Reverse Genetics of Murine Rotavirus: A Comparative Analysis of the Wild-Type and Cell-Culture-Adapted Murine Rotavirus VP4 in Replication and Virulence in Neonatal Mice.

Small-animal models and reverse genetics systems are powerful tools for investigating the molecular mechanisms underlying viral replication, virulence, and interaction with the host immune response in vivo. Rotavirus (RV) causes acute gastroenteritis in many young animals and infants worldwide. Murine RV replicates efficiently in the intestines of inoculated suckling pups, causing diarrhea, and spreads efficiently to uninoculated littermates. Because RVs derived from human and other non-mouse animal species do not replicate efficiently in mice, murine RVs are uniquely useful in probing the viral and host determinants of efficient replication and pathogenesis in a species-matched mouse model. Previously, we established an optimized reverse genetics protocol for RV and successfully generated a murine-like RV rD6/2-2g strain that replicates well in both cultured cell lines and in the intestines of inoculated pups. However, rD6/2-2g possesses three out of eleven gene segments derived from simian RV strains, and these three heterologous segments may attenuate viral pathogenicity in vivo. Here, we rescued the first recombinant RV with all 11 gene segments of murine RV origin. Using this virus as a genetic background, we generated a panel of recombinant murine RVs with either N-terminal VP8* or C-terminal VP5* regions chimerized between a cell-culture-adapted murine ETD strain and a non-tissue-culture-adapted murine EW strain and compared the diarrhea rate and fecal RV shedding in pups. The recombinant viruses with VP5* domains derived from the murine EW strain showed slightly more fecal shedding than those with VP5* domains from the ETD strain. The newly characterized full-genome murine RV will be a useful tool for dissecting virus-host interactions and for studying the mechanism of pathogenesis in neonatal mice.

Lactobacillus acidophilus Expressing Murine Rotavirus VP8 and Mucosal Adjuvants Induce Virus-Specific Immune Responses.

Rotavirus diarrhea-associated illness remains a major cause of global death in children under five, attributable in part to discrepancies in vaccine performance between high- and low-middle-income countries. Next-generation probiotic vaccines could help bridge this efficacy gap. We developed a novel recombinant Lactobacillus acidophilus (rLA) vaccine expressing rotavirus antigens of the VP8* domain from the rotavirus EDIM VP4 capsid protein along with the adjuvants FimH and FliC. The upp-based counterselective gene-replacement system was used to chromosomally integrate FimH, VP8Pep (10 amino acid epitope), and VP8-1 (206 amino acid protein) into the L. acidophilus genome, with FliC expressed from a plasmid. VP8 antigen and adjuvant expression were confirmed by flow cytometry and Western blot. Rotavirus naïve adult BALB/cJ mice were orally immunized followed by murine rotavirus strain ECWT viral challenge. Antirotavirus serum IgG and antigen-specific antibody-secreting cell responses were detected in rLA-vaccinated mice. A day after the oral rotavirus challenge, fecal antigen shedding was significantly decreased in the rLA group. These results indicate that novel rLA constructs expressing VP8 can be successfully constructed and used to generate modest homotypic protection from rotavirus challenge in an adult murine model, indicating the potential for a probiotic next-generation vaccine construct against human rotavirus.

Mucosal and systemic neutralizing antibodies to norovirus induced in infant mice orally inoculated with recombinant rotaviruses.

Rotaviruses (RVs) preferentially replicate in the small intestine and frequently cause severe diarrheal disease, and the following enteric infection generally induces variable levels of protective systemic and mucosal immune responses in humans and other animals. Rhesus rotavirus (RRV) is a simian RV that was previously used as a human RV vaccine and has been extensively studied in mice. Although RRV replicates poorly in the suckling mouse intestine, infection induces a robust and protective antibody response. The recent availability of plasmid only-based RV reverse genetics systems has enabled the generation of recombinant RVs expressing foreign proteins. However, recombinant RVs have not yet been experimentally tested as potential vaccine vectors to immunize against other gastrointestinal pathogens in vivo. This is a newly available opportunity because several live-attenuated RV vaccines are already widely administered to infants and young children worldwide. To explore the feasibility of using RV as a dual vaccine vector, we rescued replication-competent recombinant RRVs harboring bicistronic gene segment 7 that encodes the native RV nonstructural protein 3 (NSP3) protein and a human norovirus (HuNoV) VP1 protein or P domain from the predominant genotype GII.4. The rescued viruses expressed HuNoV VP1 or P protein in infected cells in vitro and elicited systemic and local antibody responses to HuNoV and RRV following oral infection of suckling mice. Serum IgG and fecal IgA from infected suckling mice bound to and neutralized both RRV and HuNoV. These findings have encouraging practical implications for the design of RV-based next-generation multivalent enteric vaccines to target HuNoV and other human enteric pathogens.

