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 revised manuscript entitled, “Selective, flexible packaging pathways of the segmented genome 
of Influenza A Virus”, the authors utilized sequential FISH staining to visualize all 8 segments within 
single cells at a single timepoint (10hpi) and quantify the colocalization of segments. Visualizing all 8 
vRNA segments is a powerful tool for studying IAV replication and assembly. The overall conclusions 
remain largely unchanged, 1) overall proportions of each segment are equal in a given cell, 2) the 
most abundant vRNP complexes are those with either 1 segment (rank 1) or 8 segments (rank 8), and 
3) higher ranked complexes are found more often in intact virions. 
Overall, the reviewers have revised their initial manuscript for clarity and have added the necessary 
caveats to the manuscript when describing the data. However, a few points should still be addressed. 
1. The authors provided clarification on the infection of A/Mallard in A549 cells and revealed “impaired 
nuclear export of NP” (lines 264-266 and in the rebuttal letter), which would assume imparired nuclear 
export of vRNP as well. In addition, the authors report a significant defect in viral replication of 
A/mallard in A459 cells compare to A/Panama (ie a 3 log reduction in viral titer at 72 hpi). If vRNA 
segments are found in the cytoplasm, but NP nuclear export is defective that would also suggest that 
the NP – vRNA binding is altered in these cells and could then impact the RNA-RNA interactions. 
Proper NP oligermization, binding, and post-translational modifications are known to be important for 
vRNP packaging, so it unclear why that authors state that host proteins may be the restrictive feature 
(lines 271-273). While host proteins may be important for IAV vRNP assembly through a variety of 
mechanisms that could impact vRNP bundling, there is no data presented in this manuscript to support 
the assertion. Given the complexity of comparing data from A/mallard and A/Panama – the authors 
should remove the data as it is misleading to compare the two when the amount of cytoplasmic vRNP 
are not equal at this time point. 
2. The authors provided the code for colocalization to the reviewers, but should make this freely 
available via github site to ensure that others can compare the colocalization algorithms. It is unclear, 
without extensive testing, whether the code uses ellipse centers for each segment spot to calculate 
the colocalization to nearby spots and whether the colocalization must fit all criteria - ie be within 
300nm xy and 1000nm z or if a spot within 1000nm in z would still be counted in the MSC even if it 
was > than 300nm in xy. 
3. In addition, the authors should acknowledge somewhere that not all 20-30 FISH probes will bind 
each segment. Chou et al (PMID: 22547828) very clearly demonstrate using photo-bleaching studies 
that only 7 FISH probes bound a single segment within a purified virion. This could be different within 
an infected cell as the accessibility of the FISH probe can change during assembly. This is the 
justification for why it is difficult to conclude that less than 2 copies of the same segment are located 
within a diffraction limited spot. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
Haralampiev et al. submitted a revised version of the manuscript titled “Selective flexible packaging 
pathways of the segmented genome of Influenza A virus” that includes improvements to data analysis 
and more comprehensive discussion. However, in its current state the narrative remains unclear. 
• The application of statistics improved the analysis of the data (seen in figure legends), but the text 
describing the presentation and interpretation of results requires revision. The results section currently 
reads like a descriptive discussion. In some cases, the authors have included statistical analyses for 
parts of the figures and include explanation in the figure legends, but they do not use the statistics to 
make meaningful conclusions about the data. See examples in lines 132-141 and 185-188. 

• Figures and figure panels should be labeled separately in the order of occurrence in the text of the 
manuscript. The authors need to restructure the text to address all panels of the figures in the order 
that they appear. It is disjointed and hard to follow in its current format. 
• Lines 53-57 seem unnecessary to include since the paper does not address reassortment. 
• Lines 62-65 appear to be contradictory and should be edited for clarity. 
• Interpretation of data should not be included in the figure legends. Lines 785-787, 799-800, 825-
827 should be moved to the appropriate results section. Statistical significance should be able to be 
determined from the figure itself (* or ns) with appropriate strata listed in the legends. 
• Fig. 5c scale is incorrect (25 instead of 20). The colored bars are distracting, unless the color 
correlates to something else to improve understanding. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
Influenza A viruses co-package eight different genome segments into virions. Previous studies 
suggested that the eight genomic segments bundle inside infected cells prior to their co-packaging 
into new virus particles, however this process has remained to be visualized in detail. The current 
revised manuscript offers a novel FISH technique to analyze genome bundling inside infected cells. 
While the manuscript does not test whether previously suspected determinants such as vRNA-vRNA 
interactions among packaging signals are required for efficient genome bundling, it suggest three 
novel mechanisms: 
1. balanced segment expression 
2. balanced accumulation of vRNPs in the cytoplasm (nuclear export) 
3. certain host factors 
On the one hand, the data on A/Panama suggest that low expression levels of particular genome 
segments (segment 1 or segment 8) may abrogate the formation of high-rank MSCs at different 
stages of their assembly. The authors frankly admit that this hypothesis is only based on two analyzed 
cells. 
Furthermore, the data on A/Mallard suggest that even in the presence of similar cytosolic levels of the 
eight segments, the formation of high-rank MSCs is inefficient. They further suggest that other 
mechanism(s) may influence genome bundling. This assumption is based on two older papers, where 
the authors identified that A/Mallard inefficiently exports the vRNPs from the nucleus to the cytoplasm 
and induces the expression of different host factors. 
Taken together, all three mechanisms could affect genome bundling, yet the current data are too 
limited and vague to draw any conclusions. The manuscript requires additional data to support either 
of these three mechanisms. 
 
The data provided by the authors in the refined manuscript suggest that the formation of high-rank 
MSCs is a dynamic process that changes with time. The authors provide a new experiment with 
A/Panama where they show that the formation of high-rank MSCs (Suppl. Fig 9) is very poor at 6 hpi, 
yet efficient at 10 hpi. In an older publication (Bogdanow et al., Nat. Com, 2019), the authors showed 
that at 6 hpi, A/Panama poorly exports its vRNPs from the nucleus, while they efficiently localize to 
the cytoplasm at 10 hpi. This suggests, that the accumulation of (certain levels of) the eight vRNPs in 
the cytoplasm triggers genome bundling. Importantly, this publication also revealed that the nuclear 
vRNP export of A/Mallard is substantially delayed compared to that of A/Panama. At 10 hpi, A/Mallard 
largely fails to export its genome from the nucleus, while at 16 hpi, the vRNPs partially accumulate in 
the cytoplasm. This may indicate that A/Mallard fails to bundle the genome as a consequence of the 
poor accumulation of vRNPs in the cytoplasm but is in principle able to bundle the genome when 
vRNPs are exported to the cytoplasm. To test this hypothesis, the authors should investigate MSC 
formation of A/Mallard at a later time point of infection. Ideally, the vRNPs should accumulate in the 

cytoplasm to similar levels as observed with A/Panama at 10 hpi. If efficient vRNP accumulation 
triggers genome bundling, the eight MSC ranks would shift towards a U-shaped distribution. The 
authors are encouraged to perform this experiment as all experimental tools should be available. 
Furthermore, reviewer 1 has raised a similar concern (concern 6, reviewer 1). 
 
Minor points: 
1) line 268-269: “the comparatively increased amount”: there is no quantification and only a picture is 
shown. 
2) line 396-398: what is the rational to compare RNA-RNA networks with MSC formation? Please 
specify. 
3) line 413: what are transient segment shortages?