In vitro synthesis of herpes simplex virus DNA in nuclei isolated from infected BSC 1 cells.

The synthesis of herpes simplex virus DNA in isolated nuclei under in vitro conditions was found to be dependent on the addition of ATP and an ATP generating system to the reaction mixture. In vitro DNA synthesis was stimulated and prolonged when p-hydroxymercuribenzoate was added to the isolated nuclei. Under these improved conditions virus DNA molecules which were initiated in vivo were completed in vitro, but most of the DNA molecules synthesized in vitro sedimented in sucrose gradients more slowly than herpes virion DNA. Denaturation of the in vitro labelled DNA molecules produced short single-stranded labelled DNA chains. Thus, under our improved in vitro conditions there was prolonged synthesis of DNA at a high rate, with the formation of both complete and incomplete virus DNA molecules.     

Analysis of herpes simplex virus DNA synthesized in infected nuclei by chromatography on benzoylated naphthoylated DEAE cellulose columns.

The nature of the DNA molecules synthesized in nuclei of herpes simplex virus (HSV)-infected cells in vivo and in vitro was studied by chromatography on BND-cellulose columns after shearing to DNA fragments of 10 to 20 X 10(6) daltons. The incorporation of labelled precursors occurs in the DNA fragments containing single-stranded regions, presumably the replication forks. Prolongation of DNA synthesis leads to the accumulation of labelled DNA fragments that lack single-stranded sequences. Analysis of the isolated DNA fragments by density centrifugation in CSCl gradients revealed that most of the labelled DNA molecules are of virus specificity and the minority are cellular DNA fragments. Double-stranded virus DNA fragments and virus DNA fragments containing single-stranded sequences band in CSCl gradients at a density of 1-718 g/ml, the density of virion DNA. This suggests that the replicating HSV DNA molecules have the same density as the virion DNA and contain relatively little single-stranded DNA. The synthesis of HSV DNA molecules under in vitro conditions in isolated nuclei occurs by incorporation of the precursors into DNA fragments with single-stranded regions. The synthesis of cellular DNA in nuclei from hydroxyurea and cytosine arabinoside treated cells also occurs by elongation of nascent DNA chains.     

Altered chromatin higher-order structure in cells infected by herpes simplex virus type 1

Summary In cells infected by herpesviruses chromosomal aberrations such as breaks and constrictions are commonly observed. Previous studies have shown that chromatin modifications in cells infected by herpes simplex virus type 1 (HSV-1) are not due to extensive breakdown of host-cell DNA or disruption of nucleosomal structure. We have previously shown that infection by HSV-1 induces single-stranded breaks in the host-cell DNA in a time-dependent fashion. Here we report that the early DNA damage observed in virus-infected cells is related to modifications in the higher-order structure of host-cell chromatin. Such modifications seem to be of a more permanent nature than the DNA single-stranded breaks.     

Replication of herpes simplex virus DNA after removal of hydroxyurea block from infected cells.

Hydroxyurea (HU) treatment of HSV-infected cells markedly inhibits the synthesis of virus DNA. Only 0-3.6 of the 3H-thymidine label was incorporated into virus DNA in the presence of HU as compared to untreated infected cells. Removal of HU resulted in a renewed synthesis of virus DNA as determined by the gradual increase in the incorporation of 3H-thymidine into HSV DNA. The labelled virus DNA molecules were isolated and chromatographed on benzoylated napthoylated DEAE (BND)-cellulose columns to separate the replicative intermediates that have single-stranded (ss) sequences from the mature double-stranded (ds) DNA genomes. Mature radioactive dsDNA molecules were found to appear at 22 min after removal of HU and gradually increased in amount thereafter. The virus DNA molecules synthesized during the initial 20 min after removal of HU, constitute the replicative intermediates of HSV DNA. It was calculated that the synthesis of HSV DNA proceeds at the rate of about 5 X 10(6) daltons per min.     

On the association of virus proteins with the nuclei of cells infected with herpes simplex virus.

