Project:  Entry Mechanism of Equine arteritis virus (EAV)  

Mathias Nitschke 

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae, which can cause severe problems in horse breeding. The outcome of arterivirus infection can range from an asymptomatic, persistent carrier state to abortion or lethal haemorrhagic fever. The primary host cells are the horse macrophages. EAV contains seven structural proteins which are essential for virus infectivity and maturation (Fig. 1). The nucleocapsid protein N forms an isometric core particle which is surrounded by the lipid envelope. N and the complex of the non-glycosylated membrane protein M and the major glycoprotein Gp5 are required for virus assembly. The putative envelope heterotrimer of the glycosylated proteins Gp2b, Gp3, Gp4 (ratio: 1:1:1) is a prime candidate for receptor-binding and for fusion of EAV envelope with the cellular target membrane. However, so far not much is known about EAV entry and fusion mechanism.

Fig.1: Structure of EAV

Results:

Infection by EAV requires the release of the viral genome by fusion with the respective target membrane of the host cell. In order to find out, whether the virus utilizes the endosomal or the non-endosomal pathway, we have investigated infection of Baby Hamster Kindey cells (BHK) by EAV.
Infection of cells assessed by the plaque reduction assay was strongly inhibited by substances which interfere with clathrin-dependent endocytosis and by lysosomotropic compounds. Furthermore, infection of BHK cells was suppressed when clathrin-dependent endocytosis was inhibited by expression of antisense RNA of the clathrin-heavy chain before infection (Fig.2). These results strongly suggest that EAV is taken up via clathrin-dependent endocytosis and is delivered to acidic endosomal compartments.
 

Fig.2: Infection of BHKasc cells by EAV. (A) Control (B) Cells with depressed clathrin-dependent endocytosis. After 24h p.i. cells were fixed and examined, stained with TRIC (red) antibodies against EAV nucleocapsid protein.

References:

Nitschke, M., Korte, T., Tielesch, C., Ter-Avetisyan, G., Tünnemann, G., Cardoso, M.C., Veit, M., Herrmann, A. (2008)
Equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis.
Virology 377, 248-254.