Precision Nuclear Run-on Analysis of post-mitotic human neurons infected with HSV-1

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-PCR analysis were then 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-PCR 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.

Importance Viral latency is a hallmark of all herpesvirus infections and arguably key to its high prevalence in human and animal populations. We found that HSV-1 transcription and viral genome replication is more restricted in LUHMES neurons compared with undifferentiated epithelial cells. Moreover, the latent viral genome is more transcriptionally active than previously appreciated, while overall lytic transcripts are in lower abundance in latency than in acute infection.

The latency-associated transcripts, in contrast, accumulate to high abundance during latency, despite only modest increases in transcription.