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

Abstract

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.

Myxoma Virus-Encoded Host Range Protein M029: A Multifunctional Antagonist Targeting Multiple Host Antiviral and Innate Immune Pathways

“…The M029-minus mutant virus was also compromised in the viral replication cycle (single step or multistep) in rabbit cell lines like RK13 and RL5 [ 47 ]. Similar results were reported in the case of the ECTVΔE3L virus, which was unable to replicate in many mammalian cell lines originating from human, monkey, and mouse sources [ 48 ]. This defect in MYXV replication in the absence of M029 and ECTV in the absence of E3 was due to defects that manifest in late gene expression.…”

“…The fact that PKR is the key target of E3 ortholog proteins is proven by the observations that the replication defect of E3L -knockout viruses can be rescued using cell lines with reduced or no PKR expression [ 78 ]. This was shown in multiple poxviruses lacking expression of E3 orthologs [ 47 , 48 , 78 ]. Absence of PKR restored the viral late protein synthesis and progeny virus formation in cell lines where the E3L mutant poxviruses were not able to replicate.…”

“…In the case of MYXV, the rabbit RK13 cell line engineered to express stably the E3 protein of VACV was used to create the vMyx-M029KO virus [ 47 ]. For ECTV, engineered BS-C-1 cells that stably express the E3 protein of VACV were used to generate the ECTVΔE3L virus [ 48 ]. Both knockout viruses were only able to be propagated in these complementing cell lines.…”

“…This defect in MYXV replication in the absence of M029 and ECTV in the absence of E3 was due to defects that manifest in late gene expression. In the infected cells, the early gene expression and DNA replication allowed the formation of a small viral factory, which indicated the abortive replication of the knockout viruses [ 48 ]. The importance of E3 in the life cycle of Modified vaccinia Ankara (MVA), a highly attenuated strain of VACV developed as a live but nonreplicating vaccine, has also been studied [ 44 ].…”

“…Similar results were reported with E3 from ECTV. Infection of susceptible BALB/c mice with ECTVΔE3L resulted in the survival of all the mice, whereas infection with wildtype (WT) ECTV and an ECTVΔE3L-revertant virus caused 100% mortality [ 48 ]. These results clearly demonstrated that E3 proteins encoded by orthopoxviruses are essential for the virulence in their pathogenic host.…”