of DNA synthesis to less than 1% of that in exponentially growing cells (Howard et al., 1974). Moreover, residual cellular DNA synthesis declines even further following infection with HSV-2 (data not shown). All the experiments described below were performed in such 'resting' BHK cells.Linbro trays containing clean, sterile, glass coverslips, were seeded at a density of 5 x 104 cells/well in Eagle's medium supplemented with tryptose phosphate and 10% (v/v) calf serum. After 24 h the cells were washed and grown for a further 7 days in Eagle's medium plus 1% (v/v) calf serum. HSV-2 (strain HG52) was added, at 5 p.f.u./cell, for 1 h, after which the inoculum was removed and replaced with 0.2 ml of the original 'resting' cell medium. Ten ~tCi of [3H]thymidine (23 Ci/mmol) was added for 2 h periods at 2 h intervals commencing 1 h postinfection. For shorter (10 or 15 min) labels, 40 ~tCi of [3H]thymidine was added. Cells were fixed by placing the coverslips in either 5% trichloroacetic acid for 10 min at 0 °C or 2-5% glutaraldehyde in phosphate-buffered saline for 20 min at room temperature. The coverslips were then attached to a microscope slide, coated with Ilford K2 emulsion and stored at 4 °C (typically 2 weeks for samples labelled 2 h, 12 weeks for samples labelled 15 min). After photograp...

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contrasting

confidence: 74%

supporting

confidence: 53%

Intranuclear Distribution of Herpes Simplex Virus Type 2 DNA Synthesis: Examination by Light and Electron Microscopy

Rixon

Atkinson

Hay

1983

Journal of General Virology

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“…Furthermore, Vaughan et al (1984) presented evidence that ICP 8, DNA polymerase, alkaline exonuclease and possibly ICP 4 may be associated in a complex. Uptake of [3H]thymidine by HSV-infected cells has shown that virus DNA synthesis is restricted to well-defined sites within the nucleus which increase in size as the infection proceeds (Roizman, 1969;Rixon et al, 1983).…”

Section: -7218 O 1986 Sgmmentioning

confidence: 99%

Intranuclear Localization of Herpes Simplex Virus Immediate-Early and Delayed-Early Proteins: Evidence that ICP 4 Is Associated with Progeny Virus DNA

Randall

Dinwoodie

1986

Journal of General Virology

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SUMMARYThe localization of ICP 4, ICP 8, DNA polymerase and alkaline exonuclease within herpes simplex virus type 1 (HSV)-infected cells has been examined by immunofluorescence using specific antibodies to these proteins. Cells were simultaneously counterstained with the DNA-binding fluorochrome 4,6-diaminido-2-phenylindole (DAPI) to reveal the intranuclear distribution of DNA. These studies showed that in the absence of virus DNA replication ICP 4, ICP 8 and DNA polymerase were diffusely distributed throughout the nucleus but during virus DNA replication these proteins accumulated at specific foci within the nucleus. Initially these foci were near the nuclear membrane but with continuing virus DNA replication they increased in size until the whole of the nucleus became affected. The increase in size of these foci was coincident with a redistribution of nuclear DNA and margination of chromatin at the nuclear membrane, as revealed by DAPI staining. The number of foci initially present in an infected cell was dependent on the multiplicity of infection. The distribution of ICP 4, ICP 8 and DNA polymerase within the nucleus was altered by treating the cells with DNase. The majority of alkaline exonuclease was diffusely distributed throughout the nucleus during virus DNA replication and did not localize at specific foci within the nucleus. Autoradiographic examination of the incorporation of [3H]thymidine in cells infected with HSV showed that viral DNA replication occurred in restricted areas within the nucleus that were similar, in terms of number, location and size, to the foci where ICP 4, ICP 8 and DNA polymerase accumulated. Furthermore, in cells blocked in mitosis following infection with HSV, ICP 4, ICP 8 and DNA polymerase, but not alkaline exonuclease, localized in areas outside the condensed chromatin structures. DAPI staining revealed the presence of DNA in these areas and, as such structures were never seen when uninfected cells had entered mitosis, it is suggested that this extrachromosomal DNA is of viral origin. These studies therefore suggest that ICP 4 is associated with progeny virus DNA and that while its intranuclear localization is initially at non-viral sites, as DNA replication proceeds so ICP 4 is recruited into areas of virus DNA transcription and replication.

