Encephalitic Alphaviruses Exploit Caveola-Mediated Transcytosis at the Blood-Brain Barrier for Central Nervous System Entry

Abstract

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.

Mayaro Virus Infects Human Brain Cells and Induces a Potent Antiviral Response in Human Astrocytes

“…Pericytes are key BBB components but are also involved in neuroinflammatory responses [ 29 ], while astrocytes are involved in immune surveillance, and their ability to produce neurotrophic factors and anti-inflammatory cytokines and to eliminate toxins highlights their prominent role in the defense of the brain against the invasion of pathogens [ 31 , 32 ]. However, those same pathogens, including arboviruses, can potentially target both astrocytes [ 32 , 33 ] and pericytes [ 33 , 34 ], thereby modulating their function.…”

Immune response and blood–brain barrier dysfunction during viral neuroinvasion

“…Not surprisingly, neurotropic virus infection also more or less affects the functional homeostasis of the BBB, changes the permeability of the BBB, and induces the BBB pro-inflammatory and anti-inflammatory innate immune responses. Many neurotropic viruses can cross the BBB via a hematogenous route and eventually invade the CNS, such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), West Nile virus (WNV), tick-borne encephalitis virus of the Flaviviridae , 6 Eastern, Western, Venezuelan equine encephalitis viruses of the Togaviridae , 7 HIV of the Retroviridae , and others. Viruses can cross the BBB through three different mechanisms ( Figure 1 ): the paracellular pathway (between cells), 8 the transcellular pathway (through cells), 9 or a ‘Trojan Horse’ mechanism through diapedesis of infected immune cells.…”

“… 10 , 11 For example, VEEV and WEEV can enter the CNS via hematogenous dissemination across an intact BBB by utilizing caveolin-mediated transcytosis. 7 The non-hematogenous routes by which viruses enter the CNS are retrograde axonal transport 12 , 13 and transynaptic trafficking. 14 Considering viruses transmitted by hematogenous routes (such as flaviviruses, HIV, etc.)…”

Potential of SARS-CoV-2 to Cause CNS Infection: Biologic Fundamental and Clinical Experience

“…The viral replication and the subsequent inflammation cytokines eventually deprive BBB of the normal function by increasing its permeability, and lead to the CNS infection. This pathway is confirmed in the cases of Encephalitic Alphaviruses (66), Hepatitis E Virus (67), and HSV (68). Considering the viral receptor, ACE2, is highly expressed in the epithelial cells, it is possible for COVID-19 to cause direct damage to the BBB, and thus enter the CNS.…”