DWV 3C Protease Uncovers the Diverse Catalytic Triad in Insect RNA Viruses

ABSTRACT Deformed wing virus (DWV) is the most prevalent Iflavirus that is infecting honey bees worldwide. However, the mechanisms of its infection and replication in host cells are poorly understood. In this study, we analyzed the structure and function of DWV 3C protease (3Cpro), which is necessary for the cleavage of the polyprotein to synthesize mature viral proteins. Thus, it is one of the nonstructural viral proteins essential for the replication. We found that the 3Cpros of DWV and picornaviruses share common enzymatic properties, including sensitivity to the same inhibitors, such as rupintrivir. The predicted structure of DWV 3Cpro by AlphaFold2, the predicted rupintrivir binding domain, and the protease activities of mutant proteins revealed that it has a Cys-His-Asn catalytic triad. Moreover, 3Cpros of other Iflaviruses and Dicistrovirus appear to contain Asn, Ser, Asp, or Glu as the third residue of the catalytic triad, suggesting diversity in insect RNA viruses. Both precursor 3Cpro with RNA-dependent RNA polymerase and mature 3Cpro are present in DWV-infected cells, suggesting that they may have different enzymatic properties and functions. DWV 3Cpro is the first 3Cpro characterized among insect RNA viruses, and our study uncovered both the common and unique characteristics among 3Cpros of Picornavirales. Furthermore, it would be possible to use the specific inhibitors of DWV 3Cpro to control DWV infection in honey bees in future. IMPORTANCE The number of managed honey bee (Apis mellifera) colonies has considerably declined in many developed countries in the recent years. Deformed wing virus (DWV) vectored by the mites is the major threat to honey bee colonies and health. To give insight into the mechanism of DWV replication in the host cells, we studied the structure–function relationship of 3C protease (3Cpro), which is necessary to cleave a viral polyprotein at the specific sites to produce the mature proteins. We found that the overall structure, some inhibitors, and processing of 3Cpro are shared between Picornavirales; however, there is diversity in the catalytic triad. DWV 3Cpro is the first viral protease characterized among insect RNA viruses and reveals the evolutionary history of 3Cpro among Picornavirales. Furthermore, DWV 3Cpro inhibitors identified in our study could also be applied to control DWV in honey bees in future.

In this study, 3 dimensional structure of the 3C proteases of several iflaviruses, including Deformed wing iflavirus (DWV), was predicated using Alphafold2. This allowed to identify potential amino acids in the catalytic site of the DWV 3C Protease and model binding of potential inhibitors of the protease activity. The recombinant DWV 3C protease expressed in E. coli and the peptide corresponding to DWV LP-VP2 interface were used to devise in vitro FRET protease assay. This assay has allowed to biochemically characterize DWV 3C protease and, using mutant versions of DWV 3C protease with alanine residues replacing the catalytic positions, to experimentally confirmed the conserved catalytic triad and mechanisms of the protease activity inhibition with known inhibitors, such as rupintrivir. The MS is in general well written, the methods are described in sufficient detail, and the results are novel. The described in vitro system fo retesting DWV 3C protease activity has a potential to be used for screening novel antiviral compounds with a potential to control DWV. I recommend to accept after addressing some minor points.
-Page 4. Include a section of expression and self-precessing of the 56 kDa of the GST-DWV 3C Protease fusion as a first section of the Results section.
-Questions to the DWV 3C protease size" -Page 6."When we purified the 56 kDa wild-type GST-DWV 3Cpro protein, a 30 kDa small band was co-purified. This band was absent in the mutant protein lacking protease activity, for example, C2307A (Fig. 6A)." -Can the the 30 kDa band (Fig, 6 A, B) be the DWC 3C protease? Explain why in Fig. 6 the proposed 3C protease has a weight of 42 kD (Fig 6C), but the recombinant is only 30 kD (Fig. 6 AB). Does it mean that the section of DWV polyprotein (positions 2094-2353, which will give approximately 30 kDa peptide) is shortened at the C-terminus that compared to the wild type 3C protease? - Fig. 6 C. Mark position of the putative 97 kDa DWV 3C protease -RdRpol precursor.
-Page 3. "DWV belongs to Iflavirus (a sister species of Dicistrovirus) in the order Picornavirales." change to "DWV belongs to the genus Iflavirus in the order Picornavirales." -Page 4 (... a 15 amino acid peptide with the potential cleavage site, AKPEMD...) Provide entire sequence of the 15 Amin acid peptide (PVQAKPEMDNPNPG) which was actually used in the assay. It was not shown if a short 6 amino acid peptide AKPEMD will act a s good substrate for the DWV 3C Protease. This proteolytic site is not "potential" -it was experimentally demonstrated that a an insertion in the DWV polyprotes (e.g. GFP) which was be flanked by PVQAKPEMDNPNPG was completely excised in the course of DWV infection (see Ryabov https://doi.org/10.3390/v12040374 ) Introduction Given that you submitted your work to a journal with broad readership interested in microbial ecology, I would suggest starting the introduction with the general information about viruses and the role of 3CPro, and then focus on honey bees. At the moment, the plan is not clear as the text shifts from hosts (paragraph 1), to viruses (paragraph 2)and then host again (paragraph 3).
First paragraph: First sentence: no references are provided about Tropilaelaps sp. here. Second sentence: the host = what host?
Second paragraph: Fourth sentence: "well characterized in picornaviruses": ss you focus on the host in the first paragraph, it might be worthwhile mentioning what host these viruses infect here too? Alternatively, moving this paragraph first might enhance clarity (see above).
Third paragraph: It is not clear at this stage why there is a need to study what you did (e.g., structure-function, protease activities of mutant proteins). Why is this study important (e.g., how will it help controlling DWV in honeybee colonies)?