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“…The virion of herpes simplex consists of a core that contains DNA (108 daltons) made in the nucleus, a multilayered capsid assembled in the nucleus from proteins made in the cytoplasm, and an envelope derived from the nuclear membrane modified by virusspecific glycoproteins (7). Of the two polyamines in the virion, spermine appears restricted to the nucleocapsid and spermidine to the viral envelope.…”

mentioning

confidence: 99%

Compartmentalization of Spermine and Spermidine in the Herpes Simplex Virion

Gibson

Roizman

1971

Proc. Natl. Acad. Sci. U.S.A.

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149

116

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Enveloped particles of herpes simplex virus produced in human cells in culture contained spermidine and spermine, in a molar ratio of 1.6 4-0.2. The spermine present within the nucleocapsid is sufficient to neutralize at least 40% of the viral DNA. Disruption of the envelope with nonionic detergent and urea resulted in the selective loss of spermidine. Exogenous ornithine can function as a precursor to host and viral polyamines only before infection.Polyamines are known to bind to nucleic acids and phospholipids (1), and may be functional (2) as well as structural (3) components of eukaryotic cell organelles. Polyamines have previously been shown to occur in appreciable amounts in the DNA-containing bacteriophages T2 and T4 (4, 5). They have not been found in poliovirus, tomato bushy stunt, cucumber, and tobacco mosaic virus, all of which contain RNA and replicate in eukaryotic cells. Interestingly, another unenveloped RNA virus, turnip yellow mosaic virus, does contain polyamines, almost exclusively in the form of spermidine (6).We report here the presence of spermine and spermidine in herpes simplex virus. The virion of herpes simplex consists of a core that contains DNA (108 daltons) made in the nucleus, a multilayered capsid assembled in the nucleus from proteins made in the cytoplasm, and an envelope derived from the nuclear membrane modified by virusspecific glycoproteins (7). Of the two polyamines in the virion, spermine appears restricted to the nucleocapsid and spermidine to the viral envelope. This result is in contrast to the finding that putrescine and spermidine are present together in the nucleocapsids of T2 and T4 phages. Purification of Virus. Purified enveloped virus was prepared by a modification of a procedure developed by Spear and Roizman (manuscript in preparation). Briefly, the infected cells were swollen in hypotonic buffer and disrupted by four strokes of a Dounce homogenizer with a tight pestle. The cytoplasm was then separated from the nuclei and large debris by centrifugation, and layered onto a 3-30% (w/w) Dextran 10 (Pharmacia Fine Chemicals, Piscataway, N.J.) gradient prepared in phosphate buffer (0.01 M, pH 7.1) in place of the Tris buffer recommended in the original procedure. The gradients were then centrifuged at 20,000 rpm for 50 min at 50C in a Beckman SW25.3 rotor. A single band present at about the midpoint of the tube contained almost exclusively enveloped nucleocapsids free of host DNA (9) with relatively little contamination by membrane fragments.

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The Herpesviruses — A Biochemical Definition of the Group

Cited by 64 publications

References 244 publications

Intranuclear Distribution of Herpes Simplex Virus Type 2 DNA Synthesis: Examination by Light and Electron Microscopy

Intranuclear Distribution of Herpes Simplex Virus Type 2 DNA Synthesis: Examination by Light and Electron Microscopy

Intranuclear Localization of Herpes Simplex Virus Immediate-Early and Delayed-Early Proteins: Evidence that ICP 4 Is Associated with Progeny Virus DNA

Compartmentalization of Spermine and Spermidine in the Herpes Simplex Virion

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