A recombinant murine-like rotavirus with Nano-Luciferase expression reveals tissue tropism, replication dynamics, and virus transmission.

Rotaviruses (RVs) are one of the main causes of severe gastroenteritis, diarrhea, and death in children and young animals. While suckling mice prove to be highly useful small animal models of RV infection and pathogenesis, direct visualization tools are lacking to track the temporal dynamics of RV replication and transmissibility in vivo. Here, we report the generation of the first recombinant murine-like RV that encodes a Nano-Luciferase reporter (NLuc) using a newly optimized RV reverse genetics system. The NLuc-expressing RV was replication-competent in cell culture and both infectious and virulent in neonatal mice in vivo. Strong luciferase signals were detected in the proximal and distal small intestines, colon, and mesenteric lymph nodes. We showed, via a noninvasive in vivo imaging system, that RV intestinal replication peaked at days 2 to 5 post infection. Moreover, we successfully tracked RV transmission to uninoculated littermates as early as 3 days post infection, 1 day prior to clinically apparent diarrhea and 3 days prior to detectable fecal RV shedding in the uninoculated littermates. We also observed significantly increased viral replication in Stat1 knockout mice that lack the host interferon signaling. Our results suggest that the NLuc murine-like RV represents a non-lethal powerful tool for the studies of tissue tropism and host and viral factors that regulate RV replication and spread, as well as provides a new tool to facilitate the testing of prophylactic and therapeutic interventions in the future.

The Role of the VP4 Attachment Protein in Rotavirus Host Range Restriction in an In Vivo Suckling Mouse Model.

The basis for rotavirus (RV) host range restriction (HRR) is not fully understood but is likely multigenic. RV genes encoding VP3, VP4, NSP1, NSP2, NSP3, and NSP4 have been associated with HRR in various studies. With the exception of NSP1, little is known about the relative contribution of the other RV genes to HRR. VP4 has been linked to HRR because it functions as the RV cell attachment protein, but its actual role in HRR has not been fully assessed. We generated a collection of recombinant RVs (rRVs) in an isogenic murine-like RV genetic background, harboring either heterologous or homologous VP4 genes from simian, bovine, porcine, human, and murine RV strains, and characterized these rRVs in vitro and in vivo. We found that a murine-like rRV encoding a simian VP4 was shed, spread to uninoculated littermates, and induced diarrhea comparably to rRV harboring a murine VP4. However, rRVs carrying VP4s from both bovine and porcine RVs had reduced diarrhea, but no change in fecal shedding was observed. Both diarrhea and shedding were reduced when VP4 originated from a human RV strain. rRVs harboring VP4s from human or bovine RVs did not transmit to uninoculated littermates. We also generated two rRVs harboring reciprocal chimeric murine or bovine VP4. Both chimeras replicated and caused disease as efficiently as the parental strain with a fully murine VP4. These data suggest that the genetic origin of VP4 partially modulates HRR in the suckling mouse and that both the VP8* and VP5* domains independently contribute to pathogenesis and transmission. IMPORTANCE Human group A rotaviruses (RVs) remain the most important cause of severe acute gastroenteritis among infants and young children worldwide despite the introduction of several safe and effective live attenuated vaccines. The lack of knowledge regarding fundamental aspects of RV biology, such as the genetic basis of host range restriction (HRR), has made it difficult to predictively and efficiently design improved, next-generation live attenuated rotavirus vaccines. Here, we engineered a collection of VP4 monoreassortant RVs to systematically explore the role of VP4 in replication, pathogenicity, and spread, as measures of HRR, in a suckling mouse model. The genetic and mechanistic bases of HRR have substantial clinical relevance given that this restriction forms the basis of attenuation for several replication-competent human RV vaccines. In addition, a better understanding of RV pathogenesis and the determinants of RV spread is likely to enhance our ability to improve antiviral drug and therapy development.

