Protein synthesis is a tightly controlled process responding to several stimuli, including viral infection. As obligate intracellular parasites, viruses depend on the translation machinery of the host and can manipulate it by affecting the availability and function of specific eukaryotic initiation factors (eIFs). Human norovirus is a member of the Caliciviridae family and is responsible for gastroenteritis outbreaks. Previous studies on feline calicivirus and murine norovirus 1 (MNV1) demonstrated that the viral protein, genome-linked (VPg), acts to direct translation by hijacking the host protein synthesis machinery. Here we report that MNV1 infection modulates the MAPK pathway to activate eIF4E phosphorylation. Our results show that the activation of p38 and Mnk during MNV1 infection is important for MNV1 replication. Furthermore, phosphorylated eIF4E relocates to the polysomes, and this contributes to changes in the translational state of specific host mRNAs. We propose that global translational control of the host by eIF4E phosphorylation is a key component of the host-pathogen interaction.
Willcocks MM, Locker N, Gomwalk Z, Royall E, Bakhshesh M, Belsham GJ, Idamakanti N, Burroughs KD, Reddy PS, Hallenbeck PL, Roberts LO (2011) Structural Features of the Seneca Valley Virus Internal Ribosome Entry Site (IRES) Element: a Picornavirus with a Pestivirus-Like IRES,JOURNAL OF VIROLOGY 85 (9) pp. 4452-4461
AMER SOC MICROBIOLOGY
Translation initiation on HIV genomic RNA relies on both cap and Internal Ribosome Entry Site (IRES) dependant mechanisms that are regulated throughout the cell cycle. During a unique phenomenon, the virus recruits initiation complexes through RNA structures located within Gag coding sequence, downstream of the initiation codon. We analyzed initiation complexes paused on the HIV-2 gag IRES and revealed that they contain all the canonical initiation factors except eIF4E and eIF1. We report that eIF3 and the small ribosomal subunit bind HIV RNA within gag open reading frame. We thus propose a novel two step model whereby the initial event is the formation of a ternary eIF3/40S/IRES complex. In a second step, dependent on most of the canonical initiation factors, the complex is rearranged to transfer the ribosome on the initiation codons. The absolute requirement of this large structure for HIV translation defines a new function for a coding region. Moreover, the level of information compaction within this viral genome reveals an additional level of evolutionary constraint on the coding sequence. The conservation of this IRES and its properties in rapidly evolving viruses suggest an important role in the virus life cycle and highlight an attractive new therapeutic target.
Enterovirus 71 (EV71) is associated with severe neurological disorders in children, and has been implicated as the infectious agent in several large-scale outbreaks with mortalities. Upon infection, the viral RNA is translated in a cap-independent manner to yield a large polyprotein precursor. This mechanism relies on the presence of an internal ribosome entry site (IRES) element within the 5'-untranslated region. Virus-host interactions in EV71-infected cells are crucial in assisting this process. We identified a novel positive IRES trans-acting factor, far upstream element binding protein 1 (FBP1). Using binding assays, we mapped the RNA determinants within the EV71 IRES responsible for FBP1 binding and mapped the protein domains involved in this interaction. We also demonstrated that during EV71 infection, the nuclear protein FBP1 is enriched in cytoplasm where viral replication occurs. Moreover, we showed that FBP1 acts as a positive regulator of EV71 replication by competing with negative ITAF for EV71 IRES binding. These new findings may provide a route to new anti-viral therapy.
Chung L, Bailey D, Leen EN, Emmott EP, Chaudhry Y, Roberts LO, Curry S, Locker N, Goodfellow IG (2014) Norovirus translation requires an interaction between the C Terminus of the genome-linked viral protein VPg and eukaryotic translation initiation factor 4G., J Biol Chem 289 (31) pp. 21738-21750
Viruses have evolved a variety of mechanisms to usurp the host cell translation machinery to enable translation of the viral genome in the presence of high levels of cellular mRNAs. Noroviruses, a major cause of gastroenteritis in man, have evolved a mechanism that relies on the interaction of translation initiation factors with the virus-encoded VPg protein covalently linked to the 5' end of the viral RNA. To further characterize this novel mechanism of translation initiation, we have used proteomics to identify the components of the norovirus translation initiation factor complex. This approach revealed that VPg binds directly to the eIF4F complex, with a high affinity interaction occurring between VPg and eIF4G. Mutational analyses indicated that the C-terminal region of VPg is important for the VPg-eIF4G interaction; viruses with mutations that alter or disrupt this interaction are debilitated or non-viable. Our results shed new light on the unusual mechanisms of protein-directed translation initiation.
Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus, the etiological agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). One of the key viral proteins that contributes to tumorigenesis is vFLIP, a viral homolog of the FLICE inhibitory protein. This KSHV protein interacts with the NFºB pathway to trigger the expression of antiapoptotic and proinflammatory genes and ultimately leads to tumor formation. The expression of vFLIP is regulated at the translational level by an internal ribosomal entry site (IRES) element. However, the precise mechanism by which ribosomes are recruited internally and the exact location of the IRES has remained elusive. Here we show that a 252-nt fragment directly upstream of vFLIP, within a coding region, directs translation. We have established its RNA structure and demonstrate that IRES activity requires the presence of eIF4A and an intact eIF4G. Furthermore, and unusually for an IRES, eIF4E is part of the complex assembled onto the vFLIP IRES to direct translation. These molecular interactions define a new paradigm for IRES-mediated translation.
Deforges J, Locker N, Sargueil B (2014) mRNAs that specifically interact with eukaryotic ribosomal subunits., Biochimie 114 pp. 48-57
The accuracy of start codon selection is determined by the translation initiation process. In prokaryotes the initiation step on most mRNAs relies on recruitment of the small ribosomal subunit onto the initiation codon by base pairing between the mRNA and the 16S rRNA. Eukaryotes have evolved a complex molecular machinery involving at least 11 initiation factors, and mRNAs do not directly recruit the small ribosomal subunit. Instead the initiation complex is recruited to the 5' end of the mRNA through a complex protein network including eIF4E that interacts with the 5' cap structure and poly-A binding protein that interacts with the 3'end. However, some viral and cellular mRNAs are able to escape this pathway by internal recruitment of one or several components of the translation machinery. Here we review those eukaryotic mRNAs that have been reported to directly recruit the 40S ribosomal subunit internally. In the well characterized cases of viral IRESes, a specific RNA structure is involved in this process, and in addition to recruitment of the ribosome, the mRNA also manipulates the ribosome structure to stimulate the first translocation step. We also review recently described IRES/ribosome interactions in cases where the molecular mechanism leading to translation initiation has yet to be described. Finally we evaluate the possibility that mRNA may recruit the 40S ribosomal subunit through base pairing with the 18S rRNA.
In this review, we provide an overview of the strategies developed by caliciviruses to subvert or regulate the host protein synthesis machinery to their advantage. As intracellular obligate parasites, viruses strictly depend on the host cell resources to produce viral proteins. Thus, many viruses have developed strategies that regulate the function of the host protein synthesis machinery, often leading to preferential translation of viral mRNAs. Caliciviruses lack a 52 cap structure but instead have a virus-encoded VPg protein covalently linked to the 52 end of their mRNAs. Furthermore, they encode 2?4 open reading frames within their genomic and subgenomic RNAs. Therefore, they use alternative mechanisms for translation whereby VPg interacts with eukaryotic initiation factors (eIFs) to act as a proteinaceous cap-substitute, and some structural proteins are produced by reinitiation of translation events. This review discusses our understanding of these key mechanisms during caliciviruses infection as well as recent insights into the global regulation of eIF4E activity.
The process of translation initiation in mammalian systems is complex and not fully understood. It is regulated by an intricate network of signalling pathways and is a significant energetic burden to the cell. Although models of initiation are available for yeast, to date, such models do not include the regulation of this process, nor do they exist for mammalian systems. Existing literature was used to reconstruct the process of translation initiation and the regulatory signalling networks in the Petri Net formalism within the software Snoopy. The final version of the model was altered to incorporate the effects of Murine Norovirus. The model was converted to a binary form and the software QSSPN was used to run Gillespie algorithm-based stochastic simulations. The predictive power of the model was established by incorporating commonly used chemical inhibitors. Using the Matthews? Correlation Coefficient, a quantitative measure of predictive power was established by comparing the model behaviour to the effects of each inhibitor recorded in existing literature. A qualitative model containing 584 reactions was constructed. The predictive power of the model was raised to MCC = 0.4558 through a series of refinements. Two predicted behaviours, an increase in eIF4E phosphorylation and a reduction of AKT phosphorylation both in response to Rapamycin, were validated with the Immunoblotting techniques, Western Blotting and Human Phospho-MAPK Arrays, in the murine monocyte/macrophage RAW 264.7 cell line. The model incorporating the effects of Murine Norovirus infection generated five testable predictions. Of these, four were verified with the Human Phospho-MAPK Arrays. The model presented here demonstrates the value of generating large-scale models using the binary model formalism and performing simulations with QSSPN. The model of the regulation of translation initiation has shown that it is capable of generating experimentally verifiable predictions. Furthermore, the incorporation of viral effects demonstrates that the model has a range of potential future uses.
