Principles of RNA recruitment to viral ribonucleoprotein condensates in a segmented dsRNA virus
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Evaluation Summary:
This study will be of interest to virologists and those working on RNA-protein particles. Strauss and colleagues studied the mechanism of RNA recruitment to ribonucleoprotein condensates using rotavirus. Using multiplexed DNA-barcorded smFISH and DNA-PAINT for direct visualization of the RNP condensates in cells, they observe the early onset of viral transcript oligomerization before the formation of viroplasms and the process of enrichment in RNP condensates. They were able to image all eleven transcripts in an RNP condensate and to quantify the amounts of these transcripts. Based on these findings, the authors suggest a selective RNA enrichment mechanism of rotavirus. The experiments are nicely executed, with good controls.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Rotaviruses transcribe 11 distinct RNAs that must be co-packaged prior to their replication to make an infectious virion. During infection, nontranslating rotavirus transcripts accumulate in cytoplasmic protein-RNA granules known as viroplasms that support segmented genome assembly and replication via a poorly understood mechanism. Here, we analysed the RV transcriptome by combining DNA-barcoded smFISH of rotavirus-infected cells. Rotavirus RNA stoichiometry in viroplasms appears to be distinct from the cytoplasmic transcript distribution, with the largest transcript being the most enriched in viroplasms, suggesting a selective RNA enrichment mechanism. While all 11 types of transcripts accumulate in viroplasms, their stoichiometry significantly varied between individual viroplasms. Accumulation of transcripts requires the presence of 3’ untranslated terminal regions and viroplasmic localisation of the viral polymerase VP1, consistent with the observed lack of polyadenylated transcripts in viroplasms. Our observations reveal similarities between viroplasms and other cytoplasmic RNP granules and identify viroplasmic proteins as drivers of viral RNA assembly during viroplasm formation.
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Evaluation Summary:
This study will be of interest to virologists and those working on RNA-protein particles. Strauss and colleagues studied the mechanism of RNA recruitment to ribonucleoprotein condensates using rotavirus. Using multiplexed DNA-barcorded smFISH and DNA-PAINT for direct visualization of the RNP condensates in cells, they observe the early onset of viral transcript oligomerization before the formation of viroplasms and the process of enrichment in RNP condensates. They were able to image all eleven transcripts in an RNP condensate and to quantify the amounts of these transcripts. Based on these findings, the authors suggest a selective RNA enrichment mechanism of rotavirus. The experiments are nicely executed, with good controls.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers …
Evaluation Summary:
This study will be of interest to virologists and those working on RNA-protein particles. Strauss and colleagues studied the mechanism of RNA recruitment to ribonucleoprotein condensates using rotavirus. Using multiplexed DNA-barcorded smFISH and DNA-PAINT for direct visualization of the RNP condensates in cells, they observe the early onset of viral transcript oligomerization before the formation of viroplasms and the process of enrichment in RNP condensates. They were able to image all eleven transcripts in an RNP condensate and to quantify the amounts of these transcripts. Based on these findings, the authors suggest a selective RNA enrichment mechanism of rotavirus. The experiments are nicely executed, with good controls.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this paper, Strauss et al. examined the molecular identity of the viroplasms that form in rotavirus infected cells. They demonstrate using smFISH and DNA-PAINT that rotavirus RNA transcripts localize to viroplasms, and that these RNA aggregates begin forming around 4 hours post infection. They use a combinatorial method, universal DNA exchange with smFISH, to visualize all eleven unique RNA transcripts in viroplasms. They use RNA-seq and smFISH analyses to determine that RV RNA transcripts comprise 17% of all coding transcripts in an infected cell, but that transcripts are not present in stoichiometric amounts. Finally, they construct a virus carrying a seg9-EGFP fusion gene with UTRs to explore the role of the 3' UTRs in transcript localization to viroplasms.
The main conclusion that most viroplasms …
Reviewer #1 (Public Review):
In this paper, Strauss et al. examined the molecular identity of the viroplasms that form in rotavirus infected cells. They demonstrate using smFISH and DNA-PAINT that rotavirus RNA transcripts localize to viroplasms, and that these RNA aggregates begin forming around 4 hours post infection. They use a combinatorial method, universal DNA exchange with smFISH, to visualize all eleven unique RNA transcripts in viroplasms. They use RNA-seq and smFISH analyses to determine that RV RNA transcripts comprise 17% of all coding transcripts in an infected cell, but that transcripts are not present in stoichiometric amounts. Finally, they construct a virus carrying a seg9-EGFP fusion gene with UTRs to explore the role of the 3' UTRs in transcript localization to viroplasms.
The main conclusion that most viroplasms contain all 11 segments of the virus is well supported, although it is somewhat expected. The work would be improved by more detailed quantification of the viroplasms examined. Specifically, is the differential stoichiometry of each segment in viroplasms true in every viroplasm in the same manner (perhaps reflecting a fundamental difference in partition mechanisms), or is there variation between individual assemblies?
An interesting conclusion of the work is that Nsp2 is required for formation of the viroplasms, however, this knockdown also reduces the levels of various viral RNAs. Thus, it remains unclear, from the work in this manuscript, if Nsp2 plays a structural role in forming viroplasms, or if Nsp2 is required for efficient expression of sufficient vRNAs to drive viroplasm assembly.
Another intriguing observation is that a viral RNA expressed from the virus efficiently assembles into viroplasms, while a viral RNA expressed from a nuclear encoded gene with a poly(A) tail is less efficiently recruited into viroplasms. This observation is used to argue that the 3' UTR drives the specificity of partitioning into viroplasms. However, this experiment should be interpreted carefully given the absence of quantification of the RNA partitioning into viroplasms, the differences in the coding sequences of the two RNAs, and the fact that one is produced in the nucleus, which could lead to additional differences (e.g. m6A modifications), and one is a cytoplasmically synthesized RNA. Improving the quantification and interpretation of this experiment will improve the manuscript.
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Reviewer #2 (Public Review):
Strauss et al. studied the mechanism of RNA recruitment to ribonucleoprotein condenstates using rotavirus. They used multiplexed DNA-barcorded smFISH and DNA-PAINT for direct visualization of the RNP condensates in cells. They observed the early onset of viral transcript oligomerization before the formation of viroplasms and the process of enrichment in RNP condensates. They imaged all eleven transcript in a RNP condensate and quantified the amount of the transcripts. Based on these findings, they suggested a selective RNA enrichment mechanism of rotavirus. The authors conducted well the experiments with good control measurement. The results look significant enough for understanding the RNA recruitment to RNP condensates and provide a potential usefulness of their approach in future work.
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