Rotavirus non-structural protein 4 usurps host cellular RIPK1-RIPK3 complex to induce MLKL-dependent necroptotic cell death.

The dynamic interface between invading viral pathogens and programmed cell death (PCD) of the host is a finely regulated process. Host cellular demise at the end of the viral life cycle ensures the release of progeny virions to initiate new infection cycles. Rotavirus (RV), a diarrheagenic virus with double-stranded RNA genome, has been reported to trigger different types of PCD such as apoptosis and pyroptosis in a highly regulated way to successfully disseminate progeny virions. Recently our lab also showed that induction of MLKL-driven programmed necroptosis by RV. However, the host cellular machinery involved in RV-induced necroptosis and the upstream viral trigger responsible for it remained unaddressed. In the present study, the signalling upstream of MLKL-driven necroptosis has been delineated where the involvement of Receptor interacting serine/threonine kinase 3 (RIPK3) and 1 (RIPK1) from the host side and RV non-structural protein 4 (NSP4) as the viral trigger for necroptosis has been shown. Interestingly, RV-NSP4 was found to be an integral component of the necrosome complex by interacting with RIPK1, thereby bypassing the requirement of RIPK1 kinase activity. Subsequently, NSP4-driven elevated cytosolic Ca2+ concentration and Ca2+-binding to NSP4 lead further to RHIM domain-dependent RIPK1-RIPK3 interaction, RIPK3-dependent MLKL phosphorylation, and eventual necroptosis. Overall, this study presents the interplay between RV-NSP4 and the host cellular necrosome complex to induce necroptotic death of host cells.

Structural basis for RAD18 regulation by MAGEA4 and its implications for RING ubiquitin ligase binding by MAGE family proteins.

MAGEA4 is a cancer-testis antigen primarily expressed in the testes but aberrantly overexpressed in several cancers. MAGEA4 interacts with the RING ubiquitin ligase RAD18 and activates trans-lesion DNA synthesis (TLS), potentially favouring tumour evolution. Here, we employed NMR and AlphaFold2 (AF) to elucidate the interaction mode between RAD18 and MAGEA4, and reveal that the RAD6-binding domain (R6BD) of RAD18 occupies a groove in the C-terminal winged-helix subdomain of MAGEA4. We found that MAGEA4 partially displaces RAD6 from the RAD18 R6BD and inhibits degradative RAD18 autoubiquitination, which could be countered by a competing peptide of the RAD18 R6BD. AlphaFold2 and cross-linking mass spectrometry (XL-MS) also revealed an evolutionary invariant intramolecular interaction between the catalytic RING and the DNA-binding SAP domains of RAD18, which is essential for PCNA mono-ubiquitination. Using interaction proteomics, we found that another Type-I MAGE, MAGE-C2, interacts with the RING ubiquitin ligase TRIM28 in a manner similar to the MAGEA4/RAD18 complex, suggesting that the MAGEA4 peptide-binding groove also serves as a ligase-binding cleft in other type-I MAGEs. Our data provide new insights into the mechanism and regulation of RAD18-mediated PCNA mono-ubiquitination.

Rotavirus activates MLKL-mediated host cellular necroptosis concomitantly with apoptosis to facilitate dissemination of viral progeny.

Reprogramming the host cellular environment is an obligatory facet of viral pathogens to foster their replication and perpetuation. One of such reprogramming events is the dynamic cross-talk between viruses and host cellular death signaling pathways. Rotaviruses (RVs) have been reported to develop multiple mechanisms to induce apoptotic programmed cell death for maximizing viral spread and pathogenicity. However, the importance of non-apoptotic programmed death events has remained elusive in context of RV infection. Here, we report that RV-induced apoptosis accompanies another non-apoptotic mode of programmed cell death pathway called necroptosis to promote host cellular demise at late phase of infection. Phosphorylation of mixed lineage kinase domain-like (MLKL) protein indicative of necroptosis was observed to concur with caspase-cleavage (apoptotic marker) beyond 6 hr of RV infection. Subsequent studies demonstrated phosphorylated-MLKL to oligomerize and to translocate to plasma membrane in RV infected cells, resulting in loss of plasma membrane integrity and release of alarmin molecules e.g., high mobility group box protein 1 (HMGB1) in the extracellular media. Moreover, inhibiting caspase-cleavage and apoptosis could not fully rescue virus-induced cell death but rather potentiated the necroptotic trigger. Interestingly, preventing both apoptosis and necroptosis by small molecules significantly rescued virus-induced host cytopathy by inhibiting viral dissemination.

