PREreview of Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome

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: 

Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome 

Jordan N Ranum, Mitchell P Ledwith, Fadi G Alnaji, Meghan Diefenbacher, Richard Orton, Elisabeth Sloan, Melissa Guereca, Elizabeth M Feltman, Katherine Smollett, Ana da Silva Filipe, Michaela Conley, Alistair B Russell, Christopher B Brooke, Edward Hutchinson, Andrew Mehle

doi: https://doi.org/10.1101/2023.12.12.570638

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: Most viral RNA-dependent RNA polymerase (RdRp) enzymes are error-prone, generating a variety of products that could be deleterious or help viruses adapt to changing environments. In addition to single-nucleotide substitutions, many RNA viruses also produce deletion-containing viral genomes (delVGs) with large internal deletions. Studies to date indicate that these products are usually unhelpful for RNA viruses as they interfere with efficient viral genome replication and can compete with full-length genomes for incorporation into nascent viral particles. They can also stimulate antiviral responses and have been associated with better clinical outcomes. Here, Andy Mehle’s team report that mRNAs derived from influenza virus DelVGs can be translated into cryptic proteins called DelVG-encoded proteins (DPRs). These DPRs can be canonical viral proteins with large deletions, or with novel carboxy-termini due to shifted reading frames. They created reporter viruses with engineered genome segments encoding carboxy-terminal V5 tags in three reading frames to corroborate the existence of these cryptic proteins. PB2 was selected for additional mechanistic investigation because PB2-derived DelVGs had a variety of large deletions, but all were predicted to retain the ability to bind to the PB1 RdRp subunit, as this function maps to the extreme amino-terminus of PB2. A series of experiments demonstrated that DPRs encoded by PB2-derived DelVGs bind and inhibit RdRp and interfere with viral replication. 

OVERALL ASSESSMENT: Recent technical advances in genomics and proteomics have provided new opportunities to discover cryptic viral proteins. This study provides a great example of how these technologies can be used for cryptic viral protein discovery and provides readers with a new appreciation for the diversity of viral protein products. The authors took the necessary steps to engineer mutant viruses with epitope tags in 3 reading frames that allow for confirmation of production of DPRs during infection. Importantly, they also investigated the function of select DPRs, showing that DPRs generated from the PB2 genome segment bind PB1 as expected and moderately inhibit RdRp activity. This investigation of DPR function is important to demonstrate the relevance and impact of the discovery of DPRs. Here, we provide the authors with feedback to make the manuscript more accessible to a broad audience, and suggestions for future mechanistic investigations.  

STRENGTHS: The authors’ claim that DelVGs encode cryptic proteins is very well supported by the data, and the research approach provides a comprehensive assessment of the protein-coding potential of DelVGs. For the most part, this comprehensive picture is properly conveyed in an attractive figure set and accompanying text. The engineering of mutant viruses to enable creation of epitope tagged DPRs is a clever approach to corroborate mass spectrometry data. Over the past decade several studies that have employed Ribo-Seq to identify cryptic viral proteins, but mechanistic studies of these products have lagged. Thus, the in-depth mechanistic studies of PB2 segment derived DPRs that inhibit RdRp function are quite welcome and informative. 

WEAKNESSES: While binding of PB2-derived DPRs to PB1 is convincingly demonstrated, the competitive binding relative to WT PB2, and overall impact on RdRp activity, are relatively modest effects. It is possible that these inhibitory effects of DPRs must be modest to be tolerated by the virus.

DETAILED U.P. ASSESSMENT: 

OBJECTIVE CRITERIA (QUALITY) 

1.   Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 2

·     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: This data is compelling and supports the authors’ conclusions regarding the transcription (via amplicon-based sequencing) and translation (via Ribo-Seq) of DelVGs. The primary challenge for our student reviewers, none of whom work on influenza viruses, was initial unfamiliarity with the concept of DelVGs and parallel coordinate mapping to visualize sequences from discontinuous templates. We suggest that the data in Figure 1 could be made more accessible to a general audience if authors include a cartoon displaying general features of the PB2 genome segment (UTRs, canonical ORF, packaging signals) and general features of the PB2 protein product (including amino-terminal PB1 binding motif and NLS), to help readers understand what features are typically retained in DPRs and what features might be lost. 

o    Minor point: we noted in the related supplemental data that the majority of PB2-derived DPRs lack the canonical NLS. What might this say about which DPRs are more important for interference with RdRp function? Does a PB2 DPR need an NLS to assemble with polymerase complexes? Do polymerases normally assemble in the cytoplasm and are imported into the nucleus intact? Some additional information about trafficking and assembly of polymerase subunits would be helpful for the readers. 

o    Minor point: The lack of DelVGs from the HA genome segment was noted in the Figure Legend and main text. This was initially intriguing (e.g. is there a mechanism to exclude HA-derived DelVGs from viral particles?), but then the readers came to understand that HA genome segments CAN make DelVGs and DPRs in later experiments. Confusion could be mitigated in main text by explaining to the reader that they can expect to see HA-derived DelVGs later in the dataset. 

·       Fig. 2: We found this data compelling, with the mapping of DPRs identified by mass spec to the appropriate parental DelVG and information about relative abundance of that DelVGs. This was another situation where we were initially unfamiliar with this kind of heatmap, but eventually were able to understand the information being presented. 

