Intracellular Low Iron Exerts Anti-BK Polyomavirus Effect by Inhibiting the Protein Synthesis of Exogenous Genes

ABSTRACT BK polyomavirus (BKPyV) is a small double-stranded DNA virus and ubiquitous human pathogen that particularly affects immunocompromised individuals. Antiviral therapy for BKPyV is urgently needed. Intracellular irons have an important role in many viral infections, yet its contribution to BKPyV and replication has not been explored. In this study, we explored the interaction between BKPyV infection and intracellular iron and the inhibitory effect of iron depletion on BKPyV infection. By creating a low-intracellular-iron environment, we demonstrated that the iron-chelating-induced iron depletion inhibits BKPyV infection in primary renal tubular epithelial cells (RPTECs) and urinary bladder cancer cells (TCCSUP cells). Iron depletion exerts an inhibitory effect after BKPyV enters the nucleus, which might be due to the inhibition of the protein synthesis of exogenous genes in iron-depleted cells. Further exploration of the target proteins of iron-regulating viral infection could potentially be used to develop new strategies for urgently needed anti-BKPyV therapies. IMPORTANCE BKPyV poses a serious threat to the health of immunocompromised patients, and there are currently no curative drugs. Understanding the relationship between the virus and intracellular environment contributes to the discovery of antiviral targets. We demonstrate here that BKPyV is inhibited in cells with a low-iron environment. We also find that iron-chelating-induced iron depletion inhibits viral and exogenous protein synthesis. Further exploration of the target proteins of iron regulation could have great potential in developing new drugs against BKPyV and other viruses.

development of anti-BKPyV therapeutics." and "However, a requirement for an intracellular iron environment and its contribution to BKPyV infection and replication has not yet been explored." The authors imply that using iron-depleting drugs would be a BKV-specific treatment, which they cite themselves is not necessarily true as that inhibits viral replication among other viruses as well.
Line 97: Methods on drug treatments. Drug concentrations are not referenced. More information on how (concentration and frequency) the drugs are currently prescribed in clinical settings and how the concentrations used in the manuscript would mirror that (or not). Hence, what is the clinical relevance of the concentrations used? Line 112: "After the surplus virus was removed, the cells were washed in PBS and treated with 5μM DFX at 0, 2, 12, 24, and 36 h post-infection (hpi), respectively." The first set of experiments were done using 20uM?
The authors start with 3 drugs, and proceed only with DFX without discussing why or the used concentrations. It is clear that DFX has the highest iron-chelating effect but also seems to 20uM kills cells (Fig.2C, few nuclei in IFA?). Are there any possible explanation of varying degrees of effect since all drugs were used at same concentration in the beginning? Line 351: "Our data suggested that iron chelators exhibited different anti-BKPyV effects due to their ability to chelate iron..." while there is a dosedependent effect its not clear all drugs worked the same.
The authors use BKPyV and BKV interchangeably. Should be streamlined. Line 401: "The next step should be to examine the anti-BKPyV effect of iron chelators in vivo." Use of clinically relevant concentrations and further studies on physiological effect of in vitro models first?
Line 402: "Moreover, possible adverse reactions should also be considered since patients with BKPyV infection after kidney transplantation are often accompanied by anemia symptoms." This is the only sentence mentioning any possible clinical sideeffects of using iron-chelaters in clinical setting, without discussing current clinical applications (except that its currently used for hyperferremia). The manuscript, Intracellular low iron exerts anti-BK polyomavirus effect by inhibiting the protein synthesis of exogenous genes, by the Zhu lab is a fairly comprehensive, rigorous, and unique study of the role cellular iron plays during a virus infection. They tested three drugs in clinical use for their ability to chelate intracellular iron and correlated this with inhibition of virus gene expression and subsequent viral loads in tissue culture supernatant. The results were impressive with each drug inhibiting infection between 27 and 98%. This correlated with drugs ability to reduce intracellular iron. The effects could be reversed by providing iron exogenously.

Major concern:
They provide no data regarding the potential cytotoxic or cytostatic effects of these drugs on the cell. These controls would be critical for proper interpretation of the results.

Summary:
This is an important and unique study that could lead to promising treatments for polyomavirus associated diseases. The experiments are well-controlled, with the exception of controls for cytotoxic/cytostatic effects, and the conclusions are supported by the data.

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1 Title: Intracellular low iron exerts anti-BK polyomavirus effect by inhibiting the protein synthesis of exogenous genes Study Summary: The study uses an in vitro model (renal tubular epithelial and urinary bladder cancer cells) to study how drug-dependent iron depletion hampers BK polyomavirus (BKV) replication. After determining that BKV replication is iron-release dependent the authors go into a lot of detail to study the effects of protein, RNA, viral genome and promoter regulation depending on drug-induced iron availability. The study suggests that iron depletion exerts a restricting effect in viral gene expression (post-mRNA transcription), after examining cell entry, replication, etc.
Overall Impression: It is a well-done study that uses a wide arrange of techniques to analyze the effect of drug-induced iron depletion on BKV replication.

