Glucocorticoid Receptor and Cell Cycle Regulator (E2F2) Cooperatively Transactivate a Cis-Regulatory Module in the HSV-1 Infected Cell Protein 0 (ICP0) Promoter
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsSummary: Herpes simplex virus type 1 (HSV-1) produces a latent infection within sensory neurons of trigeminal ganglia that can reactivate and cause recurrent infection and disease. Previous work has shown that E2F family members that regulate the mammalian cell cycle and glucocorticoid receptor (GC) can stimulate HSV-1 replication. With this background information, Jayathilake and coworkers tested the hypothesis that E2F family members and GR stimulate HSV-1 replication by activation of certain HSV-1 promotors with focus on four HSV-1 immediate early gene ICP0 cis-regulatory modules (CRM) upstream of the ICP0 promotor. This was accomplished through performance of a series of in vitro experiments using murine neuroblastoma (Neuro-2A) cells. Results revealed that E2F2 alone transactivates the ICP0-CRM-C fragment but surprisingly E2F1, E2F3a, and E2F3b has no effect on any ICP0 CRM fragments. The authors conclude that despite the ability of HSV-1 to replicate in quiescent cells, certain cell cycle factors can stimulate gene expression and thereby enhance virus spread within the host.
Review: This is a well written manuscript that provides new and important information on the fine molecular mechanism by which HSV-1 E2F2 and GR may collaborate to transactivate the ICP0 promotor activity during HSV-1 replication that will enhance virus replication in certain cell types. The in vitro studies appear to be carefully performed and produce findings that are convincing and compelling. Some minor revisions, however, could strengthen the manuscript.
- There are six HSV-1 immediate early genes (ICP4, ICP0, ICP22, ICP27, US.15, and ICP47). Why have the authors chosen to focus on one of these immediate early genes, ICP0? Importantly, why have they excluded an investigation of ICP4 because both ICP4 and ICP0 are required for the optimal expression of early genes (Fields Virology, 7th Ed)
- Line 130 states “The HSV-1 ICP0 gene is present in the repeats …” This should probably read “two repeats”.
- It is stated on Lines 172 – 175 that immortalized mouse fibroblasts (NIH-3T3 cell) were used for comparison with Neuro-2A cells, but data are not shown. Because of the important contrast of findings when compared with Neuro-2A cells, this data should be shown.
- Figure 4 with the three astericks and lines that go across the figure remain confusing despite the figure legend. The authors should provide more information to clarify the figure for the reader.
Author Response
With respect to the concerns from Reviewer #1, the following changes were made.
Concern #1 There are six HSV-1 immediate early genes (ICP4, ICP0, ICP22, ICP27, US.15, and ICP47). Why have the authors chosen to focus on one of these immediate early genes, ICP0? Importantly, why have they excluded an investigation of ICP4 because both ICP4 and ICP0 are required for the optimal expression of early genes (Fields Virology, 7th Ed).
Response: ICP4, VP16, or ICP27 CRM constructs were not transactivated by E2F1, E2F2, or the E2F3 constructs in Neuro-2A cells regardless of GR-a expression. Furthermore, we were unable to identify a consensus E2F binding sites in CRM regions and the intact promoters. This is stated in the revised manuscript (lines 285-289).
Concern #2 Line 130 states “The HSV-1 ICP0 gene is present in the repeats …” This should probably read “two repeats”.
Response: This mistake was fixed (line 107-108).
Concern #3 It is stated on Lines 172 – 175 that immortalized mouse fibroblasts (NIH-3T3 cell) were used for comparison with Neuro-2A cells, but data are not shown. Because of the important contrast of findings when compared with Neuro-2A cells, this data should be shown.
Response: The negative results for the ICP0 CRM-C fragment in NIH3T3 cells were added to the revised manuscript (Figure 5) and lines 173-190).
Concern #4 Figure 3 with the three asterisks and lines that go across the figure remain confusing despite the figure legend. The authors should provide more information to clarify the figure for the reader.
Response: In the modified manuscript, this information is now in Figure 4, the information was simplified, and the legend is explained better (lines 164-168).
Reviewer 2 Report
Comments and Suggestions for AuthorsThis manuscript presents a well-organized and convincing analysis of the transcriptional regulation of the HSV-1 ICP0 promoter by GR and E2F family members, with a notable and novel finding: the selective and cooperative transactivation of the CRM-C region by GR and E2F2. The specificity of this interaction, in contrast to the inhibitory effects of E2F1 and the lack of activity of the E2F3 isoforms, represents a significant advance in our understanding of how stress-responsive host transcription factors can differentially modulate viral immediate-early gene expression. The authors employ a comprehensive experimental approach, combining promoter dissection, site-directed mutagenesis, luciferase assays, Western blotting, and ChIP analyses in both transfected and infected Neuro-2A cells. Collectively, these data support a model in which multiple E2F binding sites and an adjacent half-GRE function cooperatively to permit GR–E2F2 occupancy and transcriptional activation, a mechanism with potential relevance to neuronal stress and HSV-1 reactivation. A few minor issues, if addressed, would further strengthen the manuscript.
- Many key conclusions are derived from transient overexpression of GR and/or E2F2 (Figures 2, 3, and 8). Additional discussion of the endogenous levels of these factors and their potential effects on CRM-C activity would be helpful.
- ChIP analyses appear to be based on only two independent experiments (Figures 7 and 8). While this is an acceptable practice, this limitation should be clearly stated.
- The lack of GR/E2F2-mediated transactivation of CRM-C in NIH-3T3 cells (compared to Neuro-2A cells) is intriguing (Figure 3, text). Additional discussion of chromatin accessibility or co-factor expression would help clarify whether this reflects neuronal specificity.
