Picornavirus security proteins promote the release of extracellular vesicle enclosed viruses via the modulation of host kinases.

The discovery that extracellular vesicles (EVs) serve as carriers of virus particles calls for a reevaluation of the release strategies of non-enveloped viruses. Little is currently known about the molecular mechanisms that determine the release and composition of EVs produced by virus-infected cells, as well as conservation of these mechanisms among viruses. We previously described an important role for the Leader protein of the picornavirus encephalomyocarditis virus (EMCV) in the induction of virus-carrying EV subsets with distinct molecular and physical properties. EMCV L acts as a 'viral security protein' by suppressing host antiviral stress and type-I interferon (IFN) responses. Here, we tested the ability of functionally related picornavirus proteins of Theilers murine encephalitis virus (TMEV L), Saffold virus (SAFV L), and coxsackievirus B3 (CVB3 2Apro), to rescue EV and EV-enclosed virus release when introduced in Leader-deficient EMCV. We show that all viral security proteins tested were able to promote virus packaging in EVs, but that only the expression of EMCV L and CVB3 2Apro increased overall EV production. We provide evidence that one of the main antiviral pathways counteracted by this class of picornaviral proteins, i.e. the inhibition of PKR-mediated stress responses, affected EV and EV-enclosed virus release during infection. Moreover, we show that the enhanced capacity of the viral proteins EMCV L and CVB3 2Apro to promote EV-enclosed virus release is linked to their ability to simultaneously promote the activation of the stress kinase P38 MAPK. Taken together, we demonstrate that cellular stress pathways involving the kinases PKR and P38 are modulated by the activity of non-structural viral proteins to increase the release EV-enclosed viruses during picornavirus infections. These data shed new light on the molecular regulation of EV production in response to virus infection.

Validating the EMCV IRES Secondary Structure with Structure-Function Analysis.

The encephalomyocarditis virus internal ribosome entry site (EMCV IRES) is a structured RNA sequence found in the 5' UTR of the genomic RNA of the encephalomyocarditis virus. The EMCV IRES structure facilitates efficient translation initiation without needing a 5' m7G cap or the cap-binding protein eIF4E. The secondary structure of IRES has been the subject of several previous studies, and a number of different structural models have been proposed. Though some domains of the IRES are conserved across the different secondary structure models, domain I of the IRES varies greatly across them. A literature comparison led to the identification of three regions of interest that display structural heterogeneity within past secondary structure models. To test the accuracy of the secondary structure models in these regions, we employed mutational analysis and SHAPE probing. Mutational analysis revealed that two helical regions within the identified regions of interest are important for IRES translation. These helical regions are consistent with only one of the structure predictions in the literature and do not form in EMCV IRES structures predicted using modern secondary structure prediction methods. The importance of these regions is further supported by multiple SHAPE protections when probing was performed after in vitro translation, indicating that these regions are involved in the IRES translation complex. This work validates a published structure and demonstrates the importance of domain I during EMCV IRES translation initiation.

[The role of the encephalomyocarditis virus type 1 proteins L and 2A in the inhibition of the synthesis of cellular proteins and the accumulation of viral proteins during infection].

INTRODUCTION: Infection of cells with encephalomyocarditis virus type 1 (EMCV-1, Cardiovirus A: Picornaviridae) is accompanied by suppression of cellular protein synthesis. The main role in the inhibition of cellular translation is assigned to the L and 2A «security¼ proteins. The mechanism of the possible influence of the L protein on cellular translation is unknown. There are hypotheses about the mechanism of influence of 2A protein on the efficiency of cap-dependent translation, which are based on interaction with translation factors and ribosome subunits. However, the available experimental data are contradictory, obtained using different approaches, and do not form a unified model of the interaction between the L and 2A proteins and the cellular translation machinery. AIM: To study the role of L and 2A «security¼ proteins in the suppression of translation of cellular proteins and the efficiency of translation and processing of viral proteins in infected cells. MATERIALS AND METHODS: Mutant variants of EMCV-1 were obtained to study the properties of L and 2A viral proteins: Zfmut, which has a defective L; Δ2A encoding a partially deleted 2A; Zfmut&Δ2A containing mutations in both proteins. Translational processes in infected cells were studied by Western-blot and the pulse method of incorporating radioactively labeled amino acids (14C) into newly synthesized proteins, followed by radioautography. RESULTS: The functional inactivation of the 2A protein does not affect the inhibition of cellular protein synthesis. A direct correlation was found between the presence of active L protein and specific inactivation of cellular protein synthesis at an early stage of viral infection. Nonspecific suppression of the translational processes of the infected cell, accompanied by phosphorylation of eIF2α, occurs at the late stage of infection. Partial removal of the 2A protein from the EMCV-1 genome does not affect the development of this process, while inactivation of the L protein accelerates the onset of complete inhibition of protein synthesis. Partial deletion of the 2A disrupts the processing of viral capsid proteins. Suppression of L protein functions leads to a decrease in the efficiency of viral translation. CONCLUSION: A study of the role of EMCV-1 L and 2A proteins during the translational processes of an infected cell, first performed using infectious viral pathogens lacking active L and 2A proteins in one experiment, showed that 2A protein is not implicated in the inhibition of cellular translation in HeLa cells; L protein seems to play an important role not only in the specific inhibition of cellular translation but also in maintaining the efficient synthesis of viral proteins; 2A protein is involved not only in primary but also in secondary processing of EMCV-1 capsid proteins.

