PREreview of A SUMO interacting motif in the Replication initiator protein of Tomato yellow leaf curl virus is required for viral replication

by Andrés Posbeyikian, Hsuan Pai, Benjamin Seager, and KODAI HONDA of the TSL Preprint Club

Published: August 28, 2024
DOI: 10.5281/zenodo.13392457
License: CC BY 4.0

Summary

This article by Gaertner et al. investigates the role of a previously uncharacterized SUMO interacting motif (SIM) within the Replication initiator protein (Rep) of Tomato yellow leaf curl virus (TYLCV), which is essential for viral replication. The study explores the molecular interaction between Rep and the host SUMO machinery, which facilitates viral genome replication through rolling-circle replication (RCR). By focusing on a non-canonical SIM within the SF3 helicase domain of Rep, the authors examine its impact on viral replication, ATPase activity, and the recruitment of SUMO proteins.

Their data revealed that the SIM is necessary for the interaction between Rep and SUMO1, as well as with the SUMO-conjugating enzyme (SCE1). Disruption of the SIM via mutation resulted in the loss of these interactions and severely impaired viral replication. Notably, this motif also influenced Rep’s ATPase activity, which is crucial for RCR elongation. The researchers observed that this motif is conserved across different geminiviruses, indicating a broader functional relevance in the viral replication process.

The study proposes that this SUMO interacting motif enables Rep to manipulate host SUMOylation pathways to enhance viral DNA replication, thereby establishing a novel connection between post-translational modifications and viral replication. These findings significantly expand the current understanding of geminivirus-host interactions and provide a potential new target for antiviral interventions aimed at disrupting viral replication.

General comments

The manuscript is concise and generally very well-written. However, in some cases, some words such as “hitherto” and “thereto” felt slightly out of place and overused.
The figures are clear and well-constructed, contributing to the overall readability of the paper.
One point of inconsistency is the use of different sets of mutants for various experiments. It would be helpful to clarify whether all mutants were tested across all assays, even if the data is not shown, to ensure a comprehensive understanding of the experimental outcomes.
It would be really beneficial if the authors provided a visual model summarizing their findings.

Introduction

The introduction is generally well written and provides the reader with a solid overview of rolling-circle replication, geminiviral Rep proteins, and their involvement with SUMOylation. It effectively introduces the necessary concepts for the focus on the Rep-SUMO interaction.
The transition to the section discussing SUMOylation (L82) could be smoother, as it feels a bit abrupt.
It would have been beneficial to include a sentence pointing out the relevance of the HUH domain for the interaction with the SUMO machinery earlier in the introduction, rather than leaving this to be inferred later when Figure 2 is presented.

Results and Discussion

Figure 1

Figure 1.B. There is a red circle with an S inscription which likely represents SUMO1 but it could be explicitly stated in the figure legend.
Figure 1.F It would be good if the authors could report the error metrics for the AF2 model they are showing here, be it coloring the model by pLDDT or showing its PAE plot. The Sumo Interacting Motif (SIM) is on a β-sheet and mutating it might affect the packing of the neighboring beta-sheets or otherwise have an effect on the overall structure, thus having effects beyond SUMO-interaction disturbance. This point isn’t really addressed in the discussion.

Figure 2

All in all very clear data. The BiFC data is coinciding with the Y2H findings. There appears to be a typo in the legends of figures C and D to the left and at the top, where SCPF should read SCFP. Otherwise very clear.

Figure 3

This figure looks very consistent. The schematics in Figure.3A. are very clear and greatly appreciated. In Figure 3.C. it looks like the I217 mutation (which was detrimental for SUMO1 and SCE1 interaction in the Y2H data shown in Fig.2.A-B) is having a significant decrease in Rep activity (as seen by the significant reduction in RFP expression in Fig.3.C,E), however it still promotes EGFP expression (as seen in Fig.3.D,F). Do the authors have an idea why this might be the case? It doesn’t seem to be the case with the double mutants I215/I217A and V216/I217A.
The bar chart shown in subplot G. would be more informative if it included the data points overlaid. Otherwise very clear. Same comment applies to other bar charts.

Figure 4

It would be good to know if the single mutants shown in previous experiments have been tested in the context of the viral infection assays. L221-222 showed strong conclusions on the mutant line Ile217, but the single mutation was not tested in the context of viral infection.
Could these SIM mutations be tested in the Rep of another virus, like the TGMV Rep mentioned in the introduction? Why is it so conserved across viruses (Fig.1.F)?

Figure 5

It would be great if some negative controls were included in the Relative ATPase activity assay, especially the single alanine mutations in positions 215 or 216. This bar chart has no error bars, suggesting it’s a single replicate.
For the size exclusion chromatography shown in subpanel B, it’s also not clear if there are replicates. The manuscript mentions that SDS-PAGE gels were ran, but it would be great to see them in the supplementary figures. It’s difficult to tell if the samples were loaded equally without that data, and it’s complicated to conclude unless the loading is controlled. An alternative method for studying this could be native PAGE.
The heading of this figure in the full length PDF document states “The SIM is required for ATPase activity and its mutation does not impact oligomerization status ” whilst the in text heading for these data states “Mutating the SIM impairs Rep ATPase activity and oligomerization”. These two headings seem contradictory and it appears there is some confusion about the interpretation of the size-exclusion chromatography data presented. The authors should be clear and consistent with their interpretations so as to not confuse readers.

Supplementary Figure 1.E.F.

This supplementary figure is only showing experiments done on the single mutants, and there seems to be an inconsistency with the mutants selected for different experiments.