# 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  
RVA protein and RNA molecules interact with each other in a variety of ways during infection and assembly processes. Incompatibilities among different genotypes resulting from these interactions may limit the diversity of genome constellations observed in nature. For example, RNA secondary structures and segment-specific sequences found in the non-translated terminal regions (NTRs) may govern the formation of the supramolecular RNA complex associated with segment packaging [94; 96; 97; 98; 99; 31] Sequence mismatches between segments from coinfecting RVAs may prevent segment interactions and co-packaging, and hence, the generation of reassortment RVAs. One of the studies [96] also suggested that VP4 has a less conserved terminal RNA structure, so the importance of these RNA interactions may vary significantly by segment. The order in which the segments associate to form the supramolecular RNA complex may be sequential. In this scenario, the smallest segments interact first, then they recruit intermediate-sized segments before finally incorporating the largest segments [97]. In addition, incompatibilities in the 3\({}^{\prime}\)-NTRs of the smaller segments may have stronger effects on segment co-segregation than do the larger segments. For instance, the smallest RVA segments, segments 10 and 11 must directly interact before they can interact with larger segments. Evidence for this supposition that the smaller segments' RNA structure is under strong selective pressure along with its protein structure (i.e., synonymous mutations are often not neutral) also comes from our observation that segments 10 and 11 co-segregated with one another more strongly than other pairs of segments (Figure 2), despite their many protein interactions with other segments. There is evidence for frequent inter-host-species reassortment of segment 10, which encodes the enterotoxin, NSP4, in nature [15; 100], so reassortment of segment 10 may be more dependent on the sequence conservation of terminal +ssRNA between segments 10 and 11, than on protein interactions. In addition to NSP5, segment 11 also encodes a second out of frame protein, NSP6, via leaky scanning. NSP6 is not required for virion function and is sometimes not expressed in rotavirus [101]; however, it is constrained due to overlapping with NSP5. Segment 11's RNA structure is thought to have some functional importance [96; 102] however segment 11 appears more tolerant of genome insertions than other segments [51; 52], likely due to packaging signal duplication [52]. The close evolutionary histories of segments 10 and 11 may also relate to NSP4-NSP5 interactions during viroplasm formation [34]. The tree spaces of both segments 4 and 9 (Figure 2) were notably distinct from the tree spaces of the rest of the genome. RVA segment 4 encodes the spike protein, VP4, which is cleaved by trypsin into VP8 * and VP5 *. VP4 interacts with different receptors depending on the strain, including sialoglycans and histo-blood glycans [103; 104; 105] These different receptors partly explain why certain P types tend to dominate in different populations, species, and age groups [105; 106]. Our results showed that the gene tree of segment 4 was distinct from the rest of the genome, suggesting that segment 4 may reassort more readily than other segments. Alternatively, segment 4's divergent history could also be explained by VP4's role in host determination (Table 1). Segment 9 (VP7) had the weakest association with host species (Table 1) which may explain its distance from VP4 and the rest of the genome. Other environmental studies have also found that segment 4 and segment 9 are more likely to appear in different genetic backgrounds [107; 108; 109].