Differential regulation of ATP hydrolysis of RIG-I-like receptors by transactivation response RNA-binding protein.

Retinoic acid inducible gene (RIG)-I-like receptors (RLRs), including RIG-I, melanoma differentiation associated-5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), play pivotal roles in viral RNA sensing to initiate antiviral interferon (IFN) responses. We previously reported that an RNA-silencing regulator, transactivation response RNA-binding protein (TRBP), up-regulates MDA5/LGP2-mediated IFN responses through interaction with LGP2. Here, we aimed to investigate the mechanism underlying the TRBP-mediated up-regulation of IFN response. Data indicated that phosphomimetic TRBP showed a modest effect, whereas the nonphosphorylated form exhibited hyperactivity in enhancing Cardiovirus-triggered IFN responses. These results suggest that encephalomyocarditis virus (EMCV) attenuates the TRBP-mediated IFN response via TRBP phosphorylation, since EMCV infection activates the kinase responsible for TRBP phosphorylation for virus replication. Furthermore, we found that TRBP-mediated up-regulation of IFN response required the ATP hydrolysis and RNA binding of LGP2. TRBP enhanced RNA-dependent ATP hydrolysis by LGP2 but not that by RIG-I or MDA5. Nonphosphorylated TRBP exhibited higher levels of activity than phosphomimetic TRBP did, suggesting its possible involvement in the mechanism underlying the up-regulation of IFN response. TRBP activated the ATP hydrolysis of LGP2 and RIG-I, but not that of MDA5, in the absence of RNA. Collectively, we showed that TRBP differentially regulated RLR-mediated ATP hydrolysis. Further elucidation of the mechanism underlying the regulation of ATP hydrolysis leading to IFN response and self- and non-self-RNA discrimination could advance the development of effective therapeutic agents against autoimmune diseases.

The Src1-PGC1α-AP1 complex-dependent secretion of substance P induces inflammation and apoptosis in encephalomyocarditis virus-infected mice.

Substance P (SP), a neuropeptide consisting of 11 amino acid residues, is involved in the pathogenesis of encephalomyocarditis virus (EMCV)-induced myocarditis by stimulating the production of proinflammatory cytokines. However, the underlying mechanism that regulates SP production is still unknown. In this study, we report the transcriptional regulation of the Tachykinin Precursor 1 (TAC1) gene that encodes SP by a transcriptional complex composed of Steroid Receptor Coactivator 1 (Src1), Peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC1α), and Activator Protein 1 (AP1) transcription factor. Infection of mice with EMCV induced the accumulation of PGC1α and increased TAC1 expression, thereby promoting the secretion of SP, initiating apoptosis, and elevating proinflammatory cytokine levels. In vitro overexpression of the Src1-PGC1α-AP1 members also induced TAC1 expression, increased the SP concentration, initiated apoptosis, and elevated proinflammatory cytokine concentrations. Depletion or inhibition of the Src1-PGC1α-AP1 complex reversed these effects. The administration of gossypol, an Src1 inhibitor, or SR1892, a PGC1α inhibitor, to EMCV-infected mice attenuated myocarditis. Taken together, our results reveal that the upregulation of TAC1 and the secretion of SP in EMCV-induced myocarditis are dependent on the Src1-PGC1α-AP1 complex. Targeting the Src1-PGC1α-AP1 complex may represent a new therapeutic strategy for myocarditis.

