Roles of Vaccinia Virus Genes E3L and K3L and Host Genes PKR and RNase L during Intratracheal Infection of C57BL/6 Mice

Abstract

The importance of the 2-5 oligoadenylate synthetase (OAS)/RNase L and double-stranded RNA (dsRNA)-dependent protein kinase (PKR) pathways in host interferon induction resulting from virus infection in response to dsRNA has been well documented. In poxvirus infections, the interactions between the vaccinia virus (VV) genes E3L and K3L, which target RNase L and PKR, respectively, serve to prevent the induction of the dsRNA-dependent induced interferon response in cell culture. To determine the importance of these host genes in controlling VV infections, mouse single-gene knockouts of RNase L and PKR and double-knockout mice were studied following intratracheal infection with VV, VV⌬K3L, or VV⌬E3L. VV caused lethal disease in all mouse strains. The single-knockout animals were more susceptible than wild-type animals, while the RNase L ؊/؊ PKR ؊/؊ mice were the most susceptible. VV⌬E3L infections of wild-type mice were asymptomatic, demonstrating that E3L plays a critical role in controlling the host immune response. RNase L ؊/؊ mice showed no disease, whereas 20% of the PKR ؊/؊ mice succumbed at a dose of 10 8 PFU. Lethal disease was routinely observed in RNase L ؊/؊ PKR ؊/؊ mice inoculated with 10 8 PFU of VV⌬E3L, with a distinct pathology. VV⌬K3L infections exhibited no differences in virulence among any of the mouse constructs, suggesting that PKR is not the exclusive target of K3L. Surprisingly, VV⌬K3L did not disseminate to other tissues from the lung. Hence, the cause of death in this model is respiratory disease. These results also suggest that an unanticipated role of the K3L gene is to facilitate virus dissemination.Small-animal (mouse) models to study poxvirus pathogenesis have been employed for decades and have provided invaluable insight into host-virus interactions and mechanistic insight as to how individual viral genes influence the host response. Infection of mice by various routes continues to be productive for study of a number of orthopoxviruses, including vaccinia virus (VV), ectromelia virus, cowpox virus, and rabbitpox virus (23, 24, 44, 45, 48-50, 67, 75, 77). The general purpose of these infected mouse studies is to provide surrogate models for human smallpox virus infection and host response to evaluate antiviral drug efficacy and the effectiveness of various smallpox vaccines. Advantages of mouse models include their relatively low cost, small size of the animals, and the fact that there are many mouse mutant strains available for genes that control responses to infection.A number of routes for the initiation of poxvirus infections in mice have been studied, including intranasal (i.n.), intratracheal (i.t.), and intradermal (i.d.) (ear pinnae, footpad) routes and scarification. Other routes, such as the intracranial (i.c.) or intraperitoneal (i.p.) route, are rarely used today because they do not effectively reproduce "natural" infection conditions. Arguably, the most relevant routes of infection in terms of human disease are the i.n. and i.t. routes, because smallpox is transmitted via th...

Variola virus E3L Zα domain, but not its Z-DNA binding activity, is required for PKR inhibition

“…As knockdown of PKR expression in HeLa cells complemented the defect in virus yield and virus-induced apoptosis by E3L-deleted vaccinia virus (Zhang et al 2008), it is clear that PKR is an important target of E3L. Consistently, the attenuated phenotype of E3L-deleted virus following intratracheal infection of wild-type mice was partially reversed upon infection of mice lacking PKR (Rice et al 2011). The fact that deletion of PKR does not fully rescue the pathogenicity of the E3L-deleted virus (Rice and Kerr 1984;Xiang et al 2002) indicates that E3L likely targets other components of the cellular antiviral defense, such as the mitochondrial anti-viral signaling (MAVS) pathway for interferon induction (Deng et al 2008).…”

“…Whereas vaccinia virus mutants lacking K3L remain competent to infect cells as well as mice, vaccinia virus mutants lacking E3L are nonpathogenic in wild-type mice (Brandt and Jacobs 2001;Rice et al 2011). Consistent with the sensitivity of pkr −/− mice to infection with ΔE3L virus (Rice et al 2011), replication of the mutant virus in HeLa cells was rescued by knockdown of PKR expression by RNA interference (Zhang et al 2008).…”

“…The fact that deletion of PKR does not fully rescue the pathogenicity of the E3L-deleted virus (Rice and Kerr 1984;Xiang et al 2002) indicates that E3L likely targets other components of the cellular antiviral defense, such as the mitochondrial anti-viral signaling (MAVS) pathway for interferon induction (Deng et al 2008). In support of this idea, the E3L-deleted virus was pathogenic in mice lacking both PKR and the anti-viral endoribonuclease RNAse L (Rice et al 2011). However, the PKR and RNase L pathways are likely not the sole targets of E3L, as E3L-deleted virus failed to replicate to high titers in mice triply lacking PKR, RNase L, and the interferon-induced Mx1 protein (Xiang et al 2002).…”

“…Consistent with the sensitivity of pkr −/− mice to infection with ΔE3L virus (Rice et al 2011), replication of the mutant virus in HeLa cells was rescued by knockdown of PKR expression by RNA interference (Zhang et al 2008). It is noteworthy that the vaccinia virus E3L protein has been reported to inhibit PKR by sequestering dsRNA activators of the kinase and by directly binding to PKR and inhibiting kinase activation (Chang et al 1992;Davies et al 1993;Romano et al 1998;Sharp et al 1998).…”

An Evolutionary View of the Arms Race between Protein Kinase R and Large DNA Viruses

“…Herpes simplex virus 1 (HSV-1) with a deletion of ␥34.5, one of its two known PKR antagonists, is avirulent in wild-type mice, even when directly inoculated into the central nervous system (CNS) (16); however, it does replicate in some cell types and, notably, is fully virulent in PKR-null mice (17). VACV lacking its PKR antagonist, E3L (VACV⌬E3L), replicates poorly in many cell types and in mice, but this defect is ameliorated at least partially in PKR-deficient cells and animals (18)(19)(20). Like HSV-1, VACV encodes at least one other PKR antagonist (K3L), which may explain why VACV⌬E3L is able to replicate at all.…”