Mammalian orthoreovirus (reovirus) is under development as a cancer virotherapy. Clinical trials demonstrate that reovirus-based therapies are safe and tolerated in patients with a wide variety of cancers. Although reovirus monotherapy has proven largely ineffective, reovirus sensitizes cancer cells to existing chemotherapeutic agents and radiation. Clinical trials are underway to test the efficacy of reovirus in combination with chemotherapeutic and radiation regimens and to evaluate the effectiveness of reovirus in conjunction with immunotherapies. Central to the use of reovirus to treat cancer is its capacity to directly kill cancer cells and alter the cellular environment to augment other therapies. Apoptotic cell death is a prominent mechanism of reovirus cancer cell killing. However, reoviruses can also kill cancer cells through nonapoptotic mechanisms. Here, we describe mechanisms of reovirus cancer cell killing, highlight how reovirus is used in combination with existing cancer treatments, and discuss what is known as to how reovirus modulates cancer immunotherapy.

Serotype 3 (T3) reoviruses induce substantially more type 1 interferon (IFN-I) secretion than serotype 1 (T1) strains. However, the mechanisms underlying differences in IFN-I production between T1 and T3 reoviruses remain undefined. Here, we found that differences in IFN-I production between T1 and T3 reoviruses correlate with activation of interferon regulatory factor 3 (IRF3), a key transcription factor for the production of IFN-I. T3 strain rsT3D activated IRF3 more rapidly and to a greater extent than the T1 strain rsT1L, in simian virus 40 (SV40) immortalized endothelial cells (SVECs). Differences in IRF3 activation between rsT1L and rsT3D were observed in the first hours of infection and were independent of viral RNA and protein synthesis. NF-κB activation mirrored IRF3 activation, with rsT3D inducing more NF-κB activity than rsT1L. We also found that IRF3 and NF-κB are activated in a mitochondrial antiviral-signaling protein (MAVS)-dependent manner. rsT1L does not suppress IRF3 activation, as IRF3 phosphorylation could be induced in rsT1L-infected cells. Transfected rsT1L and rsT3D RNA induced IRF3 phosphorylation, indicating that genomic RNA from both strains has the capacity to activate IRF3. Finally, bypassing the normal route of reovirus entry by transfecting-generated viral cores revealed that rsT1L and rsT3D core particles induced equivalent IRF3 activation. Taken together, our findings indicate that entry-related events that occur after outer capsid disassembly, but prior to deposition of viral cores into the cytoplasm, influence the efficiency of IFN-I responses to reovirus. This work provides further insight into mechanisms by which nonenveloped viruses activate innate immune responses. Detection of viral nucleic acids by the host cell triggers type 1 interferon (IFN-I) responses, which are critical for containing and clearing viral infections. Viral RNA is sensed in the cytoplasm by cellular receptors that initiate signaling pathways, leading to the activation of interferon regulatory factor 3 (IRF3) and NF-κB, key transcription factors required for IFN-I induction. Serotype 3 (T3) reoviruses induce significantly more IFN-I than serotype 1 (T1) strains. In this work, we found that differences in IFN-I production by T1 and T3 reoviruses correlate with differential IRF3 activation. Differences in IRF3 activation are not caused by a blockade of the IRF3 activation by a T1 strain. Rather, differences in events during the late stages of viral entry determine the capacity of reovirus to activate host IFN-I responses. Together, our work provides insight into mechanisms of IFN-I induction by nonenveloped viruses.