Noroviruses produce viral RNAs lacking a 5? cap structure and instead use a virus-encoded VPg protein covalently linked to viral RNA to interact with translation initiation factors and drive viral protein synthesis. Norovirus infection results in the induction of the innate response leading to interferon stimulated gene (ISG) transcription. However the translation of the induced ISG mRNAs is suppressed. A SILAC-based mass spectrometry approach was employed to analyse changes to protein abundance in both whole cell and m7GTP-enriched samples to demonstrate that diminished host mRNA translation correlates with changes to the composition of the eukaryotic initiation factor complex. The suppression of host ISG translation correlates with the activity of the viral protease (NS6) and the activation of cellular caspases leading to the establishment of an apoptotic environment. These results indicate that noroviruses exploit the differences between viral VPg-dependent and cellular cap-dependent translation in order to diminish the host response to infection.
Roth H, Magg V, Uch F, Mutz P, Klein P, Haneke K, Lohmann V, Bartenschlager R, Fackler O, Locker NS, Stoecklin G, Ruggieri A (2017) Flavivirus infection uncouples translation suppression from cellular stress responses,mBio 8 (1) e02150-16
American Society for Microbiology
As obligate parasites, viruses strictly depend on host cell translation for the production of new progeny, yet infected cells also synthesize antiviral proteins to limit virus infection. Modulation of host cell translation therefore represents a frequent strategy by which viruses optimize their replication and spread. Here we sought to define how host cell translation is regulated during infection of human cells with Dengue Virus (DENV) and Zika Virus (ZIKV), two positive-strand RNA flaviviruses. Polysome profiling and analysis of de novo protein synthesis revealed that flavivirus infection causes potent repression of host cell translation while synthesis of viral proteins remains efficient. Selective repression of host cell translation was mediated by the DENV polyprotein at the level of translation initiation. In addition, DENV and ZIKV infection suppressed host cell stress responses such as the formation of stress granules and phosphorylation of the translation initiation factor eIF2±. Mechanistic analyses revealed that translation repression was uncoupled from the disruption of stress granule formation and eIF2± signaling. Rather, DENV infection induced p38-Mnk1 signaling that resulted in the phosphorylation of the eukaryotic translation initiation factor eIF4E and was essential for the efficient production of virus particles. Together, these results identify the uncoupling of translation suppression from the cellular stress responses as a conserved strategy by which flaviviruses ensure efficient replication in human cells.
Humoud MN, Doyle N, Royall Elizabeth, Willcocks MM, Sorgeloos F, van Kuppeveld F, Roberts LO, Goodfellow IG, Langereis MA, Locker Nicolas (2016) Feline Calicivirus infection disrupts the assembly of cytoplasmic stress granules and induces G3BP1 cleavage,Journal of Virology 90 (14) pp. 6489-6501
American Society for Microbiology
In response to stress such as virus infection, cells can stall translation by storing mRNAs away in cellular compartments called stress granules (SGs). This defence mechanism favours cell survival by limiting the use of energy and nutrients until the stress is resolved. In some cases it may also block viral propagation as viruses are dependent on the host cell resources to produce viral proteins. Human norovirus is a member of the Caliciviridae family responsible for gastroenteritis outbreaks worldwide. Previous studies on caliciviruses have identified mechanisms by which they can usurp the host translational machinery, using the viral protein genome-linked VPg, or regulate host protein synthesis through the MAPK pathway. Herein we examined the effect of feline calicivirus (FCV) infection on SGs accumulation. We show that FCV infection impairs the assembly of SGs despite an increased phosphorylation of eukaryotic initiation factor eIF2±, a hallmark of stress pathway activation. Furthermore SGs did not accumulate in FCV-infected cells that are stressed with arsenite or hydrogen peroxide. FCV infection resulted in the cleavage of the SG-nucleating protein Ras-GTPase activating SH3 domain-binding protein (G3BP1), which is mediated by the viral 3C-like proteinase NS6Pro 38 . Using mutational analysis, we identified the FCV-induced cleavage site within G3BP1, which differs from the poliovirus 3C proteinase cleavage site previously identified. Finally, we showed that NS6Pro 41 -mediated G3BP1 cleavage impairs SGs assembly. In contrast, murine norovirus (MNV) infection did not impact arsenite-induced SG assembly or G3BP1 integrity suggesting that related caliciviruses have distinct effects on the stress response pathway.
