In HSV-1, the LAT Enhancer Drives Pre-IE VP16 Transcription to Initiate Reactivation
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Abstract
The HSV-1 VP16 protein, delivered to the nuclei of epithelial cells from the tegument layer of infecting virions, drives viral Immediate-Early (IE) transcription to initiate productive (lytic) infection. Latent HSV-1, established in sensory neurons, reactivates to a lytic infection in the absence of a recent de novo infection event that would deliver tegument proteins like VP16. Therefore, the role of the protein VP16 in the exit from HSV-1 latency has been hotly debated for decades. Here we show that VP16 transcription during latency is silenced by CTCF and cohesin proteins bound to a newly identified CTCF insulator site. Upon a reactivation stimulus, CTCF and cohesins are evicted and VP16 is transcribed prior to, and in the absence of, any other viral transcription. We previously identified long-range cis spatial interactions between the LAT and VP16 loci of the latent HSV-1 genome. Here, our data further indicate that the LAT enhancer serves as a neuron-specific enhancer of VP16 transcription during reactivation. Collectively, we show the HSV-1 latent genome is hard-wired for reactivation by a long-range spatial interaction between the viral gene encoding the protein that initiates lytic phase transcription (VP16) and the viral enhancer element that drives its transcription (LAT enhancer).
Author Summary
Herpes Simplex Virus 1 (HSV-1) is a significant lifelong human pathogen that infects 70% of adults worldwide. HSV-1 establishes a persistent latent infection in the peripheral nervous system where the virus can periodically reactivate in response to environmental stressors. These reactivation events result in recurrence of corneal infections that can lead to corneal blindness. It is becoming clear that CTCF insulators play a key role in the maintenance of gene silencing in DNA viruses that can establish latency. CTCF insulators are essential regulators of chromatin structure and play vital regulatory roles in transcriptional control of DNA viruses by organizing chromatin architecture during both latent and lytic stages of virus lifecycles. Here we have identified and functionally characterized a novel CTCF insulator, located at the transactivating gene VP16. We provide evidence that this insulator is involved in silencing VP16 during latent infection, while eviction of insulator protein coincides with increased VP16 gene expression during reactivation. To our knowledge, these are the first data that show that CTCF insulators is a key regulatory element in the exit from latency in a VP16-dependent manner and has important implications in understanding the heterogeneity of HSV-1 reactivation in different populations of neurons.
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/18193593.
This is an interesting and valuable preprint on the mechanism of HSV reactivation. The work is well done and address a topic that has been debated for decades. Using an in vitro model of HSV latency in differentiated neurons, the work presented suggests that the viral activator VP16 is expressed prior to immediate-early genes during viral reactivation, and may facilitate the subsequent expression of immediate-early and late genes. The work identifies a CTCF binding site close to VP16 and shows that it works as an insulator that may contribute to VP16 regulation during reactivation from latency. The authors also show that an enhancer element located near the LAT locus (LTE) is involved in …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/18193593.
This is an interesting and valuable preprint on the mechanism of HSV reactivation. The work is well done and address a topic that has been debated for decades. Using an in vitro model of HSV latency in differentiated neurons, the work presented suggests that the viral activator VP16 is expressed prior to immediate-early genes during viral reactivation, and may facilitate the subsequent expression of immediate-early and late genes. The work identifies a CTCF binding site close to VP16 and shows that it works as an insulator that may contribute to VP16 regulation during reactivation from latency. The authors also show that an enhancer element located near the LAT locus (LTE) is involved in VP16 regulation, and speculate that removal of CTFF during reactivation enables long-range interaction between the LTE and the VP16 promoter. However, the work presented does not test this hypothesis formally and more work will be needed to address it.
Major issues
On one hand, Figure 1 and 2 indicate that removal of the LTE prevent early expression of VP16, and subsequently of early and late genes. On the other hand, Figure 3 and 4 identify an insulator near VP16, and show that CTFC is evicted from the viral genome during reactivation. From this, they speculate that CTCF eviction enable long-range activation of the VP16 by the LTE. While plasmid assays support that model, the data presented does not test that hypothesis mechanistically and it remains speculative. In the present form, the claim in the abstract that "long-range spatial interaction between the viral gene encoding the protein that initiates lytic phase transcription (VP16) and the viral enhancer element that drives its transcription" is unsupported. I suggest either tempering that claim, or conducting additional experiments to address it. For example, it would be interesting to remove the insulator from HSV1 WT and 17∆A, and measure VP16 expression during latency and reactivation. If the hypothesis is correct, the insulator deletion may lead VP16 to be over-expressed during latency in HSV1-WT, while no effect should be observed in the LTE mutant.
I do not understand why in vivo experiments are only showed in the supplementary figures, and why key experiments with WT and 17∆A in vivo are shown in two separate supplementary figures (S1 and S3). Weren't these cohort infected and analyzed side by side? It makes comparing the dynamics of expression with the two virus difficult. The text writes that the dynamic in vivo agrees with their in vitro model, but i'm not sure this is supported by the data. In fig S1, both VP16 ad ICP27 are expressed at 2h and follow a similar dynamic. So this does not follow the same dynamic as in vitro, where VP16 expression occurs prior to ICP27. I suggest presenting all the in vivo data in its own figure, maybe figure 5?, and discuss how it support or deviate from the in vitro data. In addition, i suggest not expressing data as a fold change. This is often used when the underlying data is confusing. Right now it feels that the authors are trying to burry the in vivo data. This is unfortunate because this data is interesting and valuable, even though it may be harder to interpret and only partially support the overall model.
Minor issues
I find it strange that big fold changes (for example, in Fig S1, S3C) are non-significant or have a low p-value. This suggests that there is a lot of variation among replicates that the authors are not showing. I recommend showing the individual values for every figure in the paper. This is the best way to present data.
The manuscript includes an interesting paragraph justifying the use of a WT virus as a control instead of a rescue. While using both a WT and a rescue would be the best way, I believe that sequencing the viruses to identify secondary mutation, as the authors do, is appropriate. However, the authors should sequence both the 17∆A and WT isolates that they are using, and present the results in a supplementary file. I would be highly surprised if "no secondary mutations" exist between viruses isolated and propagated in different labs. Documenting these differences is important and would justify the claim that a rescue virus was not necessary.
It is unclear if the results shown in Fig. 1 and 2 were done together, or whether they represent separate experiments. If they were not done side by side, I do not think that it is appropriate to compare the dynamics between two separate infections, as there is a lot of variation just from one infection to the next, even with the same virus. If they were done separately, the results of Fig. 2D would be very hard to interpret. Clarifying the methodology would help.
Similarly, in Fig. 3C, are the pGL3 and LTE controls the same for every panel? It seems so. This is confusing to me to show the same data 5 times. Why not have just one graph?
A question about the model. Is 8 days sufficient to induce latency fully? Would things be different if HSV were reactivated after 2 or 3 weeks?
From the data presented, it seems that only a few cells are latently infected, with HSV genome copies representing 5% of GAPDH. Have the authors quantified how many cells are latently infected, and what is the number of HSV genomes per infected cell? If it were somehow different between the two viruses, that could change the interpretation of the results (for example, if one virus had fewer cells infected with more HSV copies)
Competing interests
The author declares that they have no competing interests.
Use of Artificial Intelligence (AI)
The author declares that they did not use generative AI to come up with new ideas for their review.
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