A CD22-Shp1 phosphatase axis controls integrin ß7 display and B cell function in mucosal immunity.

The integrin α4ß7 selectively regulates lymphocyte trafficking and adhesion in the gut and gut-associated lymphoid tissue (GALT). Here, we describe unexpected involvement of the tyrosine phosphatase Shp1 and the B cell lectin CD22 (Siglec-2) in the regulation of α4ß7 surface expression and gut immunity. Shp1 selectively inhibited ß7 endocytosis, enhancing surface α4ß7 display and lymphocyte homing to GALT. In B cells, CD22 associated in a sialic acid-dependent manner with integrin ß7 on the cell surface to target intracellular Shp1 to ß7. Shp1 restrained plasma membrane ß7 phosphorylation and inhibited ß7 endocytosis without affecting ß1 integrin. B cells with reduced Shp1 activity, lacking CD22 or expressing CD22 with mutated Shp1-binding or carbohydrate-binding domains displayed parallel reductions in surface α4ß7 and in homing to GALT. Consistent with the specialized role of α4ß7 in intestinal immunity, CD22 deficiency selectively inhibited intestinal antibody and pathogen responses.

An Optimized Reverse Genetics System Suitable for Efficient Recovery of Simian, Human, and Murine-Like Rotaviruses.

An entirely plasmid-based reverse genetics (RG) system was recently developed for rotavirus (RV), opening new avenues for in-depth molecular dissection of RV biology, immunology, and pathogenesis. Several improvements to further optimize the RG efficiency have now been described. However, only a small number of individual RV strains have been recovered to date. None of the current methods have supported the recovery of murine RV, impeding the study of RV replication and pathogenesis in an in vivo suckling mouse model. Here, we describe useful modifications to the RG system that significantly improve rescue efficiency of multiple RV strains. In addition to the 11 group A RV segment-specific (+)RNAs [(+)ssRNAs], a chimeric plasmid was transfected, from which the capping enzyme NP868R of African swine fever virus (ASFV) and the T7 RNA polymerase were expressed. Second, a genetically modified MA104 cell line was used in which several components of the innate immunity were degraded. Using this RG system, we successfully recovered the simian RV RRV strain, the human RV CDC-9 strain, a reassortant between murine RV D6/2 and simian RV SA11 strains, and several reassortants and reporter RVs. All these recombinant RVs were rescued at a high efficiency (≥80% success rate) and could not be reliably rescued using several recently published RG strategies (<20%). This improved system represents an important tool and great potential for the rescue of other hard-to-recover RV strains such as low-replicating attenuated vaccine candidates or low-cell culture passage clinical isolates from humans or animals.IMPORTANCE Group A rotavirus (RV) remains as the single most important cause of severe acute gastroenteritis among infants and young children worldwide. An entirely plasmid-based reverse genetics (RG) system was recently developed, opening new ways for in-depth molecular study of RV. Despite several improvements to further optimize the RG efficiency, it has been reported that current strategies do not enable the rescue of all cultivatable RV strains. Here, we described a helpful modification to the current strategies and established a tractable RG system for the rescue of the simian RRV strain, the human CDC-9 strain, and a murine-like RV strain, which is suitable for both in vitro and in vivo studies. This improved RV reverse genetics system will facilitate study of RV biology in both in vitro and in vivo systems that will facilitate the improved design of RV vaccines, better antiviral therapies, and expression vectors.

Retinoic Acid and Lymphotoxin Signaling Promote Differentiation of Human Intestinal M Cells.

