title: Orthobunyavirus entry into neurons and other mammalian cells occurs via clathrin-mediated endocytosis and requires trafficking into early endosomes.
abstract: La Crosse virus (LACV) is a leading cause of pediatric encephalitis and aseptic meningitis in the midwestern and southern United States, where it is considered an emerging human pathogen. No specific therapies or vaccines are available for LACV or any other orthobunyaviruses. Inhibition of LACV entry into cells is a potential target for therapeutic intervention, but this approach is limited by our current knowledge of the entry process. Here, we determined that clathrin-mediated endocytosis is the primary mechanism of orthobunyavirus entry and identified key cellular factors in this process. First, we demonstrated that LACV colocalized with clathrin shortly after infection in HeLa cells; we then confirmed the functional requirement of dynamin- and clathrin-mediated endocytosis for orthobunyavirus entry using several independent assays and, importantly, extended these findings to primary neuronal cultures. We also determined that macropinocytosis and caveolar endocytosis, both established routes of virus entry, are not critical for cellular entry of LACV. Moreover, we demonstrated that LACV infection is dependent on Rab5, which plays an important regulatory role in early endosomes, but not on Rab7, which is associated with late endosomes. These findings provide the first description of bunyavirus entry into cells of the central nervous system, where infection can cause severe neurological disease, and will aid in the design and development of antivirals and therapeutics that may be useful in the treatment of LACV and, more broadly, arboviral infections of the central nervous system.
authors.affiliation: Department of Neurology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Bradley S
authors.initials: BS
authors.lastname: Hollidge
authors.forename: Natalia B
authors.initials: NB
authors.lastname: Nedelsky
authors.forename: Mary-Virginia
authors.initials: MV
authors.lastname: Salzano
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Fraser
authors.forename: Francisco
authors.initials: F
authors.lastname: González-Scarano
authors.forename: Samantha S
authors.initials: SS
authors.lastname: Soldan
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D004670:Encephalitis, California / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D006367:HeLa Cells; D006801:Humans; D018062:La Crosse virus / Q000235:genetics / Q000378:metabolism*; D014709:Vero Cells; D053586:Virus Internalization; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D002966:Clathrin; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins