The Rotavirus Interferon Antagonist NSP1: Many Targets, Many Questions.

Rotavirus is a leading cause of death due to diarrhea among young children across the globe. Despite the limited coding capacity that is characteristic of RNA viruses, rotavirus dedicates substantial resources to avoiding the host innate immune response. Among these strategies is use of the interferon antagonist protein NSP1, which targets cellular proteins required for interferon production to be degraded by the proteasome. Although numerous cellular targets have been described, there remain many questions about the mechanism of NSP1 activity and its role in promoting replication in specific host species.

Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases.

UNLABELLED: The rotavirus nonstructural protein NSP1 acts as an antagonist of the host antiviral response by inducing degradation of key proteins required to activate interferon (IFN) production. Protein degradation induced by NSP1 is dependent on the proteasome, and the presence of a RING domain near the N terminus has led to the hypothesis that NSP1 is an E3 ubiquitin ligase. To examine this hypothesis, pulldown assays were performed, followed by mass spectrometry to identify components of the host ubiquitination machinery that associate with NSP1. Multiple components of cullin RING ligases (CRLs), which are essential multisubunit ubiquitination complexes, were identified in association with NSP1. The mass spectrometry was validated in both transfected and infected cells to show that the NSP1 proteins from different strains of rotavirus associated with key components of CRL complexes, most notably the cullin scaffolding proteins Cul3 and Cul1. In vitro binding assays using purified proteins confirmed that NSP1 specifically interacted with Cul3 and that the N-terminal region of Cul3 was responsible for binding to NSP1. To test if NSP1 used CRL3 to induce degradation of the target protein IRF3 or ß-TrCP, Cul3 levels were knocked down using a small interfering RNA (siRNA) approach. Unexpectedly, loss of Cul3 did not rescue IRF3 or ß-TrCP from degradation in infected cells. The results indicate that, rather than actively using CRL complexes to induce degradation of target proteins required for IFN production, NSP1 may use cullin-containing complexes to prevent another cellular activity. IMPORTANCE: The ubiquitin-proteasome pathway plays an important regulatory role in numerous cellular functions, and many viruses have evolved mechanisms to exploit or manipulate this pathway to enhance replication and spread. Rotavirus, a major cause of severe gastroenteritis in young children that causes approximately 420,000 deaths worldwide each year, utilizes the ubiquitin-proteasome system to subvert the host innate immune response by inducing the degradation of key components required for the production of interferon (IFN). Here, we show that NSP1 proteins from different rotavirus strains associate with the scaffolding proteins Cul1 and Cul3 of CRL ubiquitin ligase complexes. Nonetheless, knockdown of Cul1 and Cul3 suggests that NSP1 induces the degradation of some target proteins independently of its association with CRL complexes, stressing a need to further investigate the mechanistic details of how NSP1 subverts the host IFN response.

Psychics, aliens, or experience? Using the Anomalistic Belief Scale to examine the relationship between type of belief and probabilistic reasoning.

A growing body of research has shown people who hold anomalistic (e.g., paranormal) beliefs may differ from nonbelievers in their propensity to make probabilistic reasoning errors. The current study explored the relationship between these beliefs and performance through the development of a new measure of anomalistic belief, called the Anomalistic Belief Scale (ABS). One key feature of the ABS is that it includes a balance of both experiential and theoretical belief items. Another aim of the study was to use the ABS to investigate the relationship between belief and probabilistic reasoning errors on conjunction fallacy tasks. As expected, results showed there was a relationship between anomalistic belief and propensity to commit the conjunction fallacy. Importantly, regression analyses on the factors that make up the ABS showed that the relationship between anomalistic belief and probabilistic reasoning occurred only for beliefs about having experienced anomalistic phenomena, and not for theoretical anomalistic beliefs.

No pain no gain: The positive impact of punishment on the strategic regulation of accuracy.

