Rotavirus infection is a major cause of severe dehydrating diarrhea in infants younger than 5 y old and in particular cases of immunocompromised patients irrespective to the age of the patients. Although vaccines have been developed, antiviral therapy is an important complement that cannot be substituted. Because of the lack of specific approved treatment, it is urgent to facilitate the cascade of further understanding of the infection biology, identification of druggable targets and the final development of effective antiviral therapies. PI3K-Akt-mTOR signaling pathway plays a vital role in regulating the infection course of many viruses. In this study, we have dissected the effects of PI3K-Akt-mTOR signaling pathway on rotavirus infection using both conventional cell culture models and a 3D model of human primary intestinal organoids. We found that PI3K-Akt-mTOR signaling is essential in sustaining rotavirus infection. Thus, blocking the key elements of this pathway, including PI3K, mTOR and 4E-BP1, has resulted in potent anti-rotavirus activity. Importantly, a clinically used mTOR inhibitor, rapamycin, potently inhibited both experimental and patient-derived rotavirus strains. This effect involves 4E-BP1 mediated induction of autophagy, which in turn exerts anti-rotavirus effects. These results revealed new insights on rotavirus-host interactions and provided new avenues for antiviral drug development.

Norovirus is the main cause of viral gastroenteritis worldwide. Although norovirus gastroenteritis is self-limiting in immunocompetent individuals, chronic infections with debilitating and life-threatening complications occur in immunocompromised patients. Nitazoxanide (NTZ) has been used empirically in the clinic and has demonstrated effectiveness against norovirus gastroenteritis. In this study, we aimed at uncovering the antiviral potential and mechanisms of action of NTZ and its active metabolite, tizoxanide (TIZ), using a human norovirus (HuNV) replicon. NTZ and TIZ, collectively referred to as thiazolides (TZD), potently inhibited replication of HuNV and a norovirus surrogate, feline calicivirus. Mechanistic studies revealed that TZD activated cellular antiviral response and stimulated the expression of a subset of interferon-stimulated genes (ISGs), particularly interferon regulatory factor 1 (IRF-1), not only in a Huh7 cell-based HuNV replicon, but also in naive Huh7 and Caco-2 cells and novel human intestinal organoids. Overexpression of exogenous IRF-1 inhibited HuNV replication, whereas knockdown of IRF-1 largely attenuated the antiviral activity of TZD, suggesting that IRF-1 mediated TZD inhibition of HuNV. By using a Janus kinase (JAK) inhibitor, CP-690550, and a STAT1 knockout approach, we found that TZD induced antiviral response independently of the classical JAK-signal transducers and activators of transcription (JAK-STAT) pathway. Furthermore, TZD and ribavirin synergized to inhibit HuNV replication and completely depleted the replicons from host cells after long-term treatment. In summary, our results demonstrated that TZD combated HuNV replication through activation of cellular antiviral response, in particular by inducing a prominent antiviral effector, IRF-1. NTZ monotherapy or combination with ribavirin represent promising options for treating norovirus gastroenteritis, especially in immunocompromised patients.

Rotavirus infection has emerged as an important cause of complications in organ transplantation recipients and might play a role in the pathogenesis of inflammatory bowel disease (IBD). 6-Thioguanine (6-TG) has been widely used as an immunosuppressive drug for organ recipients and treatment of IBD in the clinic. This study aims to investigate the effects and mode-of-action of 6-TG on rotavirus replication. Human intestinal Caco2 cell line, 3D model of human primary intestinal organoids, laboratory rotavirus strain (SA11) and patient-derived rotavirus isolates were used. We have demonstrated that 6-TG significantly inhibits rotavirus replication in these intestinal epithelium models. Importantly, gene knockdown or knockout of Rac1, the cellular target of 6-TG, significantly inhibited rotavirus replication, indicating the supportive role of Rac1 for rotavirus infection. We have further demonstrated that 6-TG can effectively inhibit the active form of Rac1 (GTP-Rac1), which essentially mediates the anti-rotavirus effect of 6-TG. Consistently, ectopic over-expression of GTP-Rac1 facilitates but an inactive Rac1 (N17) or a specific Rac1 inhibitor (NSC23766) inhibits rotavirus replication. In conclusion, we have identified 6-TG as an effective inhibitor of rotavirus replication via the inhibition of Rac1 activation. Thus, for transplantation patients or IBD patients infected with rotavirus or at risk of rotavirus infection, the choice of 6-TG as a treatment appears rational.

Norovirus represents the main cause of acute nonbacterial gastroenteritis worldwide. In immunocompromised patients, it bears high risk of causing chronic infection with significant morbidity and mortality. The lack of specific treatment prompts the development of anti-norovirus agents. In this study, we have investigated the role of interferon (IFN) response and evaluated antiviral activities of different IFNs against human norovirus (HuNoV) replication using a HuNoV replicon model. We found that HuNoV RNA replication was sensitive to all types of IFNs, including IFNα (type I), IFNγ (type II), IFNλ1 and 3 (type III). IFNs canonically induce interferon-stimulated genes (ISGs) to exert their antiviral activities. By profiling a subset of important human ISGs using an overexpression approach, we have identified RTP4 and HPSE as moderate anti-norovirus ISGs, whereas IRF-1, RIG-I (also known as DDX58) and MDA5 (also known as IFIH1) were identified as potent anti-norovirus effectors. Interestingly, type I and III IFNs coordinately induced IRF-1, RIG-I and MDA5; whereas type II IFN predominantly induced IRF-1 to exhibit their anti-norovirus activities. Combination of different IFNs revealed that IFNγ worked cooperatively with type I or type III IFNs to induce ISGs and subsequently inhibit HuNoV replication. Of note, replication of HuNoV did not interfere with antiviral IFN response. In summary, we showed the potent anti-norovirus activities of different types of IFNs and identified the key anti-norovirus effectors. These findings are important for understanding norovirus-host interactions and developing antiviral therapies.

Rotavirus (RV) primarily infects enterocytes and results in severe diarrhea, particularly in children. It is known that the host immune responses determine the outcome of viral infections. Following infections, interferons (IFNs) are produced as the first and the main anti-viral cytokines to combat the virus. Here we showed that RV predominantly induced type III IFNs (IFN-λ1), and to a less extent, type I IFNs (IFN-α and IFN-β) in human intestinal cells. However, it did not produce detectable IFN proteins and thus, was not sufficient to inhibit RV replication. In contrast, we revealed the essential roles of the basal IFN signaling in limiting RV replication by silencing STAT1, STAT2 and IRF9 genes. In addition, exogenous IFN treatment demonstrated that RV replication was able to be inhibited by all types of IFNs, both in human intestinal Caco2 cell line and in primary intestinal organoids. In these models, IFNs significantly upregulated a panel of well-known anti-viral IFN-stimulated genes (ISGs). Importantly, inhibition of the JAK-STAT cascade abrogated ISG induction and the anti-RV effects of IFNs. Thus, our study shall contribute to better understanding of the complex RV-host interactions and provide rationale for therapeutic development of IFN-based treatment against RV infection.