Venezuelan and western equine encephalitis viruses (VEEV and WEEV, respectively) invade the central nervous system (CNS) early during
infection, via neuronal and hematogenous routes. While viral replication mediates host shutoff, including expression of
type I interferons (IFN), few studies have addressed how alphaviruses gain access to the CNS during established
infection or the mechanisms of viral crossing at the blood-brain barrier (BBB). Here, we show that hematogenous dissemination of VEEV and WEEV into the CNS occurs via
caveolin-1 (Cav-1)-mediated transcytosis (Cav-MT) across an intact BBB, which is impeded by IFN and inhibitors of RhoA
GTPase. Use of reporter and nonreplicative strains also demonstrates that IFN signaling mediates viral restriction within cells comprising the neurovascular unit (NVU), differentially rendering brain endothelial cells, pericytes, and astrocytes permissive to viral replication. Transmission and immunoelectron microscopy revealed early events in virus internalization and Cav-1 association within brain endothelial cells. Cav-1-deficient mice exhibit diminished CNS VEEV and WEEV titers during early
infection, whereas viral burdens in peripheral tissues remained unchanged. Our findings show that alphaviruses exploit Cav-MT to enter the CNS and that IFN differentially restricts this process at the BBB.IMPORTANCE VEEV, WEEV, and eastern equine encephalitis virus (EEEV) are
emerging infectious diseases in the Americas, and they have caused several major outbreaks in the human and horse population during the past few decades. Shortly after
infection, these viruses can infect the CNS, resulting in severe long-term neurological deficits or death. Neuroinvasion has been associated with virus entry into the CNS directly from the bloodstream; however, the underlying molecular mechanisms have remained largely unknown. Here, we demonstrate that following peripheral
infection alphavirus augments vesicular formation/trafficking at the BBB and utilizes Cav-MT to cross an intact BBB, a process regulated by activators of
Rho GTPases within brain endothelium. In vivo examination of early viral entry in Cav-1-deficient mice revealed significantly lower viral burdens in the brain than in similarly infected wild-type animals. These studies identify a potentially targetable pathway to limit neuroinvasion by alphaviruses.