Previous studies have shown that punishing people through a large penalty for volunteering incorrect information typically leads them to withhold more information (metacognitive response bias), but it does not appear to influence their ability to distinguish between their own correct and incorrect answers (metacognitive accuracy discrimination). The goal of the current study was to demonstrate that punishing people for volunteering incorrect information-versus rewarding volunteering correct information-produces more effective metacognitive accuracy discrimination. All participants completed three different general-knowledge tests: a reward test (high points for correct volunteered answers), a baseline test (equal points/penalties for volunteered correct/incorrect answers) and a punishment test (high penalty for incorrect volunteered answers). Participants were significantly better at distinguishing between their own correct and incorrect answers on the punishment than reward test, which has implications for situations requiring effective accuracy monitoring.

The battle between rotavirus and its host for control of the interferon signaling pathway.

Viral pathogens must overcome innate antiviral responses to replicate successfully in the host organism. Some of the mechanisms viruses use to interfere with antiviral responses in the infected cell include preventing detection of viral components, perturbing the function of transcription factors that initiate antiviral responses, and inhibiting downstream signal transduction. RNA viruses with small genomes and limited coding space often express multifunctional proteins that modulate several aspects of the normal host response to infection. One such virus, rotavirus, is an important pediatric pathogen that causes severe gastroenteritis, leading to ~450,000 deaths globally each year. In this review, we discuss the nature of the innate antiviral responses triggered by rotavirus infection and the viral mechanisms for inhibiting these responses.

Effects of context on recollection and familiarity experiences are task dependent.

The influence of test context on reports of recollection and familiarity depends on how these subjective recognition experiences are conceptualized and measured. Bodner and Lindsay (2003) found that critical items elicited more remember judgments but fewer know judgments in a less (vs. more) memorable context. In contrast, Tousignant and Bodner (2012) found that independent ratings of recollection and familiarity were both higher in a less memorable context. We replicated the dissociative pattern with judgments using recollect/familiar labels (Experiment 1), and in a novel R/F/B task that added a "both" option to eliminate the mutual exclusivity between the recollect and familiar options (Experiment 2). Adding a "guess" option eliminated these context effects (Experiment 3), however whether allowing guesses "cleans up" or "desensitizes" recollection and familiarity judgments remains unclear. Determining which task variants provide appropriate measures of subjective recognition experiences will require an examination of additional dissociations and triangulation with other measures.

Rotavirus NSP1 mediates degradation of interferon regulatory factors through targeting of the dimerization domain.

Rotavirus nonstructural protein NSP1 can inhibit expression of interferon (IFN) and IFN-stimulated gene products by inducing proteasome-mediated degradation of IFN-regulatory factors (IRFs), including IRF3, IRF5, and IRF7. All IRF proteins share an N-terminal DNA-binding domain (DBD), and IRF3, IRF5, and IRF7 contain a similar C-proximal IRF association domain (IAD) that mediates IRF dimerization. An autoinhibitory domain (ID) at the extreme C terminus interacts with the IAD, burying residues necessary for IRF dimerization. Phosphorylation of serine/threonine residues in the ID induces charge repulsions that unmask the IAD, enabling IRF dimerization and subsequent nuclear translocation. To define the region of IRF proteins targeted for degradation by NSP1, we generated IRF3 and IRF7 truncation mutants and transiently expressed each with simian SA11-4F NSP1. These assays indicated that the IAD represented a necessary and sufficient target for degradation. Because NSP1 did not mediate degradation of truncated forms of the IAD, NSP1 likely requires a structurally intact IAD for recognition and targeting of IRF proteins. IRF9, which contains an IAD-like region that directs interactions with signal inducer and activator of transcription (STAT) proteins, was also targeted for degradation by NSP1, while IRF1, which lacks an IAD, was not. Analysis of mutant forms of IRF3 unable to undergo dimerization or that were constitutively dimeric showed that both were targeted for degradation by NSP1. These results indicate that SA11-4F NSP1 can induce degradation of inactive and activated forms of IAD-containing IRF proteins (IRF3 to IRF9), allowing a multipronged attack on IFN-based pathways that promote antiviral innate and adaptive immune responses.