Butler Rachel, Krishnan N, Garcia-Jimenez W, Francis R, Martyn A, Mendum T, Felemban Shaza, Locker Nicolas, Salguero Bodes J, Robertson B, Stewart Graham (2017) Susceptibility of M. tuberculosis-infected host cells to phospho-MLKL driven necroptosis is dependent on cell type and presence of TNF±,Virulence 8 (8) pp. 1820-1832
Taylor & Francis
An important feature of Mycobacterium tuberculosis pathogenesis is the ability to control
cell death in infected host cells, including inhibition of apoptosis and stimulation of necrosis.
Recently an alternative form of programmed cell death, necroptosis, has been described
where necrotic cell death is induced by apoptotic stimuli under conditions where apoptotic
execution is inhibited. We show for the first time that M. tuberculosis and TNF± synergise
to induce necroptosis in murine fibroblasts via RIPK1-dependent mechanisms and
characterized by phosphorylation of Ser345 of the MLKL necroptosis death effector.
However, in murine macrophages M. tuberculosis and TNF± induce non-necroptotic cell
death that is RIPK1-dependent but independent of MLKL phosphorylation. Instead, M.
tuberculosis-infected macrophages undergo RIPK3-dependent cell death which occurs both in the presence and absence of TNF± and involves the production of mitochondrial ROS.
Immunocytochemical staining for MLKL phosphorylation further demonstrated the
occurrence of necroptosis in vivo in murine M. tuberculosis granulomas. Phosphorylated-
MLKL immunoreactivity was observed associated with the cytoplasm and nucleus of
fusiform cells in M. tuberculosis lesions but not in proximal macrophages. Thus whereas
pMLKL-driven necroptosis does not appear to be a feature of M. tuberculosis-infected
macrophage cell death, it may contribute to TNF±-induced cytotoxicity of the lung stroma
and therefore contribute to necrotic cavitation and bacterial dissemination.
Riitho V, Walters A, Somavarapu S, Lamp B, Rümenapf T, Krey T, Rey F, Oviedo-Orta E, Stewart G, Locker N, Steinbach F, Graham S (2017) Design and evaluation of the immunogenicity and efficacy of a biomimetic particulate formulation of viral antigens,Scientific Reports 7 13743
Nature Publishing Group
Subunit viral vaccines are typically not as efficient as live attenuated or inactivated vaccines at inducing
protective immune responses. This paper describes an alternative ?biomimetic? technology; whereby
viral antigens were formulated around a polymeric shell in a rationally arranged fashion with a surface
glycoprotein coated on to the surface and non-structural antigen and adjuvant encapsulated. We
evaluated this model using BVDV E2 and NS3 proteins formulated in poly-(D, L-lactic-co-glycolic
acid) (PLGA) nanoparticles adjuvanted with polyinosinic:polycytidylic acid (poly(I:C) as an adjuvant
(Vaccine-NP). This Vaccine-NP was compared to ovalbumin and poly(I:C) formulated in a similar
manner (Control-NP) and a commercial adjuvanted inactivated BVDV vaccine (IAV), all inoculated
subcutaneously and boosted prior to BVDV-1 challenge. Significant virus-neutralizing activity, and E2
and NS3 specific antibodies were observed in both Vaccine-NP and IAV groups following the booster
immunisation. IFN-³ responses were observed in ex vivo PBMC stimulated with E2 and NS3 proteins
in both vaccinated groups. We observed that the protection afforded by the particulate vaccine was
comparable to the licenced IAV formulation. In conclusion, the biomimetic particulates showed a
promising immunogenicity and efficacy profile that may be improved by virtue of being a customisable
mode of delivery.
