Abstract
BackgroundAustralian 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.MethodsABLV 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.ResultsHere 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.ConclusionsThe 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.
Highlights
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
Dynamin is required for ABLV G-mediated viral entry To examine the endocytic pathway utilized by ABLV for host cell internalization, we used maxGFP-encoding replication competent recombinant vesicular stomatitis viruses that express ABLV G glycoproteins [6]
This approach is advantageous over using WT ABLV because are recombinant vesicular stomatitis viruses (rVSV)-ABLV G viruses safer and easier to manipulate than WT ABLV, but the incorporation of GFP into the viral genome eliminates the need for traditional fluorescent antibody staining to detect infected cells
Summary
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. Australian bat lyssavirus (ABLV) is a rhabdovirus of the genus Lyssavirus endemic in Australian bat populations that is capable of causing a fatal neurological disease in humans indistinguishable from clinical rabies. There are currently 12 species of lyssaviruses and 3 additional species that have not yet been classified [7]; ABLV is most closely related to classical rabies virus (RABV), the prototype member of the Lyssavirus genus. The lyssavirus G glycoprotein is a key determinant of the neurotropic and neurovirulent properties of lyssaviruses [9]
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