Segment Self-Repulsion is the Major Driving Force of Influenza Genome Packaging

The genome of influenza A virus consists of eight separate RNA segments, which are selectively packaged into virions prior to virus budding. The microscopic mechanism of highly selective packaging involves molecular interactions between packaging signals in the genome segments and remains poorly understood. We propose that the condition of proper packaging can be formulated as a large gap between RNA-RNA interaction energies in the viable virion with eight unique segments and in improperly packed assemblages lacking the complete genome. We then demonstrate that selective packaging of eight unique segments into an infective influenza virion can be achieved by self-repulsion of identical segments at the virion assembly stage, rather than by previously hypothesized intricate molecular recognition of particular segments. Using Monte Carlo simulations to maximize the energy gap, without any other assumptions, we generated model eight-segment virions, which all display specific packaging, strong self-repulsion of the segments, and reassortment patterns similar to natural influenza. The model provides a biophysical foundation of influenza genome packaging and reassortment and serves as an important step towards robust sequence-driven prediction of reassortment patterns of the influenza virus.

Figure 1

(a) The fitness and packaging hypothesis: The virion consisting of eight unique segments has the lowest energy of segment-segment interactions. The gap between $E(V)$ and $E(U_i)$ defines the selective advantage of properly packed virions $V$ with the complete set of genes. (b) Each vRNP consists of an RNA molecule condensed on NP proteins (gray, small ellipses) and loaded to the polymerase complex (brown, large ellipses). Prior to budding, the assemblage of vRNP in the nascent virion is in thermodynamic equilibrium with free vRNP in the cytoplasm. We hypothesize that RNA-RNA interactions between packaging signals (protruding loops on the vRNP) are responsible for specific packaging; protein-protein interactions provide nonspecific attraction between the vRNP.

Figure 2

(a) Selective packaging of a segmented genome via specific segment-segment interactions. The segment interaction matrix $E_{ij}$ is overall repulsive (red or dark gray) with an intricate pattern of attraction (blue or light gray) between particular segments. (b) Alternatively, selective packaging can be achieved by self-repulsion of identical segments (dashed lines) combined with weak nonspecific attraction between the different segments; the $E_{ij}$ matrix exhibits a diagonal pattern. The diagrams represent the possible patterns of segment interactions rather than actual geometry of the virion.

Figure 3

Segment interaction energies $E_{ij}$ for the average over ${10}^4$ virions, average $\Pi =0.348$ (a), and a representative evolved virion with $\Pi =0.294$ (b). Simulations to maximize $\Pi$, without any additional assumptions, lead to the emergence of segment self-repulsion. The color key below the plots encodes for interaction energy, from attractive to repulsive. Overall change of the energies with segment number is a result of differences in the length of packaging signals.

Figure 4

Normalized packaging efficiency ${\Pi }^{*}$ for two parental strains (red, $r=0,255$) and the 254 nontrivial reassortants as a function of the reassortment index $r$. Most of the reassortants have ${\Pi }^{*}\ll 1$, well below the one of either parental strain. Inset: Probability to find a reassortant virion with a given ${\Pi }^{*}$ or above. Virions with ${\Pi }^{*}\approx 1$ have the same packaging efficiency as the parent strains and are exceedingly rare.