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.

The matrix protein of lyssavirus hijacks autophagosome for efficient egress by recruiting NEDD4 through its PPxY motif.

Lyssaviruses are well-known worldwide and often cause fatal encephalitis. Previous studies have shown that autophagy is beneficial for the replication of rabies virus (RABV), the representative lyssavirus, but the detailed mechanism remains obscure. In this study, we showed that the rabies virus matrix protein (RABV-M) used its PPxY motif to interact with the E3 ubiquitin-protein ligase NEDD4. NEDD4 then recruited MAP1LC3/LC3 via its LC3-interacting region (LIR). Interestingly, after binding to the ubiquitinated RABV-M, NEDD4 could bind more LC3 and enhance autophagosome accumulation, while NEDD4 knockdown significantly reduced M-induced autophagosome accumulation. Further study revealed that RABV-M prevented autophagosome-lysosome fusion and facilitated viral budding. Inhibition of RABV-M-induced autophagosome accumulation reduced the production of extracellular virus-like particles. We also found that M proteins of most lyssaviruses share the same mechanism to accumulate autophagosome by hijacking NEDD4. Collectively, this study revealed a novel strategy for lyssaviruses to achieve efficient viral replication by exploiting the host autophagy system.Abbreviations: ABLV: Australian bat lyssavirus; ATG5: autophagy related 5; Baf A1:bafilomycin A1;co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI:4',6-diamidino-2'-phenylindole; DMSO: dimethyl sulfoxide; EBLV:European bat lyssavirus; GFP: green fluorescent protein; GST:glutathione S-transferase; hpi: hours post-infection; hpt: hourspost-transfection; LIR: LC3-interactingregion;MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mCherry:red fluorescent protein; MOI: multiplicity of infection; NC: negativecontrol; MVB: multivesicular body; NEDD4: neural precursorcell-expressed developmentally down-regulated 4; RABV: rabies virus;SQSTM1/p62: sequestosome 1; VLP: virus-like particle; VPS4B: vacuolarprotein sorting 4B; TEM: transmission electron microscopy; WB:western blotting; WT: wild-type; µm: micrometer; µM: micromole.

An Inducible ESCRT-III Inhibition Tool to Control HIV-1 Budding.

HIV-1 budding as well as many other cellular processes require the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Understanding the architecture of the native ESCRT-III complex at HIV-1 budding sites is limited due to spatial resolution and transient ESCRT-III recruitment. Here, we developed a drug-inducible transient HIV-1 budding inhibitory tool to enhance the ESCRT-III lifetime at budding sites. We generated autocleavable CHMP2A, CHMP3, and CHMP4B fusion proteins with the hepatitis C virus NS3 protease. We characterized the CHMP-NS3 fusion proteins in the absence and presence of protease inhibitor Glecaprevir with regard to expression, stability, localization, and HIV-1 Gag VLP budding. Immunoblotting experiments revealed rapid and stable accumulation of CHMP-NS3 fusion proteins. Notably, upon drug administration, CHMP2A-NS3 and CHMP4B-NS3 fusion proteins substantially decrease VLP release while CHMP3-NS3 exerted no effect but synergized with CHMP2A-NS3. Localization studies demonstrated the relocalization of CHMP-NS3 fusion proteins to the plasma membrane, endosomes, and Gag VLP budding sites. Through the combined use of transmission electron microscopy and video-microscopy, we unveiled drug-dependent accumulation of CHMP2A-NS3 and CHMP4B-NS3, causing a delay in HIV-1 Gag-VLP release. Our findings provide novel insight into the functional consequences of inhibiting ESCRT-III during HIV-1 budding and establish new tools to decipher the role of ESCRT-III at HIV-1 budding sites and other ESCRT-catalyzed cellular processes.

Epilepsy-induced electrocardiographic alterations following cardiac ischemia and reperfusion in rats

The present study evaluated electrocardiographic alterations in rats with epilepsy submitted to an acute myocardial infarction (AMI) model induced by cardiac ischemia and reperfusion. Rats were randomly divided into two groups: control (n=12) and epilepsy (n=14). It was found that rats with epilepsy presented a significant reduction in atrioventricular block incidence following the ischemia and reperfusion procedure. In addition, significant alterations were observed in electrocardiogram intervals during the stabilization, ischemia, and reperfusion periods of rats with epilepsy compared to control rats. It was noted that rats with epilepsy presented a significant increase in the QRS interval during the stabilization period in relation to control rats (P<0.01). During the ischemia period, there was an increase in the QRS interval (P<0.05) and a reduction in the P wave and QT intervals (P<0.05 for both) in rats with epilepsy compared to control rats. During the reperfusion period, a significant reduction in the QT interval (P<0.01) was verified in the epilepsy group in relation to the control group. Our results indicate that rats submitted to an epilepsy model induced by pilocarpine presented electrical conductivity alterations of cardiac tissue, mainly during an AMI episode.