To study the establishment of latency, maintenance and virus reactivation in human neurons, we established the LUHMES in vitro model (Work package 3 – WP3). We substantially improved on the cell culture protocols so that we can now obtain millions of single (non-clustered) terminally differentiated human neurons to study HSV-1 latency and reactivation. To study the establishment of HSV-1 latency at single-cell level, we performed a time-course analysis using single-cell SLAM-seq (scSLAM-seq) covering the first 8 hours of infection (4 consecutive 2-hour windows). As 48 h of inhibition of viral DNA replication using Acyclovir is required to prevent lytic and establish latent infection, we performed the time-course +/- Aciclovir treatment. We obtained high-quality sequencing data at very high depth. Moreover, Prof. Florian Erhard (University of Regensburg) developed the computational framework for Heterogeneity-seq, which now enables us to identify cellular genes in the uninfected cells that correlate with infection outcome up to 8 hours later. The HSV-1 virus host shut-off protein (vhs) delivered by the incoming virus particles rapidly cleaves cellular RNA to free ribosomes for the translation of viral mRNAs. To avoid its detrimental effects on the old (pre-existing to the 2 h of metabolic RNA labelling) RNA profiles, we repeated the scSLAM-seq experiment comparing infection of wild-type HSV-1 with its vhs-null mutant. We just obtained high-quality sequencing data and have initiated an in-depth analysis, which will be published in a paper describing the establishment of HSV-1 latency in the LUHMES model.
In April 2024, my lab moved from Würzburg to Hannover.
Herpesvirus immediate early (IE) proteins not only play a central role in the initiation of lytic infection but also in the initiation of productive virus replication upon virus reactivation 1,42. Three HSV-1 IE proteins, namely ICP4, ICP22 and ICP27, exercise this by concerted manipulation of the host transcriptional machinery. In work package 2 (WP2) of DecipherHSV, we aim to decipher the molecular mechanisms and the functional role of these three HSV-1 IE proteins in manipulating Pol II and pervasive transcription during productive infection. We established human fibroblasts (HFF-tert) that conditionally express the three HSV-1 IE genes either individual or jointly. We could show that ICP22 is both necessary and sufficient to open up cellular chromatin downstream of genes suffering from impaired transcription termination (Djakovic et al., Nature communications 2023). Moreover, we found that the viral ICP4 protein disrupts transcription termination of canonical histone and snRNA genes. We performed PRO-seq analysis in collaboration with Prof. David Price (Iowa, USA) to study the effect of HSV-1 infection and ICP4 on cellular promoter and enhancer RNAs. Here, a postdoc of mine (Dr. Adam Whisnant) visited the lab of David Price to learn PRO-seq and perform the respective experiments. Supported by independent findings of my lab for lytic human cytomegalovirus infection, we follow the hypothesis that viral early gene expression not only requires a viral transcription factor to initiate transcription but also active recruitment of the cellular transcription elongation machinery. Employing ectopic expression of ICP4, ICP22 and ICP27, we found that isolated expression of ICP4 is both necessary and sufficient for viral early gene expression. Interestingly, the group of Prof. Markus Landthaler in Berlin found that ICP4 also harbors RNA-binding activity. Together, we are testing the hypothesis that ICP4 facilitates transcription elongation by binding to the 5’-region of viral RNAs and recruiting the cellular transcription elongation machinery. Finally, we generated cells that allow for rapid, conditional degradation of key components of the nuclear RNA degradation machinery (the Exosome). Infection of these cells will reveal highly unstable viral transcripts, i.e. viral promoter or enhancer RNAs (viral pervasive transcription).
To identify novel viral genes and genetic elements important for the establishment of latency and virus reactivation, we proposed to develop a novel MuA-based transposon mutagenesis screen (WP1). A PhD student start on the project for about 8 weeks in 2023. Unfortunately, she experienced some medical problems and dropped out after 6 months. In May 2024, a new PhD student (Lena Wolpert) joined my lab and has taken over the project. To provide her with four years for her PhD and compensate for the delay caused by the move from Würzburg to Hannover, I applied for a one-year no-cost extension for DecipherHSV.
