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Phosphoproteomics-based platforms have been widely used to identify post translational dynamics of cellular proteins in response to viral infection. The present study was undertaken to assess differential tyrosine phosphorylation during early hours of rotavirus (RV) SA11 infection. Heat shock proteins (Hsp60) were found to be enriched in the data set of RV-SA11 induced differentially tyrosine-phosphorylated proteins at 2 hr post infection (hpi). Hsp60 was further found to be phosphorylated by an activated form of Src kinase on 227th tyrosine residue, and tyrosine phosphorylation of mitochondrial chaperonin Hsp60 correlated with its proteasomal degradation at 2-2.5hpi. Interestingly, mitochondrial Hsp60 positively influenced translocation of the rotaviral nonstructural protein 4 to mitochondria during RV infections. Phosphorylation and subsequent transient degradation of mitochondrial Hsp60 during early hours of RV-SA11 infection resulted in inhibition of premature import of nonstructural protein 4 into mitochondria, thereby delaying early apoptosis. Overall, the study highlighted one of the many strategies rotavirus undertakes to prevent early apoptosis and subsequent reduced viral progeny yield.
© 2016 John Wiley & Sons Ltd.
Shared VH1-46 gene usage has been described in B cells reacting to desmoglein 3 (Dsg3) in the autoimmune disease pemphigus vulgaris (PV), as well as B cells responding to rotavirus capsid protein VP6. In both diseases, VH1-46 B cells bearing few to no somatic mutations can recognize the disease Ag. This intriguing connection between an autoimmune response to self-antigen and an immune response to foreign Ag prompted us to investigate whether VH1-46 B cells may be predisposed to Dsg3-VP6 cross-reactivity. Focused testing of VH1-46 mAbs previously isolated from PV and rotavirus-exposed individuals indicates that cross-reactivity is rare, found in only one of seven VH1-46 IgG clonotypes. High-throughput screening of IgG B cell repertoires from two PV patients identified no additional cross-reactive clonotypes. Screening of IgM B cell repertoires from one non-PV and three PV patients identified specific cross-reactive Abs in one PV patient, but notably all six cross-reactive clonotypes used VH1-46. Site-directed mutagenesis studies indicate that amino acid residues predisposing VH1-46 Abs to Dsg3 reactivity reside in CDR2. However, somatic mutations only rarely promote Dsg3-VP6 cross-reactivity; most mutations abolish VP6 and/or Dsg3 reactivity. Nevertheless, functional testing identified two cross-reactive VH1-46 Abs that both disrupt keratinocyte adhesion and inhibit rotavirus replication, indicating the potential for VH1-46 Abs to have both pathologic autoimmune and protective immune functions. Taken together, these studies suggest that certain VH1-46 B cell populations may be predisposed to Dsg3-VP6 cross-reactivity, but multiple mechanisms prevent the onset of autoimmunity after rotavirus exposure.
Copyright © 2016 by The American Association of Immunologists, Inc.
UNLABELLED - Synthesis of 2'-5'-oligoadenylates (2-5A) by oligoadenylate synthetase (OAS) is an important innate cellular response that limits viral replication by activating the latent cellular RNase, RNase L, to degrade single-stranded RNA. Some rotaviruses and coronaviruses antagonize the OAS/RNase L pathway through the activity of an encoded 2H phosphoesterase domain that cleaves 2-5A. These viral 2H phosphoesterases are phylogenetically related to the cellular A kinase anchoring protein 7 (AKAP7) and share a core structure and an active site that contains two well-defined HΦ(S/T)Φ (where Φ is a hydrophobic residue) motifs, but their mechanism of substrate binding is unknown. Here, we report the structures of a viral 2H phosphoesterase, the C-terminal domain (CTD) of the group A rotavirus (RVA) VP3 protein, both alone and in complex with 2-5A. The domain forms a compact fold, with a concave β-sheet that contains the catalytic cleft, but it lacks two α-helical regions and two β-strands observed in AKAP7 and other 2H phosphoesterases. The cocrystal structure shows significant conformational changes in the R loop upon ligand binding. Bioinformatics and biochemical analyses reveal that conserved residues and residues required for catalytic activity and substrate binding comprise the catalytic motifs and a region on one side of the binding cleft. We demonstrate that the VP3 CTD of group B rotavirus, but not that of group G, cleaves 2-5A. These findings suggest that the VP3 CTD is a streamlined version of a 2H phosphoesterase with a ligand-binding mechanism that is shared among 2H phosphodiesterases that cleave 2-5A.