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

Viruses are intracellular pathogens and are dependent on host cellular resources to carry out their cycles of perpetuation. Obtaining an integrative view of host-virus interaction is of utmost importance to understand the complex and dynamic interplay between viral components and host machineries. Besides its obvious scholarly significance, a comprehensive host-virus interaction profile also provides a platform where from host determinants of pro-viral and antiviral importance can be identified and further be subjected to therapeutic intervention. Therefore, adjunct to conventional methods of prophylactic vaccination and virus-directed antivirals, this host-targeted antiviral approach holds promising therapeutic potential. In this review, we present a comprehensive landscape of host cellular reprogramming in response to infection with rotavirus (RV) which causes profuse watery diarrhea in neonates and infants. In addition, an emphasis is given on how host determinants are either usurped or subverted by RV in course of infection and how therapeutic manipulation of specific host factors can effectively modulate the RV life cycle.

Rotavirus Induces Epithelial-Mesenchymal Transition Markers by Transcriptional Suppression of miRNA-29b.

Acute gastroenteritis (AGE) is a serious global health problem and has been known to cause millions of infant deaths every year. Rotavirus (RV), a member of the Reoviridae family, still majorly accounts for the AGE in children below 5 years of age in India and worldwide. The involvement of miRNAs in the pathogenesis of RV has been suggested to be of the proviral as well as the anti-viral nature. miRNAs that promote the RV pathogenesis are capable of targeting the cellular components to evade the host anti-viral strategies. On the other hand, miRNAs with anti-rotaviral properties are themselves incapacitated during the progression of the infection. The exploitation of the epithelial-mesenchymal transition (EMT) as a pro-rotaviral strategy has already been identified. Thus, miRNAs that proficiently target the intermediates of the EMT pathway may serve as anti-viral counterparts in the RV-host interactions. The role of microRNA-29b (miR-29b) in the majority of human cancers has been well demonstrated, but its significance in viral infections is yet to be elaborated. In this study, we have assessed the role of miR-29b in RV-induced EMT and RV replication. Our study on miR-29b provides evidence for the recruitment of RV non-structural protein NSP1 to control the trans-repression of miR-29b in a p53-dependent manner. The trans-repression of miR-29b modulates the EMT pathway by targeting tripartite motif-containing protein 44 (TRIM44) and cyclin E1 (CCNE1). SLUG and SNAIL transcription repressors (downstream of TRIM44 and CCNE1) regulate the expression of E-cadherin, an important marker of the EMT. Also, it is established that ectopic expression of miR-29b not only constrains the EMT pathway but also restricts RV replication. Therefore, miR-29b repression is a crucial event in the RV pathogenesis. Ectopic expression of miR-29b displays potential anti-viral properties against RV propagation.

Progressive Rotavirus Infection Downregulates Redox-Sensitive Transcription Factor Nrf2 and Nrf2-Driven Transcription Units.

Eukaryotic cells adopt highly tuned stress response physiology under threats of exogenous stressors including viruses to maintain cellular homeostasis. Not surprisingly, avoidance of cellular stress response pathways is an essential facet of virus-induced obligatory host reprogramming to invoke a cellular environment conducive to viral perpetuation. Adaptive cellular responses to oxidative and electrophilic stress are usually taken care of by an antioxidant defense system, core to which lies the redox-responsive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2-driven transcriptional cascade. Deregulation of host redox balance and redox stress-sensitive Nrf2 antioxidant defense have been reported for many viruses. In the current study, we aimed to study the modulation of the Nrf2-based host cellular redox defense system in response to Rotavirus (RV) infection in vitro. Interestingly, we found that Nrf2 protein levels decline sharply with progression of RV infection beyond an initial upsurge. Moreover, Nrf2 decrease as a whole was found to be accompanied by active nuclear vacuity of Nrf2, resulting in lowered expression of stress-responsive Nrf2 target genes heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1, and superoxide dismutase 1 both in the presence and absence of Nrf2-driven transcriptional inducers. Initial induction of Nrf2 concurred with RV-induced early burst of oxidative stress and therefore was sensitive to treatments with antioxidants. Reduction of Nrf2 levels beyond initial hours, however, was found to be independent of the cellular redox status. Furthermore, increasing the half-life of Nrf2 through inhibition of the Kelch-like erythroid cell-derived protein with CNC homology- (ECH-) associated protein 1/Cullin3-RING Box1-based canonical Nrf2 turnover pathway could not restore Nrf2 levels post RV-SA11 infection. Depletion of the Nrf2/HO-1 axis was subsequently found to be sensitive to proteasome inhibition with concurrent observation of increased K48-linked ubiquitination associated with Nrf2. Together, the present study describes robust downregulation of Nrf2-dependent cellular redox defense beyond initial hours of RV infection, justifying our previous observation of potent antirotaviral implications of Nrf2 agonists.