BACKGROUND & AIMS: Intestinal microfold (M) cells are a unique subset of intestinal epithelial cells in the Peyer's patches that regulate mucosal immunity, serving as portals for sampling and uptake of luminal antigens. The inability to efficiently develop human M cells in cell culture has impeded studies of the intestinal immune system. We aimed to identify signaling pathways required for differentiation of human M cells and establish a robust culture system using human ileum enteroids. METHODS: We analyzed transcriptome data from mouse Peyer's patches to identify cell populations in close proximity to M cells. We used the human enteroid system to determine which cytokines were required to induce M-cell differentiation. We performed transcriptome, immunofluorescence, scanning electron microscope, and transcytosis experiments to validate the development of phenotypic and functional human M cells. RESULTS: A combination of retinoic acid and lymphotoxin induced differentiation of glycoprotein 2-positive human M cells, which lack apical microvilli structure. Upregulated expression of innate immune-related genes within M cells correlated with a lack of viral antigens after rotavirus infection. Human M cells, developed in the enteroid system, internalized and transported enteric viruses, such as rotavirus and reovirus, across the intestinal epithelium barrier in the enteroids. CONCLUSIONS: We identified signaling pathways required for differentiation of intestinal M cells, and used this information to create a robust culture method to develop human M cells with capacity for internalization and transport of viruses. Studies of this model might increase our understanding of antigen presentation and the systemic entry of enteric pathogens in the human intestine.

Reverse Genetics Reveals a Role of Rotavirus VP3 Phosphodiesterase Activity in Inhibiting RNase L Signaling and Contributing to Intestinal Viral Replication In Vivo.

Our understanding of how rotavirus (RV) subverts host innate immune signaling has greatly increased over the past decade. However, the relative contribution of each virus-encoded innate immune antagonist has not been fully studied in the context of RV infection in vivo Here, we present both in vitro and in vivo evidence that the host interferon (IFN)-inducible 2'-5'-oligoadenylate synthetase (OAS) and RNase L pathway effectively suppresses the replication of heterologous RV strains. VP3 from homologous RVs relies on its 2'-5'-phosphodiesterase (PDE) domain to counteract RNase L-mediated antiviral signaling. Using an RV reverse-genetics system, we show that compared to the parental strain, VP3 PDE mutant RVs replicated at low levels in the small intestine and were shed less in the feces of wild-type mice, and such defects were rescued in Rnasel-/- suckling mice. Collectively, these findings highlight an important role of VP3 in promoting viral replication and pathogenesis in vivo in addition to its well-characterized function as the viral RNA-capping enzyme.IMPORTANCE Rotaviruses are significant human pathogens that result in diarrhea, dehydration, and deaths in many children around the world. Rotavirus vaccines have suboptimal efficacy in low- to middle-income countries, where the burden of the diseases is the most severe. With the ultimate goal of improving current vaccines, we aim to better understand how rotavirus interacts with the host innate immune system in the small intestine. Here, we demonstrate that interferon-activated RNase L signaling blocks rotavirus replication in a strain-specific manner. In addition, virus-encoded VP3 antagonizes RNase L activity both in vitro and in vivo These studies highlight an ever-evolving arms race between antiviral factors and viral pathogens and provide a new means of targeted attenuation for next-generation rotavirus vaccine design.

Rotavirus Reprograms Multiple Interferon Receptors and Restricts Their Intestinal Antiviral and Inflammatory Functions.