IMPORTANCE - The C-terminal domain (CTD) of rotavirus VP3 is a 2H phosphoesterase that cleaves 2'-5'-oligoadenylates (2-5A), potent activators of an important innate cellular antiviral pathway. 2H phosphoesterase superfamily proteins contain two conserved catalytic motifs and a proposed core structure. Here, we present structures of a viral 2H phosphoesterase, the rotavirus VP3 CTD, alone and in complex with its substrate, 2-5A. The domain lacks two α-helical regions and β-strands present in other 2H phosphoesterases. A loop of the protein undergoes significant structural changes upon substrate binding. Together with our bioinformatics and biochemical findings, the crystal structures suggest that the RVA VP3 CTD domain is a streamlined version of a cellular enzyme that shares a ligand-binding mechanism with other 2H phosphodiesterases that cleave 2-5A but differs from those of 2H phosphodiesterases that cleave other substrates. These findings may aid in the future design of antivirals targeting viral phosphodiesterases with cleavage specificity for 2-5A.
The innate immune response involves a broad array of pathogen sensors that stimulate the production of interferons (IFNs) to induce an antiviral state. Rotavirus, a significant cause of childhood gastroenteritis and a member of the Reoviridae family of segmented, double-stranded RNA viruses, encodes at least two direct antagonists of host innate immunity: NSP1 and VP3. NSP1, a putative E3 ubiquitin ligase, mediates the degradation of cellular factors involved in both IFN induction and downstream signaling. VP3, the viral capping enzyme, utilizes a 2H-phosphodiesterase domain to prevent activation of the cellular oligoadenylate synthase (OAS)/RNase L pathway. Computational, molecular, and biochemical studies have provided key insights into the structural and mechanistic basis of innate immune antagonism by NSP1 and VP3 of group A rotaviruses (RVA). Future studies with non-RVA isolates will be essential to understand how other rotavirus species evade host innate immune responses.
Published by Elsevier Inc.
Activators of innate immunity may have the potential to combat a broad range of infectious agents. We report that treatment with bacterial flagellin prevented rotavirus (RV) infection in mice and cured chronically RV-infected mice. Protection was independent of adaptive immunity and interferon (IFN, type I and II) and required flagellin receptors Toll-like receptor 5 (TLR5) and NOD-like receptor C4 (NLRC4). Flagellin-induced activation of TLR5 on dendritic cells elicited production of the cytokine interleukin-22 (IL-22), which induced a protective gene expression program in intestinal epithelial cells. Flagellin also induced NLRC4-dependent production of IL-18 and immediate elimination of RV-infected cells. Administration of IL-22 and IL-18 to mice fully recapitulated the capacity of flagellin to prevent or eliminate RV infection and thus holds promise as a broad-spectrum antiviral agent.
Copyright © 2014, American Association for the Advancement of Science.
UNLABELLED - Rotaviruses and orbiviruses are nonturreted Reoviridae members. The rotavirus VP3 protein is a multifunctional capping enzyme and antagonist of the interferon-induced cellular oligoadenylate synthetase-RNase L pathway. Despite mediating important processes, VP3 is the sole protein component of the rotavirus virion whose structure remains unknown. In the current study, we used sequence alignment and homology modeling to identify features common to nonturreted Reoviridae capping enzymes and to predict the domain organization, structure, and active sites of rotavirus VP3. Our results suggest that orbivirus and rotavirus capping enzymes share a domain arrangement similar to that of the bluetongue virus capping enzyme. Sequence alignments revealed conserved motifs and suggested that rotavirus and orbivirus capping enzymes contain a variable N-terminal domain, a central guanine-N7-methyltransferase domain that contains an additional inserted domain, and a C-terminal guanylyltransferase and RNA 5'-triphosphatase domain. Sequence conservation and homology modeling suggested that the insertion in the guanine-N7-methyltransferase domain is a ribose-2'-O-methyltransferase domain for most rotavirus species. Our analyses permitted putative identification of rotavirus VP3 active-site residues, including those that form the ribose-2'-O-methyltransferase catalytic tetrad, interact with S-adenosyl-l-methionine, and contribute to autoguanylation. Previous reports have indicated that group A rotavirus VP3 contains a C-terminal 2H-phosphodiesterase domain that can cleave 2'-5' oligoadenylates, thereby preventing RNase L activation. Our results suggest that a C-terminal phosphodiesterase domain is present in the capping enzymes from two additional rotavirus species. Together, these findings provide insight into a poorly understood area of rotavirus biology and are a springboard for future biochemical and structural studies of VP3.
IMPORTANCE - Rotaviruses are an important cause of severe diarrheal disease. The rotavirus VP3 protein caps viral mRNAs and helps combat cellular innate antiviral defenses, but little is known about its structure or enzymatic mechanisms. In this study, we used sequence- and structure-based alignments with related proteins to predict the structure of VP3 and identify enzymatic domains and active sites therein. This work provides insight into the mechanisms of rotavirus transcription and evasion of host innate immune defenses. An improved understanding of these processes may aid our ability to develop rotavirus vaccines and therapeutics.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
BACKGROUND - Nicaragua was the first developing nation to implement routine immunization with the pentavalent rotavirus vaccine (RV5). In this RV5-immunized population, understanding infectious etiologies of childhood diarrhea is necessary to direct diarrhea treatment and prevention efforts.
