67 research outputs found
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Mouse Ifit1b is a cap1-RNA binding protein which inhibits mouse coronavirus translation and is regulated by complexing with Ifit1c.
Knock-out mouse models have been extensively used to study the antiviral activity of interferon-induced protein with tetratricopeptide repeats (IFIT). Human IFIT1 binds to cap0 (m7GpppN) RNA, which lacks methylation on the first and second cap-proximal nucleotides (cap1, m7GpppNm, and cap2, m7GpppNmNm, respectively). These modifications are signatures of 'self' in higher eukaryotes, while unmodified cap0-RNA is recognised as foreign and, therefore, potentially harmful to the host cell. IFIT1 inhibits translation at the initiation stage by competing with the cap-binding initiation factor complex, eIF4F, restricting infection by certain viruses that possess 'non-self' cap0-mRNAs. However, in mice and other rodents the IFIT1 orthologue has been lost and the closely-related Ifit1b has been duplicated twice, yielding three paralogues: Ifit1, Ifit1b and Ifit1c. While murine Ifit1 is similar to human IFIT1 in its cap0-RNA binding selectivity, the roles of Ifit1b and Ifit1c are unknown. Here, we found that Ifit1b preferentially binds to cap1-RNA, while binding is much weaker to cap0- and cap2-RNA. In murine cells, we show that Ifit1b can modulate host translation and restrict wildtype mouse coronavirus infection. We found that Ifit1c acts as a stimulatory cofactor for both Ifit1 and Ifit1b, promoting their translation inhibition. In this way, Ifit1c acts in an analogous fashion to human IFIT3, which is a cofactor to human IFIT1. This work clarifies similarities and differences between the human and murine IFIT families, to facilitate better design and interpretation of mouse models of human infection, and sheds light on the evolutionary plasticity of the IFIT family
Use of Wearable Activity-Monitoring Technologies to Promote Physical Activity in Cancer Survivors: Challenges and Opportunities for Improved Cancer Care
The aim of this review was to explore the acceptability, opportunities, and challenges associated with wearable activity-monitoring technology to increase physical activity (PA) behavior in cancer survivors. A search of Medline, Embase, CINAHL, and SportDiscus was conducted from 1 January 2011 through 3 October 2022. The search was limited to English language, and peer-reviewed original research. Studies were included if they reported the use of an activity monitor in adults (+18 years) with a history of cancer with the intent to motivate PA behavior. Our search identified 1832 published articles, of which 28 met inclusion/exclusion criteria. Eighteen of these studies included post-treatment cancer survivors, eight were on active cancer treatment, and two were long-term cancer survivor studies. ActiGraph accelerometers were the primary technology used to monitor PA behaviors, with Fitbit as the most commonly utilized self-monitoring wearable technology. Overall, wearable activity monitors were found to be an acceptable and useful tool in improving self-awareness, motivating behavioral change, and increasing PA levels. Self-monitoring wearable activity devices have a positive impact on short-term PA behaviors in cancer survivors, but the increase in PA gradually attenuated through the maintenance phase. Further study is needed to evaluate and increase the sustainability of the use of wearable technologies to support PA in cancer survivors
A Cell-based Fluorescence Resonance Energy Transfer (FRET) Sensor Reveals Inter- and Intragenogroup Variations in Norovirus Protease Activity and Polyprotein Cleavage.
The viral protease represents a key drug target for the development of antiviral therapeutics. Because many protease inhibitors mimic protease substrates, differences in substrate recognition between proteases may affect their sensitivity to a given inhibitor. Here we use a cell-based FRET sensor to investigate the activity of different norovirus proteases upon cleavage of various norovirus cleavage sites inserted into a linker region separating cyan fluorescent protein and yellow fluorescent protein. Using this system, we demonstrate that differences in substrate processing exist between proteases from human noroviruses (genogroups I (GI) and II) and the commonly used murine norovirus (MNV, genogroup V) model. These altered the cleavage efficiency of specific cleavage sites both within and between genogroups. The differences observed between these proteases may affect sensitivity to protease inhibitors and the suitability of MNV as a model system for testing such molecules against the human norovirus protease. Finally, we demonstrate that replacement of MNV polyprotein cleavage sites with the GI or GII equivalents, with the exception of the NS6-7 junction, leads to the production of infectious virus when the MNV NS6 protease, but not the GI or GII proteases, are present.This work was funded by a Wellcome Trust Senior Fellowship awarded to IG Ref: WT097997MA) and a Marie Curie Fellowship awarded to TS (Ref: 628373).This is the final version of the article. It was first available from the American Society for Biochemistry and Molecular Biology via http://dx.doi.org/10.1074/jbc.M115.68823
Circularization of flavivirus genomic RNA inhibits de novo translation initiation.
