The identification of viral immune evasive strategies and the analysis of the molecular aspects of host–pathogen interactions are crucial to enhancing understanding of microbial pathogenesis and immunity to infection. Given the emerging importance of the TLR system in the antiviral response, understanding how viruses target this receptor family is of particular interest. During a database search for novel TIR domain–containing proteins, A46R from VV was identified (29
). This was potentially interesting because many poxviral immunomodulatory proteins, such as cytokine-binding proteins, bear sequence similarity to host factors (41
). To date, A46R is the only identified viral TIR domain–containing protein. In this paper, we show that A46R is an intracellular inhibitor of multiple TLR-dependent signaling pathways, define host signaling molecules that it targets, and demonstrate that the protein contributes to VV virulence in vivo.
The observation that A46R blocked all IL-1 signals tested (NF-κB, JNK, and ERK activation) suggested that it was acting close to the IL-1R complex. Furthermore, the presence of a TIR domain within A46R, the knowledge that TIR domains participate in homotypic interactions, and the fact that all the signals blocked by A46R were MyD88 dependent (31
), suggested that MyD88 may be sequestered by A46R. Coimmunoprecipitation and GST-pulldown experiments demonstrated an interaction between A46R and MyD88. This also suggested that A46R would antagonize TLR signaling, given the central role of MyD88 in many of these pathways. In fact, A46R inhibited every murine TLR pathway to NF-κB activation known to involve MyD88 in a mouse macrophage cell line, together with TLR4-mediated NF-κB, p38, and ERK activation, and IL-8 induction in human 293 cells.
Inhibition of TLR4-dependent NF-κB signaling in human cells was particularly potent, which led us to test the effect of A46R on other TIR domain–containing proteins with a role in this pathway. Altogether, five such proteins are known to be involved in TLR4-mediated NF-κB activation, namely the receptor itself, MyD88, Mal, TRAM, and TRIF (19
). A46R was found to be capable of associating with all five of these proteins, and hence it probably prevents the formation of the TLR4 receptor complex, thus accounting for its potent inhibition of NF-κB activation. Although these results were based on overexpression, several lines of evidence suggest that the interactions detected are specific, direct, and likely to occur in vivo. First, normally VV-expressed A46R interacted with MyD88 and Mal. Second, the interaction of A46R with MyD88, Mal, and TRIF were confirmed in yeast two-hybrid. Third, rMal interacted directly with GST-A46R in vitro. Finally, A46R displayed specificity for certain TIR domain–containing proteins and did not interact with TLR3 or SARM.
A46R is the first viral protein identified that can target host TIR domain–containing proteins. Given its ability to interact with different and diverse TIR domains (TRIF and TRAM are quite distinct in sequence from MyD88 and Mal; a and reference 20
), elucidation of the crystal structure of A46R should provide important information of general relevance as to how TIR domains interact. Because Box 2 of the TIR domain is particularly important in signaling (42
), the extra amino acids surrounding the A46R Box 2 ( a) may represent inhibitory loops that account for the ability of A46R to prevent TIR-dependent signaling. This hypothesis is being explored using mutagenesis studies.
The fact that A46R associated with all four TLR4 adaptors may suggest that TLR4 is a particularly important target for VV immune evasion. Indeed, TLR4 has been proposed to have a role in responding to fusion (F) protein of respiratory syncytial virus (43
), although the functional significance of this remains to be clarified (44
). TLR4 is also activated by envelope proteins from both mouse mammary tumor virus and Moloney murine leukemia virus, which could also be coimmunoprecipitated with TLR4 (45
). VV might interact with other or multiple TLRs that also use these adaptors. Possible VV PAMs detected by TLRs could be proteins on the surface of the intracellular mature virus or extracellular enveloped virus particles (potentially detected by TLR2 or TLR4; references 8
), intracellular dsRNA produced from the bidirection transcription of the VV genome (potentially detected by TLR3; reference 11
), or the dsDNA genome itself (potentially detected by TLR9, which responds to the dsDNA genome of herpes simplex virus; reference 15
). The role of TLRs in responding to VV PAMs is currently being investigated.
