Conserved Rotavirus NSP5 and VP2 Domains Interact and Affect Viroplasm

Abstract

One step of the life cycle common to all rotaviruses (RV) studied so far is the formation of viroplasms, membrane-less cytosolic inclusions providing a microenvironment for early morphogenesis and RNA replication. Viroplasm-like structures (VLS) are simplified viroplasm models consisting of complexes of nonstructural protein 5 (NSP5) with the RV core shell VP2 or NSP2. We identified and characterized the domains required for NSP5-VP2 interaction and VLS formation. VP2 mutations L124A, V865A, and I878A impaired both NSP5 hyperphosphorylation and NSP5/VP2 VLS formation. Moreover, NSP5-VP2 interaction does not depend on NSP5 hyperphosphorylation. The NSP5 tail region is required for VP2 interaction. Notably, VP2 L124A expression acts as a dominant-negative element by disrupting the formation of either VLS or viroplasms and blocking RNA synthesis. In silico analyses revealed that VP2 L124, V865, and I878 are conserved among RV species A to H. Detailed knowledge of the protein interaction interface required for viroplasm formation may facilitate the design of broad-spectrum antivirals to block RV replication. IMPORTANCE Alternative treatments to combat rotavirus infection are a requirement for susceptible communities where vaccines cannot be applied. This demand is urgent for newborn infants, immunocompromised patients, adults traveling to high-risk regions, and even for the livestock industry. Aside from structural and physiological divergences among RV species studied before now, all replicate within cytosolic inclusions termed viroplasms. These inclusions are composed of viral and cellular proteins and viral RNA. Viroplasm-like structures (VLS), composed of RV protein NSP5 with either NSP2 or VP2, are models for investigating viroplasms. In this study, we identified a conserved amino acid in the VP2 protein, L124, necessary for its interaction with NSP5 and the formation of both VLSs and viroplasms. As RV vaccines cover a narrow range of viral strains, the identification of VP2 L124 residue lays the foundations for the design of drugs that specifically block NSP5-VP2 interaction as a broad-spectrum RV antiviral.

Rotavirus Replication Factories Are Complex Ribonucleoprotein Condensates

“…Furthermore, these compounds did not dissolve viroplasmic condensates after chemical cross-linking. Importantly, the observed condensates are formed exclusively when the untagged version of NSP5 is co-expressed in cells along with either NSP2, or VP2 17,46 , as well as during RV infection. Thus, the formation of the observed condensates reflects the unique physicochemical properties of NSP5/NSP2, and is not artifactual due to overexpression or fluorescent tagging 47,48 .…”

“…Previous studies uncovered protein composition of viroplasms, suggesting that these cytoplasmic inclusions are formed when NSP5 is co-expressed with NSP2 and/or the viral capsid protein VP2 14,15,17,23,46 , even in the absence of RV infection. Here, from multiple lines of evidence, we revealed that rotavirus viroplasms represent ribonucleoprotein condensates that are formed via phase separation of NSP5 and NSP2: First, cytosolic inclusions formed by NSP5/NSP2 are initially spherical; second, two or more NSP5-rich droplets can fuse and relax into a sphere; third, infection with the NSP5-defficient rotavirus mutant does not yield these droplets, unless NSP5 is produced in trans; fourth, these droplets are formed upon NSP5 and NSP2 mixing in vitro, and when these proteins are co-expressed in vivo; finally, NSP2/NSP5 droplets can be instantly dissolved when treated with aliphatic alcohols known to disrupt multivalent interactions driving liquid-liquid phase separation.…”

“…Indeed, previous observations 49 suggesting that the kinetics of viroplasm formation increase in direct correlation with the multiplicity of infection fully support our model. Current views of replication factory formation in RVs are dominated by the idea of multiple viral proteins being recruited into viroplasms in a specific order 15,17,23,46,50 resulting in their particular organisation 51 . Here, we propose a unifying model for viroplasm assembly (Fig.…”

Mammalian orthoreovirus core protein μ2 reorganizes host microtubule-organizing center components

“…Cells were incubated twice with quenching buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl) for 3 min on ice. The cell lysates were prepared in 180 μl of TNN buffer (100 mM Tris-HCl pH 8.0, 250 mM NaCl, 0.5% NP-40, 1X HALT phosphatase inhibitor cocktail (Thermo Fischer Scientific) and cOmplete™ protease inhibitor cocktail (Roche)) as described previously ( Buttafuoco et al, 2020 ). The cellular extract was loaded onto 50 μl of PerfectPro Ni-NTA agarose (5 PRIME, Germany) equilibrated in 25 mM imidazole in PBS.…”

Rotavirus A Genome Segments Show Distinct Segregation and Codon Usage Patterns

“…NSP2 protein interactions with the +ssRNA are therefore critical to stabilizing +ssRNA contacts (30). NSP2 also interacts with NSP5 to form viroplasms (along with VP1, VP2, VP3, V6 and NSP4) (31) where genome packaging, replication, assembly of progeny cores and double-layered particles (DLPs) occurs (32)(33)(34). To effect transmission, RVA encodes an enterotoxin, NSP4, which elevates cytosolic Ca 2+ in cells.…”