# Rotavirus A Genome Segments Show Distinct Segregation and Codon Usage Patterns
## 4 Discussion
### Potential Impact of Rotavirus RNA and Protein Interactions on Segment Co-Segregation
Based on the high genetic diversity of segments 4 and 9, and segment 4 seeming to have a less conserved role in RVA +ssRNA assortment, reassortment into new genetic backgrounds may confer a selective advantage and a broader host range, as strains can have an opportunity to evolve in a novel host, they may otherwise be unable to infect, due to a barrier caused by another segment (e.g., poor receptor-binding). Segment 4's larger geodesic distance from the rest of the genome and high diversity in RNA secondary structure, suggests that segment 4's synonymous sites may be less critical to the segment's function. Segment 9, on the other hand, is critical for the formation/stabilization of the supramolecular RNA complex and for packaging the genome. Both segments 4 and 9 have been shown to tolerate homologous recombination among highly divergent genotypes, including recombination events resulting in the disruption of many amino acids, whilst still maintaining overall tertiary structure [110]. Due to the importance of VP1, VP2, and VP3 during the formation of the virion, synthesis of dsRNA, and associating with the 11 +ssRNA segments, one might expect these segments to be the least likely to reassort independently. However, the critical VP1, VP2, and VP3 interactions are mostly protein-protein interactions, so even a genetically distant strain could maintain a conserved amino acid sequence allowing these segments some flexibility with their genetic background. Our results showed that, while these three segments are generally associated with the larger \"gene tree\" of the rest of the genome, they have a more independent evolutionary history than for example, segments 10 and 11, which almost entirely overlap in tree space. Interestingly, segment 7 (encoding NSP3) shared the closest evolutionary history with segment 1 (Figure 1). We expected segment 1 to have the closest evolutionary history with segments 2 or 3 given their proteins' interactions, however, VP1's high degree of structural conservation [5] and having less functionally important RNA structure than the other segments may explain its tolerance for novel genetic backbones. Segment 7/NSP3 may have less strain-specific interactions with other segments resulting in less fitness variation following a reassortment event. Segment 7 may have endured a significant reassortment event around 1970 (Figure 5, and SI file S10) which also may explain its geodesic from the other segments. NSP3's primary function is to recognize a conserved group-specific sequence present on all group A RV segments and interact with host eIF4G, a protein that is highly conserved among orthologs [111]. This suggests NSP3 genes could be flexible to many RVA genetic backgrounds and hosts. Segment 5 (NSP1) was only particularly distinct in the tree space (Figure 2) from segments 1, 4, and 9. This was somewhat surprising as NSP1 has relatively low conservation, can tolerate insertions and deletions, and is not required for rotavirus replication in vitro (although the RNA is still required for packaging). NSP1's important role in targeting the host's antiviral response as an interferon antagonist [112], inhibiting apoptosis [113; 114], and activating NFkappaB [115] may explain a stronger host-association. NSP1 reassortment with different host strains may confer a deleterious effect in vivo, despite the reassortment's ability to compete in vitro (i.e., in the absence of a significant immune response).