Encephalitic alphaviruses exploit caveolae-mediated transcytosis at the blood-brain barrier for CNS entry

Abstract

21Venezuelan and Western equine encephalitis viruses (VEEV and WEEV) invade the CNS early during 22 infection, via neuronal and hematogenous routes (1, 2). While viral replication mediates host-shut off, 23 including expression of type I interferons (IFN) (3, 4), few studies have addressed how alphaviruses 24 gain access to the CNS during established infection or the mechanisms of viral crossing at the blood-25 brain barrier (BBB). Here, we show that hematogenous dissemination of VEEV and WEEV into the 26 CNS occurs via caveolin (Cav)-1-mediated transcytosis (Cav-MT) across an intact BBB, which is 27 impeded by IFN and inhibitors of RhoA GTPase. Use of reporter and non-replicative strains also 28 demonstrates that IFN signaling mediates viral restriction within cells comprising the neurovascular unit 29 (NVU), differentially rendering brain endothelial cells, pericytes and astrocytes permissive to viral 30 replication. Transmission and immunoelectron microscopy revealed early events in virus internalization 31and Cav-1-association within brain endothelial cells. Cav-1-deficient mice exhibit diminished CNS 32 VEEV and WEEV titers during early infection, whereas viral burdens in peripheral tissues remained 33 unchanged. Our findings show that alphaviruses exploit Cav-MT to enter the CNS, and that IFN 34 differentially restricts this process at the BBB. 35 36 3 Importance 37 VEEV, WEEV and EEEV are emerging infectious diseases in the Americas, and they have caused 38 several major outbreaks in the human and horse population during the past few decades. Shortly after 39 infection, these viruses can infect the CNS, resulting in severe long-term neurological deficits or death. 40Neuroinvasion has been associated with virus entry into the CNS directly from the blood-stream, 41 however the underlying molecular mechanisms have remained largely unknown. Here we demonstrate 42 that following peripheral infection alphavirus augments vesicular formation/trafficking at the BBB and 43 utilizes Cav-MT to cross an intact BBB, a process regulated by activators of Rho GTPAses within brain 44 endothelium. In vivo examination of early viral entry in Cav-1-deficient mice revealed significantly lower 45 viral burdens than in similarly infected wild-type animals. These studies identify a potentially targetable 46 pathway to limit neuroinvasion by alphaviruses. 47 50

Members of Venezuelan Equine Encephalitis Complex entry into host cells by clathrin-mediated endocytosis in a pH-dependent manner

“…In line with this idea, Lee and collaborators show that entry of CHIKV into human muscle cells is mediated by macropinocytosis [19]. Besides, new findings show that VEEV and Western equine encephalitis virus exploit the caveolin-1-mediated transcytosis via to enter the central nervous system [33], providing alternative pathways for virus entry in different types of cells. For example, as described for Influenza virus, clathrin-endocytosis and macropinocytosis can operate simultaneously in HeLa, A549 and other non-human cells [34].…”