Results
As the methods are last, it might be helpful for the reader to add some explanations about why each experiment was conducted at the beginning of each subsection.
Enzymatic properties of DWV 3C Why is it important to know the temperature and PH?
Inhibitors of DWV 3C "Rupintrivir was the most effective compound and the IC50 value (0.36 μM) was lower than that of enterovirus 3Cpro (1.65-7.3 μM) but higher than that of human rhinovirus 3Cpro (5 nM) (Tan et al., 2016;Wang et al., 2011;Dragovich et al., 1999)." I would avoid providing citations in the results section, this relates more to the discussion.
Predicted structure of DWV 3Cpro by Alphafold2 "We obtained multiple models that are very similar to each other": this is a bit vague, please provide details or remove.

Discussion
Before starting with the details, I suggest starting this section by summarizing the aim and major findings of the study in a few sentences.
In general, the discussion and results parts are very similar. Citations are provided in the results, and the discussion is providing many details. I miss the bigger picture in the discussion, why was this study conducted and what are the major findings. For instance, as the introduction is focused on honey bee health, I would expect more details about how the findings can help improving their health.

Methods
This section should provide more details about the origin of the samples and viruses analyzed. For instance, where do the viruses used come from for the "Expression and purification" part?

Supplementary
Do you plan to submit the relevant information on public repositories? Staff Comments:

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Introduction
Given that you submitted your work to a journal with broad readership interested in microbial ecology, I would suggest starting the introduction with the general information about viruses and the role of 3CPro, and then focus on honey bees. At the moment, the plan is not clear as the text shifts from hosts (paragraph 1), to viruses (paragraph 2)and then host again (paragraph 3).
Authors: In the first paragraph, we introduce about the negative impact of DWV on honey bee colony and health. In the second paragraph, we describe that DWV belongs to the genus Iflavirus in the order Picornavirales which contains 3C protease in common. We then explain about the essential roles of 3C protease for replication of picornavirus. In the third paragraph, we briefly describe our study to characterize the structure and function of DWV 3C pro to give insight into the mechanism of viral replication. We believe this is logical order for the Introduction section. Second paragraph: Fourth sentence: "well characterized in picornaviruses": ss you focus on the host in the first paragraph, it might be worthwhile mentioning what host these viruses infect here too? Alternatively, moving this paragraph first might enhance clarity (see above).
Authors: Picornaviruses infect a wide range of vertebrates. This has been added in the revised manuscript (line 69).
Third paragraph: It is not clear at this stage why there is a need to study what you did (e.g., structure-function, protease activities of mutant proteins). Why is this study important (e.g., how will it help controlling DWV in honeybee colonies)?
Authors: Although DWV has been best studied among honey bee viruses, very little is known about the mechanisms of infection and replication in the host cells. Since 3C protease is crucial for viral replication and well characterized with picornaviruses regarding the structure-function relationship and inhibitors, our study on DWV 3C pro should give insight into the mechanism of viral replication. This has been described in the revised manuscript (line 89-91).