The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy.

Naked viruses can escape host cells before the induction of lysis via release in extracellular vesicles (EVs). These nanosized EVs cloak the secreted virus particles in a host-derived membrane, which alters virus-host interactions that affect infection efficiency and antiviral immunity. Currently, little is known about the viral and host factors regulating this form of virus release. Here, we assessed the role of the encephalomyocarditis virus (EMCV) Leader protein, a 'viral security protein' that subverts the host antiviral response. EV release upon infection with wildtype virus or a Leader-deficient mutant was characterized at the single particle level using high-resolution flow cytometry. Inactivation of the Leader abolished EV induction during infection and strongly reduced EV-enclosed virus release. We demonstrate that the Leader promotes the release of virions within EVs by stimulating a secretory arm of autophagy. This newly discovered role of the EMCV Leader adds to the variety of mechanisms via which this protein affects virus-host interactions. Moreover, these data provide first evidence for a crucial role of a non-structural viral protein in the non-lytic release of picornaviruses via packaging in EVs.

Rapid detection of porcine encephalomyocarditis virus (EMCV) by isothermal reverse transcription recombinase polymerase amplification assays.

In this study, we combined reverse transcription recombinase polymerase amplification assay with the fluorescence detection platform (qRT-RPA) and lateral flow biosensor (LFB RT-RPA) to allow for rapid detection of porcine encephalomyocarditis virus (EMCV). Primers and probes were designed to target the highly conserved region of 3D gene of porcine EMCV. The optimal reaction condition of qRT-RPA and LFB RT-RPA was set as 42 °C for 20 min. The assays were highly specific to EMCV and no cross-reactions were observed with seven other porcine viruses. With a 10-fold serially diluted EMCV genomic RNA as template, the limit of detection was 1.0 × 102 and 1.0 × 101 copies for qRT-RPA assay and LFB RT-RPA assay, respectively. A total of 92 samples from different sources were examined using qRT-RPA, LFB RT-RPA and qRT-PCR. We found 100% diagnostic agreement between qRT-RPA (23/92) and qRT-PCR (23/92), and 97.83% diagnostic agreement between LFB RT-RPA (25/92) and qRT-PCR (23/92). There was no significant difference in performance between the RT-RPA assays developed in this study and a previously described qRT-PCR. However, RT-RPA assays were rapid and easy to perform while LFB RT-RPA exhibited higher sensitivity for EMCV than qRT-PCR. Therefore, the developed EMCV RT-RPA assays provide an attractive and promising tool for effective detection of EMCV in low-resource settings.

Genes for tRNA recycling are upregulated in response to infection with Theiler's mouse encephalitis virus.

The concept of tRNA recycling has recently emerged from the studies of ribosome-associated quality control. Therein tRNase ZS removes the 2', 3'>p from the ANKZF1-cleaved tRNA and the subsequent TRNT1 action re-generates the intact tRNA. To know the roles of the tRNA recycling in vivo, we investigated how viral infection affects the tRNA recycling system by analyzing the mRNA levels of tRNase ZS and TRNT1. We found that both genes in HeLa cells are upregulated in response to infection of Theiler's mouse encephalitis virus but not to that of an influenza A virus. Upregulation was also observed in cells infected with encephalomyocarditis virus with reduced efficiency. The levels of the IFN-ß mRNA appeared to positively correlate with those of the tRNase ZS and TRNT1 mRNAs. The tRNase ZS gene may be regulated post-transcriptionally in the cells infected with Theiler's mouse encephalitis virus.

Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch.

Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection.

Encephalomyocarditis Virus Abrogates the Interferon Beta Signaling Pathway via Its Structural Protein VP2.

