Rotavirus morphogenesis starts in intracellular inclusion bodies called viroplasms. RNA replication and
packaging are mediated by several viral proteins, of which VP1, the RNA-dependent RNA polymerase, and VP2,
the core scaffolding protein, were shown to be sufficient to provide replicase activity in vitro. In vivo, however,
viral replication complexes also contain the nonstructural proteins NSP2 and NSP5, which were shown to be
essential for replication, to interact with each other, and to form viroplasm-like structures (VLS) when
coexpressed in uninfected cells. In order to gain a better understanding of the intermediates formed during
viral replication, this work focused on the interactions of NSP5 with VP1, VP2, and NSP2. We demonstrated
a strong interaction of VP1 with NSP5 but only a weak one with NSP2 in cotransfected cells in the absence of
other viral proteins or viral RNA. By contrast, we failed to coimmunoprecipitate VP2 with anti-NSP5 antibodies
or NSP5 with anti-VP2 antibodies. We constructed a tagged form of VP1, which was found to colocalize
in viroplasms and in VLS formed by NSP5 and NSP2. The tagged VP1 was able to replace VP1 structurally by
being incorporated into progeny viral particles. When applying anti-tag-VP1 or anti-NSP5 antibodies, coimmunoprecipitation
of tagged VP1 with NSP5 was found. Using deletion mutants of NSP5 or different fragments
of NSP5 fused to enhanced green fluorescent protein, we identified the 48 C-terminal amino acids as the region
essential for interaction with VP1.
