Biology subjects

Gebert, J. T.

Publications and source records attributed to Gebert, J. T..

3 recordsLinked to original sources

bioRxiv2024

Viroporin activity from rotavirus nonstructural protein 4 induces intercellular calcium waves that contribute to pathogenesis

Acute gastroenteritis remains the second leading cause of death among children under the age of 5 worldwide. While enteric viruses are the most common etiology, the drivers of their virulence remain incompletely understood. We recently found that cells infected with rotavirus, the most prevalent enteric virus in infants and young children, initiate hundreds of intercellular calcium waves that enhance both fluid secretion and viral spread. Understanding how rotavirus triggers intercellular calcium waves may allow us to design safer, more effective vaccines and therapeutics, but we still lack a mechanistic understanding of this process. In this study, we used existing virulent and attenuated rotavirus strains, as well as reverse engineered recombinants, to investigate the role of rotavirus nonstructural protein 4 (NSP4) in intercellular calcium wave induction using in vitro, organoid, and in vivo model systems. We found that the capacity to induce purinergic intercellular calcium waves (ICWs) segregated with NSP4 in both simian and murine-like rotavirus backgrounds, and NSP4 expression alone was sufficient to induce ICWs. NSP4s ability to function as a viroporin, which conducts calcium out of the endoplasmic reticulum, was necessary for ICW induction. Furthermore, viroporin activity and the resulting ICWs drove transcriptional changes indicative of innate immune activation, which were lost upon attenuation of viroporin function. Multiple aspects of RV disease severity in vivo correlated with the generation of ICWs, identifying a critical link between viroporin function, intercellular calcium waves, and enteric viral virulence.

microbiology↗

bioRxiv2024

Purinergic Signaling Drives Multiple Aspects of Rotavirus Pathophysiology

Rotavirus causes life-threatening diarrhea in children, resulting in [~]200,000 deaths/year. The current treatment during infection is Oral Rehydration Solution which successfully replenishes fluids but does not alleviate diarrhea volume or severity. As a result, there is an urgent need to better understand rotavirus pathophysiology and develop more effective pediatric therapeutics. Rotavirus primarily infects the tips of small intestinal villi, yet has far-reaching effects on cell types distant from infected cells. We recently identified that rotavirus infected cells release the purinergic signaling molecule ADP, which activates P2Y1 receptors on nearby uninfected cells in vitro. To elucidate the role of purinergic signaling via P2Y1 receptors during rotavirus infection in vivo, we used the mouse-like rotavirus strain D6/2 which generates a severe infection in mice. C57BL/6J mouse pups were given an oral gavage of D6/2 rotavirus and assessed over the course of 5-7 days. Beginning at day 1 post infection, infected pups were treated daily by oral gavage with saline or 4 mg/kg MRS2500, a selective P2Y1 antagonist. Mice were monitored for diarrhea severity, diarrhea incidence, and viral shedding. Neonatal mice were euthanized at days 3 and 5 post-infection and small intestine was collected to observe infection. MRS2500 treatment decreased the severity, prevalence, and incidence of rotavirus diarrhea. Viral stool shedding, assessed by qPCR for rotavirus gene levels, revealed that MRS2500 treated pups had significantly lower viral shedding starting at day 4 post infection compared to saline treated pups, which suggests P2Y1 signaling may enhance rotavirus replication. Finally, we found that inhibition of P2Y1 with MRS2500 limited transmitted rotavirus diarrhea to uninfected pups within a litter. Together, these results suggest that P2Y1 signaling is involved in the pathogenesis of a homologous murine rotavirus strain, making P2Y1 receptors a promising anti-diarrheal, anti-viral therapeutic target to reduce rotavirus disease burden.

microbiology↗

bioRxiv2023

The Inositol Trisphosphate Receptor (IP3R) is Dispensable for Rotavirus-induced Ca2+ Signaling and Replication but Critical for Paracrine Ca2+ Signals that Prime Uninfected Cells for Rapid Virus Spread

Rotavirus is a leading cause of viral gastroenteritis. A hallmark of rotavirus infection is an increase in cytosolic Ca2+ caused by the nonstructural protein 4 (NSP4). NSP4 is a viral ion channel that releases Ca2+ from the endoplasmic reticulum (ER) and the increase in Ca2+ signaling is critical for rotavirus replication. In addition to NSP4 itself, host inositol 1,4,5- trisphosphate receptor (IP3R) ER Ca2+ channels may contribute to rotavirus-induced Ca2+ signaling and by extension, virus replication. Thus, we set out to determine the role of IP3R Ca2+ signaling during rotavirus infection using IP3R-knockout MA104-GCaMP6s cells (MA104- GCaMP6s-IP3R-KO), generated by CRISPR/Cas9 genome editing. Live Ca2+ imaging showed that IP3R-KO did not reduce Ca2+ signaling in infected cells but eliminated rotavirus-induced intercellular Ca2+ waves (ICWs) and therefore the increased Ca2+ signaling in surrounding, uninfected cells. Further, MA104-GCaMP6s-IP3R-TKO cells showed similar rotavirus susceptibility, single-cycle replication, and viral protein expression as parental MA104- GCaMP6s cells. However, MA104-GCaMP6s-IP3R-TKO cells exhibited significantly smaller rotavirus plaques, decreased multi-round replication kinetics, and delayed virus spread, suggesting that rotavirus-induced ICW Ca2+ signaling stimulates virus replication and spread. Inhibition of ICWs by blocking the P2Y1 receptor also resulted in decreased rotavirus plaque size. Conversely, exogenous expression of P2Y1 in LLC-MK2-GCaMP6s cells, which natively lack P2Y1 and rotavirus ICWs, rescued the generation of rotavirus-induced ICWs and enabled plaque formation. In conclusion, this study shows that NSP4 Ca2+ signals fully support rotavirus replication in individual cells; however, IP3R is critical for rotavirus-induced ICWs and virus spread by priming Ca2+-dependent pathways in surrounding cells. ImportanceMany viruses exploit host Ca2+ signaling to facilitate their replication; however, little is known about how distinct types of Ca2+ signals contribute to the overall dysregulation of Ca2+ signaling or promote virus replication. Using cells lacking IP3R, a host ER Ca2+ channel, we could differentiate between intracellular Ca2+ signals within virus-infected cells and intercellular Ca2+ waves (ICWs), which increase Ca2+ signaling in neighboring, uninfected cells. In infected cells, IP3R was dispensable for rotavirus-induced Ca2+ signaling and replication, suggesting the rotavirus NSP4 viroporin supplies these signals. However, IP3R-mediated ICWs increase rotavirus replication kinetics and spread, indicating that the Ca2+ signals from the ICWs may prime nearby uninfected cells to better support virus replication upon eventual infection. This "pre-emptive priming" of uninfected cells by exploiting host intercellular pathways in the vicinity of virus-infected cells represents a novel mechanism for viral reprogramming of the host to gain a replication advantage.

microbiology↗