Type I interferon (IFN)-mediated antiviral responses are critical for modulating host-virus responses, and indeed, viruses have evolved strategies to antagonize this pathway. Encephalomyocarditis virus (EMCV) is an important zoonotic pathogen, which causes myocarditis, encephalitis, neurological disease, reproductive disorders, and diabetes in pigs. This study aims to understand how EMCV interacts with the IFN pathway. EMCV circumvents the type I IFN response by expressing proteins that antagonize cellular innate immunity. Here, we show that EMCV VP2 is a negative regulator of the IFN-ß pathway. This occurs via the degradation of the MDA5-mediated cytoplasmic double-stranded RNA (dsRNA) antiviral sensing RIG-I-like receptor (RLR) pathway. We show that structural protein VP2 of EMCV interacts with MDA5, MAVS, and TBK1 through its C terminus. In addition, we found that EMCV VP2 could significantly degrade RLRs by the proteasomal and lysosomal pathways. For the first time, EMCV VP2 was shown to play an important role in EMCV evasion of the type I IFN signaling pathway. This study expands our understanding that EMCV utilizes its capsid protein VP2 to evade the host antiviral response.IMPORTANCE Encephalomyocarditis virus is an important pathogen that can cause encephalitis, myocarditis, neurological diseases, and reproductive disorders. It also causes huge economic losses for the swine industry worldwide. Innate immunity plays an important role in defending the host from pathogen infection. Understanding pathogen microorganisms evading the host immune system is of great importance. Currently, whether EMCV evades cytosolic RNA sensing and signaling is still poorly understood. In the present study, we found that viral protein VP2 antagonized the RLR signaling pathway by degrading MDA5, MAVS, and TBK1 protein expression to facilitate viral replication in HEK293 cells. The findings in this study identify a new mechanism for EMCV evading the host's innate immune response, which provide new insights into the virus-host interaction and help develop new antiviral approaches against EMCV.

Quantifying the dynamics of IRES and cap translation with single-molecule resolution in live cells.

Viruses use internal ribosome entry sites (IRES) to hijack host ribosomes and promote cap-independent translation. Although they are well-studied in bulk, the dynamics of IRES-mediated translation remain unexplored at the single-molecule level. Here, we developed a bicistronic biosensor encoding distinct repeat epitopes in two open reading frames (ORFs), one translated from the 5' cap, and the other from the encephalomyocarditis virus IRES. When combined with a pair of complementary probes that bind the epitopes cotranslationally, the biosensor lights up in different colors depending on which ORF is translated. Using the sensor together with single-molecule tracking and computational modeling, we measured the kinetics of cap-dependent versus IRES-mediated translation in living human cells. We show that bursts of IRES translation are shorter and rarer than bursts of cap translation, although the situation reverses upon stress. Collectively, our data support a model for translational regulation primarily driven by transitions between translationally active and inactive RNA states.

Unpaired Guanosines in the 5' Untranslated Region of HIV-1 RNA Act Synergistically To Mediate Genome Packaging.

The viral protein Gag selects full-length HIV-1 RNA from a large pool of mRNAs as virion genome during virus assembly. Currently, the precise mechanism that mediates the genome selection is not understood. Previous studies have identified several sites in the 5' untranslated region (5' UTR) of HIV-1 RNA that are bound by nucleocapsid (NC) protein, which is derived from Gag during virus maturation. However, whether these NC binding sites direct HIV-1 RNA genome packaging has not been fully investigated. In this report, we examined the roles of single-stranded exposed guanosines at NC binding sites in RNA genome packaging using stable cell lines expressing competing wild-type and mutant HIV-1 RNAs. Mutant RNA packaging efficiencies were determined by comparing their prevalences in cytoplasmic RNA and in virion RNA. We observed that multiple NC binding sites affected RNA packaging; of the sites tested, those located within stem-loop 1 of the 5' UTR had the most significant effects. These sites were previously reported as the primary NC binding sites by using a chemical probe reverse-footprinting assay and as the major Gag binding sites by using an in vitro assay. Of the mutants tested in this report, substituting 3 to 4 guanosines resulted in <2-fold defects in packaging. However, when mutations at different NC binding sites were combined, severe defects were observed. Furthermore, combining the mutations resulted in synergistic defects in RNA packaging, suggesting redundancy in Gag-RNA interactions and a requirement for multiple Gag binding on viral RNA during HIV-1 genome encapsidation.IMPORTANCE HIV-1 must package its RNA genome during virus assembly to generate infectious viruses. To better understand how HIV-1 packages its RNA genome, we examined the roles of RNA elements identified as binding sites for NC, a Gag-derived RNA-binding protein. Our results demonstrate that binding sites within stem-loop 1 of the 5' untranslated region play important roles in genome packaging. Although mutating one or two NC-binding sites caused only mild defects in packaging, mutating multiple sites resulted in severe defects in genome encapsidation, indicating that unpaired guanosines act synergistically to promote packaging. Our results suggest that Gag-RNA interactions occur at multiple RNA sites during genome packaging; furthermore, there are functionally redundant binding sites in viral RNA.