Willcocks M, Zaini S, Chamond N, Ulryck N, Allouche D, Rajagopalan N, Davids N, Fahnøe U, Hadsbjerg J, Rasmussen T, Roberts L, Sargueil B, Belsham G, Locker N (2017) Distinct roles for the IIId2 sub-domain in pestivirus and picornavirus Internal Ribosome Entry Sites,Nucleic Acids Research 45 (22) pp. 13016-13028
Oxford University Press
Viral internal ribosomes entry site (IRES) elements coordinate the recruitment of the host translation
machinery to direct the initiation of viral protein synthesis. Within hepatitis C virus (HCV)-like IRES
elements, the sub-domain IIId(1) is crucial for recruiting the 40S ribosomal subunit. However, some
HCV-like IRES elements possess an additional sub-domain, termed IIId2, whose function remains
unclear. Herein we show that IIId2 sub-domains from divergent viruses have different functions. The
IIId2 sub-domain present in Seneca valley virus (SVV), a picornavirus, is dispensable for IRES activity,
while the IIId2 sub-domains of two pestiviruses, classical swine fever virus (CSFV) and border
disease virus (BDV), are required for 80S ribosomes assembly and IRES activity. Unlike in SVV, the
deletion of IIId2 from the CSFV and BDV IRES elements impairs initiation of translation by inhibiting
the assembly of 80S ribosomes. Consequently, this negatively affects the replication of CSFV and
BDV. Finally, we show that the SVV IIId2 sub-domain is required for efficient viral RNA synthesis and
growth of SVV, but not for IRES function. This study sheds light on the molecular evolution of viruses
by clearly demonstrating that conserved RNA structures, within distantly related RNA viruses, have
acquired different roles in the virus life cycles.
The role of m6A methylation of RNA has remained elusive for decades, however recent technological advances are now allowing the mapping of the m6A methylation landscape at nucleotide level. This has spurred an explosion in our understanding of the role played by RNA epigenetics in RNA biology. m6A modifications have been tied to almost every aspects of the mRNA life cycle and it is now clear that RNA virus genomes are subject to m6A methylation. These modifications play various roles in the viral replication cycle. This review will summarize recent breakthroughs concerning m6A RNA modification and their implications for cellular and viral RNAs.
Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus, the etiological agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). One of the key viral proteins that contribute to tumorigenesis is vFLIP, a viral homolog of the FLICE inhibitory protein. This KSHV protein interacts with the NFºB pathway to trigger the expression of antiapoptotic and proinflammatory genes and ultimately leads to tumor formation. The expression of vFLIP is regulated at the translational level by an internal ribosomal entry site (IRES) element. However, the precise mechanism by which ribosomes are recruited internally and the exact location of the IRES has remained elusive. The aims of this study were to confirm the previously identified 252-nt fragment directly upstream of vFLIP as the location of the vFLIP IRES in cellulo and to determine the structure and mechanism of action of the vFLIP IRES. Here we show that a 252-nt, within a coding region, directs translation in HEK293 cells. We have also established its RNA structure using chemical and enzymatic probing of RNA structure in solution and mutational analysis studies revealed that the domain If of the vFLIP IRES is crucial for its activity. Also, we demonstrate that IRES activity requires the presence of eIF4A and the eIF4E-eIF4G interaction. These interactions may define a new paradigm for IRES-mediated translation. Finally, we attempted to identify cellular proteins that may interact with the vFLIP IRES using several types of protein affinity chromatography, but we could detect a protein interacting with vFLIP IRES but yet to be confirmed.
Culicoides biting midges are the biological vector for Bluetongue virus (BTV), the aetiological agent of bluetongue disease (BT) in ruminants. Like all other hematophagous arthropods, Culicoides midges
secrete pharmacologically active saliva during the acquisition of a blood meal. This saliva acts to
counteract the mammalian host blood coagulation and local immune responses that are initiated as a
result of vector blood feeding. As shown for numerous other arthropod-pathogen systems, Culicoides
saliva may play a role in BTV infection, beyond simple virus transmission during blood meal acquisition.
