PREreview of Nuclear body reorganization by the viral RNAkaposinpromotes Kaposi’s sarcoma-associated herpesvirus gene expression

We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: 

Nuclear body reorganization by the viral RNA kaposin promotes Kaposi’s sarcoma-associated herpesvirus gene expression

Mariel Kleer, Michael J. Johnston, Jennifer A. Corcoran

bioRxiv 2024.09.20.614208; doi: https://doi.org/10.1101/2024.09.20.614208

We will adhere to the Universal Principled (UP) Review guidelines proposed in: 

Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029 

SUMMARY: Kleer and Corcoran previously generated a collection of KSHV recombinant viruses with alterations in the multi-cistronic kaposin locus to understand the relative contributions of different protein products to key phenotypes. A major conclusion of these studies was that re-coding the kaposin RNA to reduce its GC-rich and repetitive nature without changing the protein products decreased viral fitness. This led them to hypothesize that kaposin RNA has an important role to play in supporting KSHV infection. Here, using RNA-FISH, they demonstrate that kaposin RNA accumulates in nuclear foci following reactivation from latency in epithelial and B lymphocyte infection models. By probing known protein markers of nuclear bodies, they demonstrate that these kaposin foci are nuclear speckles (NSs), rather than paraspeckles, Cajal bodies, or PML bodies. As lytic replication progresses, these kaposin NS foci change, becoming fewer and larger and more round, with substantially reduced levels of the MALAT1 ncRNA present as kaposin levels increase, suggesting that kaposin is displacing MALAT1 from its niche. A kaposin ectopic expression model confirmed that kaposin localizes to NSs and alters their character, making them larger and more numerous, and once again, MALAT1 was displaced. Employing a collection of kaposin mutant viruses and kaposin expression constructs, they demonstrate that kaposin proteins are dispensable for the kaposin NS localization phenotype, whereas the central DR1 repeats are essential; this is the first function ascribed to the DR1 repeats. Transcriptome analysis of kaposin-deficient virus infections demonstrated its broad role in supporting lytic viral gene expression. By contrast, kaposin deficiency alters latent gene expression by increasing or decreasing gene expression in a position-specific manner, suggestive of topologically associated domains (TADs) normally regulated by the CTCF chromatin insulator. However, CHIP experiments suggest that CTCF deposition remains unchanged in kaposin mutant virus infections. NSs have been shown to regulate local transcription and mRNA processing outcomes. Kaposin deficient viruses displayed alterations in host pre-mRNA splicing, with reduced usage of alternative 5’-splice sites, increased usage of alternative transcription termination sites, and increased intron retention; however, no clear consensus emerged across the mutant virus collection. Some alterations in KSHV pre-mRNA splicing were observed as well. Using a de novo endothelial cell infection model, they demonstrated that kaposin is required for efficient production of many viral mRNAs. Finally, the authors used a luciferase reporter assay to determine that kaposin’s contribution to transcriptional output operates in cis and cannot boost transcription in trans when supplied on a separate plasmid. This finding is consistent with the positional effects of NSs in supporting transcription and post-transcriptional processing in the nucleus.  

OVERALL ASSESSMENT: This manuscript identifies a new viral ncRNA with a novel role as an architectural RNA that supports the formation of NSs and thereby provides positional control of transcription and mRNA processing, supporting viral gene expression and altering host gene expression. Generally, the central claims of the manuscript are well supported by the data, and we appreciated the high-quality data presentation and quantitation, as described below. While the reviewers appreciated this thorough investigation of kaposin function, the primary weakness of this manuscript was in the storytelling; the methodical approach to guiding the reader through a large dataset, toggling between main and supplementary figures and discussing each finding at length, undermined the readability of the manuscript. We recommend revising the manuscript to focus on the key novel findings and generate a leaner product to capture the reader’s attention throughout.

STRENGTHS: The major claims are well supported by an ample dataset. From the abstract: “Kaposin acts in cis as an architectural RNA to rebuild cellular nuclear speckles (NSs) to sites proximal to the viral genome to optimize viral gene expression. We show kaposin RNA is both necessary and sufficient for NS remodeling, and ablating kaposin colocalization with NSs using kaposin-deficient recombinant viruses impairs viral gene expression”. The authors clearly demonstrate that kaposin operates in cis and not in trans. They demonstrate that it is sufficient to remodel NSs in the absence of infection, and kaposin-deficient viruses fail to do so, providing a rigorous proof. The absence of kaposin results in dysregulated latent and lytic viral gene expression and poor replication, and they demonstrate that the genome is positioned nearby these NSs, so it seems logical that kaposin plays a role in optimizing viral gene expression.