Members of the Flaviviridae family, including dengue virus (DENV) and yellow fever virus, cause serious disease in humans, whilst maternal infection with Zika virus (ZIKV) can induce microcephaly in newborns. Following infection, flaviviral RNA genomes are translated to produce the viral replication machinery but must then serve as a template for the transcription of new genomes. However, the ribosome and viral polymerase proceed in opposite directions along the RNA, risking collisions and abortive replication. Whilst generally linear, flavivirus genomes can adopt a circular conformation facilitated by long-range RNA-RNA interactions, shown to be essential for replication. Using an in vitro reconstitution approach, we demonstrate that circularization inhibits de novo translation initiation on ZIKV and DENV RNA, whilst the linear conformation is translation-competent. Our results provide a mechanism to clear the viral RNA of ribosomes in order to promote efficient replication and, therefore, define opposing roles for linear and circular conformations of the flavivirus genome
Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5'-terminal regions of cap0-, cap1- and 5'ppp- mRNAs.
Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA's 5'-terminal 'cap'. The minimal 'cap0' consists of N7-methylguanosine linked to the first nucleotide via a 5'-5' triphosphate (ppp) bridge. Cap0 is further modified by 2'-O-methylation of the next two riboses, yielding 'cap1' (m7GpppNmN) and 'cap2' (m7GpppNmNm). However, some viral RNAs lack 2'-O-methylation, whereas others contain only ppp- at their 5'-end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0- and 5'ppp- mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K1/2,app ∼9 to 23 nM). The 2'-O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K1/2,app ∼450 nM). The 5'-terminal regions of 5'ppp-mRNAs were recognized by IFIT5 (K1/2,app ∼400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5'-terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1- and 5'ppp- mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations
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Ifit1 regulates norovirus infection and enhances the interferon response in murine macrophage-like cells.
Background: Norovirus, also known as the winter vomiting bug, is the predominant cause of non-bacterial gastroenteritis worldwide. Disease control is predicated on a robust innate immune response during the early stages of infection. Double-stranded RNA intermediates generated during viral genome replication are recognised by host innate immune sensors in the cytoplasm, activating the strongly antiviral interferon gene programme. Ifit proteins (interferon induced proteins with tetratricopeptide repeats), which are highly expressed during the interferon response, have been shown to directly inhibit viral protein synthesis as well as regulate innate immune signalling pathways. Ifit1 is well-characterised to inhibit viral translation by sequestration of eukaryotic initiation factors or by directly binding to the 5' terminus of foreign RNA, particularly those with non-self cap structures. However, noroviruses have a viral protein, VPg, covalently linked to the 5' end of the genomic RNA, which acts as a cap substitute to recruit the translation initiation machinery. Methods: Ifit1 knockout RAW264.7 murine macrophage-like cells were generated using CRISPR-Cas9 gene editing. These cells were analysed for their ability to support murine norovirus infection, determined by virus yield, and respond to different immune stimuli, assayed by quantitative PCR. The effect of Ifit proteins on norovirus translation was also tested in vitro. Results: Here, we show that VPg-dependent translation is completely refractory to Ifit1-mediated translation inhibition in vitro and Ifit1 cannot bind the 5' end of VPg-linked RNA. Nevertheless, knockout of Ifit1 promoted viral replication in murine norovirus infected cells. We then demonstrate that Ifit1 promoted interferon-beta expression following transfection of synthetic double-stranded RNA but had little effect on toll-like receptor 3 and 4 signalling. Conclusions: Ifit1 is an antiviral factor during norovirus infection but cannot directly inhibit viral translation. Instead, Ifit1 stimulates the antiviral state following cytoplasmic RNA sensing, contributing to restriction of norovirus replication
The RNA Helicase eIF4A is required for Sapovirus translation
The eukaryotic initiation factor (eIF) 4A is a DEAD-box helicase that unwinds RNA structure in the 5´-untranslated region (UTR) of mRNAs. Here, we investigated the role of eIF4A in porcine sapovirus VPg-dependent translation. Using inhibitors and dominant negative mutants, we found that eIF4A is required for viral translation and infectivity, suggesting that despite the presence of a very short 5´-UTR, eIF4A is required to unwind RNA structure in the sapovirus genome to facilitate virus translation.This work was supported by Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Science, ICT and Future Planning (2014R1A2A2A01004292), and by funding to IG (Wellcome Senior Fellow) from the Wellcome Trust (Ref: 097997/Z/11/Z) and Biological and Biotechnology Research Council (BBSRC) (Ref: BB/I012303/1) and by BBSRC funding to SC (Ref: BB/J001708/1). TS is a Marie Curie Fellow (Ref:628373). We thank Professor Jerry Pelletier (McGill University, Canada) for providing hippuristanol
Insights into Cleavage Specificity from the Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease Complexed with a Peptide Substrate.
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