Herpes simplex virus 1 DNA is in unstable nucleosomes throughout the lytic infection cycle, and the instability of the nucleosomes is independent of DNA replication.

Herpes simplex virus 1 (HSV-1) DNA is chromatinized during latency and consequently regularly digested by micrococcal nuclease (MCN) to nucleosome-size fragments. In contrast, MCN digests HSV-1 DNA in lytically infected cells to mostly heterogeneous sizes. Yet HSV-1 DNA coimmunoprecipitates with histones during lytic infections. We have shown that at 5 h postinfection, most nuclear HSV-1 DNA is in particularly unstable nucleoprotein complexes and consequently is more accessible to MCN than DNA in cellular chromatin. HSV-1 DNA was quantitatively recovered at this time in complexes with the biophysical properties of mono- to polynucleosomes following a modified MCN digestion developed to detect potential unstable intermediates. We proposed that most HSV-1 DNA is in unstable nucleosome-like complexes during lytic infections. Physiologically, nucleosome assembly typically associates with DNA replication, although DNA replication transiently disrupts nucleosomes. It therefore remained unclear whether the instability of the HSV-1 nucleoprotein complexes was related to the ongoing viral DNA replication. Here we tested whether HSV-1 DNA is in unstable nucleosome-like complexes before, during, or after the peak of viral DNA replication or when HSV-1 DNA replication is inhibited. HSV-1 DNA was quantitatively recovered in complexes fractionating as mono- to polynucleosomes from nuclei harvested at 2, 5, 7, or 9 h after infection, even if viral DNA replication was inhibited. Therefore, most HSV-1 DNA is in unstable nucleosome-like complexes throughout the lytic replication cycle, and the instability of these complexes is surprisingly independent of HSV-1 DNA replication. The specific accessibility of nuclear HSV-1 DNA, however, varied at different times after infection.

During lytic infections, herpes simplex virus type 1 DNA is in complexes with the properties of unstable nucleosomes.

The genomes of herpes simplex virus type 1 (HSV-1) are regularly chromatinized during latency such that their digestion with micrococcal nuclease (MCN) releases nucleosome-sized DNA fragments. In lytically infected cells, in contrast, MCN releases HSV-1 DNA in primarily heterogeneously sized fragments. Consistently, only a small percentage of this HSV-1 DNA coimmunoprecipitates with histones. Most current models propose that histones associate with HSV-1 DNA during lytic infections at low occupancy. However, histone modification or occupation is also proposed to regulate HSV-1 transcription. It remains unclear how the histones associated with a small percentage of HSV-1 DNA may regulate transcription globally. Moreover, the physical properties of the complexes containing histones and HSV-1 DNA are unknown. We evaluated the HSV-1 DNA-containing complexes at 5 h after (lytic) infection by biochemical fractionations. Nuclear HSV-1 DNA did not fractionate as protein-free HSV-1 DNA but as DNA in cellular nucleosomes. Moreover, MCN released HSV-1 DNA in complexes that fractionate as cellular mono- and dinucleosomes by centrifugation followed by sucrose gradients and size-exclusion chromatography. The HSV-1 DNA in such complexes was protected to heterogeneous sizes and was more accessible to MCN than DNA in most cellular chromatin. Using a modified MCN digestion to trap unstable digestion intermediates, HSV-1 DNA was quantitatively recovered in discrete mono- to polynucleosome sizes in complexes fractionating as cellular mono- to polynucleosomes. The HSV-1 DNAs in complexes fractionating as mono- to dinucleosomes were stabilized by cross-linking. Therefore, most HSV-1 DNA forms particularly unstable nucleosome-like complexes at 5 h of lytic infection.

Five years of progress on cyclin-dependent kinases and other cellular proteins as potential targets for antiviral drugs.

In 1997-1998, the pharmacological cyclin-dependent kinase (CDK) inhibitors (PCIs) were independently discovered to inhibit replication of human cytomegalovirus, herpes simplex virus type 1 and HIV-1. The results from small clinical trials against cancer were then suggesting that PCIs could be safe enough to be used clinically. It was thus hypothesized that PCIs could have the potential to be developed as novel antivirals targeting cellular proteins. Consequently, Antiviral Chemistry & Chemotherapy published in 2001 the first review on the potential of CDKs, and cellular proteins in general, as potential targets for antivirals. The viral functions inhibited by PCIs, or their cellular targets, were then just starting to be characterized. The antiviral spectrum of PCIs and their effects on viral disease were still mostly untested. Even their actual specificity was not yet completely characterized. In addition, cellular proteins were not accepted as valid targets for antivirals. Significant progress has been made in the last 5 years in understanding the antiviral activities of PCIs and the potential roles of cellular proteins in general as targets for antivirals. The first clinical trials of the antiviral activities of PCIs and other inhibitors of cellular protein kinases have now been scheduled. Herein, we review the progress made since the publication of the first review on PCIs as potential antiviral drugs and on CDKs, and cellular proteins in general, as potential targets for antiviral drugs. We also highlight the major issues that still need to be addressed before PCIs or other drugs targeting cellular proteins can be developed as clinical antivirals.

Roscovitine inhibits activation of promoters in herpes simplex virus type 1 genomes independently of promoter-specific factors.

Flavopiridol, roscovitine, and other inhibitors of Cyclin-Dependent Kinases (CDK) inhibit the replication of a variety of viruses in vitro while proving nontoxic in human clinical trials of their effects against cancer. Consequently, these and other Pharmacological CDK inhibitors (PCIs) have been proposed as potential antivirals. Flavopiridol potently inhibits all tested CDKs and inhibits the transcription of most cellular and viral genes. In contrast, roscovitine and other purine PCIs inhibit with high potency only CDK1, CDK2, CDK5, and CDK7, and they specifically inhibit the expression of viral but not cellular genes. The levels at which purine PCIs inhibit gene expression are unknown, as are the factors which determine their specificity for expression of viral but not cellular genes. We show herein that roscovitine prevents the initiation of transcription of herpes simplex virus type 1 (HSV-1) genes but has no effect on transcription elongation. We further show that roscovitine does not inhibit the initiation or elongation of cellular transcription and that its inhibitory effects are specific for promoters in HSV-1 genomes. Therefore, we have identified a novel biological activity for PCIs, i.e., their ability to prevent the initiation of transcription. We have also identified genome location as one of the factors that determine whether the transcription of a given gene is inhibited by roscovitine. The activities of roscovitine on viral transcription resemble one of the antiherpesvirus activities of alpha interferon and could be used as a model for the development of novel antivirals. The genome-specific effects of roscovitine may also be important for its development against virus-induced cancers.