·       Fig. 3: We appreciated the clever approach to viral genome engineering to demonstrate DPR accumulation. The cartoon in Fig. 3A could be a little clearer if the zone that encompasses all DelVG internal deletions were included as well, so the reader could easily appreciate the placement of the 3’-junction zone. Harmonizing annotation of the western blots could make it more clear to the reader which of the 3 bands marked with arrows on the IP-anti-V5 western blot are supposed to match the 2 bands marked with asterisks on the anti-PB2 whole cell lysate western blot (or the WBs could be re-run to make them more similar to facilitate direct comparisons). Also, it would be helpful to better understand the limitations of this western blotting analysis; what epitopes are recognized by the PB2 antibody and are they absent from some of the DPRs? Would the banding pattern be the same if the anti-V5 immunoprecipitates were probed with anti-PB2 antibody instead of anti-V5 antibody? 

·       Fig. 4: We appreciated the importance of testing the function of individual PB2-derived DelVGs. The first experiment in Figure 4B shows a moderate but significant inhibitory effect in a standard replicon assay; the authors are careful to state that these results are significant, but they do not overstate the magnitude of the effect. Appropriate controls are in place, although the Y-axis should display units rather than just ‘polymerase activity’, and the data for each PB2-derived DelVG should be normalized to the negative control as well as Renilla luciferase. 

o    Fig 4C also needs a more informative y-axis label with units, and is missing a negative control and normalization to that negative control. Inclusion of a negative control would allow the reader to better appreciate how ‘broken’ the PB2 416/2189 ‘no stop’ construct is, and the relative contribution of protein to RNA to the inhibitory effect. The western blot is not very informative in its compressed state, as it doesn’t convey molecular weight of the protein product or provide any insight into residual bands in the ‘stop’ construct. Overall, Fig. 4C might be improved by providing a supporting cartoon that displays the construct and introduced stop codons, and it also might be easier to interpret if it were set apart from Fig. 4B, so the reader does not try to make direct comparisons between 4B and 4C, which are quite different experiments with different y-axis scales. The western blot needs annotation. 

·       Fig. 5: This figure is generally convincing, but the dose-dependent inhibition of the PA-PB2 co-IP, reflecting polymerase assembly, is relatively subtle. This conclusion might be strengthened using another DPR derived from PB1 or PA subunit that would be predicted to similarly compete with WT subunits and inhibit polymerase assembly.  

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, appropriate technical expertise is demonstrated throughout. 

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

·   Replicon luciferase data could have been normalized to negative control, in addition to the Renilla transfection control. 

·   Minor suggestion: The amplion sequencing had a quality filtering step, but the direct RNA sequencing did not. Direct RNA sequencing was done with an Oxford Nanopore Minion. Considering the inherently higher error rates of Minion sequencing, a tool such as filtlong (https://github.com/rrwick/Filtlong), skipping the size filtering or filtering to 90 nucleotides which was subsequently done, could have improved the accuracy of each base call which could have improved the confidence of deletion site identification. 

·     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.  

·   Generally strong throughout the manuscript. 

2.   Quality: Completeness (1–3 scale) SCORE = 1.5

·     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 conclusion from the title “Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome” is certainly very well supported. Interestingly, the title doesn’t say anything about DPR function, which turns out to be a major component of the dataset, even if it is less well developed and supported than the primary conclusion.

o    Minor point: our student group read the abstract and introduction and were anticipating that the dataset would include some investigation of DelVG-induced immune responses. Later in the manuscript, some students were confused about the model in Figure 5D that talked about ‘non-productive replication’ driven by DelVGs, which is not really addressed by the dataset and instead relates to established data in the field. We think that some of these reader expectations could be managed in an expanded Introduction, to make the scope of the current study clear and provide more context about the many past studies of influenza virus DelVGs that firmly established the prior model. 

·     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. 

·   N/A

3. Quality: Reproducibility (1–3 scale) SCORE = 1

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

·   Yes. "Experiments were performed in at least biological triplicate, with each including at least three technical replicates” (p. 9).

·     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

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

·   Yes

4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1.5

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

·     Yes. The study is put in proper context and the data is clearly described. 

·     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? 

·     Yes, although the discussion of other studies of cryptic influenza virus proteins comes quite late in the Discussion. Some more coverage of these other studies in the Introduction would be quite welcome and would not undermine the impact of the current study. 

·     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.

·       Overall, the manuscript is very well written. We made some comments on data presentation above in Section 1. We also found: 

·       Two typos in the Abstract: “DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes as they.” “Here, we identify an additionally inhibitory mechanism….” 

·       One typo in the Introduction: “Indeed, the preferential replication of DelVGs is seen in high-throughput sequencing data where they cause they cause an under-representation of the deleted regions in the middle of viral genome segments (15)” 

MORE SUBJECTIVE CRITERIA (IMPACT): 

1.   Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE= 2

A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ‘‘1’’ would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.] 

How big of an advance would you consider the findings to be if fully supported but not extended? 

·       This preprint greatly expands our understanding of the influenza virus proteome with these new cryptic viral proteins, and conservation of DelVGs and DPRs across IAV strains is an important extension of initial findings. 

·       As stated above, the impact of this study could be enhanced by further study of the inhibitory mechanisms of DPRs. 

·       We also think the reader would benefit from some broader discussion or speculation about why these DPRs are tolerated and maintained in the genome despite seemingly deleterious effects. Could there be some advantages to DPR expression that counterbalance the polymerase poisoning phenotype, for example? How might they affect antigen presentation?  

2.   Impact: Extensibility (1–4 or N/A scale) SCORE = 2

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)?  This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn’t apply. 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)

·       Yes, in the sense that the DPRs were shown to be made by diverse IAVs, not just the WSN laboratory strain that was the primary focus of the study. 

Competing interests

The authors declare that they have no competing interests.