Loss of Cytoskeletal Transport during Egress Critically Attenuates Ectromelia Virus Infection In Vivo

Abstract

d Egress of wrapped virus (WV) to the cell periphery following vaccinia virus (VACV) replication is dependent on interactionswith the microtubule motor complex kinesin-1 and is mediated by the viral envelope protein A36. Here we report that ectromelia virus (ECTV), a related 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 comets, 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 ⌬A36R 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 ⌬A36R 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. Furthermore, despite the attenuation of the mutant virus, we found that infection nonetheless induced protective immunity in mice, suggesting that orthopoxvirus vectors with A36 deletions may be considered another safe vaccine alternative.T he crowded, densely packed cytoplasm presents a significant hurdle to the subcellular transport of viruses, both in the translocation of viruses to their site of replication following cell entry and in the subsequent egress of progeny viruses to the cell periphery (16,18,29,69,70). This hurdle is most acute for large double-stranded DNA (dsDNA) viruses such as those belonging to the orthopoxvirus genus, which includes variola virus (VARV) and ectromelia virus (ECTV), the causative agents of smallpox and mousepox, respectively, and the prototypical orthopoxvirus, vaccinia virus (VACV). These viruses have a complicated replication cycle that has been studied best at the cellular level with VACV. Vaccinia virus replication produces two infectious forms: mature intracellular virus (MV), which has a single membrane and is generated at the so-called virus factory; and wrapped virus (WV), which is derived from MV and acquires an additional double membrane at the trans-Golgi network or early endosome compartment (33, 58). While it is apparent that microtubule transport plays a critical role at multiple stages during VACV replication, the stage best characterized at the molecular level is the transport of WV from the trans-Golgi network to the cell periphery (27,32,57,75,76). Once there, WV fuse with the plasma membrane and are released either directly or following the transient activation of actin-based motility ...

Ectromelia virus accumulates less double-stranded RNA compared to vaccinia virus in BS-C-1 cells

“…post-infection that factory formation for ECTV was equivalent to the levels observed with VACV. Overall, these findings are similar to a prior report (Lynn et al, 2012). Therefore, ECTV exhibits delayed replication kinetics relative to VACV, which may have marked effects on viral dsRNA accumulation within cells.…”

“…(Roper, 2006). Conversely, ECTV plaques are commonly not visualized until after a minimum of 4 or 5 days of incubation (Chen et al, 1992; Hand et al, 2015; Lynn et al, 2012; Panchanathan et al, 2005). To demonstrate the differences in plaque formation kinetics between ECTV and VACV, representative images are shown in Figure 2A for GFP-expressing viruses.…”

Characterization of Ectromelia Virus Deficient in EVM036 , the Homolog of Vaccinia virus F13L , and Its Application for Rapid Generation of Recombinant Viruses

“…Moreover, following ECTV-⌬036 immunization, BALB/c mice were fully protected from ECTV-WT challenge. These results are consistent with previous reports showing protective immunity after immunization with ECTV deficient in the ortholog of A36R (22) and with VACV LC16m8, which has a frameshift mutation in B5R (20,30). Neither of these viruses produces EV.…”

The role of signalling and the cytoskeleton during Vaccinia Virus egress

“…Consequently, an absence of A36 during infection reduces the cell-to-cell spread of vaccinia, as the virus is less likely to reach and fuse with the plasma membrane (Ward and Moss, 2001a). Deletion of the gene encoding ECTV-Mos-42, an A36 orthologue in Ectromelia virus, the causative agent of mousepox, also results in reduced cell-to-cell spread (Lynn et al, 2012). Immunofluorescence images of HeLa cells infected for 8 h with WR or the recombinant B5R virus, which does not form IEV as it lacks the viral protein B5.…”

“…A36 is highly conserved in orthopoxvirus genomes, suggesting that virus-induced actin polymerization at the plasma membrane is widely used by mammalian poxviruses to enhance their cell-to-cell spread. Indeed, monkeypox and variola viruses induce Src and Abl dependent actin tails, while the A36 homologue of ectromelia virus enhances viral spread by inducing actin tail formation (Lynn et al, 2012;Reeves et al, 2011). Moreover, the ability to stimulate actin polymerization is not just restricted to orthopoxviruses, as other divergent vertebrate poxviruses (Chordopoxviridae) can also induce actin tails Duteyrat et al, 2006;Law et al, 2004).…”