Effect of encephalomyocarditis virus 3'Untranslated region on GFP transient expression: implications in recombinant protein production / Efecto de la Región 3'-no traducida del virus de la encefalomiocarditis sobre la expresión transitoria de GFP: implicaciones en la producción de proteínas recombinantes

The enrichment of therapeutic protein production yield in mammalian cell cultures by modulating mRNA stability is a fairly new strategy in biotechnological applications. Here, we describe the application of 3'-untranslated region (3'UTR) from RNA viral genome to modulate mRNA stability.The data obtained showed that the use of the 3 'UTR sequence of the encephalomyocarditis virus (EMCV 3'UTR) downstream of the target gene was not able to significantly modulate the free energy density indicators of the RNA. However, the sequence influenced the stability of the mRNA (and, therefore, the amount of protein production) in a cell type and time-dependent manner, indicating a central role of mRNA-stabilizing binding sites/cellular factors in this process. Our data might be of interest for the biotechnology community to improve recombinant protein production in mammalian cell cultures and RNA-based therapy/vaccination approaches.

El enriquecimiento de la producción terapéutica de proteínas en cultivos de células de mamíferos mediante la modulación de la estabilidad del ARNm es una estrategia nueva en aplicaciones biotecnológicas. Se describe la aplicación de la región 3'-no traducida (3'UTR) del genoma viral ARN para modular la estabilidad del ARNm. Los datos obtenidos mostraron que el uso de la secuencia 3'UTR del virus de la encefalomiocarditis (EMCV 3'UTR) aguas abajo del gen objetivo no pudo modular significativamente los indicadores de densidad de energía libre del ARN. Sin embargo, la secuencia influyó en la estabilidad del ARNm (y, por lo tanto, en la cantidad de producción de proteínas) dependiente de la célula y del tiempo, lo que indica un papel central de los sitios de unión estabilizadores de ARNm/factores celulares en este proceso. Nuestros datos podrían ser de interés para la comunidad biotecnológica para mejorar la producción de proteínas recombinantes en cultivos de células de mamíferos y en enfoques de terapia/vacunación basados en ARN.

Palmitoylation of Hepatitis C Virus NS2 Regulates Its Subcellular Localization and NS2-NS3 Autocleavage.

Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a multifunctional protein implicated in both HCV RNA replication and virus particle assembly. NS2-encoded cysteine protease is responsible for autoprocessing of NS2-NS3 precursor, an essential step in HCV RNA replication. NS2 also promotes HCV particle assembly by recruiting envelope protein 2 (E2) to the virus assembly sites located at the detergent-resistant membranes (DRM). However, the fundamental mechanism regulating multiple functions of NS2 remains unclear. In this study, we discovered that NS2 is palmitoylated at the position 113 cysteine residue (NS2/C113) when expressed by itself in cells and during infectious-HCV replication. Blocking NS2 palmitoylation by introducing an NS2/C113S mutation reduced NS2-NS3 autoprocessing and impaired HCV RNA replication. Replication of the NS2/C113S mutant was restored by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between NS2 and NS3 to separate the two proteins independently of NS2-mediated autoprocessing. These results suggest that NS2 palmitoylation is critical for HCV RNA replication by promoting NS2-NS3 autoprocessing. The NS2/C113S mutation also impaired infectious-HCV assembly, DRM localization of NS2 and E2, and colocalization of NS2 with Core and endoplasmic reticulum lipid raft-associated protein 2 (Erlin-2). In conclusion, our study revealed that two major functions of NS2 involved in HCV RNA replication and virus assembly, i.e., NS2-NS3 autoprocessing and E2 recruitment to the DRM, are regulated by palmitoylation at NS2/C113. Since S-palmitoylation is reversible, NS2 palmitoylation likely allows NS2 to fine tune both HCV RNA replication and infectious-particle assembly.IMPORTANCE Chronic infection with hepatitis C virus (HCV) is a major cause of severe liver diseases responsible for nearly 400,000 deaths per year. HCV NS2 protein is a multifunctional regulator of HCV replication involved in both viral-genome replication and infectious-virus assembly. However, the underlying mechanism that enables the protein to participate in multiple steps of HCV replication remains unknown. In this study, we discovered that NS2 palmitoylation is the master regulator of its multiple functions, including NS2-mediated self-cleavage and HCV envelope protein recruitment to the virus assembly sites, which in turn promote HCV RNA replication and infectious-particle assembly, respectively. This newly revealed information suggests that NS2 palmitoylation could serve as a promising target to inhibit both HCV RNA replication and virus assembly, representing a new avenue for host-targeting strategies against HCV infection.