Recruitment of cellular clathrin to viral factories and disruption of clathrin-dependent trafficking.

The viral factories of mammalian reovirus (MRV) are cytoplasmic structures that serve as sites of viral genome replication and particle assembly. A 721-aa MRV non-structural protein, µNS, forms the factory matrix and recruits other viral proteins to these structures. In this report, we show that µNS contains a conserved C-proximal sequence (711-LIDFS-715) that is similar to known clathrin-box motifs and is required for recruitment of clathrin to viral factories. Clathrin recruitment by µNS occurs independently of infecting MRV particles or other MRV proteins. Ala substitution for a single Leu residue (mutation L711A) within the putative clathrin-binding motif of µNS inhibits clathrin recruitment, but does not prevent formation or expansion of viral factories. Notably, clathrin-dependent cellular functions, including both endocytosis and secretion, are disrupted in cells infected with MRV expressing wild-type, but not L711A, µNS. These results identify µNS as a novel adaptor-like protein that recruits cellular clathrin to viral factories, disrupting normal functions of clathrin in cellular membrane trafficking. To our knowledge, this is the only viral or bacterial protein yet shown to interfere with clathrin functions in this manner. The results additionally establish a new approach for studies of clathrin functions, based on µNS-mediated sequestration.

Rotavirus variant replicates efficiently although encoding an aberrant NSP3 that fails to induce nuclear localization of poly(A)-binding protein.

The rotavirus (RV) non-structural protein NSP3 forms a dimer that has binding domains for the translation initiation factor eIF4G and for a conserved 3'-terminal sequence of viral mRNAs. Through these activities, NSP3 has been proposed to promote viral mRNA translation by directing circularization of viral polysomes. In addition, by disrupting interactions between eIF4G and the poly(A)-binding protein (PABP), NSP3 has been suggested to inhibit translation of host polyadenylated mRNAs and to stimulate relocalization of PABP from the cytoplasm to the nucleus. Herein, we report the isolation and characterization of SA11-4Fg7re, an SA11-4F RV derivative that contains a large sequence duplication initiating within the genome segment (gene 7) encoding NSP3. Our analysis showed that mutant NSP3 (NSP3m) encoded by SA11-4Fg7re is almost twice the size of the wild-type protein and retains the capacity to dimerize. However, in comparison to wild-type NSP3, NSP3m has a decreased capacity to interact with eIF4G and to suppress the translation of polyadenylated mRNAs. In addition, NSP3m fails to induce the nuclear accumulation of PABP in infected cells. Despite the defective activities of NSP3m, the levels of viral protein and progeny virus produced in SA11-4Fg7re- and SA11-4F-infected cells were indistinguishable. Collectively, these data are consistent with a role for NSP3 in suppressing host protein synthesis through antagonism of PABP activity, but also suggest that NSP3 functions may have little or no impact on the efficiency of virus replication in widely used RV-permissive cell lines.

Diversity of interferon antagonist activities mediated by NSP1 proteins of different rotavirus strains.

Studies involving limited numbers of rotavirus (RV) strains have shown that the viral gene 5 product, NSP1, can antagonize beta interferon (IFN-ß) expression by inducing the degradation of IFN-regulatory factors (IRFs) (IRF3, IRF5, and IRF7) or a component of the E3 ubiquitin ligase complex responsible for activating NF-κB (ß-transducin repeat-containing protein [ß-TrCP]). To gain a broader perspective of NSP1 activities, we examined various RV strains for the ability to inhibit IFN-ß expression in human cells. We found that all strains encoding wild-type NSP1 impeded IFN-ß expression but not always through IRF3 degradation. To identify other degradation targets involved in suppressing IFN-ß expression, we used transient expression vectors to test the abilities of a diverse collection of NSP1 proteins to target IRF3, IRF5, IRF7, and ß-TrCP for degradation. The results indicated that human RVs rely predominantly on the NSP1-induced degradation of IRF5 and IRF7 to suppress IFN signaling, whereas NSP1 proteins of animal RVs tended to target IRF3, IRF5, and IRF7, allowing the animal viruses a broader attack on the IFN-ß signaling pathway. The results also suggested that the NSP1-induced degradation of ß-TrCP is an uncommon mechanism of subverting IFN-ß signaling but is one that can be shared with NSP1 proteins that induce IRF degradation. Our analysis reveals that the activities of NSP1 proteins are diverse, with no obvious correlations between degradations of pairs of target proteins. Thus, RVs have evolved functionally distinct approaches for subverting the host antiviral response, a property consistent with the immense sequence variation noted for NSP1 proteins.

