Identification of a Small Molecule That Compromises the Structural Integrity of Viroplasms and Rotavirus Double-Layered Particles

Abstract

Despite the availability of two attenuated vaccines, rotavirus (RV) gastroenteritis remains an important cause of mortality among children in developing countries causing about 215,000 infant deaths annually. Currently, there are no specific antiviral therapies available. RV is a non-enveloped virus with a segmented double-stranded RNA genome. Viral genome replication and assembly of transcriptionally active double-layered particles (DLPs) take place in cytoplasmic viral structures called viroplasms. In this study, we describe strong impairment of the early stages of RV replication induced by a small molecule known as RNA polymerase III inhibitor, ML-60218 (ML). This compound was found to disrupt already assembled viroplasms and hamper the formation of new ones without the need of transcription of cellular RNAs. This phenotype correlated with reduction in accumulated viral proteins and newly made viral genome segments, disappearance of the hyperphosphorylated isoforms of the viroplasm-resident protein NSP5 and inhibition of infectious progeny virus production. In transcription assays with purified DLPs, ML showed a dose-dependent inhibitory activity indicating the viral nature of its target. ML was found to interfere with the formation of higher order structures of VP6, the protein forming the DLP outer layer, without compromising its ability to trimerize. Electron microscopy of ML-treated DLPs showed a dose-dependent structural damage. Our data suggest that interactions between VP6 trimers are essential not only for DLP stability but also for the structural integrity of viroplasms in infected cells. Rotavirus gastroenteritis is responsible for a large number of infant deaths in developing countries. Unfortunately, in those countries where effective vaccines are urgently needed, the efficacy of the available vaccines is particularly low. Therefore, the development of antivirals is an important goal, as they might complement the available vaccines or represent an alternative option. Moreover, they may be decisive in fighting the acute phase of infection. This work describes the inhibitory effect on rotavirus replication of a small molecule initially reported as an RNA polymerase III inhibitor. The molecule is the first chemical compound identified able to disrupt viroplasms, the viral replication machinery, and to compromise the stability of DLPs by targeting the viral protein VP6. This molecule thus represents a starting point towards the development of more potent and less cytotoxic compounds against rotavirus infection.

Conserved Rotavirus NSP5 and VP2 Domains Interact and Affect Viroplasm

“…Guinea pig anti-NSP5, guinea pig anti-NSP2, guinea pig anti-VP2, and mouse scFV anti-NSP5 clone 1F2 were described previously (8,19,23,66). Guinea pig anti-VP6 was described previously (10,67). Mouse monoclonal anti-VP6 (clone 2F) was a gift from N. Mattion (CEVAN, Buenos Aires, Argentina).…”

Recombinant Rotaviruses Rescued by Reverse Genetics Reveal the Role of NSP5 Hyperphosphorylation in the Assembly of Viral Factories

“…NSP5 hyperphosphorylation is a complex process which gives rise to multiple phosphorylation states ranging from the most abundant 28-kDa phosphoisoform up to the hyperphosphorylated 32- to 34-kDa states (19, 20). All of these forms have been found to be more stable in viroplasms, while chemical disruption of viroplasms results in NSP5 dephosphorylation (21). The mechanism of NSP5 phosphorylation is not yet wholly understood, but it involves interactions with other viral proteins.…”

Treading a HOSTile path: Mapping the dynamic landscape of host cell–rotavirus interactions to explore novel host-directed curative dimensions

“…Notable examples are antiviral drug therapy with nitazoxanide which reduced duration of diarrheal episodes in children suffering from acute rotaviral diarrhea possibly by interfering with the viral morphogenesis [ 299–302 ] and with smectite (diosmectite; a natural alumino-silicate clay) that adsorbs infectious viral particles [ 303–305 ]. Moreover, host factor-independent targeting of RV has also been reported in vitro using other small molecules with viroplasm/DLP disintegrating potency [ 306 ] and viral transcription inhibitory effects [ 307 ]. With the recent advances in technological refinement such as adopting human intestinal organoids as infection model [ 86 , 123 , 140 , 141 , 213 , 257–259 , 261 , 263 , 276 , 308 , 309 ] and using rotaviral reverse genetics [ 187 , 245 , 249 , 310–313 ], future research should be propelled toward unraveling novel mechanistic aspects of host–RV interactions, and assessing therapeutic potential of reported anti-RV small molecules (host-targeted, virus-targeted, or in potential synergistic combination) in clinical settings.…”