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

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/10539235. 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 specifi

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