The work of this thesis highlights for the first time, that Culicoides sonorensis saliva can enhance BTV
infection and replication within bovine monocytes, most likely via a mechanism that influences the early
stages of virus infection. Furthermore, the undertaken studies identify that the saliva of C. sonorensis
naturally contains bacterial lipopolysaccharides (LPS), and suggest that this non-protein salivary
component is likely responsible for the observed saliva induced enhancement of BTV infection and
replication. C. sonorensis saliva induced modification of the bovine host cytokine response was also
assessed in this thesis. The presence of saliva during BTV infection was shown to enhance the
production of the immunomodulatory cytokine IL-10 and also reduce the BTV induced production of
IFN-g from PBMC cultures from some, but not all, cattle.
Collectively the results of this thesis highlight the active properties of C. sonorensis saliva in the context
of enhancing BTV infection and the complex relationships between BTV, Culicoides midges, their
salivary material and the bovine host, indicating the continuing need to elucidate these multifaceted
virus-vector-host interactions. Furthermore, the study of these interactions will enable a greater
understanding of the underlying mechanisms of BTV pathogenesis thus leading to potential applications
to novel methods of viral control.
Phillips Jack O, Butt Louise E, Henderson Charlotte A, Devonshire Martin, Healy Jess, Conway Stuart J, Locker Nicolas, Pickford Andrew R, Vincent Helen A, Callaghan Anastasia J (2018) High-density functional-RNA arrays as a versatile platform for studying RNA-based interactions,Nucleic Acids Research 46 (14) pp. 1-10
Oxford University Press (OUP)
We are just beginning to unravel the myriad of interactions in which non-coding RNAs participate. The intricate RNA interactome is the foundation of many biological processes, including bacterial virulence and human disease, and represents unexploited resources for the development of potential therapeutic interventions. However, identifying specific associations of a given RNA from the multitude of possible binding partners within the cell requires robust high-throughput systems for their rapid screening. Here, we present the first demonstration of functional-RNA arrays as a novel platform technology designed for the study of such interactions using immobilized, active RNAs. We have generated high-density RNA arrays by an innovative method involving surface-capture of in vitro transcribed RNAs. This approach has significant advantages over existing technologies, particularly in its versatility in regards to binding partner character. Indeed, proof-of-principle application of RNA arrays to both RNA?small molecule and RNA?RNA pairings is demonstrated, highlighting their potential as a platform technology for mapping RNA-based networks and for pharmaceutical screening. Furthermore, the simplicity of the method supports greater user-accessibility over currently available technologies. We anticipate that functional-RNA arrays will find broad utility in the expanding field of RNA characterization.
Riitho Victor, Larska Magdalena, Strong Rebecca, La Rocca Anna, Locker Nicolas, Alenius Stefan, Steinbach Falko, Liu Lihong, Uttenthal Åse, Graham Simon P. (2018) Comparative analysis of adaptive immune responses following experimental infections of cattle with bovine viral diarrhoea virus-1 and an Asiatic atypical ruminant pestivirus,Vaccine 36 (30) pp. 4494-4500
Atypical ruminant pestiviruses are closely related to the two bovine viral diarrhoea virus (BVDV) species, BVDV-1 and BVDV-2. While there is evidence of cross-protective immune responses between BVDV-1 and BVDV-2, despite antigenic differences, there is little information on the antigenic cross-reactivity with atypical ruminant pestiviruses. The aim of this study was therefore to assess the specificity of antibody and T cell responses induced by experimental infection of calves with BVDV-1 strain Ho916, Th/04_KhonKaen (TKK), an Asiatic atypical ruminant pestivirus, or co-infection with both viruses. Homologous virus neutralization was observed in sera from both single virus infected and co-infected groups, while cross-neutralization was only observed in the TKK infected group. T cell IFN-³ responses to both viruses were observed in the TKK infected animals, whereas Ho916 infected calves responded better to homologous virus. Specifically, IFN-³ responses to viral non-structural protein, NS3, were observed in all infected groups while responses to viral glycoprotein, E2, were virus-specific. Broader antigen-specific cytokine responses were observed with similar trends between inoculation groups and virus species. The limited T cell and antibody immune reactivity of Ho916 inoculated animals to TKK suggests that animals vaccinated with current BVDV-1-based vaccines may not be protected against atypical ruminant pestiviruses.
