A role of hypoxia-inducible factor 1 alpha in Murine Gammaherpesvirus 68 (MHV68) lytic replication and reactivation from latency

The hypoxia-inducible factor 1 alpha (HIF1α) protein and the hypoxic microenvironment are critical for infection and pathogenesis by the oncogenic gammaherpesviruses (γHV), Kaposi sarcoma herpes virus (KSHV) and Epstein-Barr virus (EBV). However, understanding the role of HIF1α during the virus life cycle and its biological relevance in the context of host has been challenging due to the lack of animal models for human γHV. To study the role of HIF1α, we employed the murine gammaherpesvirus 68 (MHV68), a rodent pathogen that readily infects laboratory mice. We show that MHV68 infection induces HIF1α protein and HIF1α-responsive gene expression in permissive cells. siRNA silencing or drug-inhibition of HIF1α reduce virus production due to a global downregulation of viral gene expression. Most notable was the marked decrease in many viral genes bearing hypoxia-responsive elements (HREs) such as the viral G-Protein Coupled Receptor (vGPCR), which is known to activate HIF1α transcriptional activity during KSHV infection. We found that the promoter of MHV68 ORF74 is responsive to HIF1α and MHV-68 RTA. Moreover, Intranasal infection of HIF1αLoxP/LoxP mice with MHV68 expressing Cre- recombinase impaired virus expansion during early acute infection and affected lytic reactivation in the splenocytes explanted from mice. Low oxygen concentrations accelerated lytic reactivation and enhanced virus production in MHV68 infected splenocytes. Thus, we conclude that HIF1α plays a critical role in promoting virus replication and reactivation from latency by impacting viral gene expression. Our results highlight the importance of the mutual interactions of the oxygen-sensing machinery and gammaherpesviruses in viral replication and pathogenesis.

1. Fig1A, the band of HIF1a is weak, especially when there is a stronger non-specific band above. It would be more convincing if author could include positive control on the same gel.
We now include western blot data for positive HIF1 protein expression by CoCl2 treatment and is represented as a single band with anti-HIF1 antibody by Abcam.

Fig1D. Although the list of metabolic related genes are known to be induced by HIF1a, authors still need to confirm that the induction of these genes is dependent on HIF1a with MHV68 infection.
Figure 3D comparing the expression of metabolic genes in presence or absence of HIF1 now shows that expression of metabolic genes by MHV68 is dependent on HIF1 activity.

In Fig3A, in HIF1a Null cells, hypoxia condition still strongly decreased viral production, implying factors other than HIF1a contributes to the reduction. Authors may provide some explanations, Similarly, ORF9 and ORF25 without HRE were strongly reduced in HIF1a Null cells under hypoxia, which is apparently non related to HIF1a.
The goal of the experiment at 3% oxygen levels (now in Figure 4) was to show that in lower-yet physiological-oxygen level conditions in which upregulation of HIF1 was increased there was a stronger effect noticed when HIF1 was deleted. This is why these set of experiments are now showcased in Figure 4 for clarity. Regarding the regulation of non-HRE containing genes: in the current version of the manuscript we decided to include only HRE containing genes since-as now discussed in the current manuscript-we could not dissociate indirect effects due to the decreased transcription of genes because the viral gene regulating its expression was an HRE containing gene such as the case of the transactivator RTA. We agree with the problem in the figure. In the current version we are providing better labeling for the scheme of animal infection.

Fig. 5, authors showed the induction of glycolytic enzymes was abolished in
6D, it is also a little choppy here to show IL1b. Since authors present a series of IFNs in figure 5, why did the author show IL-1b production instead of IFNs in vivo? This reviewer also would like to remind the authors IL1b and TNFa at day 7 may not represent innate immune responses.
We have analyzed IL1 IFN, IL-6, TNF and IFN in lungs lysates on 3, 5 and 7 dpi but only IL1 levels in lungs from HIF1LoxP mice showed decreased at day 7 when compared to WT infection. We also include an explanation for this result in the discussion. Line 475: It is also likely that a reduction in viral expansion during the initial lytic phases in the lung could affect the extent of inflammation explaining the significant decrease of IL1 production in lungs lysates on day 7 ( Figure 5D). In the original version there was not significant differences in latency establishment at day 42 and differences in reactivation were not very marked and very difficult to explain with do-able experiments. Therefore, we decided to focus and expand only on the results at Day 16 showing an impact of HIF1 deletion in reactivation which we complemented with the new Figure 7.

Reviewer 2
We appreciate that this reviewer found that our study "adds new information in direct measurement of lytic virus infection and in vivo latency and reactivation using the mouse gammaherpesvirus system". We appreciate this reviewers' criticisms and suggestions that have helped us to improve our manuscript.