WEAKNESSES: The manuscript reports on a very thorough investigation of novel RNA biology in KSHV infected cells, but the overlong text undermines readability. A leaner approach to writing that puts more emphasis on the main conclusions and their novelty would help get the message across to readers. At the same time, providing more information about nuclear speckle biology from the outset would help readers appreciate the novelty and importance of these findings. Some conclusions require additional support, including Fig. 4, where they claim increased cytoplasmic localization of kaposin RNA in the absence of SRRM2, but without a nuclear stain, this remains somewhat inconclusive.

DETAILED U.P. ASSESSMENT: 

OBJECTIVE CRITERIA (QUALITY) 

1.   Quality: Experiments  

·     Figure by figure, do experiments, as performed, have the proper controls? [note: we use this ‘figure-by-figure' section for broader detailed critiques, rather than only focusing on controls]

·       Fig. 1: Overall, the experiments described in Figure 1 are well controlled and easily interpreted. This figure prompted a group discussion about the kinetics of KSHV lytic replication. The authors use a 48 h post-dox timepoint to monitor kaposin localization in epithelial cells and a 24 h post-dox timepoint in B lymphocytes, but do not provide rationale for these choices. Some additional description of the kinetics of a full KSHV lytic replication cycle in these two infection models should be sufficient to provide rationale and address this question (when do genome replication and virion release occur? Some of this information may be found in their previous J. Virology paper). If the authors have images from other stages of lytic replication, that would be helpful as well, but not necessary to address concerns.

·       Fig. 2: These RNA-FISH experiments are properly controlled, and readers appreciated the clear presentation of quantitative data that accompanied representative images.

·       Fig. 3: These experiments nicely demonstrate dose-dependent effects of kaposin RNA on NS characteristics, supporting the idea of NS remodeling, and once again, the quantitative data is well presented. Our only critique is that the representative images (e.g. Fig. 3A, 4000 ng kaposin) do not always reflect the findings of the quantitative data. Could the authors select images that more accurately reflect the median observation?

·       Fig. 4: Figures 4A-D, describing the mapping of determinants of kaposin localization to NSs, are very well presented and described. No notes. By contrast, the important experiment investigating kaposin nuclear retention in Figs 4E-G is a little underdeveloped. We thought that the reader would benefit from seeing controls presented in the main figures without the necessity of consulting the supplement, to show the reader that the fractionation was well controlled and successful. Furthermore, the authors claim that kaposin accumulates in the cytoplasm in Fig. 4G is difficult to verify, as there is no nuclear stain like DAPI included in this experiment. Altogether, we think that this important experiment deserves a more exposition and demonstration of controls, and likely deserves its own standalone figure rather than being an add-on to Figs 4A-D.   

·       Fig. 5: This figure nicely describes the findings of transcriptome studies. One of the important findings of Fig. 5A was that in the lytic cycle, kaposin deficiency had an impact on the expression of host mRNA processing genes. This finding could be better highlighted in the text. Figs. 5B and 5D do a good job of presenting the effects of kaposin deficiency on KSHV latent and lytic gene expression, respectively, providing the curious finding of positional dysregulation of gene expression. The one aspect of this figure that is a bit underdeveloped is Fig. 5C, which shows the results of CHIP experiments, and no significant effect of kaposin deficiency on CTCF binding at several loci. This experiment would benefit from additional controls – could CTCF function be altered by chemical or genetic interference in these experiments? This would help provide confidence in the findings and help the reader gauge how well the assay reports on CTCF biology. Overall, we thought that this experiment was not sufficient to conclusively rule out chromatin insulation in the mechanism of kaposin dependent alterations in latent viral gene expression.

·       Fig. 6: For the most part, these experiments appear to be properly controlled. The readers really appreciated the high-quality data presentation in Fig. 6B, which nicely demonstrated which alterations in splicing patterns were significant across a very complex dataset. We acknowledge that the experiments performed in Figs. 6C and 6D are challenging, as it can be difficult to capture pre-cursors or intermediates for rapidly processed viral mRNAs. However, once again here, additional controls would strengthen this data. Could a pre-mRNA splicing inhibitor be used as a control to demonstrate the integrity of these experiments?  

·       Fig. 7: This figure reports on two quite distinct experiments, which should be presented in separate figures to ensure the reader fully appreciates the remarkable findings in Figs. 7F/G. Figs. 7A-E provide ample support for the Figure Title “Kaposin-mediated NS seeding alters viral transcription after primary infection”. The data is clearly presented and nicely quantitated in Figs. 7D-E. However, this figure title does not address the most important finding, found in Figs. 7F/G, that kaposin supports robust viral gene expression in cis, not in trans. As mentioned previously, giving Figs. 7F/G their own figure would help the reader appreciate this clever approach and important finding. With some extra space, these analyses could easily be expanded to include other kaposin mutant expression vectors described in this study.

Are specific analyses performed using methods that are consistent with answering the specific question?  