Choe Junho, Lin Shuibin, Zhang Wencai, Liu Qi, Wang Longfei, Ramirez-Moya Julia, Du Peng, Kim Wantae, Tang Shaojun, Sliz Piotr, Santisteban Pilar, George Rani E, Richards William G, Wong Kwok-Kin, Locker Nicolas, Slack Frank J, Gregory Richard I (2018) mRNA circularization by METTL3-eIF3h enhances translation and promotes oncogenesis,Nature 561 pp. 556-560
Nature Publishing Group
N6-Methyladenosine (m6A), the most abundant posttranscriptional messenger RNA (mRNA)
modification, is emerging as an important regulator of gene expression1. Manipulation of m6A
impacts different developmental and biological processes, and altered m6A homeostasis is
linked to cancer2-5. m6A is catalyzed by METTL3 and enriched in the 3? untranslated region (3?
UTR) of a large subset of mRNAs at sites close to the stop codon1. METTL3 can promote
translation but the mechanism and widespread relevance remain unknown2. Here we show that
METTL3 enhances translation only when tethered to reporter mRNA at sites close to the stop
codon supporting a mRNA looping mechanism for ribosome recycling and translational
control. Electron microscopy reveals the topology of individual polyribosomes with single
METTL3 foci found in close proximity to 5? cap-binding proteins. We identify a direct physical and functional interaction between METTL3 and the eukaryotic translation initiation factor 3
subunit h (eIF3h). METTL3 promotes translation of a large subset of oncogenic mRNAs,
including Bromodomain-containing protein 4 (BRD4) that are also m6A-modified in human
primary lung tumors. The METTL3-eIF3h interaction is required for enhanced translation,
formation of densely packed polyribosomes, and oncogenic transformation. METTL3 depletion
inhibits tumorigenicity and sensitizes lung cancer cells to BRD4 inhibition. These findings
uncover a mRNA looping mechanism of translation control and identify METTL3-eIF3h as a
potential cancer therapeutic target.
Bovine viral diarrhoea virus (BVDV) is an important pathogen that causes infectious disease of cattle worldwide and results in significant economic losses. Vaccination has long been used as a tool for control of BVDV but inadequacies of existing vaccines have hampered eradication efforts. Attempts to develop sub-unit vaccines have focused on the structural envelope protein E2, which is a dominant target of neutralising antibodies and as well as CD4 T cell responses. This study aimed to rationally address the development of more efficacious vaccines by characterising the kinetics and specificity of T cell responses to a BVDV type 1 peptide library in calves rendered immune to BVDV following recovery from experimental infection. Upon identification of E2 and NS3 as the dominant targets of CD4 T cell responses, we assessed whether T cells induced by one virus genotype were capable of responding to a heterologous virus genotype and to identified E2 and NS3 as targets of genotype-specific and genotype transcending responses, respectively. This finding strengthened the argument for inclusion of both antigens in a subunit vaccine formulation. A nanoparticulate formulation of E2 and NS3 adjuvanted with poly(I:C) was shown to induce protective responses comparable to a commercial available BVDV vaccine in a vaccination and challenge experiment. It is hoped that the data generated will have implications for the design of improved vaccines against BVD.
Equine infectious anaemia virus (EIAV) is a retrovirus with worldwide distribution which is notifiable to the OIE. Despite its importance to the equine industry, most information regarding its biology have been obtained using only two strains (EIAVWYO and EIAVLIA) from the USA and China, respectively. Recently full genome sequences from Ireland, Italy and Japan have been published; however, this is still not representative of the number of EIAV outbreaks experienced globally each year. The limited availability of published sequences makes design of a universal EIAV PCR difficult, hence diagnosis is solely reliant on serology. Accordingly, it is important to further investigate the re?emerging cases in other areas of the world. Here, we provide information regarding the outbreaks of EIA in England in 2010 and 2012 including the molecular characterization of strains. Full genome was obtained for two symptomatic cases but could not be resolved for the asymptomatic cases. The two British genomes from 2010 (EIAVDEV) and 2012 (EIAVCOR) each represent a new phylogenetic group, each differing genetically from the other available full genome sequences by 21.1%?25.5%. That the majority of new EIAV full genome sequences to be published adds another phylogenetic group indicates that the surface of EIAV global diversity is just being scratched. These data highlight that further work is needed to fully understand EIAV genetic diversity, namely the full genome sequencing of EIAV cases from a variety of locations and time points. This would aid both the use of phylogenetics in parallel with horse tracing as the epidemiological tool of disease tracking and the design of a universally applicable molecular diagnostic method.
