Peer review of: Selective flexible packaging pathways of the segmented genome of influenza A virus (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
In the manuscript entitled, “Selective, flexible packaging pathways of the segmented genome of 
Influenza A Virus”, the authors utilized sequential FISH staining to visualize all eight influenza viral 
segments within a single infected cell at a single timepoint during infection. In this approach, the 
authors used two dyes per vRNA segment in random order to validate their staining. Importantly, the 
authors imaged each segment twice with FISH probe sets conjugated to two different fluorophores to 
isolate the reproducible intracellular localization of each segment. The overall conclusions of this study 
are that, 1) the most abundant vRNP complexes are those with either 1 segment (rank 1) or 8 
segments (rank 8) while Intermediate ranks are found at lower abundances, 2) higher ranked 
complexes are found more often in intact virions, 3) complexes rarely contain multiple copies of the 
same segment, and 4) disruption of multi-segment complexes (MSC) in human cells infected with an 
avian virus. 
Uncovering the mechanism by which IAV genome segments assemble is crucial to understanding 
reassortment and virion production. Visualizing these vRNA segments by FISH, especially all 8, is a 
powerful tool to study influenza selective assembly. The data presented supports the current model of 
IAV genome assembly where vRNP segments form bundles, the study also supports a newer model 
that the vRNP interaction clusters are flexible in nature as demonstrated with RNA-RNA interaction 
mapping in purified virions (PMID: 31332385). However, the study presented here has the advantage 
of knowing the precise three-dimensional location and composition of each vRNP bundle with in a cell 
yet they do not utilize this information to further refine the vRNP networks at a cellular level. Given 
the recent data demonstrating that heterogeneity in viral infection at a single cell level (PMID 
29451492 and PMID 30181264), the authors should consider whether the multiple bundling routes are 
a consequence of including cells that do not contain all 8 segments into their analysis. Overall the 
visualization of all 8 segments within infected cells and in bound virions is unique and this technique 
serves as an additional strategy to confirm a model of influenza assembly where the vRNP form 
subcomplexes that are flexible as demonstrated by biochemical and recent machine learning 
approaches (PMID: 30689627). 
Overall, the clarity of the study could be improved with attention to the following issues. 
1. The authors should provide experimental validation of the specificity for each probe set against 
other segments as well as any cRNA/vmRNA. Precise probe specificity is critical for accurate 
conclusions to be drawn from these data sets. It is also important to note that the authors state that 
the colocalization algorithm was a custom script but have not publicly shared the code or imaging data 
sets. 
2. How do the networks defined by the authors in Fig 6 compare to the recently published RNA-RNA 
interactions for the Udorn or Wyoming/03/2003 H3N2 virus in PMID: 31332385. In particular, the lack 
of segments 7 and 8 in any of the initial complexes is intriguing and it may be due to the use of “all 
cells” in this analysis rather than cells that had an equal proportion of all 8 segments. Based on the 
data in Fig 3, segment 8 was under represented more often compared to the other segments and that 
may bias the networks constructed in Fig 6. 
3. On page 8, the authors stated that, “we never obtained evidence for more than two copies of the 
same segment in an MSC; less than 5% of the MSCs contained two copies of the same segment”. 
However, volume binning for vRNA spots in this study was 300 x 1000 nm, when they reference that a 
vRNP is 50-150 nm. If this statement was derived from the FISH experiments, it would be impossible 
that single vRNA of a specific segment could be differentiated given the limits of their methods and 
imaging resolution of standard confocal microscopy. This conclusion should be experimentally 
validated using approaches similar to PMID: 22547828 or else do not make such a claim. 
4. The current model of IAV MSC assembly suggests that segments assemble as MSCs translocate 
from the nuclei to the plasma membrane. If so, lower ranked complexes would be observed near the 

nucleus with higher ranked complexes near the plasma membrane. The authors should analyze ranks 
of complexes within various distances from the nuclei and/or plasma membrane. 
5. In Fig. 5C, it appears that the frequency of MSCs containing S1 is greater than S2 and so on 
(frequency of S1>S2>S3>…S8). In virions with MSCs ranked less than 8, is the probability of which 
vRNP segments make it into these particles linked to segment size? In this study, are larger segments 
found more often in lower ranked MSCs compared to smaller segments during infection? This could be 
an artifact of the number of probes capable of binding to a given segment and should be 
acknowledged in the text. 
6. In this study, the authors used a low-pathogenic avian IAV, A/Mallard, to study MSC assembly 
under non-permissive IAV infections. Given that infection with this virus is abortive in human A549 
cells, the rationale for this experimental design is unclear. The replication kinetics of A/Mallard versus 
A/Panama are likely different and thus a 10 hpi cross-section between these infections may not be 
comparable. The complexity of this comparison makes these data difficult to interpret why the pattern 
of vRNP colocalization are different between these two virus infections. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
Haralampiev, Prisner, et al. used a multiplexed FISH assay to investigate all 8 influenza vRNA 
segments in parallel within A549 cells infected with human or avian influenza A virus. These data 
support the prevailing model of influenza virus genome packaging in which each one of eight-influenza 
virus vRNPs are selectively bundled into multi-segment complexes and then subsequently incorporated 
into newly forming virions. 
 
