In this report, we demonstrate that a tick-borne flavivirus, LGTV, antagonizes IFN signaling by inhibiting the JAK-STAT signal transduction pathway in response to both IFN-α/β and IFN-γ. Furthermore, we have demonstrated that LGTV NS5 alone can suppress STAT1 phosphorylation in response to IFN-β or IFN-γ stimulation (Fig. and ). Thus, NS5 is a direct antagonist of IFN signaling. This antagonistic function of NS5 is most likely to result from its association with the IFN receptor complexes (Fig. ).
Human DC are important early target cells for flavivirus replication (24
). During LGTV replication in human DC, NS5 associated with the IFN-α/β receptor subunit, IFNAR2 (Fig. ). The direct association of NS5 with IFNAR2 in LGTV-infected DC is likely to result in the functional inhibition of JAK-STAT signal transduction observed in these cells. Consequently, there may be a dual role for LGTV NS5 in pathogenesis. First, inhibition of IFN will facilitate early virus replication at the level of the individual cell. Second, the inhibition of signaling in these cells may disrupt the bridge between innate and adaptive immunity by compromising DC activation and/or maturation, as IFN-α/β have critical roles in DC maturation, survival, and function (12
). This hypothesis is supported by recent findings demonstrating inhibited maturation and T-cell-stimulatory capabilities of human DC infected with DEN-2 in vitro (41
). If similar interactions occur between DC and the highly virulent TBE viruses, this may contribute significantly to the severity of some TBE virus infections.
Since both tick- and mosquito-borne flaviviruses prevent JAK activation in response to IFN-α/β and IFN-γ (16
), it is possible that a common mechanism of inhibition exists. NS5 has 80 to 90% amino acid identity among members of the TBE virus serogroup, suggesting that the function of NS5 in IFN antagonism might be conserved in this group. However, previous studies investigating JAK-STAT signaling following DEN or WNV infection have implicated nonstructural proteins other than NS5 (32
), and NS4B in particular (38
). The fact that we did not identify NS4B as an antagonist may be due to the expression in these studies of the mature LGTV NS4B that lacks the 23-amino-acid signal sequence. The presence of this sequence has recently been shown to be crucial to the inhibitory function of NS4B (38
). Given the conservation in function of various proteins throughout the flaviviruses, it is surprising that NS5 has not been identified as an antagonist for other viruses. In particular, WNV infection prevented Jak1 and Tyk2 activation in response to IFN-α (33
) in a manner very similar to that described here for LGTV, suggesting that the two viruses may utilize similar mechanisms to suppress JAK-STAT signaling pathways. Furthermore, while the polyprotein of LGTV shares only approximately 40% amino acid identity with WNV or DEN, LGTV NS5 shares a relatively high degree of amino acid identity (approximately 57 to 59%) with both WNV and DEN-2 NS5 proteins. The role of NS5 as an antagonist for LGTV may simply reflect a divergence in the IFN evasion strategies used by tick- and mosquito-borne flaviviruses. Therefore, it will be of interest to determine the relative contribution of NS5 in the IFN resistance of the different vector-borne flaviviruses.
This study suggests that NS5 has a role in viral pathogenesis as well as in virus replication. NS5 is the largest of the flavivirus NS proteins. It has a central function in RNA replication as it contains the viral RNA-dependent RNA polymerase and methyltransferase/RNA capping functions (10
). The primary cellular location of NS5 is the endoplasmic reticulum, where virus replication occurs (48
). However, the additional role for this protein in IFN antagonism suggests that some NS5 may localize to a position proximal to the plasma membrane of infected cells. The regulation of NS5 distribution most likely occurs through posttranslational modification, including phosphorylation (21
). Thus, further studies examining flavivirus NS5 expression, regulation, and distribution will be required to fully elucidate NS5 functions in both virus replication and IFN resistance.