Localization of mammalian orthoreovirus proteins to cytoplasmic factory-like structures via nonoverlapping regions of microNS.

Virally induced structures called viral factories form throughout the cytoplasm of cells infected with mammalian orthoreoviruses (MRV). When expressed alone in cells, MRV nonstructural protein microNS forms factory-like structures very similar in appearance to viral factories, suggesting that it is involved in forming the structural matrix of these structures. microNS also associates with MRV core particles; the core proteins mu2, lambda1, lambda2, lambda3, and sigma2; and the RNA-binding nonstructural protein sigmaNS. These multiple associations result in the recruitment or retention of these viral proteins or particles at factory-like structures. In this study, we identified the regions of microNS necessary and sufficient for these associations and additionally examined the localization of viral RNA synthesis in infected cells. We found that short regions within the amino-terminal 220 residues of microNS are necessary for associations with core particles and necessary and sufficient for associations with the proteins mu2, lambda1, lambda2, sigma2, and sigmaNS. We also found that only the lambda3 protein associates with the carboxyl-terminal one-third of microNS and that viral RNA is synthesized within viral factories. These results suggest that microNS may act as a cytoplasmic scaffolding protein involved in localizing and coordinating viral replication or assembly intermediates for the efficient production of progeny core particles during MRV infection.

SARS-CoV-2 infection, COVID-19 pathogenesis, and exposure to air pollution: What is the connection?

Exposure to air pollutants has been previously associated with respiratory viral infections, including influenza, measles, mumps, rhinovirus, and respiratory syncytial virus. Epidemiological studies have also suggested that air pollution exposure is associated with increased cases of SARS-CoV-2 infection and COVID-19-associated mortality, although the molecular mechanisms by which pollutant exposure affects viral infection and pathogenesis of COVID-19 remain unknown. In this review, we suggest potential molecular mechanisms that could account for this association. We have focused on the potential effect of exposure to nitrogen dioxide (NO2 ), ozone (O3 ), and particulate matter (PM) since there are studies investigating how exposure to these pollutants affects the life cycle of other viruses. We have concluded that pollutant exposure may affect different stages of the viral life cycle, including inhibition of mucociliary clearance, alteration of viral receptors and proteases required for entry, changes to antiviral interferon production and viral replication, changes in viral assembly mediated by autophagy, prevention of uptake by macrophages, and promotion of viral spread by increasing epithelial permeability. We believe that exposure to pollutants skews adaptive immune responses toward bacterial/allergic immune responses, as opposed to antiviral responses. Exposure to air pollutants could also predispose exposed populations toward developing COIVD-19-associated immunopathology, enhancing virus-induced tissue inflammation and damage.

Cognitive mechanisms underlying recovered-memory experiences of childhood sexual abuse.

People sometimes report recovering long-forgotten memories of childhood sexual abuse. The memory mechanisms that lead to such reports are not well understood, and the authenticity of recovered memories has often been challenged. We identified two subgroups of people reporting recovered memories of childhood sexual abuse. These subgroups differed dramatically in their cognitive profiles: People who recovered memories of abuse through suggestive therapy exhibited a heightened susceptibility to the construction of false memories, but showed no tendency to underestimate their prior remembering. Conversely, people who recovered memories of abuse spontaneously showed a heightened proneness to forget prior incidences of remembering, but exhibited no increased susceptibility to false memories. This double dissociation points to mechanisms that underlie recovered-memory experiences and indicates that recovered memories may at times be fictitious and may at other times be authentic.

