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.

Modulation of nucleotide metabolism by picornaviruses.

Viruses actively reprogram the metabolism of the host to ensure the availability of sufficient building blocks for virus replication and spreading. However, relatively little is known about how picornaviruses-a large family of small, non-enveloped positive-strand RNA viruses-modulate cellular metabolism for their own benefit. Here, we studied the modulation of host metabolism by coxsackievirus B3 (CVB3), a member of the enterovirus genus, and encephalomyocarditis virus (EMCV), a member of the cardiovirus genus, using steady-state as well as 13C-glucose tracing metabolomics. We demonstrate that both CVB3 and EMCV increase the levels of pyrimidine and purine metabolites and provide evidence that this increase is mediated through degradation of nucleic acids and nucleotide recycling, rather than upregulation of de novo synthesis. Finally, by integrating our metabolomics data with a previously acquired phosphoproteomics dataset of CVB3-infected cells, we identify alterations in phosphorylation status of key enzymes involved in nucleotide metabolism, providing insight into the regulation of nucleotide metabolism during infection.

Assessment of Kinome-Wide Activity Remodeling upon Picornavirus Infection.

Picornaviridae represent a large family of single-stranded positive RNA viruses of which different members can infect both humans and animals. These include the enteroviruses (e.g., poliovirus, coxsackievirus, and rhinoviruses) as well as the cardioviruses (e.g., encephalomyocarditis virus). Picornaviruses have evolved to interact with, use, and/or evade cellular host systems to create the optimal environment for replication and spreading. It is known that viruses modify kinase activity during infection, but a proteome-wide overview of the (de)regulation of cellular kinases during picornavirus infection is lacking. To study the kinase activity landscape during picornavirus infection, we here applied dedicated targeted mass spectrometry-based assays covering ∼40% of the human kinome. Our data show that upon infection, kinases of the MAPK pathways become activated (e.g., ERK1/2, RSK1/2, JNK1/2/3, and p38), while kinases involved in regulating the cell cycle (e.g., CDK1/2, GWL, and DYRK3) become inactivated. Additionally, we observed the activation of CHK2, an important kinase involved in the DNA damage response. Using pharmacological kinase inhibitors, we demonstrate that several of these activated kinases are essential for the replication of encephalomyocarditis virus. Altogether, the data provide a quantitative understanding of the regulation of kinome activity induced by picornavirus infection, providing a resource important for developing novel antiviral therapeutic interventions.

Safety concern of recombination between self-amplifying mRNA vaccines and viruses is mitigated in vivo.

Self-amplifying mRNA (SAM) vaccines can be rapidly deployed in the event of disease outbreaks. A legitimate safety concern is the potential for recombination between alphavirus-based SAM vaccines and circulating viruses. This theoretical risk needs to be assessed in the regulatory process for SAM vaccine approval. Herein, we undertake extensive in vitro and in vivo assessments to explore recombination between SAM vaccine and a wide selection of alphaviruses and a coronavirus. SAM vaccines were found to effectively limit alphavirus co-infection through superinfection exclusion, although some co-replication was still possible. Using sensitive cell-based assays, replication-competent alphavirus chimeras were generated in vitro as a result of rare, but reproducible, RNA recombination events. The chimeras displayed no increased fitness in cell culture. Viable alphavirus chimeras were not detected in vivo in C57BL/6J, Rag1-/- and Ifnar-/- mice, in which high levels of SAM vaccine and alphavirus co-replicated in the same tissue. Furthermore, recombination between a SAM-spike vaccine and a swine coronavirus was not observed. In conclusion we state that although the ability of SAM vaccines to recombine with alphaviruses might be viewed as an environmental safety concern, several key factors substantially mitigate against in vivo emergence of chimeric viruses from SAM vaccine recipients.

The activation of the adaptor protein STING depends on its interactions with the phospholipid PI4P.

Activation of the endoplasmic reticulum (ER)-resident adaptor protein STING, a component of a cytosolic DNA-sensing pathway, induces the transcription of genes encoding type I interferons (IFNs) and other proinflammatory factors. Because STING is activated at the Golgi apparatus, control of the localization and activation of STING is important in stimulating antiviral and antitumor immune responses. Through a genome-wide CRISPR interference screen, we found that STING activation required the Golgi-resident protein ACBD3, which promotes the generation of phosphatidylinositol 4-phosphate (PI4P) at the trans-Golgi network, as well as other PI4P-associated proteins. Appropriate localization and activation of STING at the Golgi apparatus required ACBD3 and the PI4P-generating kinase PI4KB. In contrast, STING activation was enhanced when the lipid-shuttling protein OSBP, which removes PI4P from the Golgi apparatus, was inhibited by the US Food and Drug Administration-approved antifungal itraconazole. The increase in the abundance of STING-activating phospholipids at the trans-Golgi network resulted in the increased production of IFN-ß and other cytokines in THP-1 cells. Furthermore, a mutant STING that could not bind to PI4P failed to traffic from the ER to the Golgi apparatus in response to a STING agonist, whereas forced relocalization of STING to PI4P-enriched areas elicited STING activation in the absence of stimulation with a STING agonist. Thus, PI4P is critical for STING activation, and manipulating PI4P abundance may therapeutically modulate STING-dependent immune responses.

Neutralizing antibodies reveal cryptic vulnerabilities and interdomain crosstalk in the porcine deltacoronavirus spike protein.

Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen that has recently been detected in humans. Despite this zoonotic concern, the antigenic structure of PDCoV remains unknown. The virus relies on its spike (S) protein for cell entry, making it a prime target for neutralizing antibodies. Here, we generate and characterize a set of neutralizing antibodies targeting the S protein, shedding light on PDCoV S interdomain crosstalk and its vulnerable sites. Among the four identified antibodies, one targets the S1A domain, causing local and long-range conformational changes, resulting in partial exposure of the S1B domain. The other antibodies bind the S1B domain, disrupting binding to aminopeptidase N (APN), the entry receptor for PDCoV. Notably, the epitopes of these S1B-targeting antibodies are concealed in the prefusion S trimer conformation, highlighting the necessity for conformational changes for effective antibody binding. The binding footprint of one S1B binder entirely overlaps with APN-interacting residues and thus targets a highly conserved epitope. These findings provide structural insights into the humoral immune response against the PDCoV S protein, potentially guiding vaccine and therapeutic development for this zoonotic pathogen.