The Alpha and the Gamma of latency establishment: Deciphering early events after nuclear entry of herpesvirus genomes

Abstract

Herpesviruses are pathogens that can infect humans and animals and are well adapted to their host. Primary infection with herpesviruses results in lifelong persistence of the herpesvirus, with the possibility of reactivation. After a first phase of lytic replication, most herpesviruses enter a state known as latency. During latency, very few viral genes are transcribed and the genomes can remain in the infected cell without being eliminated by the immune system. During latency, the herpesviral deoxyribonucleic acid (DNA) is modified by epigenetic modifications, i.e. DNA methylation and posttranslational repressive histone modifications. A recent study showed that the cytidine guanosine dinucleotide (CpG)-rich genome of the gammaherpesvirus Kaposi’s Sarcoma-associated herpesvirus (KHSV) rapidly attracts Polycomb repressive complexes (PRCs) via the non-canonical (nc) recruitment pathway. This mechanism normally serves to silence CpG islands of cellular DNA via recognition of unmethylated CpG motifs. Interestingly, alphaherpesviruses such as herpes simplex virus-1 (HSV-1) also have a very high content of CpG sequences. Previous results from our group suggest that PRC-mediated repression may be a default pathway to repress CpG-rich viral DNA (vDNA).

Therefore, we hypothesize that herpesviruses with high CpG content might generally exploit this pathway to support latency. Therefore, the study presented here aims to further validate this hypothesis. To this end, a direct comparative analysis was performed between KSHV and lytic-cycle-deficient mutants of the alphaherpesviruses HSV-1 and pseudorabies virus, which also have a very high CpG content. Genome-wide chromatin analyses were performed by chromatin-immunoprecipitation-sequencing (ChIP-seq), early after infection, to elucidate the epigenome of the lytic-cycle-deficient alphaherpesviruses. Furthermore, a fluorescence-based visualization method, which makes use of bioorthogonal chemistry, was utilized to track the spatiotemporal interaction of individual viral episomes with host factors that might be involved in herpesvirus silencing. In addition, knock-down of the host factor ATRX was performed with subsequent infection experiments to investigate its impact on herpesvirus reactivation.

The data presented here show for the first time rapid acquisition of the PRC-associated facultative heterochromatin mark H2AK119ub by lytic-cycle-deficient pseudorabies virus (PRV) and HSV-1 genomes. Interestingly, both viruses also acquired constitutive heterochromatin, via H3K9me3. Further analysis of the underlying mechanism revealed that the acquisition of the different forms of heterochromatin appears to be dependent on the infected cell type and thus very likely on distinct host factors. In particular, evidence was provided that the host factor Alpha-thalassemia/mental retardation syndrome X-linked (ATRX), a component of promyelocytic leukemia-nuclear bodies (PML-NBs), plays a role in the establishment of a tightly repressed condition of the virus genome and might influence constitutive heterochromatin formation.

These results suggest that alphaherpesvirus genomes, similar to KSHV, can attract PRCs via the ncPRC-recruitment pathway. Unlike latently replicating KSHV episomes, non-replicating alphaherpesvirus genomes did not acquire H3K27me3. Since H3K27me3 is preferentially formed on replicated DNA, the lack of vDNA replication in latent alphaherpesviruses may be related to the fact that H3K27me3 could not be detected on these genomes. Hence, vDNA replication may play an important role during the acquisition of the different forms of heterochromatin of herpesvirus genomes. Moreover, the data presented in this dissertation suggest a model in which alphaherpesviruses might establish two differentially silenced subpopulations. In this model, PRC-mediated repression leads to the establishment of authentic alphaherpesvirus latent reservoirs with the ability to reactivate, whereas PML-NB-mediated repression can lead to permanent silencing and, in dividing cells, even to subsequent elimination of the virus. Together, these results show a new aspect of alphaherpesvirus heterochromatinization and partly generalize the PRC-mediated repression mechanism among two herpesvirus subfamilies. Therefore, this study enhances the understanding of the early repression of incoming herpesviral DNA.