Mature Rotavirus Particles Contain Equivalent Amounts of 7meGpppG-Capped and Noncapped Viral Positive-Sense RNAs.
Journal of virology
Viruses have evolved different strategies to overcome their recognition by the host innate immune system. The addition of caps at their 5' RNA ends is an efficient mechanism not only to ensure escape from detection by the innate immune system but also to ensure the efficient synthesis of viral proteins. Rotavirus mRNAs contain a type 1 cap structure at their 5' end that is added by the viral capping enzyme VP3, which is a multifunctional protein with all the enzymatic activities necessary to add the cap and also functions as an antagonist of the 2'-5'-oligoadenylate synthetase (OAS)/RNase L pathway. Here, the relative abundances of capped and noncapped viral RNAs during the replication cycle of rotavirus were determined. We found that both classes of rotaviral plus-sense RNAs (+RNAs) were encapsidated and that they were present in a 1:1 ratio in the mature infectious particles. The capping of viral +RNAs was dynamic, since different ratios of capped and noncapped RNAs were detected at different times postinfection. Similarly, when the relative amounts of capped and uncapped viral +RNAs produced in an in vitro transcription system were determined, we found that the proportions were very similar to those in the mature viral particles and in infected cells, suggesting that the capping efficiency of VP3, both in vivo and in vitro, might be close to 50%. Unexpectedly, when the effect of simultaneously knocking down the expression of VP3 and RNase L on the cap status of viral +RNAs was evaluated, we found that, even though at late times postinfection there was an increased proportion of capped viral RNAs in infected cells, the viral particles isolated from this condition contained equal ratios of capped and noncapped viral RNA, suggesting that there might be selective packaging of capped and noncapped RNAs. IMPORTANCE Rotaviruses have a genome composed of 11 segments of double-stranded RNA. Whether all 5' ends of the positive-sense genomic RNAs contained in the mature viral particles are modified by a cap structure is unknown. In this work, we characterized the relative proportions of capped and noncapped viral RNAs in rotavirus-infected cells and in viral particles by using a direct quantitative assay. We found that, independent of the relative proportions of capped/noncapped RNAs present in rotavirus-infected cells, there were similar proportions of these two kinds of 5'-modified positive-sense RNAs in the viral particles.
View details for DOI 10.1128/jvi.01151-22
View details for PubMedID 36000838
The Role of the VP4 Attachment Protein in Rotavirus Host Range Restriction in an In Vivo Suckling Mouse Model
Journal of Virology
View details for DOI 10.1128/jvi.00550-22
A recombinant murine-like rotavirus with Nano-Luciferase expression reveals tissue tropism, replication dynamics, and virus transmission.
Frontiers in immunology
2022; 13: 911024
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.
View details for DOI 10.3389/fimmu.2022.911024
View details for PubMedID 35967392
An optimized reverse genetics system suitable for efficient recovery of simian, human and murine-like rotaviruses.
Journal of virology
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 RVA segment-specific (+)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. Secondly, a genetically modified MA104 cell line was used in which several compounds of the innate immune 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 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.
View details for DOI 10.1128/JVI.01294-20
View details for PubMedID 32759316
Reverse genetics reveals a role of the rotavirus VP3 phosphodiesterase activity in inhibiting RNase L signaling and contributing to intestinal viral replication in vivo.
Journal of virology
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 IFN-inducible 2'-5'-oligoadenylate synthetase (OAS) and ribonuclease L (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 a RV reverse genetics system, we show that compared to the parental strain, VP3 PDE mutant RVs replicated at a lower level in the small intestine and 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.ImportanceRotaviruses 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 to improve current vaccines, we aim to better understand how rotavirus interacts with the host innate immune system in the small intestine. Here, we demonstrate that the 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 the next-generation rotavirus vaccine design.
View details for DOI 10.1128/JVI.01952-19
View details for PubMedID 32051268
Retinoic Acid and Lymphotoxin Signaling Promote Differentiation of Human Intestinal M Cells.
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.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.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.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.
View details for DOI 10.1053/j.gastro.2020.03.053
View details for PubMedID 32247021