Whereas the VP6-Wa-encoding plasmid was generated without problems, sequencing analyses showed that the plasmid containing the VP7-Wa genome segment was unstable upon cloning in bacteria. Plasmids encoding VP6-Wa and VP7-Wa were generated by DNA synthesis and cloning. A plasmid encoding VP4-Wa was already available. In order to test the hypothesis that strain-specific interactions between the outer and intermediate capsid proteins are important for the generation of viable reassortants, all possible combinations of VP4, VP7, and VP6 should be tested using a well-established plasmid-based RG system. have shown that human RVA genome constellations are influenced by viral protein interactions and identified genotype specific changes at viral VP2/VP6, VP7/VP6, and VP4/VP6 interfaces. In line with this observation, Heiman et al. Analyses of circulating human RVA strains also indicate that there are preferred genome constellations. While this could theoretically allow members of RVA to freely exchange genome segments, experiments using reverse genetics (RG) systems suggest that not all genome segments of different RVA strains can readily reassort. Differences in the viral UTRs at the genome segment ends have been shown to limit reassortment between different rotavirus species, but within one rotavirus species, at least the outmost genome segment termini are highly conserved. However, little is known about the factors that influence reassortment. Therefore, reassortment plays a major role in increasing the genetic diversity of RVA. Progeny virions generated by reassortment can acquire new phenotypic properties, including improved immune evasion or altered host range. Upon superinfection of the same target cell with another RVA strain, an exchange of genome segments is possible, a process referred to as reassortment. Zoonotic transmission of RVA between animals and humans has been documented, but viral replication in heterologous hosts is often inefficient. However, it was recently shown that VP6-specific IgG has intracellular neutralization activity and protects mice against rotavirus infection. In contrast to the surface-exposed VP7 and VP4, antibodies directed against the intermediate capsid layer protein VP6 are unable to neutralize cell-free mature virus particles. Both VP7 and VP4 are involved in viral entry and contain antigenic sites that induce neutralizing antibody responses. To date, 42 VP7 genotypes (G1-42) and 58 different VP4 genotypes (P) have been described. Especially the RVA genome segments encoding VP7 and VP4 have a very high genetic diversity. In this system, the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genotypes are represented by Gx-P-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx, in which x indicates the number of the genotype. Based on the complete nucleotide sequence of all eleven genome segments, a classification system has been established. Each segment contains one to two open reading frames that are flanked by untranslated regions (UTRs). The inner capsid layer encloses the viral genome, which comprises eleven different dsRNA segments. The base of the spike is embedded in the virus capsid and in extensive contact with both VP7 and VP6. Based on the atomic model of an infectious RVA particle, each viral spike is comprised of three VP4 molecules. The protease-sensitive VP4 forms the viral spike and mediates attachment to target cells. The outer capsid layer comprises VP4 and VP7, the intermediate layer VP6, and the inner layer VP2. Rotaviruses are non-enveloped viruses with a triple-layered capsid. Restoring the natural VP4/VP7/VP6 interactions may therefore improve the rescue of RVA reassortants by reverse genetics, which could be useful for the development of next generation RVA vaccines. Analysis of the predicted structural protein interfaces identified amino acid residues, which might influence protein interactions. The replication kinetics of the triple-reassortant and its parent strain Wa were comparable, while the replication of all other rescued reassortants was similar to SA11. However, a VP4/VP7/VP6-Wa triple-reassortant was successfully generated, indicating that the presence of homologous VP7 and VP6 enabled the incorporation of VP4-Wa into the SA11 backbone. VP7-Wa, VP6-Wa, and VP7/VP6-Wa reassortants were effectively rescued, but the VP4-Wa, VP4/VP7-Wa, and VP4/VP6-Wa reassortants were not viable, suggesting a limiting effect of VP4-Wa. To gain insight into the factors that restrict reassortment, we utilized reverse genetics and tested the generation of simian RVA strain SA11 reassortants carrying the human RVA strain Wa capsid proteins VP4, VP7, and VP6 in all possible combinations. However, not all reassortants are viable, which limits the ability to generate customized viruses for basic and applied research. Rotavirus A (RVA) genome segments can reassort upon co-infection of target cells with two different RVA strains.
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