Rotavirus vaccines: why continued investment in research is necessary.

PURPOSE OF REVIEW: Rotavirus vaccines were first introduced more than a decade ago and have had a tremendous impact on reducing the number of hospitalizations and deaths due to rotavirus-associated diarrhea. This review will discuss current rotavirus vaccines, post-licensure surveillance, progress in non-replicating vaccine development, and why continued research is important for understanding a virus that remains a globally leading cause of death due to diarrhea. RECENT FINDINGS: Research advances have enhanced our understanding of how vaccines induce protection against subsequent severe disease, how the virus replicates and spreads in the face of the host immune system, and basic mechanisms governing the viral life cycle. SUMMARY: Much remains to be learned about how to improve vaccine success, what are the molecular determinants of host range and virulence, and what are the interactions of the virus with the host that drive its replicative success, among many other important questions.

The relationship between anomalistic belief and biases of evidence integration and jumping to conclusions.

Biases in the assessment and integration of evidence are likely contributors to anomalistic (e.g., paranormal, extra-terrestrial) beliefs because of the non-evidence based nature of these beliefs. However, little research has examined the relationship between anomalistic beliefs and evidence integration biases. The current study addressed this gap by examining the relationship between anomalistic belief and four such biases; bias against disconfirmatory evidence (BADE), bias against confirmatory evidence (BACE), liberal acceptance bias, and the jumping to conclusions bias (JTC). Standard BADE scenarios were used to measure BADE, BACE, and the liberal acceptance bias: Participants were given three pieces of evidence, one at a time, and required to rate several alternative explanations. The JTC was measured using two draws-to-decisions tasks (beads and emotionally salient), and participants also completed measures of anomalistic belief and delusion-proneness. Results showed that liberal acceptance was the only evidence integration bias that significantly predicted greater overall anomalistic belief. However, this relationship was no longer significant once delusion proneness was controlled for. Additionally, BADE significantly predicted experiential (but not other types of) anomalistic beliefs even after controlling for delusion proneness. We propose that liberal acceptance may lead people to form anomalistic beliefs on the basis of little evidence, and that stronger BADE may make these beliefs highly resistant to change.

Increased hindsight bias in schizophrenia.

An underlying theme common to prominent theoretical accounts of cognition in schizophrenia is that information processing is disproportionately influenced by recently/currently encountered information relative to the influence of previously learned information. In this study, the authors tested this account by using the hindsight bias or knew-it-all-along (KIA) paradigm, which demonstrates that newly acquired knowledge influences recall of past events. In line with the account that patients with schizophrenia display a disproportionately strong influence of recently encountered information relative to the influence of previously learned information, patients displayed a KIA effect that was significantly greater than in controls. This result is discussed in the context of the cognitive underpinnings of the KIA effect and delusion formation.

Comparative difficulty and the strategic regulation of accuracy: the impact of test-list context on monitoring and meta-metacognition.

A growing body of research has shown that context manipulations can have little or no impact on accuracy performance, yet still significantly influence metacognitive performance. For example, participants in a test-list context paradigm study one list of words with a medium levels-of-processing task and a second word list with either a shallow or deep task: Recognition for medium words does not differ across conditions, however medium words are significantly more likely to be labeled as "remembered" (vs. merely familiar) if they had been studied with a shallow word list (Bodner & Lindsay, 2003). The goal of the current studies was to extend the test-list context paradigm to strategic regulation (report/withhold recognition test), and broaden it to incorporate different types of stimuli (i.e., face stimuli in place of a medium word list). The paradigm also was modified to include separate answer (studied/new) confidence and decision (report/withhold) confidence ratings at test. Results showed that context did not impact recognition accuracy for faces across the context conditions, however participants were more likely to report (i.e., volunteer) their face responses if they had studied the shallow word list. The results also demonstrated a difference between answer confidence and decision confidence, and the pattern of this difference depended on whether responses were reported or withheld (Experiment 1). Overall, the data are presented as support for the functional account of memory, which views memory states as inferential and attributional rather than static categories.

