Analysis of host and viral nascent and steady-state RNA levels in a human neuronal model of herpes simplex virus 1 infection

We used precision nuclear run-on with sequencing (PRO-seq) in conjunction with quantitative RT-qPCR to map transcription, and transcript abundance in terminally differentiated LUHMES neurons before (mock) and after infection with Herpes Simplex Virus 1 (HSV-1). Initial PRO-seq analysis of cellular transcription demonstrated that the mock-infected differentiated LUHMES neurons expressed genes associated with differentiated neurons. This gene set included transcription of genes related to dopaminergic signaling, cell adhesion, and neuronal cell differentiation. By contrast, epithelial cells transcribed genes involved in cell cycle progression and DNA damage repair. PRO-Seq analysis of HSV-1-infected LUHMES neurons indicated only modest transcription of viral genomes. Additionally, genes of LUHMES neurons maintained RNA polymerase II (Pol II) promoter proximal pausing whereas we previously showed HSV-1 infection decreases promoter-proximal pausing on genes of epithelial cells. PRO-Seq and RT-qPCRanalysis were performed during a time course of infection, spanning both acute and latent phases. The results from the acute infection demonstrated slower rates of transcription, viral mRNA accumulation, and viral genome replication compared to undifferentiated, non-neuronal cells. RT-qPCR indicated that steady-state transcripts from all viral gene kinetic classes increased in abundance from 1 h to 24 h post-infection. This accumulation correlated with an increase in transcription on the same genes as demonstrated by PRO-seq. In contrast, the latent phase of infection was distinguished by high levels of transcription on all viral genes, but very low viral transcript abundance. These results indicate that viral gene transcription initiation does not correlate with viral transcript abundance during the latent phase, and suggest that blocks to RNA polymerase processivity and other components of the RNA life cycle likely contribute to maintenance of latency in LUHMES neurons.Author summary: Viral latency is a hallmark of all herpesvirus infections and arguably key to its high prevalence in human and animal populations. Previous studies showed minimal transcription of the Herpes Simplex Virus 1 genome during latency except for the latency associated transcript or LAT. Here we used a novel technique to identify transcriptional activity in a LUHMES neuronal culture model of HSV-1 latency. We found that HSV-1 transcription and viral genome replication were more restricted in LUHMES compared with those activities in undifferentiated infected epithelial cells. Surprisingly, the latent viral genome was more transcriptionally active than previously appreciated. While stable viral transcripts were in lower abundance in latency than in acute infection, transcriptional activity was higher. Unlike other viral transcripts, the LAT accumulated to high abundance during latency, despite only modest increases in transcription. These data suggest that HSV-1 latency is regulated through events following transcriptional initiation on many viral genes to limit production of viral mRNA.