The present study contributes to our understanding of the interface of poxviruses with the skin immune system by demonstrating that (i) vaccinia virus replicates productively in an epidermal LC cell line, XS52; (ii) virus infection of XS52 cells suppresses their ability to produce TNF-α and IL-6 in response to stimulation by LPS and poly(I:C); (iii) loss of the E3L dsRNA binding protein causes infection of XS52 cells to abort at a stage prior to the onset of late gene expression, which results in induction of TNF-α secretion by ΔE3L-infected cells and significant amelioration of the suppressive effects of WT virus on LPS- or poly(I:C)-induced cytokine production; (iv) loss of the K1L protein, which has no effect on virus replication in XS52 cells, also partially reverses the virus inhibition of LPS-induced cytokine production; and (v) abortive infection of primary epidermal LCs by WT vaccinia virus inhibits their ability to present antigen and activate T cells.
These findings extend previous studies of poxvirus interactions with DCs, most of which have focused on monocyte-derived or bone marrow-derived DCs. Whereas WT vaccinia virus or vaccinia virus recombinants have been reported to infect such DCs abortively (14
), we found that the XS52 cells line supports virus vaccinia virus replication to a high titer. The kinetics of virus production during a synchronous infection of XS52 cells and the temporal pattern of viral protein synthesis are similar to those seen in BSC40 cells, which are a standard laboratory host for vaccinia virus. It appears that adaptation of XS52 cells to continuous culture endows them with a permissiveness for vaccinia virus infection that is not found in primary LCs harvested freshly from mouse epidermis, which we find are infected abortively. The latter result agrees with the recent report by Liu et al. (33
) that vaccinia virus abortively infects primary LCs from human skin. In our study and theirs, there is evidence that at least some early viral gene expression occurs in vaccinia virus-infected primary LCs, which echoes the findings that early gene expression is detectable in monocyte-derived DCs abortively infected with vaccinia virus (14
The immune effector functions of XS52 cells and primary skin LCs are actively suppressed by vaccinia virus infection. Vaccinia virus-infected XS52 cells are strongly attenuated in their response to LPS and poly(I:C), which signal cytokine production via the TLR4 and TLR3 pathways, respectively. Recent studies have shown that vaccinia virus and other mammalian poxviruses encode several proteins that inhibit the intracellular phase of TLR signaling, especially events involving NF-κB (6
). The present study implicates E3L and K1L as contributors to the interdiction of immune signaling pathways in XS52 cells by vaccinia virus.
The vaccinia virus E3L protein binds avidly to dsRNA (10
). E3L blocks IFN induction and antagonizes the IFN-regulated antiviral enzymes dsRNA-activated protein kinase PKR and RNase L (42
). Deletion of E3L sensitizes vaccinia virus replication to IFN in permissive RK13 cells and results in a host range phenotype whereby ΔE3L cannot replicate in HeLa or BSC40 cells (11
). Abortive infection with ΔE3L triggers apoptosis of HeLa cells, apparently in response to dsRNA accumulation after the onset of late viral transcription (30
). We find here that ΔE3L abortively infects XS52 cells. However, the restriction point in XS52 cells appears to differ from that reported for HeLa cells, where ΔE3L triggers a complete shutoff of the synthesis of both viral and host proteins by 6 h postinfection (31
). ΔE3L-infected XS52 cells continue to synthesize host proteins for at least 12 h without any apparent synthesis of viral late proteins. We suspect that some early viral genes are expressed in ΔE3L-infected XS52 cells, insofar as the abortive infection triggers secretion of TNF-α and IL-6, which are not produced during infection with WT vaccinia virus. Moreover, ΔE3L is less effective than WT vaccinia virus in attenuating the production of TNF-α and IL-6 by XS52 cells in response to LPS and poly(I:C), which might be pertinent to its prospects as a safer vaccine for smallpox prophylaxis and inducing mucosal immunity against other pathogens (8
The activation of cytokine production in ΔE3L-infected cells in the absence of exogenous stimuli is apparently a consequence of virus activation of the NF-κB pathway through the canonical mechanism of IκBα degradation. Decay of IκBα is observed at late times in ΔE3L-infected BSC40 or XS52 cells but not when these cells are infected with WT vaccinia virus. These results suggest a novel role for E3L as an antagonist of NF-κB signaling. This new function imputed to E3L is context dependent, insofar as a permissive infection of RK13 cells with ΔE3L did not result in decay of IκBα, suggesting that other viral proteins (e.g., K1L) might suffice to block IκBα turnover in RK13 cells. We find that K1L plays a role in viral attenuation of the LPS cytokine response in XS52 cells because deletion of K1L increases TNF-α and IL-6 secretion after LPS treatment.
The findings here that vaccinia virus attenuates immune signaling in epidermis-derived DCs resonate with previous studies with abortively infected monocyte-derived DCs, where WT vaccinia virus inhibited maturation of DCs exposed to proinflammatory cytokines or poly(I:C) and consequently inhibited their ability to activate T cells (16
). Also, Li et al. (32
) reported that WT vaccinia virus infection of human bone marrow-derived DCs, macrophages, and B cells decreased MHC class II-restricted antigen presentation to CD4+
T cells. It is noteworthy that whereas WT vaccinia virus infection of monocyte-derived DCs fails to trigger their maturation, infection with attenuated modified vaccinia virus Ankara activates DCs to secrete proinflammatory cytokines (15
), a situation analogous to our observation that infection with ΔE3L triggers cytokine production by epidermal DCs.
The permissiveness of XS52 cells for vaccinia virus replication provides a valuable model for future studies of the interface of poxviruses with skin immune cells. Systematic testing of other single-gene knockout viruses and viruses with multigene deletions should aid in identifying additional viral mediators of the suppression of cytokine production. XS52 cells should also prove useful in evaluating antiviral drugs as agents for treatment of poxvirus skin infections and complications of vaccination. We show that infection of these LC-like cells is not sensitive to an anti-poxvirus agent, araC, that is widely used in laboratory studies to arrest viral DNA replication. However, ANO emerges as a more plausible agent for topical treatment of poxvirus infections because it retains potency in XS52 cells.