Equine hepacivirus (EHcV) (now also classified as hepacivirus A) is the closest genetic relative to hepatitis C virus (HCV) and is proposed to have diverged from HCV within the last 1000 years. The 52 untranslated regions (UTRs) of both HCV and EHcV exhibit internal ribosome entry site (IRES) activity, allowing cap-independent translational initiation, yet only the HCV 52UTR has been systematically analysed. Here, we report a detailed structural and functional analysis of the EHcV 52UTR. The secondary structure was determined using selective 22 hydroxyl acylation analysed by primer extension (SHAPE), revealing four stem?loops, termed SLI, SLIA, SLII and SLIII, by analogy to HCV. This guided a mutational analysis of the EHcV 52UTR, allowing us to investigate the roles of the stem?loops in IRES function. This approach revealed that SLI was not required for EHcV IRES-mediated translation. Conversely, SLIII was essential, specifically SLIIIb, SLIIId and a GGG motif that is conserved across the Hepaciviridae. Further SHAPE analysis provided evidence that this GGG motif mediated interaction with the 40S ribosomal subunit, whilst a CUU sequence in the apical loop of SLIIIb mediated an interaction with eIF3. In addition, we showed that a microRNA122 target sequence located between SLIA and SLII mediated an enhancement of translation in the context of a subgenomic replicon. Taken together, these results highlight the conservation of hepaciviral translation mechanisms, despite divergent primary sequences.
Hosmillo Myra, Lu Jia, McAllaster Michael R, Eaglesham James B, Wang Xinjie, Emmott Edward, Domingues Patricia, Chaudhry Yasmin, Fitzmaurice Tim J, Tung Matthew KH, Panas Marc Dominik, McInerney Gerald, Locker Nicolas, Wilen Craig B, Goodfellow Ian G (2019) Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation,eLife 8 e46681 pp. 1-35
eLife Sciences Publications
Knowledge of the host factors required for norovirus replication has been hindered by the challenges associated with culturing human noroviruses. We have combined proteomic analysis of the viral translation and replication complexes with a CRISPR screen, to identify host factors required for norovirus infection. The core stress granule component G3BP1 was identified as a host factor essential for efficient human and murine norovirus infection, demonstrating a conserved function across the Norovirus genus. Furthermore, we show that G3BP1 functions in the novel paradigm of viral VPg-dependent translation initiation, contributing to the assembly of translation complexes on the VPg-linked viral positive sense RNA genome by facilitating ribosome recruitment. Our data uncovers a novel function for G3BP1 in the life cycle of positive sense RNA viruses and identifies the first host factor with pan-norovirus pro-viral activity.
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.
Brocard Michele, Ladevaia Valentina, Klein Philipp, Hall Belinda, Lewis Glenys, Lu Jia, Burke James, Willcocks Margaret, Parker Roy, Goodfellow Ian G., Ruggieri Alessia, Locker Nicolas (2020) Norovirus infection results in eIF2± independent host translation shut-off and remodels the G3BP1 interactome evading stress granule formation,PLoS Pathogens
Public Library of Science
Viral infections impose major stress on the host cell. In response, stress pathways can rapidly deploy defence mechanisms by shutting off the protein synthesis machinery and triggering the accumulation of mRNAs into stress granules to limit the use of energy and nutrients. Because this threatens viral gene expression, viruses need to evade these pathways to propagate. Human norovirus is responsible for gastroenteritis outbreaks worldwide. Here we examined how norovirus interacts with the eIF2± signaling axis controlling translation and stress granules. While norovirus infection represses host cell translation, our mechanistic analyses revealed that eIF2± signaling mediated by the stress kinase GCN2 is uncoupled from translational stalling. Moreover, infection results in a redistribution of the RNA-binding protein G3BP1 to replication complexes and remodelling of its interacting partners, allowing the avoidance from canonical stress granules. These results define novel strategies by which norovirus undergo efficient replication whilst avoiding the host stress response and manipulating the G3BP1 interactome.