The present study provides new insights to the interaction of poxviruses with the skin immune system. Prior analyses focused on understanding the etiology of eczema vaccinatum, a fulminant vaccinia virus skin infection that occurs in the setting of AD. Howell et al. (25
) showed that cathelicidin, an antimicrobial peptide produced by injured or infected skin, inactivates vaccinia virus infectivity when the peptide is incubated with virions prior to inoculation of cell monolayers. Cathelicidin-deficient mice developed larger and more numerous skin lesions when infected by scarification with vaccinia virus (25
). Cathelicidin production rises in response to vaccinia virus infection of skin biopsies; notably, this response is attenuated in vaccinia virus-infected AD skin (26
). Cathelicidin induction in vaccinia virus-infected skin was apparently mediated via TLR3 and could be elicited in uninfected skin biopsies by the TLR3 agonist poly(I:C) (26
As shown here, antimicrobial peptides and TLR3 signaling are not the full story with respect to the poxvirus-skin dynamic. Indeed, we find that murine keratinocytes can mount a vigorous antiviral cytokine response when they sense a vaccinia virus infection. However, this response is thwarted completely by the virus-encoded RNA-binding protein E3, which acts in keratinocytes as an inhibitor of a signaling pathway through MAVS and IRF3. Little information was available previously concerning the capacity of primary mouse keratinocytes to mount an innate immune response. We find here that they do not respond to agonists of TLR4 and TLR9, as gauged by secretion of any of the proinflammatory mediator proteins assayed. However, they are stimulated by exogenous poly(I:C) to produce CCL5 via a signaling pathway requiring TLR3, TRIF, and IRF3.
Vaccinia ΔE3L infection of keratinocytes results in secretion of IFN-β, IL-6, and two CC chemokines: CCL4 (MIP1-β) and CCL5 (RANTES). The CC chemokines attract and activate macrophages and T cells. There are obvious advantages to a dermatotrophic virus to suppress this arm of the host's innate immune response in the skin. A remarkable feature of poxviruses is that they evade the CC chemokines at multiple levels: (i) by E3-mediated inhibition of CCL4 and CCL5 production in the infected skin cell (see above) and (ii) by encoding a secreted 35-kDa CC chemokine inhibitor protein (vCCI) that binds with high affinity to CC-type chemokines (4
) and blocks their productive interaction with chemokine receptors on the surface of leukocytes (2
). Most poxviruses that infect mammalian species (including variola, cowpox, and the Lister strain of vaccinia) produce vCCI, the exception being the vaccinia WR strain used here and in most other studies of viral replication. Reading et al. (54
) showed that introducing the vaccinia virus Lister vCCI gene into vaccinia virus WR attenuated lung inflammatory responses in a mouse model of lethal intranasal infection. Thus, poxviruses do not only interdict production of CC chemokines by virus-infected cells (at least in skin cells) but also impede the action of CC chemokines elaborated by proinflammatory bystander cells in other organs such as lung (54
IL-6 is a cytokine with multiple immune functions that plays a critical role in defending mice against vaccinia virus infection (35
). We find that IL-6 is secreted by ΔE3L-infected mouse keratinocytes but not by cells infected with wild-type vaccinia virus. These results are consistent with a prior report that deletion of E3L leads to increased levels of IL-6 mRNA in HeLa cells infected with modified vaccinia Ankara (44
IFN-β is a key mediator of innate antiviral immunity that exerts many diverse effects on both poxvirus-infected cells and uninfected immune responder cells; hence, poxviruses take pains to disrupt IFN signaling pathways at multiple levels, both extracellular and intracellular (57
). Vaccinia virus interdicts this process at the earliest possible step during infection of mouse keratinocytes by suppressing the very production of IFN-β. Our finding that ΔE3L-infected keratinocytes secrete IFN-β is in accord with earlier reports that (i) ΔE3L infection of cultured human HT10180 cells induced the accumulation of IFN-β mRNA, whereas no mRNA response was seen in cell infected with wild-type vaccinia virus (70
); and (ii) chicken embryo fibroblasts secreted type I IFNs when abortively infected with modified vaccinia Ankara-ΔE3L virus (23
By exploiting keratinocytes derived from knockout mice, we have identified the pathway components that sense vaccinia virus infection and are blocked by E3 (MAVS and IRF3) as well as those that are irrelevant to the keratinocytes’ antipoxviral response (TLR3, TRIF, TLR9, and MyD88). We see that IRF3 phosphorylation, a marker for its transcriptionally active state, is triggered in ΔE3L-infected primary keratinocytes but not in cells infected with wild-type vaccinia virus. E3L blockade of IRF3 phosphorylation and/or IRF3-mediated transcriptional responses was noted previously in a variety of continuous cell lines (38
). Although consideration of E3's effects on virus host dynamics and cell physiology have most often focused on PKR as the major target, this view has been challenged by studies showing (i) that E3 interdicts a PKR-independent IRF3 activation pathway (60
) and (ii) that the avirulent vaccinia ΔE3L mutant remains avirulent in mice knocked out for PKR and RNase L (70
). A more nuanced view is emerging whereby E3's blockade of PKR is critical for a productive cellular infection by virtue of preventing PKR-induced apoptosis and PKR-induced inhibition of viral protein synthesis (75
), but another cellular pathway is targeted by E3 to suppress the innate immune response to poxvirus infection.