Rotaviruses (RV) cause acute severe diarrhea in the absence of substantial intestinal inflammation. They are also highly infectious in their homologous host species. The replication capacity of RV in the small bowel is substantially due to its ability to inhibit different types of interferons (IFNs). Here, we found that during RV infection in vitro, both virus-infected and uninfected bystander cells resist STAT1 phosphorylation and interferon regulatory factor 7 (IRF7) induction in response to exogenous interferon (IFN). Functionally, cellular transcription in response to stimulation with IFN, but not intracellular double-stranded RNA (dsRNA), was inhibited by RV. Further, IFNAR1 stimulation during RV infection significantly repressed a set of virus-induced transcripts. Regulation of IFN signaling in vivo was studied in suckling mice using the highly infectious murine EW RV strain. Kinetic studies indicated that sustained EW RV replication and IFN induction in the small intestine are accompanied by significant decreases in IFN-stimulated transcripts. Lipopolysaccharide (LPS)-mediated intestinal damage, driven by STAT1-induced inflammation, was also prevented in EW RV-infected mice. Remarkably, by ectopically stimulating either IFNAR1 or IFNGR1 in EW RV-infected mice, we could eliminate several intestinal antiviral and inflammatory transcriptional responses to RV. In contrast to infection with homologous RV, infection with a STAT1-sensitive heterologous RV strain induced IFN-stimulated transcripts, inflammatory cytokines, and intestinal expression of STAT1-pY701. Finally, RV strain-specific STAT1 regulation also likely determines the intestinal activation of multiple caspases. The simian RRV strain, but not murine EW RV, uniquely triggers the cleavage of both extrinsic and intrinsic caspases (caspases 8, 9, and 3) in a STAT1-mediated manner. Collectively, our findings reveal efficient reprograming of multiple IFN receptors toward a negative-feedback mode of signaling, accompanied by suppression of IFN-mediated antiviral, apoptotic, and inflammatory functions, during natural RV intestinal infection.IMPORTANCE Rotavirus is a highly infectious pathogen that causes severe diarrhea. Replication of RV in the small intestine is restricted to homologous host species, and host range restriction is substantially determined by the interferon response. In this study, we demonstrate that during infection, RV bystander cells resist exogenous IFN-mediated STAT1 signaling and transcription. In a suckling mouse model, ectopically stimulating different intestinal interferon receptors during RV infection eliminates several innate and inflammatory antiviral responses. Different intestinal inflammatory cytokines were also suppressed by homologous RV, as was intestinal damage in response to endotoxin. The ability of RV to suppress IFN-mediated receptors likely impacts intestinal cell homeostasis, as the cleavage of multiple intestinal caspases during RV infection is mediated by the IFN-STAT1 signaling pathway. Together, our results provide a mechanism underlying both the remarkable interferon resistance of homologous RV and its ability to prevent substantial inflammatory damage to the small bowel.

Human VP8* mAbs neutralize rotavirus selectively in human intestinal epithelial cells.

We previously generated 32 rotavirus-specific (RV-specific) recombinant monoclonal antibodies (mAbs) derived from B cells isolated from human intestinal resections. Twenty-four of these mAbs were specific for the VP8* fragment of RV VP4, and most (20 of 24) were non-neutralizing when tested in the conventional MA104 cell-based assay. We reexamined the ability of these mAbs to neutralize RVs in human intestinal epithelial cells including ileal enteroids and HT-29 cells. Most (18 of 20) of the "non-neutralizing" VP8* mAbs efficiently neutralized human RV in HT-29 cells or enteroids. Serum RV neutralization titers in adults and infants were significantly higher in HT-29 than MA104 cells and adsorption of these sera with recombinant VP8* lowered the neutralization titers in HT-29 but not MA104 cells. VP8* mAbs also protected suckling mice from diarrhea in an in vivo challenge model. X-ray crystallographic analysis of one VP8* mAb (mAb9) in complex with human RV VP8* revealed that the mAb interaction site was distinct from the human histo-blood group antigen binding site. Since MA104 cells are the most commonly used cell line to detect anti-RV neutralization activity, these findings suggest that prior vaccine and other studies of human RV neutralization responses may have underestimated the contribution of VP8* antibodies to the overall neutralization titer.

Rotavirus VP3 targets MAVS for degradation to inhibit type III interferon expression in intestinal epithelial cells.

Rotaviruses (RVs), a leading cause of severe diarrhea in young children and many mammalian species, have evolved multiple strategies to counteract the host innate immunity, specifically interferon (IFN) signaling through RV non-structural protein 1 (NSP1). However, whether RV structural components also subvert antiviral response remains under-studied. Here, we found that MAVS, critical for the host RNA sensing pathway upstream of IFN induction, is degraded by the RV RNA methyl- and guanylyl-transferase (VP3) in a host-range-restricted manner. Mechanistically, VP3 localizes to the mitochondria and mediates the phosphorylation of a previously unidentified SPLTSS motif within the MAVS proline-rich region, leading to its proteasomal degradation and blockade of IFN-λ production in RV-infected intestinal epithelial cells. Importantly, VP3 inhibition of MAVS activity contributes to enhanced RV replication and to viral pathogenesis in vivo. Collectively, our findings establish RV VP3 as a viral antagonist of MAVS function in mammals and uncover a novel pathogen-mediated inhibitory mechanism of MAVS signaling.

STAG2 deficiency induces interferon responses via cGAS-STING pathway and restricts virus infection.