The precise mechanism by which NS5 associates with the IFN receptor complexes and inhibits JAK-STAT signaling is not yet understood, although two broad possibilities exist. The simplest explanation is that NS5 prevents the receptors from functioning. This could be achieved by preventing ligation of receptor subunits through steric hindrance. Alternatively, NS5 may lock up or freeze receptor complexes through multivalent interactions with receptor components such that further signaling cannot occur. In unstimulated cells, the receptor components are preassembled. For example, IFNAR2 is associated with Jak1, STAT1, and STAT2; IFNAR1 is associated with Tyk2. Therefore, binding to a receptor subunit as well as additional components either preceding or following IFN stimulation may prevent further signal transduction.
The second possibility for NS5-mediated inhibition is that NS5 associates with receptors to specifically prevent JAK function. During normal JAK-STAT signal transduction, negative feedback mechanisms exist to regulate signaling. One mechanism operates through the expression of SOCS (suppressor of cytokine signaling proteins (1
). SOCS proteins can bind IFN receptor subunits in order to access the catalytic domain of JAKs, thus preventing their activation. SOCS proteins can also bind phosphorylated JAKs and target them for degradation via the proteasome. In LGTV-infected cells, inhibition of the proteasome does not restore JAK-STAT signaling (S. M. Best and M. E. Bloom, unpublished), suggesting that this mechanism is not involved. However, LGTV NS5 may bind IFN receptors and function in an analogous way to SOCS proteins to prevent the phosphorylation of JAKs. Alternatively, NS5 might function as a virus equivalent of another cellular negative regulator, a protein tyrosine phosphatase, to remove phosphate groups from JAK catalytic domains (3
Both RNA and DNA viruses utilize a variety of mechanisms to inhibit IFN-stimulated JAK-STAT signaling (5
). The paramyxoviruses interfere directly with STAT proteins, preventing their nuclear translocation, targeting them for degradation via the proteasome, or sequestering them in high-molecular-mass cytoplasmic complexes (17
). Human cytomegalovirus degrades Jak1 and IRF-9 via the proteasome (36
), while the murine polyomavirus T antigen binds to and inhibits Jak1 activity (53
). The E6 protein of human papillomavirus 18 binds to Tyk2 and prevents its normal association with the IFNAR1 subunit, thereby inhibiting JAK-STAT signaling (29
). Inhibition of JAK phosphorylation has also been associated with suppression of JAK-STAT signaling following infection with human herpes simplex virus type 1 (inhibited phosphorylation of Jak1, Jak2, and Tyk2) (7
), Sendai virus (Tyk2) (23
), and measles virus (Jak1) (55
). The precise mechanism(s) by which flaviviruses circumvent JAK-STAT signaling remains to be determined.
The identification of NS5 as an inhibitor of JAK-STAT signaling may have implications for vaccine design and development of therapeutics. Currently, LGTV is being developed as a backbone for live-attenuated chimeric virus vaccines for protection against TBEV infection (44
). If NS5 of these chimeric viruses also interferes with IFN signaling, identification and alteration of the NS5 sequences responsible might further attenuate the viruses and increase immune responsiveness, thus reducing the risk of possible complications associated with a live vaccine. In addition, IFN-α/β are potential therapeutics for infection with various flaviviruses (34
). In this context, identification of the NS5 sequences that interact with the IFN receptor could lead to the design of additional therapeutic inhibitors for use in combination with, or instead of, IFN-α/β.
This newly defined role of NS5 as an IFN antagonist may promote virus survival by preventing the antiviral state from being established in infected cells. Furthermore, a consequence of the protein-protein interactions described, particularly in infected DC, may be to perturb adaptive immune responses to infection, thus delaying virus clearance. Because this interaction occurs in cells from mice and from humans, it may aid virus replication in hosts that serve to amplify the virus in the environment as well as in human hosts. Hence, in addition to its central role in viral RNA replication, NS5 is potentially an important virus virulence factor.