Investigating the intracellular trafficking of mature African horse sickness virus particles

Abstract

African horse sickness virus (AHSV) is an arbovirus in the genus Orbivirus and the family Reoviridae known to successfully propagate in different species, such as mammalian hosts and insect vectors. In mammalian cells, infection by AHSV results in cell lysis and release of progeny virions, while insect cells establish persistent infection with no visible cytopathic effect and progeny virions escape infected cells by non-lytic processes. The events leading to AHSV release from infected cells, mammalian or insect, is unknown and is thought to contribute to the cytopathic effect observed in mammalian cells and absence thereof in insect cells. This study aimed to investigate and compare AHSV trafficking in mammalian and insect cells, with the hope of detecting underlying differences between mammalian and insect cells. Transmission electron microscopy revealed that particles distributed either to inside the lumen of vesicles were associated with the cytoplasmic face of vesicles, or were present in the cytoplasm without close proximity to any intracellular structures or organelles. In mammalian cells, the majority of particles distributed inside the lumen of vesicles at early times post infection and to the cytoplasm at later times post infection. In insect cells, the majority of particles distributed to the cytoplasm throughout the course of infection. The presence of mature AHSV particles in the lumen of small smooth-surfaced membrane-bound structures in both mammalian cells and insect cells implicated a role for membrane trafficking pathways in virus transport. We subsequently targeted cellular factors involved in membrane trafficking in cells to elucidate the cellular pathways involved in AHSV replication. The targets were: vesicle formation along the secretory route, the ubiquitin-proteasome system regulating the levels of free ubiquitin, and lipid kinases involved in multivesicular body (MVB) biogenesis. Inhibition of vesicle-dependent transport along the secretory route significantly decreased virus release in mammalian but not insect cells. Together with TEM findings, these results suggest AHSV transport in mammalian cells occurs by vesicle-dependent mechanisms early after infection, and support NS3-mediated transport models for the non-lytic release of particles. From our TEM analysis, AHSV transport later after infection in mammalian cells seems not to predominantly involve membrane trafficking. The mechanism of transport and non-lytic release in insect cells does not involve the secretory pathway and remains to be elucidated. Other components of the exocytic and MVB pathways are involved in AHSV replication in mammalian and insect cells, but not directly involved in AHSV transport to the cell surface. Our findings demonstrate the involvement of membrane trafficking pathways in events leading to AHSV release. Inhibition of cellular factors affected virus replication differently in mammalian and insect cells and shows a difference in the cell-mediated events leading to release from infected mammalian cells versus infected insect cells. These results demonstrate the relevance of elucidating cell-mediated events leading to AHSV release in understanding viral pathogenesis and describe the involvement of additional host proteins/pathways in AHSV egress which provide new insights to understand orbivirus interactions with their hosts.