·     Is there appropriate technical expertise in the collection and analysis of data presented?

·   Yes

·     Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?  

·   We really appreciated the use of the panel of kaposin-deficient viruses and expression constructs as a clever and rigorous approach to map determinants of kaposin function. We also appreciated the use of IF-FISH as a robust method, as well as the use of different targets to mark nuclear speckles (NEAT1, SON, SRRM2).

·   When describing how their CellProfiler pipeline was used to quantify nuclear speckle data, the authors stated that “any cell that did not stain positive for kaposin was removed or “masked” from the remainder of the analysis.” This would be to exclude cells that did not respond to dox-induced lytic reactivation of KSHV. Would the result be the same if they used a different lytic cycle marker? Is it possible to have a cell that enters lytic replication that displays little or no kaposin staining?

·     Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship.  

·   Controls and experimental foundations are well within norms for this field.

2.   Quality: Completeness  

·     Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. 

·   The major title- and abstract-level conclusions are: (1) kaposin acts in cis to “rebuild” NSs (2) kaposin is necessary & sufficient for NS remodeling (3) preventing colocalization of kaposin & NSs has an impact on viral gene expression. We carefully considered these conclusions and agreed that the data provided good support for these conclusions.  

·     Are there experiments or analyses that have not been performed but if ‘‘true’’ would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ‘‘completeness’’ here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. 

·   Further investigation of certain aspects of the study may change some conclusions (e.g. the relatively underdeveloped investigation of positional effects of kaposin on latent viral gene expression), but all major conclusions are very well supported by the data.

3. Quality: Reproducibility  

·     Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?

·   For Figure 1, the number of replicates is not clearly shown in the Figure Legend. For Figure 3E, only 2 biological replicates were performed, so a 3^rd should be completed. 

·     Is there sufficient raw data presented to assess the rigor of the analysis? 

·   Yes.

·     Are methods for experimentation and analysis adequately outlined to permit reproducibility? 

·   Yes. The methods are sufficient.

·     If a ‘‘discovery’ dataset is used, has a ‘‘validation’ cohort been assessed and/or has the issue of false discovery been addressed?  

·   False discovery rate was clearly indicated in the mRNA processing data in Fig. 6.

4. Quality: Scholarship  

·     Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? 

·     N/A. This is the first report of a ncRNA function for kaposin, so there are no other relevant studies to cite. This speaks to the novelty of their findings.  

·     Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? 

·     We think that the reader would benefit from more relevant background information about NSs (function, composition, etc.) to provide a better foundation for understanding the significance of kaposin localization of these structures and subsequent remodeling.

·     Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not be significant basis for decisions.

·     The manuscript would benefit from some restructuring. The methodical approach to guiding the reader through a large dataset, toggling between main and supplementary figures and discussing each finding at length, undermined the readability of the manuscript. We recommend revising the manuscript to focus on the key novel findings and generate a leaner product to capture the reader’s attention throughout. Also, as a rule of thumb, if the Results section has a paragraph that only has callouts to supplemental data, then at least some of that data should be in the main text.

·     There was good agreement that the reader would benefit from more information about NSs and architectural RNAs (a new term for this audience) in the Introduction. Similarly, if there is relevant information to share about the functions of MALAT1 or SRRM2 or SON in the context of cell biology or virus infection, that would be helpful for the reader. Could MALAT1 displacement contribute to the differences in mRNA processing observed in Fig. 6? (minor: we really appreciated the small figure describing alternative mRNA processing outcomes – clear and concise).

·     Some unclear naming conventions, i.e. “kaposin-recombinant” –we think it would be clearer to say something like “kaposin-deficient”. Likewise, there were several words used to describe the changes in NSs: “rebuild, seed, scaffold, remodel”. It would be wise to decide on a single term and stick with it. After some debate, we settled on ‘remodel’. Minor: be consistent in italicization of kaposin when referring to the RNA.

·     The graphical abstract might be difficult to understand for a non-expert and should be revised for further clarity. Minor: Subtly different colour combinations should be avoided.

·     Could the Discussion provide some clues about the next logical steps in investigating loose threads, like the positional effect of kaposin on latent viral gene expression?

MORE SUBJECTIVE CRITERIA (IMPACT): 

Impact: Novelty/Fundamental and Broad Interest  

How big of an advance would you consider the findings to be if fully supported but not extended? Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ‘‘repeat a mouse-based experiment in humans’’) and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2)

·       This manuscript presents important novel findings about a newly discovered viral ncRNA. The authors mention that similar ncRNAs are present in other herpesvirus genomes, and extending their findings to investigate the localization of these other viral ncRNAs and their effects on NS remodeling in ectopic expression models or infection models would certainly be an attractive way to extend their findings.

Competing interests

The authors declare that they have no competing interests.