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

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.

ICAM-1 Binding Rhinoviruses A89 and B14 Uncoat in Different Endosomal Compartments

“…Although a number of viruses traffic through the ERC during entry (55)(56)(57), their infection was not inhibited by Rab11-S25N, which is indicative of the Twenty-four hours later, the respective virus (at 100 TCID 50 /cell) was allowed to internalize for 60 min; unbound virus was washed away with cold PBSϩ containing 25 mM NH 4 Cl for 15 min, and replication was allowed to proceed for 6.5 h at 37°C in infection medium containing NH 4 Cl. Cells were fixed, quenched, permeabilized, and blocked, and de novo-synthesized viral protein was detected with the respective antibodies, followed by suitable fluorescent secondary antibodies.…”

Status of antiviral therapeutics against rabies virus and related emerging lyssaviruses

“…Identifying pursuable host targets thus remains to be a challenging endeavor [93,94]. RABV entry is mediated by clathrin-coated endocytosis which is dependent on actin cytoskeleton reorganization for internalization [95]. A closely related Rhabdovirus, vesicular stomatitis virus (VSV), employs a comparable entry strategy [96,97].…”

“…A closely related Rhabdovirus, vesicular stomatitis virus (VSV), employs a comparable entry strategy [96,97]. Dynamin is a critical component of multiple endocytotic pathways and plays a role in actin reassembly and organization [95,97]. Dynasore, a membrane-permeable inhibitor of dynamin GTPase activity, has been shown to inhibit the entry process for several viruses, including RABV and VSV [95,98].…”

Multifactorial Regulation of G Protein-Coupled Receptor Endocytosis

“…Rab5 is a component of clathrin-coated vesicles, and a complex of Rab5 and Rab-guanine nucleotide-dissociation inhibitor is necessary for the invagination of clathrin-coated pits (Bucci et al ., 1992; McLauchlan et al ., 1998; Seachrist et al ., 2000; Weir et al ., 2014). A possible isotypespecific interaction of Rab5 with clathrin was recently reported in Leishmania donovani (Rastogi et al ., 2016).…”