Results
As the methods are last, it might be helpful for the reader to add some explanations about why each experiment was conducted at the beginning of each subsection.

Enzymatic properties of DWV 3C
Why is it important to know the temperature and PH?
Authors: These are important to determine the optimum condition to measure the protease activity. This has been mentioned in the revised manuscript (line 100-102).
Inhibitors of DWV 3C "Rupintrivir was the most effective compound and the IC50 value (0.36 μM) was lower than that of enterovirus 3Cpro (1.65-7.3 μM) but higher than that of human rhinovirus 3Cpro (5 nM) (Tan et al., 2016;Wang et al., 2011;Dragovich et al., 1999)." I would avoid providing citations in the results section, this relates more to the discussion.
Authors: This sentence has been moved to the Discussion section in the revised manuscript (line 228-231).
Predicted structure of DWV 3Cpro by Alphafold2 "We obtained multiple models that are very similar to each other": this is a bit vague, please provide details or remove.
Authors: This has been removed in the revised manuscript (line 128-129).

Discussion
Before starting with the details, I suggest starting this section by summarizing the aim and major findings of the study in a few sentences.
Authors: These are already explained in the Abstract and Introduction sections so that we think it is better to avoid repeating them in the Discussion section.
In general, the discussion and results parts are very similar. Citations are provided in the results, and the discussion is providing many details. I miss the bigger picture in the discussion, why was this study conducted and what are the major findings. For instance, as the introduction is focused on honey bee health, I would expect more details about how the findings can help improving their health.
Authors: We have added the following sentences, "Rupintrivir and quercetin (Wu et al., 2021) as well as ebselen inhibited DWV replication in cultured honey bee cells. Thus, these compounds and the other 3C pro inhibitors we identified in this study could be used to control DWV in honey bee colony." in the revised manuscript (line 239-242).

Methods
This section should provide more details about the origin of the samples and viruses analyzed. For instance, where do the viruses used come from for the "Expression and purification" part?
Authors: We did not use DWV for the experiments described in "Expression and purification of GST-DWV 3C pro " section. This explains how a plasmid DNA to express GST-DWV 3C pro in E. coli was constructed and how the protein was purified. However, as pointed out by the reviewer, we used DWV for the experiments described in "Infection of honey bee pupal head cells with DWV" section. Thus, this section has been modified with the origin of DWV we used in the revised manuscript (line 356-357).

Supplementary
Do you plan to submit the relevant information on public repositories?
Authors: Since we did not sequence any novel DNA/RNA sequences, the Supplementary information will not be deposited to public data base. Thank you for your responses and revisions. The reviewers have more questions that need to be addressed in the revised version.
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This study experimentally demonstrated proteolytic activity of the proposed 3C protease of DWV, model its 3D structure, and identified catalytic amino acid in the active site. Be more specific how this information "may help to control DWV" -e.e. by developing specific inhibitors of DWV 3C protease activity?
Reviewer #3 (Comments for the Author): The author should indicate that "the specific inhibitors of DWV 3Cpro could be used to control DWV infection in honey bees" is a prospect, but the statement in the manuscript seems it is a confirmed result. Importance: I think the objectives "To understand the mechanism of DWV replication in the host cells," seems indistinct.
Introduction: PAGE3 LINE83: I think it is better to use the reason why you focus on the catalytic triad and enzymatic properties of DWV 3CPro as the intro of the statement of your work. PAGE3 LINE86: "the structure-function relationship", the "-" is in an inconsistent format.
Results-Inhibitors of DWV 3Cpro

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The author should indicate that "the specific inhibitors of DWV 3Cpro could be used to control DWV infection in honey bees" is a prospect, but the statement in the manuscript seems it is a confirmed result. Importance: I think the objectives "To understand the mechanism of DWV replication in the host cells," seems indistinct.
Introduction: PAGE3 LINE83: I think it is better to use the reason why you focus on the catalytic triad and enzymatic properties of DWV 3C Pro as the intro of the statement of your work. PAGE3 LINE86: "the structure-function relationship", the "-" is in an inconsistent format. Page5 line157 & Page6 line173: "the structure of DWV 3Cpro predicted by AlphaFold2 is correct"-this statement seems overgeneralizing.