Here, we identified MAVS as an essential component of the poxvirus-sensing pathway in murine keratinocytes, acting upstream of IRF3. MAVS is an obligate mediator of signaling through the cytoplasmic viral RNA sensors RIG-I/MDA5 that drive the innate immune responses to many RNA viruses. Ours is the first demonstration that the MAVS-dependent RNA-sensing pathway is triggered by infection with a poxvirus (a DNA virus) and then actively suppressed by a poxvirus gene product. RIG-I binds and responds to either dsRNA or 5′-triphosphate single-stranded RNA (12
). It is most likely that the agonist for innate immune signaling in ΔE3L-infected cells is dsRNA formed by vaccinia virus intermediate mRNAs (45
). Single-stranded viral RNA is an unlikely agonist in this setting because the 5′-triphosphate ends of nascent vaccinia virus mRNAs are quickly converted to a 5′-diphosphate and then further modified to m7
GpppRNA by the vaccinia virus mRNA capping enzyme.
Our finding that the C-terminal dsRNA binding domain of E3 suffices to suppress the keratinocyte response to vaccinia virus infection further implicates dsRNA as the agonist, insofar as this domain binds avidly to dsRNA duplexes but not appreciably to duplex DNA or an RNA/DNA hybrid (21
). A single K167A mutation in the E3 C-terminal domain that disrupts dsRNA binding (20
) abolishes the ability of E3 to inhibit PKR-independent IRF phosphorylation in cultured cells (60
). Moreover, E3 point mutations that disrupt dsRNA binding also eliminate the ability of E3 to block the RIG-I-dependent activation of the IFN-β promoter triggered in mouse fibroblasts infected with Sendai viruses that were engineered to produce cytoplasmic dsRNA with capped 5′ ends (61
). The latter scenario, entailing viral transcription of capped mRNAs from complementary sense/antisense templates, is analogous to the mechanism by which dsRNA is formed in poxvirus-infected cells. A simplistic view is that E3 blocks the MAVS-dependent cytoplasmic viral RNA sensing pathway in poxvirus-infected cells by competing with RIG-I and/or MDA5 for binding to dsRNA. We do not rule out more complex scenarios in which E3 engages in inhibitory protein-protein interactions (55
) with one or more of the components of the MAVS-dependent RNA sensing pathway.
Vaccinia viruses purposefully deleted of genes that affect replication fitness in mammalian cells are considered plausible candidates for the next generation of safer live vaccines. Vaccination of mice with a relatively modest dose of ΔE3L by the intranasal route resulted in limited replication in the nasal mucosa (with no spread to lung or brain) and elicited protective immunity against subsequent challenge with a lethal dose of wild-type vaccinia virus (67
). Our observations concerning how ΔE3L infection activates innate immunity in skin epithelial cells suggests that the MAVS pathway could be a brake on viral spread in nasal epithelium, and they raise the prospect that scarification with ΔE3L might be an effective immunization strategy with lower risk of unchecked spread in the skin than wild-type vaccinia virus.