title: Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5.
abstract: BACKGROUND
  Australian bat lyssavirus (ABLV), a rhabdovirus of the genus Lyssavirus which circulates in both pteropid fruit bats and insectivorous bats in mainland Australia, has caused three fatal human infections, the most recent in February 2013, manifested as acute neurological disease indistinguishable from clinical rabies. Rhabdoviruses infect host cells through receptor-mediated endocytosis and subsequent pH-dependent fusion mediated by their single envelope glycoprotein (G), but the specific host factors and pathways involved in ABLV entry have not been determined.
  
  
  METHODS
  ABLV internalization into HEK293T cells was examined using maxGFP-encoding recombinant vesicular stomatitis viruses (rVSV) that express ABLV G glycoproteins. A combination of chemical and molecular approaches was used to investigate the contribution of different endocytic pathways to ABLV entry. Dominant negative Rab GTPases were used to identify the endosomal compartment utilized by ABLV to gain entry into the host cell cytosol.
  
  
  RESULTS
  Here we show that ABLV G-mediated entry into HEK293T cells was significantly inhibited by the dynamin-specific inhibitor dynasore, chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and the actin depolymerizing drug latrunculin B. Over expression of dominant negative mutants of Eps15 and Rab5 also significantly reduced ABLV G-mediated entry into HEK293T cells. Chemical inhibitors of caveolae-dependent endocytosis and macropinocytosis and dominant negative mutants of Rab7 and Rab11 had no effect on ABLV entry.
  
  
  CONCLUSIONS
  The predominant pathway utilized by ABLV for internalization into HEK293T cells is clathrin-and actin-dependent. The requirement of Rab5 for productive infection indicates that ABLV G-mediated fusion occurs within the early endosome compartment.
authors.forename: Dawn L
authors.initials: DL
authors.lastname: Weir
authors.forename: Eric D
authors.initials: ED
authors.lastname: Laing
authors.forename: Ina L
authors.initials: IL
authors.lastname: Smith
authors.forename: Lin-Fa
authors.initials: LF
authors.lastname: Wang
authors.affiliation: Department of Microbiology, Uniformed Services University, Bethesda, MD 20814, USA. christopher.broder@usuhs.edu.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Broder
mesh_terms: D000199:Actins / Q000378:metabolism*; D001315:Australia; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis; D004847:Epithelial Cells / Q000821:virology; D054884:Host-Pathogen Interactions; D006801:Humans; D018114:Lyssavirus / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000378:metabolism*; D053586:Virus Internalization; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D000199:Actins; D002966:Clathrin; D014759:Viral Envelope Proteins; D020696:rab5 GTP-Binding Proteins