Identification of the Cell-Surface Protease ADAM9 as an Entry Factor for Encephalomyocarditis Virus.

Encephalomyocarditis virus (EMCV) is an animal pathogen and an important model organism, whose receptor requirements are poorly understood. Here, we employed a genome-wide haploid genetic screen to identify novel EMCV host factors. In addition to the previously described picornavirus receptors sialic acid and glycosaminoglycans, this screen unveiled important new host factors for EMCV. These factors include components of the fibroblast growth factor (FGF) signaling pathway, such as the potential receptors FGFR1 and ADAM9, a cell-surface metalloproteinase. By employing various knockout cells, we confirmed the importance of the identified host factors for EMCV infection. The largest reduction in infection efficiency was observed in cells lacking ADAM9. Pharmacological inhibition of the metalloproteinase activity of ADAM9 did not affect virus infection. Moreover, reconstitution of inactive ADAM9 in knockout cells restored susceptibility to EMCV, pointing to a proteinase-independent role of ADAM9 in mediating EMCV infection. Using neutralization assays with ADAM9-specific antiserum and soluble receptor proteins, we provided evidence for a role of ADAM9 in EMCV entry. Finally, binding assays showed that ADAM9 facilitates attachment of EMCV to the cell surface. Together, our findings reveal a role for ADAM9 as a novel receptor or cofactor for EMCV.IMPORTANCE EMCV is an animal pathogen that causes acute viral infections, usually myocarditis or encephalitis. It is thought to circulate mainly among rodents, from which it is occasionally transmitted to other animal species, including humans. EMCV causes fatal outbreaks of myocarditis and encephalitis in pig farms and zoos, making it an important veterinary pathogen. Although EMCV has been widely used as a model to study mechanisms of viral disease in mice, little is known about its entry mechanism. Here, we employ a haploid genetic screen for EMCV host factors and identify an essential role for ADAM9 in EMCV entry.

Real-Time Temporal Dynamics of Bicistronic Expression Mediated by Internal Ribosome Entry Site and 2A Cleaving Sequence.

Multicistronic elements, such as the internal ribosome entry site (IRES) and 2A-like cleavage sequence, serve crucial roles in the eukaryotic ectopic expression of exogenous genes. For utilization of multicistronic elements, the cleavage efficiency and order of elements in multicistronic vectors have been investigated; however, the dynamics of multicistronic element-mediated expression remains unclear. Here, we investigated the dynamics of encephalomyocarditis virus (EMCV) IRES- and porcine teschovirus-1 2A (p2A)-mediated expression. By utilizing real-time fluorescent imaging at a minute-level resolution, we monitored the expression of fluorescent reporters bridged by either EMCV IRES or p2A in two independent cultured cell lines, HEK293 and Neuro2a. We observed significant correlations for the two fluorescent reporters in both multicistronic elements, with a higher correlation coefficient for p2A in HEK293 but similar coefficients for IRES-mediated expression and p2A-mediated expression in Neuro2a. We further analyzed the causal relationship of multicistronic elements by convergent cross mapping (CCM). CCM revealed that in all four conditions examined, the expression of the preceding gene causally affected the dynamics of the subsequent gene. As with the cross correlation, the predictive skill of p2A was higher than that of IRES in HEK293, while the predictive skills of the two multicistronic elements were indistinguishable in Neuro2a. To summarize, we report a significant temporal correlation in both EMCV IRES- and p2A-mediated expression based on the simple bicistronic vector and real-time fluorescent monitoring. The current system also provides a valuable platform to examine the dynamic aspects of expression mediated by diverse multicistronic elements under various physiological conditions.

Picornavirus infection induces temporal release of multiple extracellular vesicle subsets that differ in molecular composition and infectious potential.