Biphasic regulation of RNA interference during rotavirus infection by modulation of Argonaute2.

RNA interference (RNAi) is an evolutionary ancient innate immune response in plants, nematodes, and arthropods providing natural protection against viral infection. Viruses have also gained counter-defensive measures by producing virulence determinants called viral-suppressors-of-RNAi (VSRs). Interestingly, in spite of dominance of interferon-based immunity over RNAi in somatic cells of higher vertebrates, recent reports are accumulating in favour of retention of the antiviral nature of RNAi in mammalian cells. The present study focuses on the modulation of intracellular RNAi during infection with rotavirus (RV), an enteric virus with double-stranded RNA genome. Intriguingly, a time point-dependent bimodal regulation of RNAi was observed in RV-infected cells, where short interfering RNA (siRNA)-based RNAi was rendered non-functional during early hours of infection only to be reinstated fully beyond that early infection stage. Subsequent investigations revealed RV nonstructural protein 1 to serve as a putative VSR by associating with and triggering degradation of Argonaute2 (AGO2), the prime effector of siRNA-mediated RNAi, via ubiquitin-proteasome pathway. The proviral significance of AGO2 degradation was further confirmed when ectopic overexpression of AGO2 significantly reduced RV infection. Cumulatively, the current study presents a unique modulation of host RNAi during RV infection, highlighting the importance of antiviral RNAi in mammalian cells.

Synchronized Orchestration of miR-99b and let-7g Positively Regulates Rotavirus Infection by Modulating Autophagy.

Rotavirus (RV), the major etiological agent of viral gastroenteritis in young children, kills over 200 thousand infants each year. In spite of available vaccines, rotaviral diarrhoea is still a major problem in developing countries of Asia and Africa. Therefore, the studies on RV infection and host antiviral responses are warranted. The active correlation between virus infection and activation of autophagy machinery and positive influence of autophagy on RV replication have been documented recently. Previous study from our group showed dysregulation of several cellular miRNAs during RV infection, though their significance remained largely unknown. Since cellular microRNAs (miRNAs) have been implicated in the control of several fundamental biological processes including stress response and autophagy, we focused on two miRNAs, miR-99b and let-7g, and analyzed their function to gain insight into the miRNA-autophagy crosstalk during RV infection. This study shows that RV suppresses let-7g expression but enhances miR-99b that in turn augment major autophagy regulators. Ectopic expression of let-7g and knockdown of miR-99b resulted in inhibition of autophagy, hence, reduction of RV replication. Overall, our study highlights new mechanistic insights for understanding the role of miRNAs in modulating RV infection and possibility of using RNA interference as an antiviral therapeutic target.

RA-839, a selective agonist of Nrf2/ARE pathway, exerts potent anti-rotaviral efficacy in vitro.

Acute watery diarrhea due to Rotavirus (RV) infection is associated with high infantile morbidity and mortality in countries with compromised socio-economic backgrounds. Although showing promising trends in developed countries, the efficacy of currently licensed RV vaccines is sub-optimal in socio-economically poor settings with high disease burden. Currently, there are no approved anti-rotaviral drugs adjunct to classical vaccination program. Interestingly, dissecting host-rotavirus interaction has yielded novel, non-mutable host determinants which can be subjected to interventions by selective small molecules. The present study was undertaken to evaluate the anti-RV potential of RA-839, a recently discovered small molecule with potent and highly selective agonistic activity towards cellular redox stress-sensitive Nuclear factor erytheroid-derived-2-like 2 (Nrf2)/Antioxidant Response Element (ARE) pathway. In vitro studies revealed that RA-839 inhibits RV RNA and protein expression, viroplasm formation, yield of virion progeny and virus-induced cytopathy independent of RV strains, RV-permissive cell lines and without bystander cytotoxicity. Anti-RV potency of RA-839 was subsequently identified to be independent of stochastic Interferon (IFN) stimulation but to be dependent on RA-839's ability to stimulate Nrf2/ARE signaling. Interestingly, anti-rotaviral effects of RA-839 were also mimicked by 2-Cyano-3, 12-dioxo-oleana-1, 9(11)-dien-28-oic acid methyl ester (CDDO-Me) and Hemin, two classical pharmacological activators of Nrf2/ARE pathway. Overall, this study highlights that RA-839 is a potent antagonist of RV propagation in vitro and can be developed as anti-rotaviral therapeutics.