Learning the sequence of influenza A genome assembly during viral replication using point process models and fluorescence in situ hybridization

Abstract

15Within influenza virus infected cells, viral genomic RNA are selectively packed into progeny 16 virions, which predominantly contain a single copy of 8 viral RNA segments. Intersegmental 17 RNA-RNA interactions are thought to mediate selective packaging of each viral 18 ribonucleoprotein complex (vRNP). Clear evidence of a specific interaction network culminating 19 in the full genomic set has yet to be identified. Using multi-color fluorescence in situ 20 hybridization to visualize four vRNP segments within a single cell, we developed image-based 21 models of vRNP-vRNP spatial dependence. These models were used to construct likely 22 be useful for studying other assembly processes, especially for complexes with modest affinities 40 and more components than can be visualized simultaneously. 41 42 43 44 595 All images, derived data and source code will be available upon publication at 596

Frustration and Fidelity in Influenza Genome Assembly

“…We will need a principled approach to incorporate information from disparate data sources. Nevertheless, multiple lines of evidence (measurements of segment co-localization [27]; non-random nearest-neighbor propensities in packaged virions [8]; viral growth rates upon swapping packaging signals [15]) support our hypothesis that the core interaction network underlying influenza genome assembly is tree-like. These results not only provide insight into the dynamics of infection, but also have implications for understanding how new influenza strains emerge via genomic re-assortment and evolution.…”

“…Our approach is distinct from the challenge of finding the order of aggregation [27], which by definition is a tree whose nodes are clustered states and whose directed edges are reactions. The objects of our study are networks that could be cyclic or tree-like, whose nodes are segments and whose undirected edges are physical interactions.…”

“…We reasoned that segment pairs predicted to directly interact are more likely to co-localize during infection. The co-localization of all 28 segment pairs has been measured at eight hours post-infection [27]; this allows us to compare the co-localization of seven interacting segment pairs in a given tree with that of the remaining 21 segment pairs. We find that six of the eight Pareto trees are significantly colocalized (two-tailed Mann Whitney U-test, Holm-Bonferroni correction for multiple comparisons, significance level 0.05; Fig.…”

“…One way to prevent this is to use a fixed order of assembly, by tuning the aggregation rates (rapidly make X-Y, and then slowly make X-Y-Z). However vRNPs appear to aggregate in many possible orders (though some might be preferred [26], [27]). In this situation cyclic interaction networks will always show low fidelity, due to vRNPs being trapped in futile or frustrated off-pathway clusters.…”

“…In addition to the equilibrium RNA-RNA binding measured in vitro, livecell imaging reveals that some inter-segment interactions are irreversible on the timescale of an infection [26]. Distinct vRNP segments are seen to co-localize inside the cytoplasm of infected cells [26], [27]. EM tomography of virions shows that the eight vRNPs are arranged as parallel rods, with electrondense regions that suggest tight lateral interactions [19].…”