Reovirus non-structural protein σ1s is required for the establishment of viremia and hematogenous viral dissemination. However, the function of the σ1s protein during the reovirus replication cycle is not known. In this study, we found that σ1s was required for efficient reovirus replication in SV40-immortalized endothelial cells (SVECs), mouse embryonic fibroblasts, human umbilical vein endothelial cells (HUVECs), and T84 human colonic epithelial cells. In each of these cell lines, wild type reovirus produced substantially higher viral titers than a σ1s-deficient mutant. The σ1s protein was not required for early events in the reovirus infection, as no difference in infectivity between the wild type and σ1s-null viruses was observed. However, wild type virus produced markedly higher viral protein levels than the σ1s-deficient strain. The disparity in viral replication did not result from differences in viral transcription or protein stability. We further found that the σ1s protein was dispensable for cell killing and induction of type-1 interferon responses. In the absence of σ1s, viral factory (VF) maturation was impaired, but sufficient to support low levels of reovirus replication. Together, our results indicate that σ1s is not absolutely essential for viral protein production, but rather potentiates reovirus protein expression to facilitate reovirus replication. Our findings suggest that σ1s enables hematogenous reovirus dissemination by promoting efficient viral protein synthesis, and thereby reovirus replication, in cells that are required for reovirus spread to the blood.Hematogenous dissemination is critical a step in the pathogenesis of many viruses. For reovirus, nonstructural protein σ1s is required for viral spread via the blood. However, the mechanism by σ1s promotes reovirus dissemination is unknown. Here, we identified σ1s as a viral mediator of reovirus protein expression. We found several cultured cell lines in which σ1s is required for efficient reovirus replication. In these cells, wild type virus produced substantially higher levels of viral protein than a σ1s-deficient mutant. The σ1s protein was not required for viral mRNA transcription or viral protein stability. Owing to reduced levels of viral protein synthesized in the absence of σ1s, maturation of viral factories was impaired and significantly fewer viral progeny were produced. Taken together, our findings indicate that σ1s is required for optimal reovirus protein production, and thereby viral replication, in cells required hematogenous reovirus dissemination.

Acetaminophen (APAP)‐induced liver injury is the most common cause of acute liver failure (ALF) in the Western world. APAP toxicity progresses to multiorgan dysfunction and thus has broader whole‐body implications. Importantly, greater 30‐day mortality has been observed in liver transplant recipients following ALF due to APAP‐related versus non‐APAP‐related causes. Reasons for this discrepancy have yet to be determined. Extrahepatic toxicities of APAP overdose may represent underappreciated and unaddressed comorbidities within this patient population. In the present study, rapid induction of apoptosis following APAP overdose was observed in the intestine, an organ that greatly influences the physiology of the liver. Strikingly, apoptotic cells appeared to be strictly restricted to the intestinal crypts. The use of leucine‐rich repeat‐containing G protein–coupled receptor 5 (LGR5) reporter mice confirmed that the LGR5‐positive (+) crypt base stem cells were disproportionately affected by APAP‐induced cell death. Although the apoptotic cells were cleared within 24 hours after APAP treatment, potentially long‐lived consequences on the intestine due to APAP exposure were indicated by prolonged deficits in gut barrier function. Moreover, small intestinal cell death was found to be independent of tumor necrosis factor receptor signaling and may represent a direct toxic insult to the intestine by exposure to high concentrations of APAP. Conclusion: APAP induces intestinal injury through a regulated process of apoptotic cell death that disproportionately affects LGR5+ stem cells. This work advances our understanding of the consequences of APAP toxicity in a novel organ that was not previously considered as a significant site of injury and thus presents potential new considerations for patient management.

Hematopoietic stem and progenitor cells (HSPCs) function to replenish the immune cell repertoire under steady-state conditions, and in response to inflammation due to infection or stress. While the bone marrow serves as the primary niche for hematopoiesis, extramedullary mobilization and differentiation of HSPCs occurs in the spleen during acute Plasmodium infection –n a critical step in the host immune response. Here, we identified an atypical HSPC population in the spleen of C57BL/6 mice, with a Lineage−Sca-1+c-kit− (LSK−) phenotype that proliferates in response to infection with non-lethal Plasmodium yoelii 17X. Infection-derived LSK− cells upon transfer into naïve congenic mice were found to differentiate predominantly into mature follicular B cells. However, when transferred into infection-matched hosts, infection-derived LSK− cells gave rise to B cells capable of entering into a germinal center reaction, and developed into memory B cells and antibody-secreting cells that were capable of producing parasite-specific antibodies. Differentiation of LSK− cells into B cells in vitro was enhanced in the presence of parasitized RBC lysate, suggesting that LSK− cells expand and differentiate in direct response to the parasite. However, the ability of LSK− cells to differentiate into B cells was not dependent on MyD88 as myd88−/− LSK− cell expansion and differentiation remained unaffected after Plasmodium infection. Collectively, these data identify a population of atypical lymphoid progenitors that differentiate into B-lymphocytes in the spleen, and are capable of contributing to the ongoing humoral immune response against Plasmodium infection.