Development of fluorescent and affinity tagged variants of rotavirus nonstructural protein 4 using the reverse genetics system

Rotavirus nonstructural protein 4 (NSP4) is a crucial component of the virus, playing both a role in virion assembly and as a potential target for therapeutic intervention. Due to RNA packaging signals and a DLP-binding domain at the end of gene segment 10, studying NSP4 within infectious rotaviruses has been challenging. To address this, researchers employed a plasmid-based reverse genetics system to create six recombinant rotaviruses (rRVs) with tagged or reporter-fused NSP4 using three methods.

These approaches included expression from an alternative segment (gs7) in a bicistronic format, N-terminal tagging of gs10, and fusion of the first 20 amino acids with a reporter, followed by bicistronic full-length NSP4 expression.

All six rRVs were capable of replication, with gs10-based constructs closely replicating the wild-type virus. However, gs7-based constructs showed a slight attenuation and, in one instance, genetic instability upon passage. The engineered NSP4 proteins displayed proper glycosylation and oligomerization. Through live-cell imaging, the tagged NSP4 proteins were observed to track accurately during synthesis and were linked to intercellular calcium signals that occur due to NSP4's action.

In addition, affinity purification of the tagged NSP4 recovered viral proteins and potential host interactors, such as ANP32A, ANP32E, PPM1G, and H2AC1. These rRVs provide a robust toolkit for investigating NSP4 function during genuine infection and offer a versatile method for engineering restricted rotavirus gene segments.