Ectromelia virus lacking the E3L ortholog is replication-defective and nonpathogenic but does induce protective immunity in a mouse strain susceptible to lethal mousepox.

All known orthopoxviruses, including ectromelia virus (ECTV), contain a gene in the E3L family. The protein product of this gene, E3, is a double-stranded RNA-binding protein. It can impact host range and is used by orthopoxviruses to combat cellular defense pathways, such as PKR and RNase L. In this work, we constructed an ECTV mutant with a targeted disruption of the E3L open reading frame (ECTVΔE3L). Infection with this virus resulted in an abortive replication cycle in all cell lines tested. We detected limited transcription of late genes but no significant translation of these mRNAs. Notably, the replication defects of ECTVΔE3L were rescued in human and mouse cells lacking PKR. ECTVΔE3L was nonpathogenic in BALB/c mice, a strain susceptible to lethal mousepox disease. However, infection with ECTVΔE3L induced protective immunity upon subsequent challenge with wild-type virus. In summary, E3L is an essential gene for ECTV.

Ectopic expression of vaccinia virus E3 and K3 cannot rescue ectromelia virus replication in rabbit RK13 cells.

As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV.