The ability of many viruses to successfully induce a productive infection involves evasion or modulation of host antiviral responses. Our previous work reported that both WNV-infected and WNV replicon-bearing cells are deficient in TLR3-induced IFN-β promoter activation and activation of IRF3 in HeLa cells (53
). In the present study we identified the WNV NS1 protein as responsible for TLR3 inhibition. Using our approach of separately expressing individual NS proteins, we did not observe TLR3 inhibition with any of the other NS protein expression constructs tested. As alluded to above, this approach does not completely rule out possible contributions of other NS proteins when they are expressed in their natural context as part of the viral polyprotein, and we did observe a low level of residual stimulation in cells transfected with NS1 alone (Fig. ).
Compared to all of our constitutively NS1-expressing cell lines, HeLa 1.1.1 replicon cells consistently exhibited stronger TLR3 inhibition. Western blot analysis demonstrated that HeLa 1.1.1 cells express approximately 20 times as much NS1 as either HeLa D-24 or HeLa G. These findings are in line with our observations that TLR3 inhibition is dependent on the level of NS1 expression (Fig. ).
NS1 inhibited TLR3-stimulated IFN-β promoter and NF-κB-dependent promoter activation in reporter assays, nuclear translocation of NF-κB and IRF3, expression of IL-6, and establishment of an antiviral state. Interestingly, we noted a marked discrepancy between activation of the IFN-β promoter in reporter assays and activation of the endogenous IFN-β promoter in HeLa cells. We were not able to observe significant secretion of type I IFN by bioassay in culture supernatants of pIC-treated HeLa cells (data not shown). However, in coculture experiments HeLa cells were able to protect non-TLR3-expressing cells (Huh7) from VRP infection after pIC treatment (results not shown). It is therefore possible that low levels of type I IFN are produced by HeLa cells in response to TLR3 engagement and are able to act in a para-and/or autocrine fashion to establish an antiviral state. Alternatively, other TLR3-induced genes might be responsible for conferring an antiviral state independent of type I IFN. The identification of the exact mechanism of establishment of an antiviral state in HeLa cells by TLR3 is beyond the scope of this investigation and will be pursued separately.
Interference with TLR3 signaling by NS1 and WNV replicons was not only observed in HeLa cells but also in TLR3-expressing HEK293 cells and in WNV replicon-bearing mouse embryo fibroblasts (F. Gilfoy and P. Mason, personal communication). Our findings are in direct contrast to a previous report describing no interference of WNV infection with TLR3 signal transduction in several different cell lines (14
). Some of these discrepancies might be attributed to the use of different cell lines in that study. However, TLR3-expressing HEK293 cells were used both in the former study and in our experiments. It is possible that amounts of NS1 insufficient to inhibit TLR3 were expressed in the former study since the pIC challenges of infected cells were conducted anywhere from 3 to 6 h postinfection (14
). We previously described TLR3 inhibition in WNV-infected cells at 10 to 12 h postinfection (53
) and demonstrate in the present study that the degree of TLR3 inhibition is dependent on the expression level of NS1.
The role of TLR3 in the control of virus infections is somewhat controversial. In vitro studies have demonstrated that flaviviruses can be recognized by several different PRRs, including RIG-I, mda-5, and protein kinase R (7
). Flaviviruses have also been reported to activate the ssRNA PRR TLR7 in plasmacytoid dendritic cells (56
). Interestingly, flaviviruses do not seem to engage TLR3 during infection in cell culture systems, as described by Fredericksen et al. (14
) and according to our own unpublished observations. Most of these studies were performed using common cell lines that respond to TLR3 ligands. It is reasonable to speculate that TLR3 engagement might be different in vivo in the context of specific cell-cell interactions. As an example, a role for TLR3 in cross-priming by TLR3-expressing CD8+
dendritic cells in the activation of cytotoxic T lymphocytes has been described (54
), and several studies clearly demonstrate an involvement of TLR3 in the innate response to virus infection in vivo.
While Edelmann et al. described no differences in the pathogenesis of several viruses in TLR3-deficient mice compared to wild-type animals (11
), several other studies, conducted with a variety of viruses, demonstrate that TLR3 can have either protective or detrimental influences on viral pathogenesis. In most cases, TLR3-deficient mice displayed reduced production of proinflammatory cytokines in response to virus infection including tumor necrosis factor alpha, IL-6, or monocyte chemotactic protein 1 (18
). However, the outcome in terms of pathogenesis can be quite distinct, depending on the virus. Influenza A virus infection in TLR3-deficient mice yields higher virus titers and reduced cytokine responses, yet these mice have a survival advantage (29
). Similarly, TLR3 signaling in WNV-infected mice leads to increased permeability of the blood-brain barrier and increased WNV invasion into the central nervous system, whereas TLR3-deficient mice showed decreased central nervous system invasion (61
). In both cases these results were attributed to TLR3-dependent production of proinflammatory cytokines, which cause detrimental effects when they are produced at high levels. In both influenza A virus and WNV infection, higher levels of virus production could be detected in the absence of functional TLR3. In contrast, encephalomyocarditis virus infection in TLR3-deficient mice led to decreased cytokine production accompanied by higher viral loads and increased mortality. Interestingly, levels of IFN-β were not decreased in encephalomyocarditis virus-infected TLR3-deficient animals (21
). It is evident from these examples that the role of TLR3 in viral infection can vary with the virus species and the cell type(s) infected.
NS1 is a glycoprotein that is required for flavivirus replication (37
) and is also secreted to high levels during flavivirus infection in vivo (24
). Two recent studies have addressed a potential role of NS1 in immunomodulation. NS1 was shown to interfere with activation of complement factor H in vitro (8
), and cell surface-associated NS1 was found to mediate phagocytosis of WNV-infected cells and thus might play a role in clearance of virally infected cells (9
). The mechanism of NS1-mediated inhibition of TLR3 is currently under investigation. Both TLR3 and NS1 associate with membranes of the endoplasmic reticulum for maturation and targeting to their respective destination(s). It is possible that direct interactions of NS1 and TLR3 are responsible for TLR3 inhibition, or, alternatively, NS1 could interfere with downstream signaling such as recruitment of the adaptor protein TRIF or activation of TBK-1. Dengue NS1 protein can be endocytosed by several different cell types (1
), and an intriguing alternative is that secreted NS1 might inhibit TLR3 signaling after being endocytosed by target cells, possibly by interfering with TLR3 signaling in the endosome. This hypothesis is currently under investigation.
Several viruses have evolved independent mechanisms to interfere with TLR3. The hepatitis C virus NS3/4A protease interferes with TLR3-dependent IRF3 activation by cleaving the TLR3 adaptor molecule TRIF (30
), and vaccinia virus interferes with TLR3 through expression of its AR46 protein (57
). Our results add another example to this list and delineate a potentially different mechanism of targeting TLR3. The fact that several different viruses and, in particular, two small RNA viruses containing genomes with limited coding capacity have evolved ways to interfere with this innate immune signaling pathway strongly argues for an interplay between TLR3 and the virus. These interactions are bound to have important implications for the biology of these viruses while their significance might not be fully understood at present.