Innate Immune Response of Human Plasmacytoid Dendritic Cells to Poxvirus Infection Is Subverted by Vaccinia E3 via Its Z-DNA/RNA Binding Domain

Abstract

Plasmacytoid dendritic cells (pDCs) play important roles in antiviral innate immunity by producing type I interferon (IFN). In this study, we assess the immune responses of primary human pDCs to two poxviruses, vaccinia and myxoma virus. Vaccinia, an orthopoxvirus, was used for immunization against smallpox, a contagious human disease with high mortality. Myxoma virus, a Leporipoxvirus, causes lethal disease in rabbits, but is non-pathogenic in humans. We report that myxoma virus infection of human pDCs induces IFN-α and TNF production, whereas vaccinia infection does not. Co-infection of pDCs with myxoma virus plus vaccinia blocks myxoma induction effects. We find that heat-inactivated vaccinia (Heat-VAC; by incubating the virus at 55°C for 1 h) gains the ability to induce IFN-α and TNF in primary human pDCs. Induction of IFN-α in pDCs by myxoma virus or Heat-VAC is blocked by chloroquine, which inhibits endosomal acidification required for TLR7/9 signaling, and by inhibitors of cellular kinases PI3K and Akt. Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1. These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway. Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists. The myxoma virus ortholog of vaccinia E3 (M029) lacks the N-terminal Z-DNA/RNA binding domain, which might contribute to the immunostimulating properties of myxoma virus.

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

“…In this study, we further explored the role of the E3L NTD and found that the ZBD, but not its nucleic acid-binding function, is important for PKR inhibition. It is noteworthy that the E3L ZBD, but not its Z-DNA binding activity, is likewise required to block the induction of the innate immune response and cytokine production in poxvirus-infected plasmacytoid dendritic cells (Dai et al 2011;Cao et al 2012). In contrast, the Z-DNA binding activity of E3L was previously shown to be critical for viral pathogenicity (Kim et al 2003).…”

“…Likewise, substitution of alanine in place of vaccinia E3L residues Y48 and P63 markedly reduced viral lethality following intracranial inoculation in mice (Kim et al 2003). In contrast to these studies revealing the importance of the E3L Z-DNA binding activity, the ability of E3L to antagonize the innate immune response in plasmacytoid dendritic cells is dependent on the ZBD, but not its Z-DNA binding activity (Dai et al 2011;Cao et al 2012). Taken together, these data reveal important roles for the E3L ZBDand its Z-DNA binding activity; however, the contribution of Z-DNA binding to E3L inhibition of PKR is not known.…”

Innate immune activation of NFκB and its antagonism by poxviruses

“…2). Poxvirus infection is detected by TLR7 [47], TLR8 [48] and TLR9 [49,50] in endosomes of conventional and plasmacytoid dendritic cells (cDCs and pDCs). Survival of mice after a lethal ECTV infection was critically reliant on TLR9, while MVA infection protected mice from ECTV in a manner dependent on TLR9 [49].…”

“…Survival of mice after a lethal ECTV infection was critically reliant on TLR9, while MVA infection protected mice from ECTV in a manner dependent on TLR9 [49]. Recognition of poxvirus by TLR7 was proposed to involve detection of viral RNA transcripts [47], while surprisingly, TLR8-dependent responses were suggested to be mediated by recognition of poly(A)/T-rich poxviral DNA sequences [48]. Interestingly, although TLR3 and TLR9 are not normally expressed in skin [51,52], MCV-infected skin lesions exhibited increased expression of TLR3 and TLR9 in infected tissues, suggesting that these TLRs may be involved in the local host response to MCV in infected keratinocytes [52,53].…”

“…E3 is a key virulence factor for VACV, that has multiple activities in suppressing the host immune response, and contains an N-terminal Z-DNA/RNA binding motif as well as a C-terminal dsRNA binding domain [66]. The Z-DNA/RNA binding motif of E3 was shown to block TLR7 activation in pDCs [47]. This domain is not retained in the myxoma ortholog of E3, thus preventing it from evading TLR7, which suggests significant gain or loss of function between orthologous proteins in different poxviruses.…”