In cells infected with herpes simplex virus, HSV-I, newly synthesized polypeptides accumulated in the nucleus at different rates, which did not change during the first 6 h after infection. Canavanine, an arginine analogue, prevented the nuclear accumulation of ICP (infected cell polypeptides) 5 and 8 and azetidine, a proline analogue, prevented that of ICP 5 and 7. The transfer of polypeptides to the nucleus was inhibited at 4 degrees C but not by dinitrophenol. Some of the nuclear polypeptides could be released by washing isolated nuclei with hypertonic salt solutions. ICP 17 was particularly sensitive to high salt treatment while ICP 5 and II were resistent. ICP 4b, a modified form of the alpha polypeptide ICP 4, was released by EDTA, and the detergent NP40 removed ICP II. Treatment of nuclei with DNase selectively reduced the amount of bound alpha polypeptides ICP 4c (the second modified form of ICP 4), 0 and 27 as well as ICP 8 and 25. Nuclei isolated from infected or uninfected cells and incubated in labelled cytoplasmic extracts took up primarily ICP 8 and 32. Alpha polypeptides were taken up to a lesser extent and ICP 6 and 10 were excluded. It is concluded that affinities for various constituents of host cell nuclei are likely to determine the nuclear accumulation of specific virus polypeptides.     

Immunogenicity of subviral herpes simplex virus preparations

Summary Production of neutralizing antibodies was followed in guinea pigs, rabbits hamsters and mice immunized with crude antigen extracts (AM) from human diploid cells infected with herpes simplex virus type 1. The AM induced relatively high levels of neutralizing antibodies in all four species. The antibodies were predominantly complement-requiring and remained so even after administration of repeated AM doses. With the strains used, the antibody response was predominantly type specific and, surprisingly, the type specificity of sera usually increased after administration of repeated doses of AM. Guinea pigs seemed to be the best responsive animal species. They developed the highest levels of antibodies and complement-nonrequiring antibodies were seen in them earlier than in the other animal species. The dose-response experiments carried out in guinea pigs indicated that after a single dose administration the ratio between complement-requiring and complement-nonrequiring antibodies was dependent on the amount of antigen administered. When AM was given without adjuvant less efficient antibody production was observed than after the administration of the same amount of antigen with adjuvant.With 3 Figures     

DNA repair replication in human embryonic lung cells infected with herpes simplex virus.

Some aspects of repair replication of cellular DNA in herpes simplex virus (HSV)-infected human embryonic lung (HEL) cells were investigated. The buoyant density of the radioactive DNA, after incorporation of radioactive bromodeoxyuridine, was used as the criterion of whether semiconservative or repair replication of the host DNA had occurred. The results showed: HSV-infected cells were able to repair ultra violet-damaged cellular DNA. The HSV-infected cells did not induce repair replication of cellular DNA until 14 hr after infection. Twenty-four hours after infection, however, at least 10 to 15% of the cellular DNA replication was of the repair type. The onset of inhibition of cellular DNA replication, after infection with HSV, was not concomitant with the onset of repair replication of cellular DNA.     

Transcription of herpes simplex type 1 DNA in nuclei isolated from infected HEp-2 and KB cells

Viral RNA synthesis in nuclei isolated from cells infected with Herpes simplex virus type 1 (HSV-1) has been examined for sensitivity to the fungal toxin α-amanitin. In both HEp-2 and KB cell lines, the production of HSV-1 specific RNA is inhibited 90% or greater by the administration of the toxin at 0.32 μg/ml. These results suggest that an α-amanitin sensitive, DNA-dependent RNA polymerase, possibly the host cell nucleoplasmic polymerase, is responsible for HSV-1 RNA synthesis in the infected cell.     

Analysis of Herpes simplex virus DNA synthesized in vitro in nuclei isolated from infected BSC-1 cells.

Herpes simplex virus (HSV) DNA molecules synthesized in isolated nuclei in vitro were analyzed by centrifugation in sucrose gradients and by chromatography on benzoylated naphthoylated DEAE (BND)-cellulose columns. Some of the in vitro synthesized viral DNA molecules were found to have a high molecular weight when cosedimented with a HSV-DNA marker. Over 60% of the in vitro labeled DNA molecules were eluted from the BND-cellulose columns by 1.0 M NaCl containing 2% caffeine, indicating the presence of single-stranded sequences in the DNA molecules, a property of replicating DNA molecules. It is concluded that under in vitro conditions, the elongation of nascent viral DNA molecules takes place.     

Polyamine metabolism in cells infected with herpes simplex virus.

Ifection of LS cells with HSV-1 resulted in an inhibition of spermidine and spermine synthesis from putrescine, possibly through inhibition of host cell protein synthesis. The rate of putrescine uptake increased soon after infection, and later, polyamines were lost from the cells. Inhibition of spermidine and spermine synthesis by methylglyoxal bis (amidinohydrazone) did not affect virus replication.     