"Stabilization" of HIF1a protein is stated repeatedly, yet no experiments are
presented that support the claim in this system. The conclusion needs to be excised from the paper or data should be added to support.
We agree and appreciate this reviewer's remark and suggestion. Since the focus on the manuscript was not enquiring into the nature of the HIF1 upregulation induced by MHV68 infection we modified the conclusion of the experiments according to this reviewers' suggestion to accumulation/ increased levels etc. We choose the HIF1 system vs Cre-virus in vitro because we found that Cre was expressed after HIF was upregulated by de novo infection. Although this was not the ideal solution, we found it was suitable for the needed experiments and that the results we obtained with the HIF1 Exon 2 deleted cells were compatible with the ones we obtained with the HIF1LoxP animals and Cre-virus. To further support the validity of our in vivo Cre-induced deletion system, we now provide the proof of excision of HIF1 exon 2 by MHV68-Cre in splenocytes and lung tissues at day 16 (now, Figure 6C)

Data analysis is not transparent: blanket statement assures that all experiments were conducted at least 3 times, but report does not state whether figures represent averages or representatives;
We agree with the lack of consistency and accurate description pointed out by the reviewer. We now include in all figure legends how many times the experiment was conducted and whether the result represents an average or representatives.

comparison of infected cells to uninfected cells for luciferase activity is likely unfair because virus infection is likely to impact transcription at both specific and nonspecific levels, better would be an internal control such as dual luciferase.
We agree with this reviewer on the importance of using Dual luciferase to gauge nonspecific transcriptional effects of viral infection. We thank this reviewer that identified the lack of proper descriptions for our Dual luciferase experiments already done in the first manuscript. In this version we clearly state the use of dual luciferase assay was used in figures 1C and 2C.

RTA data mentioned and listed in the figure legend was not included. Inclusion of additional viral gene expression profiles would also be a big improvement.
We agree that showing actual in vivo evidence of viral gene expression would strengthen the manuscript. Thus, we are now showing definitive evidence of MHV-68 Cre expression in the lungs by showing HIF1 exon2 deletion.

Host cytokine response profile would benefit from greater context, such as discussion of other published studies or demonstration of expression and better justification for the small set analyzed in vivo.
Done in the discussion. We focalized in having at least a cytokine marker of inflammation (see answer to question 6 of Reviewer 1)

Editorial improvements in grammar (commas, spelling) could be made.
We have address these in the attached manuscript.

Reviewer 3
We are glad that this reviewer found that our "results support a role of HIF1a during MHV-68 infection both in vitro and in vivo". We are thankful for this reviewer's comments that have been instrumental in improving our manuscript. We are particularly grateful for this reviewer's idea on using ex vivo reactivation in lower oxygen conditions which now constitute the new Figure 7 which provides further experimental support of Figure 6 results on the role of HIF1 in reactivation from latency. (Fig. 1A). However, the authors did not get into details about this increase. This is what was described in the paper, line 115-117, "HIF1a stabilization was observed until 24 hours, which was also correlated with a 6-fold increase in HIF1a mRNA levels (Fig1B) We thank this reviewer for his/her in-depth analysis and suggestions. Please see answer to question # 1 of Reviewer 2.

Under both normoxia and hypoxia, virion production was reduced when functional
HIF1a protein is absent (Fig. 3A). This data would indicate that HIF1a plays a positive role in in WT cells under hypoxia (3% O2), where HIF1a protein is stabilized and allowed to perform its function, viral gene expression seemed to increase (Fig. 4), and yet virion production was reduced compared to 21% O2 (Fig. 3A). How do authors reconcile these pieces of data?
We agree with the reviewer that there was a slight difference at 5 MOI and no difference at 0.5 MOI. However; the goal of the experiment at 3% oxygen levels (now in Figure 4) was to show that in lower-yet physiological-oxygen level conditions in which upregulation of HIF1 was increased there was a stronger effect noticed when HIF1 was deleted. This is why these set of experiments are now showcased in Figure 4 for clarity. In following with the new Figure structure and its content/ interpretation the current gene expression data represents fold decrease comparing HIF1 null to WT and as explained in the text showed a higher impact on HRE containing genes when the experiment was done in 3%. We sincerely thank this reviewer's interesting idea, we have now generated data showing that low oxygen levels do not affect frequency of reactivation of latently infected splenocytes, however it accelerates virus replication and increases virus production significantly.

One potential issue for moderate phenotypes observed in vivo is
-And it is not clear whether latently infected cells already have HIF1a exon 2 removed or the removal happens during reactivation.
We have provided evidence for MHV68-Cre activity in latently infected splenocytes derived from day 16 infection by excision assay.

Fig5. There is no data IFIT8 (line 263). If there is little reduction of IFN-related genes in knockout cells, one would expect that viral replication increases in knockout cells both in vivo and in vitro. And yet the opposite was observed. Why?
We have made edited the manuscript and IFN-related studies in vitro have been eliminated. Moreover, we provide explanation in the discussion as follows in line 475, It is also likely that a reduction in viral expansion during the initial lytic phases in