- Although ICP0 is a critical immediate-early gene, the Results do not examine whether disruption of CRM-C, E2F binding sites, or the half-GRE alters viral gene expression kinetics or productive replication during infection (Figures 5, 7, and 8). This limitation could be acknowledged as a future research direction.
- The basis for detecting GR and E2F2 at the ICP0 promoter at 0 h post-infection is not entirely clear (Figure 7). A more explicit explanation distinguishing viral entry from early release dynamics would strengthen this interpretation.
- The Western blot data demonstrate dynamic effects of DEX treatment and HSV-1 infection on E2F2 abundance (Figure 6), but these findings are not fully integrated with the transcriptional (Figure 3) and ChIP results (Figures 7 and 8). A more detailed discussion would improve coherence across these datasets.
Author Response
With respect to the concerns from Reviewer #2, the following changes were made.
Concern #1 Many key conclusions are derived from transient overexpression of GR and/or E2F2 (Figures 2, 3, and 8). Additional discussion of the endogenous levels of these factors and their potential effects on CRM-C activity would be helpful.
Response: If HSV-1 infects dividing cells, one would expect E2F2 protein levels would be higher, which we suspect enhances viral replication. This is discussed in the revised manuscript (lines 287-289).
Concern #2 ChIP analyses appear to be based on only two independent experiments (Figures 7 and 8). While this is an acceptable practice, this limitation should be clearly stated.
Response: This important information is stated in the legends of Figures 7 and 8 (lines 222-223 and 254-255). Furthermore, the results from the ChIP studies were consistent.
Concern #3 The lack of GR/E2F2-mediated transactivation of CRM-C in NIH-3T3 cells (compared to Neuro-2A cells) is intriguing (Figure 3, text). Additional discussion of chromatin accessibility or co-factor expression would help clarify whether this reflects neuronal specificity.
Response: This is mentioned in the Discussion (lines 299-304).
Concern #4 Although ICP0 is a critical immediate-early gene, the results do not examine whether disruption of CRM-C, E2F binding sites, or the half-GRE alters viral gene expression kinetics or productive replication during infection (Figures 5, 7, and 8). This limitation could be acknowledged as a future research direction.
Response: We have plans to generate a mutant virus that lacks the ½ GRE and E2F consensus binding sites, and this is mentioned in the Discussion of the revised manuscript (lines 302-304).
Concern #5 The basis for detecting GR and E2F2 at the ICP0 promoter at 0 h post-infection is not entirely clear (Figure 7). A more explicit explanation distinguishing viral entry from early release dynamics would strengthen this interpretation.
Response: Agreed, this is explained better in the revised manuscript (lines 228-233).
Concern #6 The Western blot data demonstrate dynamic effects of DEX treatment and HSV-1 infection on E2F2 abundance (Figure 6), but these findings are not fully integrated with the transcriptional (Figure 3) and ChIP results (Figures 7 and 8). A more detailed discussion would improve coherence across these datasets.
Response: The Western Blot was removed because Invitrogen told me the lot of this antibody had problems with Western Blots. Future studies are being planned to examine E2F1 and E2F2 in cultured cells and compare these results to determine if E2F2 is induced in TG neurons during explant-induced reactivation from latency. We are currently testing different E2F2 antibodies to identify one that consistently works for Western Blots.
Reviewer 3 Report
Comments and Suggestions for AuthorsPlease see the attachment
Comments for author File: 
Comments.pdf
Author Response
With respect to the concerns raised by Reviewer #3, the following changes were made.
Concern #1: In Fig. 1B, what do the three orange ovals (near -95, middle of -281, and -485, next to -635) represent? The light blue oval for NF-1 looks different between Fig. 1B and Fig. 1C. It is hard to tell the black oval from the dark green oval.
Response: Thank you for pointing this out. This is NF-Y. NF-Y is a transcription factor that can interact with CCAAT boxes. Since this motif was not in the CRM-C fragment, we did not discuss it in the manuscript. I have “stretched” the promoter so that it is longer from left to right in the revised Figure 1. Consequently, I can now see the black oval from the dark green oval. The problem is the ICP0 contains many transcription factor binding sites, and the density of transcription factors from -458 to -800 leads to partial overlap of the respective symbols for certain transcription factors.
Concern #2 In Figs. 2 and 3, the bar graph can be presented in the same style, since each treatment was labeled on the X-axis. The Figure looks busy with different filling styles. The # is not explained in the figure legend.
Response: The # denotes promoter activity was signficantly reduced compared to basal transcription of the respective ICP0 CRM constructs (lines127-128 and 155-157). The color schemes are now the same. Despite there is a lot of information in A-D, I think these studies can be interpreted.
Concern #3 In Fig. 4, "panel A" can be removed since only one panel is presented in the Figure. The difference between all E2F3 treatment groups and GR or GR+Dex can be marked with a bracket.
Response: This panel A was deleted.
Concern #4 In Figs. 5 and 8, the * is too small to see in the figures.
Response: Agreed. The size of the asterisks (*) for Figures 5 and 8 are now larger.
Concern #5 In Fig. 7, the E2F2 is still present at 8hpi, while the western blot (Fig. 6) showed a significant reduction of E2F2 protein at 8hpi. How do you explain the discrepancy? Only R0 primers are indicated in Fig. 7a, but not the ICP0 CRM-C primers.
Response: As stated above, we removed the Western Blot figure because the E2F2 antibody had problems with Western Blots. Future studies will examine E2F1 and E2F2 in cultured cells and compare it to TG during explant-induced reactivation from latency.
