Transcription of the herpes simplex virus latency-associated transcript promotes the formation of facultative heterochromatin on lytic promoters.

An important question in virology is the mechanism(s) by which persistent viruses such as the herpesviruses and human immunodeficiency virus (HIV) establish a latent infection in specific types of cells. In the case of herpesviruses, herpes simplex virus (HSV) infection of epithelial cells results in a lytic infection, whereas latent infection is established in sensory neurons. Recent studies have shown the importance of chromatin structure in the regulation of latent infection for both HSV and HIV. For HSV, we have shown previously that the viral latency-associated transcript (LAT) promotes lytic gene silencing and the association of one heterochromatin marker, dimethylation of lysine 9 on histone H3 (H3K9me2), with viral lytic genes. In this study, we further defined the structure of latent viral chromatin by examining the heterochromatin markers on histones associated with the HSV latent genome. We detected the H3K9me2, H3K9me3, and H3K27me3 modifications, with H3K27me3, which is indicative of facultative heterochromatin, exhibiting the highest enrichment on all viral promoters tested. A modification associated with cellular centromeric heterochromatin, H4K20me3, was not detected. A mutant virus containing a 1.8-kbp deletion within the LAT region showed reduced levels of the facultative heterochromatin marker (H3K27me3) along with H3K9me3 during latency, whereas a viral mutant defective for the LAT promoter showed a specific reduction in H3K27me3. Cellular long, noncoding RNAs induce facultative heterochromatin, and this study shows that transcription of a viral noncoding RNA can also induce facultative heterochromatin to promote lytic gene silencing during latency.

Role for A-type lamins in herpesviral DNA targeting and heterochromatin modulation.

Posttranslational modification of histones is known to regulate chromatin structure and transcriptional activity, and the nuclear lamina is thought to serve as a site for heterochromatin maintenance and transcriptional silencing. In this report, we show that the nuclear lamina can also play a role in the downregulation of heterochromatin and in gene activation. Herpes simplex virus DNA initiates replication in replication compartments near the inner edge of the nucleus, and histones are excluded from these structures. To define the role of nuclear lamins in HSV replication, we examined HSV infection in wild-type and A-type lamin-deficient (Lmna-/-) murine embryonic fibroblasts (MEFs). In Lmna-/- cells, viral replication compartments are reduced in size and fail to target to the nuclear periphery, as observed in WT cells. Chromatin immunoprecipitation and immunofluorescence studies demonstrate that HSV DNA is associated with increased heterochromatin in Lmna-/- MEFs. These results argue for a functional role for A-type lamins as viral gene expression, DNA replication, and growth are reduced in Lmna-/- MEFs, with the greatest effect on viral replication at low multiplicity of infection. Thus, lamin A/C is required for targeting of the viral genome and the reduction of heterochromatin on viral promoters during lytic infection. The nuclear lamina can serve as a molecular scaffold for DNA genomes and the protein complexes that regulate both euchromatin and heterochromatin histone modifications.

Retinoic acid receptor beta silences human papillomavirus-18 oncogene expression by induction of de novo methylation and heterochromatinization of the viral control region.

Retinoic acid receptor beta2 (RAR beta2) is often down-regulated during the multistep process to cervical cancer. In that way, its inhibitory function on the transcription factor AP-1, indispensable to maintain human papillomavirus (HPV) gene expression is relieved. Using HPV-18 positive HeLa cells as a model system, we show that ectopic expression of RAR beta2 is able to down-regulate HPV-18 transcription by selectively abrogating the binding of AP-1 to the viral regulatory region in a ligand-independent manner. This resulted in down-regulation of the viral mRNAs at the level of initiation of transcription. Decreased oncogene expression was accompanied by a re-induction of cell cycle inhibitory proteins such as p53, p21(CIP1), and p27(KIP) as well as by a cessation of cellular growth. Reduced transcriptional activity as a consequence of AP-1 reduction by selective c-Jun degradation apparently targets the HPV-18 regulatory region for epigenetic modification such as de novo methylation and nucleosomal condensation. This mechanism is otherwise counterbalanced by active and abundant viral transcription in malignant cells, because RAR beta2 itself becomes inactivated during cervical carcinogenesis. Hence, our study shows that the temporal co-existence of a potential repressor and viral oncoproteins is mutually exclusive and provides evidence of a cross-talk between a nuclear receptor, AP-1, and the epigenetic machinery.