Although Mal and TRAM are yet to be directly implicated in responding to viruses, MyD88 (for TLR7; references 8
) and TRIF (for TLR3) have been shown to have a role, particularly in relation to type I IFN production (15
). TRIF is essential for both TLR4- and TLR3-mediated IRF3 activation and IFN-β production (21
) and probably has a key role in the initiation of the IFN-based antiviral response, leading to the inhibition of viral replication and spread. Although the relevance of TLR3 in responding to viruses in vivo has recently been questioned (46
), there is evidence that TRIF is important in controlling VV replication. Hoebe et al. (22
) showed that macrophages from mice in which trif
was disrupted supported the replication of VV to a higher titre than did macrophages from normal mice. Thus, the ability of A46R to inhibit TRIF-mediated IRF3 activation may be of primary importance to VV infection.
Consistent with its proposed role in antagonizing early innate TLR responses, A46R was expressed early during infection. Furthermore, deletion of A46R from VV caused attenuation in a murine intranasal model, and also led to enhanced levels of cells on day 2 in lungs of infected animals. The use of a revertant virus in which the A46R gene was reinserted into the deletion virus confirmed that the attenuation observed in the deletion virus, and the difference in lung cell numbers, were due solely to the absence of A46R. The ability of A46R to target intracellular TIR-dependent signaling most likely accounts for its role in virulence, probably by blocking the induction of immune response genes downstream of TLRs, as has been shown here for the chemokines IL-8 and RANTES. Possibly, inhibition of chemokine induction by A46R might account for the early enhanced levels of cells in the absence of A46R expression.
Previously, we identified another VV TLR antagonist, A52R, which could inhibit TLR-dependent NF-κB activation. There are several lines of evidence that A46R and A52R are not functionally redundant. First, they target distinct TLR signaling molecules (30
). Second, although they do have some overlapping effects (such as the inhibition of MyD88-dependent NF-κB activation), their overall effects on TLR signaling are quite distinct. Although A52R is a good NF-κB inhibitor, it has no inhibitory effect on MAP kinase activation (not depicted), nor on TLR3-mediated IRF3 activation ( b). In contrast, A46R blocks both MAP kinase activation and, importantly, TLR3- and TRIF-mediated IRF3 activation. Furthermore, A46R has little effect on TLR3-mediated NF-κB activation, which A52R blocks potently ( a). This was surprising because A46R interacts with TRIF. But, presumably, the interaction of A46R with TRIF has a greater effect on downstream IRF3 activation compared with NF-κB because these two pathways bifurcating from TRIF have been shown to be quite distinct (48
). Finally and crucially, the deletion of either A46R
from VV causes attenuation ( b and reference 30
) and, thus, both contribute to virulence and are nonredundant.
N1L is another intracellular VV protein that contributes to virulence (50
), which has also been shown recently to function by antagonizing TLR signaling, but at the level of IκB kinases and related kinases involved in IRF3 activation (51
). Thus, the importance of blocking TLR signaling is demonstrated by the retention by VV of at least three distinct mechanisms of disrupting these pathways. Furthermore, the attenuated phenotypes seen in the absence of A46R, A52R, or N1L provide evidence for a role for TLRs in containing VV infections. The action of these intracellular TLR inhibitory proteins would be expected to be restricted to infected cells, whereas immunomodulatory proteins secreted from VV-infected cells, such as cytokine, chemokine, and IFN-binding proteins, can act on ligands produced from both infected and uninfected cells.
Finally, A46R is also found in variola virus, the causative agent of smallpox. Concern about the threat of the use of variola as a bioweapon has led to a renewed desire to understand this human pathogen. However, little is known about the role of human TLRs in sensing variola virus. Given that VV A46R targets human adaptors, the knowledge that A52R is truncated in variola virus, together with the fact that the VV and variola virus A46R amino acid sequences differ by only eight residues ( a), it is likely that variola virus A46R would have an important role in interactions with the human TLR system.