Several naked virus species, including members of the Picornaviridae family, have recently been described to escape their host cells and spread infection via enclosure in extracellular vesicles (EV). EV are 50-300 nm sized lipid membrane-enclosed particles produced by all cells that are broadly recognized for playing regulatory roles in numerous (patho)physiological processes, including viral infection. Both pro- and antiviral functions have been ascribed to EV released by virus-infected cells. It is currently not known whether this reported functional diversity is a result of the release of multiple virus-containing and non-virus containing EV subpopulations that differ in composition and function. Using encephalomyocarditis virus infection (EMCV, Picornaviridae family), we here provide evidence that EV populations released by infected cells are highly heterogeneous. Virus was contained in two distinct EV populations that differed in physical characteristics, such as sedimentation properties, and in enrichment for proteins indicative of different EV biogenesis pathways, such as the plasma membrane resident proteins Flotillin-1 and CD9, and the autophagy regulatory protein LC3. Additional levels of EV heterogeneity were identified using high-resolution flow cytometric analysis of single EV. Importantly, we demonstrate that EV subsets released during EMCV infection varied largely in potency of transferring virus infection and in their kinetics of release from infected cells. These data support the notion that heterogeneous EV populations released by virus-infected cells can exert diverse functions at distinct time points during infection. Unraveling the compositional, temporal and functional heterogeneity of these EV populations using single EV analysis technologies, as employed in this study, is vital to understanding the role of EV in virus dissemination and antiviral host responses.

An analysis by metabolic labelling of the encephalomyocarditis virus ribosomal frameshifting efficiency and stimulators.

Programmed -1 ribosomal frameshifting is a mechanism of gene expression whereby specific signals within messenger RNAs direct a proportion of ribosomes to shift -1 nt and continue translating in the new reading frame. Such frameshifting normally depends on an RNA structure stimulator 3'-adjacent to a 'slippery' heptanucleotide shift site sequence. Recently we identified an unusual frameshifting mechanism in encephalomyocarditis virus, where the stimulator involves a trans-acting virus protein. Thus, in contrast to other examples of -1 frameshifting, the efficiency of frameshifting in encephalomyocarditis virus is best studied in the context of virus infection. Here we use metabolic labelling to analyse the frameshifting efficiency of wild-type and mutant viruses. Confirming previous results, frameshifting depends on a G_GUU_UUU shift site sequence and a 3'-adjacent stem-loop structure, but is not appreciably affected by the 'StopGo' sequence present ~30 nt upstream. At late timepoints, frameshifting was estimated to be 46-76 % efficient.

Cellular cap-binding protein, eIF4E, promotes picornavirus genome restructuring and translation.

Picornaviruses use internal ribosome entry sites (IRESs) to translate their genomes into protein. A typical feature of these IRESs is their ability to bind directly to the eukaryotic initiation factor (eIF) 4G component of the eIF4F cap-binding complex. Remarkably, the hepatitis A virus (HAV) IRES requires eIF4E for its translation, but no mechanism has been proposed to explain this. Here we demonstrate that eIF4E regulates HAV IRES-mediated translation by two distinct mechanisms. First, eIF4E binding to eIF4G generates a high-affinity binding conformation of the eIF4F complex for the IRES. Second, eIF4E binding to eIF4G strongly stimulates the rate of duplex unwinding by eIF4A on the IRES. Our data also reveal that eIF4E promotes eIF4F binding and increases the rate of restructuring of the poliovirus (PV) IRES. This provides a mechanism to explain why PV IRES-mediated translation is stimulated by eIF4E availability in nuclease-treated cell-free extracts. Using a PV replicon and purified virion RNA, we also show that eIF4E promotes the rate of eIF4G cleavage by the 2A protease. Finally, we show that cleavage of eIF4G by the poliovirus 2A protease generates a high-affinity IRES binding truncation of eIF4G that stimulates eIF4A duplex unwinding independently of eIF4E. Therefore, our data reveal how picornavirus IRESs use eIF4E-dependent and -independent mechanisms to promote their translation.

Protein-directed ribosomal frameshifting temporally regulates gene expression.

Programmed -1 ribosomal frameshifting is a mechanism of gene expression, whereby specific signals within messenger RNAs direct a proportion of translating ribosomes to shift -1 nt and continue translating in the new reading frame. Such frameshifting normally occurs at a set ratio and is utilized in the expression of many viral genes and a number of cellular genes. An open question is whether proteins might function as trans-acting switches to turn frameshifting on or off in response to cellular conditions. Here we show that frameshifting in a model RNA virus, encephalomyocarditis virus, is trans-activated by viral protein 2A. As a result, the frameshifting efficiency increases from 0 to 70% (one of the highest known in a mammalian system) over the course of infection, temporally regulating the expression levels of the viral structural and enzymatic proteins.