Comparisons of the M1 genome segments and encoded mu2 proteins of different reovirus isolates.

BACKGROUND: The reovirus M1 genome segment encodes the mu2 protein, a structurally minor component of the viral core, which has been identified as a transcriptase cofactor, nucleoside and RNA triphosphatase, and microtubule-binding protein. The mu2 protein is the most poorly understood of the reovirus structural proteins. Genome segment sequences have been reported for 9 of the 10 genome segments for the 3 prototypic reoviruses type 1 Lang (T1L), type 2 Jones (T2J), and type 3 Dearing (T3D), but the M1 genome segment sequences for only T1L and T3D have been previously reported. For this study, we determined the M1 nucleotide and deduced mu2 amino acid sequences for T2J, nine other reovirus field isolates, and various T3D plaque-isolated clones from different laboratories. RESULTS: Determination of the T2J M1 sequence completes the analysis of all ten genome segments of that prototype. The T2J M1 sequence contained a 1 base pair deletion in the 3' non-translated region, compared to the T1L and T3D M1 sequences. The T2J M1 gene showed approximately 80% nucleotide homology, and the encoded mu 2 protein showed approximately 71% amino acid identity, with the T1L and T3D M1 and mu2 sequences, respectively, making the T2J M1 gene and mu2 proteins amongst the most divergent of all reovirus genes and proteins. Comparisons of these newly determined M1 and mu2 sequences with newly determined M1 and mu2 sequences from nine additional field isolates and a variety of laboratory T3D clones identified conserved features and/or regions that provide clues about mu2 structure and function. CONCLUSIONS: The findings suggest a model for the domain organization of mu2 and provide further evidence for a role of mu2 in viral RNA synthesis. The new sequences were also used to explore the basis for M1/mu2-determined differences in the morphology of viral factories in infected cells. The findings confirm the key role of Ser/Pro208 as a prevalent determinant of differences in factory morphology among reovirus isolates and trace the divergence of this residue and its associated phenotype among the different laboratory-specific clones of type 3 Dearing.

Remembering remembering.

We developed a laboratory analogue of the "forgot-it-all-along" effect that J. W. Schooler, M. Bendiksen, and Z. Ambadar (1997) proposed for cases of "recovered memories" in which individuals had forgotten episodes of talking about the abuse when they were supposedly amnestic for it. In Experiment 1, participants studied homographs with disambiguating context words; in Test 1 they received studied- or other-context words as cues; and in Test 2 they received studied-context cues and judged whether they had recalled each item during Test 1. In Experiment 2, retrieval cues were manipulated on both tests. In Experiment 3, both the studied- and other-context cues corresponded to the same meaning of each homograph. In Experiment 4, Test 1 was free recall, and studied- versus other-context cues were presented in Test 2. Participants more often forgot that they had previously recalled an item if they were cued to think of it differently on the two tests.

Monitoring and meta-metacognition in the own-race bias.

Although there is a great deal of research focused on identification issues related to own-versus other-race faces very few experiments have explored whether metacognitive monitoring contributes to the own-race bias. In the current experiment the typical own-race bias paradigm was modified so that type-2 signal detection measures (e.g. Higham & Arnold, 2007a,b) could be used to directly measure metacognitive monitoring at retrieval. A second goal of the experiment was to explore whether self-reported confidence ratings differed depending on whether they were directed at answer accuracy (e.g., judging a face as "studied") versus at decisions about that answer (e.g., volunteering vs. withholding that answer). Overall the results demonstrated that monitoring does contribute to the own-race bias, in that participants were better at monitoring their memory for own-race faces. Further, there was a significant difference between the two confidence measures, and the pattern of this difference depended on whether responses had been volunteered or withheld.