Cohesin is a multi-subunit nuclear protein complex that coordinates sister chromatid separation during cell division. Highly frequent somatic mutations in genes encoding core cohesin subunits have been reported in multiple cancer types. Here, using a genome-wide CRISPR-Cas9 screening approach to identify host dependency factors and novel innate immune regulators of rotavirus (RV) infection, we demonstrate that the loss of STAG2, an important component of the cohesin complex, confers resistance to RV replication in cell culture and human intestinal enteroids. Mechanistically, STAG2 deficiency results in spontaneous genomic DNA damage and robust interferon (IFN) expression via the cGAS-STING cytosolic DNA-sensing pathway. The resultant activation of JAK-STAT signaling and IFN-stimulated gene (ISG) expression broadly protects against virus infections, including RVs. Our work highlights a previously undocumented role of the cohesin complex in regulating IFN homeostasis and identifies new therapeutic avenues for manipulating the innate immunity.

Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells.

Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.

VP4- and VP7-specific antibodies mediate heterotypic immunity to rotavirus in humans.

Human rotaviruses (RVs) are the leading cause of severe diarrhea in young children worldwide. The molecular mechanisms underlying the rapid induction of heterotypic protective immunity to RV, which provides the basis for the efficacy of licensed monovalent RV vaccines, have remained unknown for more than 30 years. We used RV-specific single cell-sorted intestinal B cells from human adults, barcode-based deep sequencing of antibody repertoires, monoclonal antibody expression, and serologic and functional characterization to demonstrate that infection-induced heterotypic immunoglobulins (Igs) primarily directed to VP5*, the stalk region of the RV attachment protein, VP4, are able to mediate heterotypic protective immunity. Heterotypic protective Igs against VP7, the capsid glycoprotein, and VP8*, the cell-binding region of VP4, are also generated after infection; however, our data suggest that homotypic anti-VP7 and non-neutralizing VP8* responses occur more commonly in people. These results indicate that humans can circumvent the extensive serotypic diversity of circulating RV strains by generating frequent heterotypic neutralizing antibody responses to VP7, VP8*, and most often, to VP5* after natural infection. These findings further suggest that recombinant VP5* may represent a useful target for the development of an improved, third-generation, broadly effective RV vaccine and warrants more direct examination.

Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis.

Despite the wide administration of several effective vaccines, rotavirus (RV) remains the single most important etiological agent of severe diarrhea in infants and young children worldwide, with an annual mortality of over 200,000 people. RV attachment and internalization into target cells is mediated by its outer capsid protein VP4. To better understand the molecular details of RV entry, we performed tandem affinity purification coupled with high-resolution mass spectrometry to map the host proteins that interact with VP4. We identified an actin-binding protein, drebrin (DBN1), that coprecipitates and colocalizes with VP4 during RV infection. Importantly, blocking DBN1 function by siRNA silencing, CRISPR knockout (KO), or chemical inhibition significantly increased host cell susceptibility to RV infection. Dbn1 KO mice exhibited higher incidence of diarrhea and more viral antigen shedding in their stool samples compared with the wild-type littermates. In addition, we found that uptake of other dynamin-dependent cargos, including transferrin, cholera toxin, and multiple viruses, was also enhanced in DBN1-deficient cells. Inhibition of cortactin or dynamin-2 abrogated the increased virus entry observed in DBN1-deficient cells, suggesting that DBN1 suppresses dynamin-mediated endocytosis via interaction with cortactin. Our study unveiled an unexpected role of DBN1 in restricting the entry of RV and other viruses into host cells and more broadly to function as a crucial negative regulator of diverse dynamin-dependent endocytic pathways.

Comparative Proteomics Reveals Strain-Specific ß-TrCP Degradation via Rotavirus NSP1 Hijacking a Host Cullin-3-Rbx1 Complex.