The authors developed significant improvements to an existing approach in order to answer an 
important and interesting question pertinent to the influenza field with relevance to other viruses with 
segmented genomes. The data have the potential to advance current understaning of influenza A virus 
genome assembly, an area of high interest in the field. However, a more rigorous analysis and 
systematic presentation of the data is greatly needed. The current version of the manuscript is unclear 
and poorly organized, to the extent that it is difficult to fully evaluate the results. The authors should 
consider rectifying the following major and minor concerns: 
 
1. The introduction would benefit from a more comprehensive summary regarding several topics: i) if 
you are going to present reassortment as an evolutionary benefit of genome segmentation, you should 
also mention fitness costs of reassortment ii) packaging model—if you are going to mention both 
random vs selective packaging models, you should present evidence for the prevailing model 
(selective) but note the limitations of the previous studies, iii) expand on the limitations of current 
understanding of influenza virus genome packaging mechanism and the signals that direct packaging. 
2. Quantification of observed differences were included throughout the manuscript. For example, avoid 
vague phrasing such as “mostly a rather balanced ratio”, “approximately equal”, “slightly lower than 
average” and “highly similar” and quantify these differences using statistics. 
3. In sub-section 2 of the results section, expand on the rationale for determining vRNA spot 
dimensions and exclusion of nuclear spots. Also reference data to support the statement that <5% of 
MSCs showed 2 copies of the same segment. 
4. An important aspect of this study is the inclusion of human vs avian influenza A strains infecting a 
human cell line. The results would be more meaningful if presented in a way that cohesively and 
linearly presents these data (i.e., the figures should be presented chronologically in the text, e.g., 
figure 2C/D should be interpreted before analyzing Figure 3) with emphasis on comparing/contrasting 
human vs avian influenza A-infected cells. 
5. Figure 3. Presentation of the data that shows the variance among the different cells (with statistical 

analysis) is needed. It is unclear whether row 1 represents an average over all cells (as mentioned in 
the text) or total number of MSCs or segments. Showing averages, with bars including error bars, 
would allow the authors to make meaningful comparisons between ranks rather than referring to the 
u-shaped distribution 
6. Fig. 4. Rather than showing segment counts for each MSC for each cell, it would be more impactful 
to consolidate these data to show major trends (or lack thereof) to more clearly articulate the 
conclusion that there is no dominant MSC composition within each rank. 
7. ‘Reliability of colocalization analysis’ sub-section of the results should be included earlier on in the 
text. These are important controls that should be presented before the data, but do not necessarily 
need a separate section and can be integrated throughout the text. 
8. ‘Implications for IAV genome assembly’ sub-section should be moved to the Discussion/Conclusions 
section of the manuscript. 
9. Please include panel letters on all figures for clarity throughout the figure legends and text. 
10. Figure legends should not contain results or interpretation of the data shown, except for a 
descriptive title if warranted. 
11. Include more descriptive y axis titles for Figure 2A/C (e.g., Cells infected with A/Panama or Cells 
infected with A/Mallard). 
12. Include axis labels for Fig. 5C. If represented on the same graph, scale of y-axis needs to be 
consistent. Purple color for NP vRNA is not distinguishable in image in Panel A. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
The paper by Haralampiev et al. investigates the bundling of the 8 influenza A virus (IAV) genome 
segments by a multiplexed FISH assay. The authors could provide first insight into the cytoplasmic 
segment composition and possible genome complex intermediates. 
 
The paper describes for the first time a robust FISH-based assay to study genome bundling of all IAV 
segments and thus provides an excellent basis for future studies. However, besides developing a 
robust FISH assays, the authors did not really apply their method to address some open questions in 
the field of genome packaging. 
 
Major points: 
 
1) To convincingly demonstrate that the avian IAV used in this study is able to bundle correctly the 
authors should study the vRNP composition also in avian cells. 
 
2) Packaging sequences in the viral genome are required for correct packaging of the 8 different 
genome segments into a virus particle. It is believed that these sequences orchestrate genome 
bundling. Thus, it is not clear why the authors did not investigate a packaging mutant virus. This 
would provide valuable information. 
 
3) There are published data available suggesting that vRNP bundling increases en route to the plasma 
membrane. This information is lacking and if technical possible should be included. 
 
4) There are Panama virus-infected cells were 8 bundles are not efficiently formed, because some 
segments are poorly expressed. This suggests that balanced genome expression levels are important. 
To address this point experimentally the authors might consoder to generate a virus that express less 
efficiently only one segment. 
 

Minor points: 
 
1) Control experiments (page 15: Reliability….) is very difficult to understand. 
2) In the supplement all analyzed cells (Panama and Mallard) can be shown. 
3) Show pictures of genome distribution in Mallard-infected cells 
4) Infection of human cells with the Mallard virus: are semi-infectious virus particles released from the 
cells? 
5) Is the introduction of the human PB2 signature in the Mallard strain sufficient to restore genome 
bundling? 
6) ...mediating interactions between vRNPs (10,13-23)Ref https://doi.org/10.1073/pnas.0437772100 
is missing. 
7) ...Fluorescence in situ hybridization (FISH) studies (24-26). Ref 24 is not accurate because that 
was FISH in particles. 
8) Definition of packaging in the footnote: For the reviewer packaging is the incorporation of the 
genomes into viral particles. 
9) ...In fact, considering all possible combinations of vRNPs, the random packaging model predicts 
that 98% of the MSCs would contain two or more copies of a distinct segment species: How do the 
authors come to 98%? 
10) ... To assess the vRNP composition of mature A/Panama virions by MuSeq-FISH, virions present in 
virus stocks were allowed to bind to the surface of human A549 cells on ice followed by immediate 
chemical fixation. Interestingly, the frequency distribution of MSCs in A/Panama virions was clearly 
different to that found in A/Panama-infected cells being 
shifted to MSCs of high rank (Fig. 5): How valid is the comparison? Which virus stock was used? One 
after infection with a high MOI or one after infection with a low MOI? 
11) The authors analyzed only one time point post infection and one infection dose. Is the vRNP 
composition different at earlier time points and at lower MOIs?