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

Abstract

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.

Sphingomyelins in mosquito saliva modify the host lipidome to enhance transmission of flaviviruses by promoting viral protein levels

“…Concentrated EVs were labelled with a lipid dye and purified through density gradient to remove unbound dye 52 . Hepatocytes were then exposed to labelled EVs at 37°C before quantifying the proportion of cells that contained the dye and therefore internalized EVs, using high resolution flowcytometry 53,54 . We observed an increasing proportion of cells containing the EV label from 5% at 2.5h post exposition, 8% at 5h up to 50% at 24h, whereas control cells exposed to material prepared from equal volumes of unconditioned culture medium and subjected to the same staining and purification steps had barely detectable level of labelled cells (Figure 1C).…”

“…Density gradients were centrifuged at 190,000 x g for 16 h in SW60 rotor. EV-containing gradient fractions (1.06 g/ml -1.08 g/ml) of 308 µl were collected and EVs were detected by high-resolution flow cytometry using a Cytek Aurora flow cytometer with Enhanced Small Particle module as previously detailed 53,54 . The EV-containing gradient fractions were pooled, diluted with PBS + 0.1% EV-depleted BSA and centrifuged at 190,000 x g for 65 min at 4°C.…”