A recent study In Press at the Journal of Cell Biology reports that herpes simplex virus 1 (HSV-1) exploits heterochromatin to successfully exit the nucleus. The heterochromatin markers macroH2A1 and H3K27me3 are necessary for heterochromatin redistribution in the nuclear periphery during HSV-1 infection to promote nuclear egress. The loss of macroH2A1 and H3K27me3 resulted in significantly lower infectious progeny, but did not impair HSV-1 viral transcription, protein production, or replication. This suggests that macroH2A1 and H3K27me3-dependent heterochromatin is necessary for efficient nuclear egress of the HSV-1 capsids.
Dr. Daphne Avgousti, an assistant professor in the Human Biology Division at Fred Hutch and senior author of the study, and her team hypothesized that HSV-1 exploits host heterochromatin dynamics to egress from the nuclear compartment. To test this, the authors infected primary human foreskin fibroblasts (HFFs) with HSV-1. Following infection, transmission electron microscopy (TEM) was used to visualize heterochromatin formation in infected HFFs. The authors found that uninfected cells showed dark staining at the nuclear periphery and infected cells showed regions of lighter staining amongst larger domains of heterochromatin, suggesting the formation of ‘channels’. Viral capsids were associating with the inner nuclear membrane only at regions of less dense stain. This indicated that viral capsids interact with chromatin in low-density areas of the nuclear envelope upon infection. As macroH2A1 and H3K27me3 are required for heterochromatin localization, the authors investigated whether HSV-1 infection required macroH2A1 and H3K27me3. To accomplish this, macroH2A1 was knocked out (KO) in HFFs, and H3K27me3 was chemically inhibited. In uninfected cells loss of macroH2A1 and H3K27me3, less heterochromatin was observed in the nuclear periphery than in wild type (WT) cells, suggesting that heterochromatin localization is disrupted in macroH2A1 KO and H3K27me3-depleted conditions.
During HSV-1 infection, macroH2A1 and H3K27me3 levels were previously observed to increase in WT HFFs cells using mass-spectrometry by Dr. Avgousti. The authors next used CUT&RUN to profile the genomic localization of macroH2A1 and H3K27me3 during viral infection and identified the regions of the genome where macroH2A1 changed after HSV-1 infection. Further, they found that H3K27me3 is also largely enriched in the same broad regions as macroH2A1. These results suggested redistribution of both macroH2A1 and H3K27me3 on host chromatin during HSV-1 infection. The authors then tested the transcriptional output of the newly formed macroH2A1 and H3K27me3 bound regions. They found that the presence of macroH2A1 and H3K27me3 correlates with a decrease in transcription in transcriptionally active regions during HSV-1 infection. This suggests that macroH2A1 and H3K27me3 are forming “new” heterochromatin during HSV-1 infection.
Next, Dr. Avgousti's team investigated the effect of macroH2A1 and H3K27me3 during HSV-1 infection by infecting HSV-1 WT and macroH2A1 KO HFFs cells. The authors found that macroH2A1 loss did not affect viral genome, viral RNA, or viral protein production. This suggests that neither HSV-1 replication nor protein production is affected by macroH2A1 loss. The same results were observed by chemical inhibition of H3K27me3. The authors then measured infectious progeny using plaque assays. The authors found a significant reduction of infectious HSV-1 in macroH2A1 KO HFF and H3K27me3 chemical depletion supernatant compared with WT cells. These results indicated that the loss of macroH2A1 and H3K27me3 leads to a significant defect in infectious viral progeny, but not viral protein, RNA, or genome accumulation. These results were further validated using clinical HSV-1 isolates supplied by Dr. Keith Jerome’s lab (Vaccine and Infectious Disease Division).
The authors infected WT and macroH2A1 KO HFFs cells and observed capsids’ localization in the nucleus compartment using TEM. There was an accumulation of capsids in macroH2A1 KO nuclei when compared with WT nuclei, indicating that the loss of macroH2A1-dependent heterochromatin adversely affects HSV-1 nuclear egress. When H3K27me3 was inhibited in infected cells HSV-1 capsids were found to line up along the nuclear membrane. These results suggest that macroH2A1 and H3K27me3-dependent heterochromatin are necessary for efficient nuclear egress of the HSV-1 capsids. These results demonstrate that HSV-1 infection induces chromatin channels the nuclear periphery that are dependent on macroH2A1 and H3K27me3. The authors conclude host heterochromatin chromatin redistribution is essential for successful HSV-1 capsid egress.
This research was supported by funding from the Deutsche Forschungsgemeinschaf and the Wellcome Trust through a Collaborative Award. This research was also supported by start-up funds from the RNA Bioscience Initiative at the University of Colorado School of Medicine, the Fred Hutch and National institutes of Health.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Daphne Avgousti contributed to this work.
Hannah C Lewis, Laurel E Kelnhofer-Millevolte, Mia R Brinkley, Hannah E Arbach, Edward A Arnold, Saskia Sanders, Jens B Bosse, Srinivas Ramachandran, and Daphne C Avgousti. 2023. HSV-1 exploits host heterochromatin for nuclear egress. JCB.