Faculty of Science, School of Molecular Bioscience
Abstract
The subcellular space is a dense, complex environment that viral pathogens must efficiently navigate for their survival and dissemination. The prototypal poxvirus member, vaccinia virus (VACV) has been shown to subvert both host microtubule-based and actin-based transport mechanisms to this end. The utilisation of fluorescent protein technology has been at the crux of many of these discoveries, but addressing remaining questions is made difficult by the various morphological forms of VACV and the sheer number of viruses being transported at any one time in a single infected cell. We have used the development of photoswitchable fluorescent protein Dendra2 to create a cutting edge imaging system that allows us to gain insight into the dynamics of subcellular virus dissemination. We have constructed recombinant viruses expressing core protein A3 and envelope proteins B5 and F13 fused to Dendra2 in both parental and deletion strains of the Western Reserve strain and have examined the envelopment at and egress from the trans-Golgi network. Egress of wrapped virus to the cell periphery following vaccinia virus (VACV) replication is dependent on interactions with the microtubule motor complex kinesin-1 and is mediated by the viral envelope proteins A36 and F12. We have utilised a mouse-based infection model to validate the important role these transport events have during an in vivo, endemic infection. Ectromelia virus, an orthopoxvirus and the causative agent of mousepox, encodes an A36 homologue (ECTV-Mos-142) that is highly conserved despite a large truncation at the C terminus. Deleting the ECTV A36R gene leads to a reduction in the number of extracellular viruses formed and to a reduced plaque size, consistent with a role in microtubule transport. We also observed a complete loss of virus-associated actin tails, another phenotype dependent on A36 expression during VACV infection. ECTV A36R was severely attenuated when used to infect the normally susceptible BALB/c mouse strain. ECTV ΔA36R replication and spread from the draining lymph nodes to the liver and spleen were significantly reduced in BALB/c mice and in Rag-1-deficient mice, which lack T and B lymphocytes. The dramatic reduction in ECTV ΔA36R titers early during the course of infection was not associated with an augmented immune response. Taken together, these findings demonstrate the critical role that subcellular transport pathways play not only in orthopoxvirus infection in an in vitro context but also during orthopoxvirus pathogenesis in a natural host