Major Comments:
Line 72 and 83: "Therefore, understanding the relationship between BKPyV and the host cell environment is critical for the development of anti-BKPyV therapeutics." and "However, a requirement for an intracellular iron environment and its contribution to BKPyV infection and replication has not yet been explored." The authors imply that using iron-depleting drugs would be a BKV-specific treatment, which they cite themselves is not necessarily true as that inhibits viral replication among other viruses as well.
Line 97: Methods on drug treatments. Drug concentrations are not referenced. More information on how (concentration and frequency) the drugs are currently prescribed in clinical settings and how the concentrations used in the manuscript would mirror that (or not). Hence, what is the clinical relevance of the concentrations used? Line 112: "After the surplus virus was removed, the cells were washed in PBS and treated with 5μM DFX at 0, 2, 12, 24, and 36 h postinfection (hpi), respectively." The first set of experiments were done using 20uM?
The authors start with 3 drugs, and proceed only with DFX without discussing why or the used concentrations. It is clear that DFX has the highest iron-chelating effect but also seems to 20uM kills cells (Fig.2C, few nuclei in IFA?). Are there any possible explanation of varying degrees of effect since all drugs were used at same concentration in the beginning? Line 351: "Our data suggested that iron chelators exhibited different anti-BKPyV effects due to their ability to chelate iron…" while there is a dose-dependent effect its not clear all drugs worked the same.
The authors use BKPyV and BKV interchangeably. Should be streamlined. Line 402: "Moreover, possible adverse reactions should also be considered since patients with BKPyV infection after kidney transplantation are often accompanied by anemia symptoms." This is the only sentence mentioning any possible clinical side-effects of using iron-chelaters in clinical setting, without discussing current clinical applications (except that its currently used for hyperferremia). Reviewer #1 (Comments for the Author): Title: Intracellular low iron exerts anti-BK polyomavirus effect by inhibiting the protein synthesis of exogenous genes Study Summary: The study uses an in vitro model (renal tubular epithelial and urinary bladder cancer cells) to study how drug-dependent iron depletion hampers BK polyomavirus (BKV) replication. After determining that BKV replication is iron-release dependent the authors go into a lot of detail to study the effects of protein, RNA, viral genome and promoter regulation depending on drug-induced iron availability. The study suggests that iron depletion exerts a restricting effect in viral gene expression (post-mRNA transcription), after examining cell entry, replication, etc. Overall Impression: It is a well-done study that uses a wide arrange of techniques to analyze the effect of drug-induced iron depletion on BKV replication.

Minor Comments:
Major Comments: Line 72 and 83: "Therefore, understanding the relationship between BKPyV and the host cell environment is critical for the development of anti-BKPyV therapeutics." and "However, a requirement for an intracellular iron environment and its contribution to BKPyV infection and replication has not yet been explored." The authors imply that using iron-depleting drugs would be a BKV-specific treatment, which they cite themselves is not necessarily true as that inhibits viral replication among other viruses as well.
Reply: Thank you for your rigorous consideration. In fact, we intended to describe the importance of the host cell environment for BKPyV infection and to introduce the idea of studying the correlation between BKPyV and intracellular iron. Our expression in the manuscript was not rigorous, and we have modified.
Line 97: Methods on drug treatments. Drug concentrations are not referenced. More information on how (concentration and frequency) the drugs are currently prescribed in clinical settings and how the concentrations used in the manuscript would mirror that (or not). Hence, what is the clinical relevance of the concentrations used? Line 112: "After the surplus virus was removed, the cells were washed in PBS and treated with 5μM DFX at 0, 2, 12, 24, and 36 h post-infection (hpi), respectively." The first set of experiments were done using 20uM?
Reply: Thank you for your rigorous consideration. As clinically approved drugs, iron chelators are limited to the treatment of iron overload. However, there is no uniform standard for blood concentration of drugs and no data on the clinical treatment of viral infections by these drugs. In this paper, we refer to the concentration of drugs used to inhibit HIV infection in vitro in previous literature (Virology 367 (2007) 324-333;The Journal of Infectious Diseases 2000;181:484-90). Based on above research, we decided to use 20μM drugs (24h subcutaneous infusion of 100 mg/kg DFO in patient with iron overload) as a preliminary demonstration of the results of "Iron chelators inhibit BKPyV infection", because the drug has little effect on cell proliferation at this concentration.
In subsequent dose-inhibition assay, we further found that 5μM was the lowest concentration at which DFX exerted strongest inhibitory effect (as shown in Figure 3A-D), so we considered 5μM DFX was a more reasonable choice in time-inhibition experiments and studies on mechanism. Sorry for not explaining this point, we have added it in the fourth part of result.
The authors start with 3 drugs, and proceed only with DFX without discussing why or the used concentrations. It is clear that DFX has the highest iron-chelating effect but also seems to 20uM kills cells (Fig.2C, few nuclei in IFA?). Are there any possible explanation of varying degrees of effect since all drugs were used at same concentration in the beginning? Line 351: "Our data suggested that iron chelators exhibited different anti-BKPyV effects due to their ability to chelate iron..." while there is a dose-dependent effect its not clear all drugs worked the same.
Reply: Thank you for your rigorous consideration. We demonstrated that the iron chelators play an anti-BKPyV role through iron depletion by using iron supplement assay ( Figure 3E-H). As the reviewer said, DFX has the highest iron-chelating effect, and we verified DFX had the strongest anti-BKPyV effect at the same concentration ( Figure  2A-D). Therefore, DFX was used as a representative drug to deplete iron in the subsequent study on mechanism. We have added explain in the fourth part of result. As for whether 20μM DFX has killing effect on cells, we conducted CCK-8 assay and apoptosis assay. We found that 50μM DFX had an inhibitory effect on cell proliferation, but did not induce an increase in apoptosis ( Figure S2). Therefore, we think that 20μM DFX has an inhibitory effect on cell proliferation, leading to the reduction of nucleus. We maintained the same concentration of the three iron chelators (DFX, DFO and DFP) in the initial experiment in order to initially reveal that the effect of anti-BKPyV may be related to iron chelation and to guide the subsequent dose-inhibition experiment and iron supplement experiment. To be sure, "Our data suggested that iron chelators exhibited different anti-BKPyV effects due to their ability to chelate iron..." is indeed imprecise and can only be drawn in combination with subsequent experimental results. We have made correction according to the reviewer's comments.