METHODS - We followed a population-based sample of children <5 years in León, Nicaragua for diarrhea episodes through household visits. Information was obtained on RV5 history and sociodemographics. Stool samples collected during diarrhea episodes and among healthy children underwent laboratory analysis for viral, bacterial and parasitic enteropathogens. Detection frequency and incidence of each enteropathogen was calculated.
RESULTS - The 826 children in the cohort experienced 677 diarrhea episodes during 607.5 child-years of exposure time (1.1 episodes per child-year). At least 1 enteropathogen was detected among 61.1% of the 337 diarrheal stools collected. The most common enteropathogens among diarrheal stools were: norovirus (20.4%), sapovirus (16.6%), enteropathogenic Escherichia coli (11.3%), Entamoeba histolytica/dispar (8.3%), Giardia lamblia (8.0%) and enterotoxigenic E. coli (7.7%), with rotavirus detected among 5.3% of diarrheal stools. Enteropathogenic Escherichia coli and enterotoxigenic E. coli were frequently detected among stools from healthy children. Among children with diarrhea, norovirus was more commonly detected among younger children (< 2 years) and G. lamblia was more commonly detected among older children (2-4 years). The mean age of rotavirus detection was 34.6 months.
CONCLUSIONS - In this Central American community after RV5 introduction, rotavirus was not commonly detected among children with diarrhea. Prevention and appropriate management of norovirus and sapovirus should be considered to further reduce the burden of diarrheal disease.
UNLABELLED - Group A rotaviruses (RVs) remain a leading cause of childhood gastroenteritis worldwide. Although the G/P types of locally circulating RVs can vary from year to year and differ depending upon geographical location, those with G1P, G2P, G3P, G4P, G9P, and G12P specificities typically dominate. Little is known about the evolution and diversity of G2P RVs and the possible role that widespread vaccine use has had on their increased frequency of detection. To address these issues, we analyzed the 12 G2P RV isolates associated with a rise in RV gastroenteritis cases at Vanderbilt University Medical Center (VUMC) during the 2010-2011 winter season. Full-genome sequencing revealed that the isolates had genotype 2 constellations typical of DS-1-like viruses (G2P-I2-R2-C2-M2-A2-N2-T2-E2-H2). Phylogenetic analyses showed that the genome segments of the isolates were comprised of two or three different subgenotype alleles; this enabled recognition of three distinct clades of G2P viruses that caused disease at VUMC in the 2010-2011 season. Although the three clades cocirculated in the same community, there was no evidence of interclade reassortment. Bayesian analysis of 328 VP7 genes of G2 viruses isolated in the last 39 years indicate that existing G2 VP7 gene lineages continue to evolve and that novel lineages, as represented by the VUMC isolates, are constantly being formed. Moreover, G2 lineages are characteristically shaped by lineage turnover events that introduce new globally dominant strains every 7 years, on average. The ongoing evolution of G2 VP7 lineages may give rise to antigenic changes that undermine vaccine effectiveness in the long term.
IMPORTANCE - Little is known about the diversity of cocirculating G2 rotaviruses and how their evolution may undermine the effectiveness of rotavirus vaccines. To expand our understanding of the potential genetic range exhibited by rotaviruses circulating in postvaccine communities, we analyzed part of a collection of rotaviruses recovered from pediatric patients in the United States from 2010 to 2011. Examining the genetic makeup of these viruses revealed they represented three segregated groups that did not exchange genetic material. The distinction between these three groups may be explained by three separate introductions. By comparing a specific gene, namely, VP7, of the recent rotavirus isolates to those from a collection recovered from U.S. children between 1974 and 1991 and other globally circulating rotaviruses, we were able to reconstruct the timing of events that shaped their ancestry. This analysis indicates that G2 rotaviruses are continuously evolving, accumulating changes in their genetic material as they infect new patients.
AIMS - Antibodies are the principal mediator of immunity against reinfection with viruses. Antibodies typically neutralize viruses by binding to virion particles in solution prior to attachment to susceptible cells. Once viruses enter cells, conventional antibodies cannot inhibit virus infection or replication. It is desirable to develop an efficient and nontoxic method for the introduction of virus-inhibiting antibodies into cells.
MATERIALS & METHODS - In this article, we report a new method for the delivery of small recombinant antibody fragments into virus-infected cells using a dendrimer-based molecular transporter.
RESULTS & CONCLUSION - The construct penetrated virus-infected cells efficiently and inhibited virus replication. This method provides a novel approach for the immediate delivery of inhibitory antibodies directed to virus proteins that are exposed only in the intracellular environment. This approach circumvents the current and rather complicated expression of inhibitory antibodies in cells following gene transfer.