Mitochondrial DNA synthesis in Raji cells infected by herpes simplex virus or Epstein-Barr virus

Mitochondrial DNA synthesis in herpes simplex virus infected Raji cells appears to be stimulated early in the infectious cycle in contrast to nuclear DNA synthesis which is progressively inhibited. This phenomenon cannot be observed in Raji cells abortively infected with Epstein-Barr virus.

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Zusammenfassung Die mitochondriale DNA-Synthese in Herpes-simplex-Virus-infizierten Raji-Zellen ist während der frühen Phase des Infektionscyclus gesteigert. Im Gegensatz dazu wird die nucleäre DNA-Synthese zunehmend gehemmt. Dieses Phänomen ist nicht in Raji-Zellen zu beobachten, die abortiv mit Epstein-Barr-Virus infiziert sind.

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This investigation was supported by the Deutsche Forschungsgemeinschaft.     

DNA synthesis in chromatin preparations from human fibroblasts infected by cytomegalovirus

Summary Chromatin prepared from (¹⁴C)-thymidine pulse labelled cytomegalovirus-infected human fibroblasts 72 hours postinfection exhibited under appropriate conditions endogenous activity of (³H)-thymidine triphosphate incorporation which was relatively salt-resistant and phosphonoacetic acid-sensitive. Isopycnic centrifugation of the doubly labelled DNA in CsCl revealed that cell-free incorporation occurred into viral as well as into host cell DNA. Density labelling experiments with bromodeoxyuridine triphosphate suggested the incorporation into viral DNA to be due to replicative DNA synthesis. Chromatin from infected cells contained, in addition to cellular, viral DNA polymerase activity.With 5 Figures     

Replication intermediates of herpes simplex virus DNA.

The DNA synthesized in herpes simplex virus (HSV)-infected nuclei in vivo was analyzed by chromatography on benzoylated naphthoylated DEAE-cellulose columns. Viral-DNA molecules that elute with caffeine contain single-stranded DNA sequences sensitive to a micrococcal endonuclease that cleaves single-stranded DNA only. These viral-DNA molecules behave kinetically as precursors to mature virion DNA, and their transition to the mature double-stranded form occurs within a period of 10 to 20 min. Electron microscopy revealed viral-DNA molecules at different stages of replication. The mechanism of HSV-DNA replication is discussed.     

Electron microscopy of herpes simplex virus DNA molecules isolated from infected cells by centrifugation in CsCl density gradients.

Herpes simplex virus (HSV) DNA molecules were isolated from infected BSC 1 cells and centrifuged in CsCl-ethidium bromide density gradients. Both newly labelled and mature virus DNA molecules were found to have a linear conformation. The morphology of virus DNA molecules at different stages of the virus growth cycle in BSC 1 cells, was studied by electron microscopy after separation of virus DNA from cellular DNA by centrifugation in CsCl gradients. In each sample, about 200 virus DNA molecules were photographed and the different morphological forms were studied. Four classes of virus DNA molecules were observed: (a) mature linear DNA molecules, 52-4 +/- 3-3 micronm in length, (b) DNA intermediates, (c) virus DNA molecules having one or more single-stranded filaments attached to them and (d) molecules with collapsed regions or with branches. A few circular molecules as well as linear DNA molecules longer than unit length were also observed. The virus DNA molecules resembling replicative intermediates gradually increased in number and reached a maximal amount of about 5% of the virus DNA population at 12 h after infection. The other forms of virus DNA were found to persist after the number of replicating DNA molecules decreased.     

Rapid DNA diagnosis of herpes simplex virus serotypes.

The presence of nucleotide sequences specific for each of herpes simplex virus (HSV) serotypes was demonstrated. These sequences were applied for dot DNA-DNA hybridization and for PCR for rapid DNA diagnosis of HSV infections. These sequences were found by molecular cloning of HSV-DNA fragments after digestion of DNA by KpnI enzyme. The type 1-specific sequence was found around the 5' end of BamHI B-fragment in the L region of type 1 DNA (corresponds to alpha gene 27, promoter-regulatory region) and the type 2-specific sequence was around the junction region of the L and S of type 2 DNA (corresponds to a' sequence). Both simple dot blot hybridization and PCR of HSV DNA's, employing these type-specific nucleotide sequences, were proven to be much more useful than immunofluorescence in terms of type-specific diagnosis of HSV infections.