A recombinant rotavirus harboring a spike protein with a heterologous peptide reveals a novel role of VP4 in viroplasm stability

Abstract

The rotavirus (RV) VP4 spike protrudes as a trimeric structure from the five-fold axes of the virion triple-layer. Infectious RV particles need to be proteolytically cleaved in VP4 into two subunits, VP8* and VP5*, constituting both the distal part and central body of the virus spike. Modification of VP4 has been challenging as it is involved in biological processes such as the interaction with sialic acid and integrins, cell tropism and hemagglutinin activity. Using RV reverse genetics, four loops in the lectin domain of the VP8* subunit were engineered independently to harbor a small biotin acceptor peptide (BAP) tag and then tested for their ability to rescue virus. Only a single recombinant virus, rRV/VP4-BAP, harboring VP4 with a modified loop at position K145-G150 was rescued. This rRV/VP4-BAP internalizes, replicates, and generates virus progeny, demonstrating that the VP4 spike of RV particles can be genetically manipulated by the incorporation of at least 15 exogenous amino acids. VP4-BAP had a similar distribution as VP4 in infected cells by localizing in the cytoskeleton and surrounding viroplasms. However, compared to wild-type RV, rRV/VP4-BAP featured a reduced replication fitness and impaired viroplasm stability. Upon treatment with 1,6-hexanediol, a drug disrupting liquid-liquid phase-separated condensates, the kinetic of rRV/VP4-BAP viroplasm recovery was delayed, and their size and numbers reduced when compared to viroplasms of wild type RV. Moreover, siRNA silencing of VP4 expression in RV strain SA11 showed similar recovery patterns as rRV/VP4-BAP, revealing a novel function of VP4 in viroplasm stability.