Rotaviruses (RVs) are the leading cause of severe gastroenteritis in young children, accounting for half a million deaths annually worldwide. RV encodes non-structural protein 1 (NSP1), a well-characterized interferon (IFN) antagonist, which facilitates virus replication by mediating the degradation of host antiviral factors including IRF3 and ß-TrCP. Here, we utilized six human and animal RV NSP1s as baits and performed tandem-affinity purification coupled with high-resolution mass spectrometry to comprehensively characterize NSP1-host protein interaction network. Multiple Cullin-RING ubiquitin ligase (CRL) complexes were identified. Importantly, inhibition of cullin-3 (Cul3) or RING-box protein 1 (Rbx1), by siRNA silencing or chemical perturbation, significantly impairs strain-specific NSP1-mediated ß-TrCP degradation. Mechanistically, we demonstrate that NSP1 localizes to the Golgi with the host Cul3-Rbx1 CRL complex, which targets ß-TrCP and NSP1 for co-destruction at the proteasome. Our study uncovers a novel mechanism that RV employs to promote ß-TrCP turnover and provides molecular insights into virus-mediated innate immunity inhibition.

Correction: Distinct Roles of Type I and Type III Interferons in Intestinal Immunity to Homologous and Heterologous Rotavirus Infections.

[This corrects the article DOI: 10.1371/journal.ppat.1005600.].

Distinct Roles of Type I and Type III Interferons in Intestinal Immunity to Homologous and Heterologous Rotavirus Infections.

Type I (IFN-α/ß) and type III (IFN-λ) interferons (IFNs) exert shared antiviral activities through distinct receptors. However, their relative importance for antiviral protection of different organ systems against specific viruses remains to be fully explored. We used mouse strains deficient in type-specific IFN signaling, STAT1 and Rag2 to dissect distinct and overlapping contributions of type I and type III IFNs to protection against homologous murine (EW-RV strain) and heterologous (non-murine) simian (RRV strain) rotavirus infections in suckling mice. Experiments demonstrated that murine EW-RV is insensitive to the action of both types of IFNs, and that timely viral clearance depends upon adaptive immune responses. In contrast, both type I and type III IFNs can control replication of the heterologous simian RRV in the gastrointestinal (GI) tract, and they cooperate to limit extra-intestinal simian RRV replication. Surprisingly, intestinal epithelial cells were sensitive to both IFN types in neonatal mice, although their responsiveness to type I, but not type III IFNs, diminished in adult mice, revealing an unexpected age-dependent change in specific contribution of type I versus type III IFNs to antiviral defenses in the GI tract. Transcriptional analysis revealed that intestinal antiviral responses to RV are triggered through either type of IFN receptor, and are greatly diminished when receptors for both IFN types are lacking. These results also demonstrate a murine host-specific resistance to IFN-mediated antiviral effects by murine EW-RV, but the retention of host efficacy through the cooperative action by type I and type III IFNs in restricting heterologous simian RRV growth and systemic replication in suckling mice. Collectively, our findings revealed a well-orchestrated spatial and temporal tuning of innate antiviral responses in the intestinal tract where two types of IFNs through distinct patterns of their expression and distinct but overlapping sets of target cells coordinately regulate antiviral defenses against heterologous or homologous rotaviruses with substantially different effectiveness.

Permissive replication of homologous murine rotavirus in the mouse intestine is primarily regulated by VP4 and NSP1.

Homologous rotaviruses (RV) are, in general, more virulent and replicate more efficiently than heterologous RV in the intestine of the homologous host. The genetic basis for RV host range restriction is not fully understood and is likely to be multigenic. In previous studies, RV genes encoding VP3, VP4, VP7, nonstructural protein 1 (NSP1), and NSP4 have all been implicated in strain- and host species-specific infection. These studies used different RV strains, variable measurements of host range, and different animal hosts, and no clear consensus on the host range restriction determinants emerged. We used a murine model to demonstrate that enteric replication of murine RV EW is 1,000- to 10,000-fold greater than that of a simian rotavirus (RRV) in suckling mice. Intestinal replication of a series of EW × RRV reassortants was used to identify several RV genes that influenced RV replication in the intestine. The role of VP4 (encoded by gene 4) in enteric infection was strain specific. RRV VP4 reduced murine RV infectivity only slightly; however, a reassortant expressing VP4 from a bovine RV strain (UK) severely restricted intestinal replication in the suckling mice. The homologous murine EW NSP1 (encoded by gene 5) was necessary but not sufficient for promoting efficient enteric growth. Efficient enteric replication required a constellation of murine genes encoding VP3, NSP2, and NSP3 along with NSP1.