A major mechanism by which WNV evades the host antiviral response is to suppress IFN-stimulated JAK-STAT signaling (11
). In this report, we have demonstrated that NS5 from the virulent NY99 strain of WNV is a potent inhibitor of IFN-mediated signal transduction. WNV NS5 expression prevented the development of the cellular antiviral state, as demonstrated by its ability to augment NDV-GFP replication in IFN-treated cells (Fig. ). As observed during infection (11
), IFN antagonism mediated by WNV-NY99 NS5 was associated with failure of STAT1 to be phosphorylated (Fig. ), translocate to the nucleus (Fig. ), and participate in the expression of ISRE-dependent genes (Fig. ). This work adds WNV-NY99 to the number of highly pathogenic flaviviruses that utilize NS5 as an efficient IFN antagonist (including TBEV, DENV, and JEV), suggesting that this function of NS5 is essential to the success of flaviviruses as emerging and reemerging pathogens (28
Effective host IFN responses are critical to recovery from flavivirus infection. Thus, the relative ability of these viruses to subvert the IFN response may be a decisive factor in their virulence. In support of this concept, we found that NS5 from WNV-NY99 was a potent suppressor of IFN responses, whereas NS5 from the closely related but attenuated KUN was not (Fig. ). These results are consistent with previous work that examined the ability of individual KUN proteins to suppress ISRE-dependent responses and did not find a role for NS5 (26
). A single residue at position 653 is largely responsible for this difference since its mutation in KUN NS5 to the corresponding NY99 residue (S653F) conferred an ability to antagonize signaling similar to that of WT NY99 NS5 (Fig. ). Furthermore, introduction of F653S to NY99 NS5 compromised the ability of this protein to prevent pY-STAT1 accumulation, suggesting that this residue is more generally important for WNV NS5 function in IFN antagonism. Incorporation of the NS5 mutation S653F into a recombinant KUN increased the virus's ability to suppress IFN-β-mediated STAT1 phosphorylation and ISRE-dependent gene expression. Strikingly, KUN NS5 bearing the S653F mutation during transient expression demonstrated only a 2-fold increase in its ability to inhibit pY-STAT1, yet replication of a recombinant KUN bearing this mutation resulted in a 30-fold increase in inhibition of signaling compared to WT virus (Fig. ). This more potent antagonism was associated with greater resistance to the antiviral effects of IFN during WNV replication (Fig. ). The importance of S653F during virus replication provides definitive evidence for the biological relevance of NS5 and, specifically, the residue at position 653, in IFN antagonism.
Interestingly, we found that viral proteins accumulated to higher levels at 24 hpi in KUN NS5:S653F-infected cells than in cells infected with WT virus (Fig. ) without an increase in infectious virus (Fig. ). Because E and NS5 protein levels were greater in both IFN-competent and -incompetent cells infected with KUN NS5:S653F at 24 hpi, it is possible that the S653F mutation not only increases resistance to IFN (Fig. ) but also stabilizes NS5 expression. This may also result in a general acceleration of protein expression (and perhaps RNA replication) by stabilizing the replication complex. Incorporation of S653F into KUN NS5 expressed ectopically did not alter its expression level (Fig. ). However, NS5 turnover is likely to be more complex during virus replication, as exemplified by the fact that DEN NS5-mediated degradation of STAT2 was observed only when NS5 was expressed as part of a cleavable polyprotein (2
). Thus, the mutation may affect NS5 stability only after cleavage. Alternatively, NS5 may be stabilized through increased binding to a cellular target induced during virus replication. Future experiments will more precisely address the mechanism of IFN antagonism and its relationship to WNV NS5 turnover.
Residue 653 lies within not only the IFN antagonism domain previously identified for LGTV NS5 but also the three-dimensional pocket we previously proposed to mediate much of LGTV NS5's function in IFN resistance (44
). In addition, mutagenesis studies demonstrated that at least three WNV NS5 residues located in this site, W382, VI631/632, and W651, were important for IFN antagonism. Hence, this site appears more broadly important to NS5 function, suggesting that the mechanism of STAT1 inhibition, at least in part, may be common to NS5 proteins from both TBEV and JEV serogroups. NS5 proteins from JEV-N and JEV-SA also demonstrated significantly different abilities to prevent pY-STAT1 accumulation (Fig. ) and differ from each other at eight amino acids. Based on the experiments presented here, we predict that residue 640 within JEV NS5, located within the same site of NS5 and divergent between JEV-N and JEV-SA strains, is responsible for these differences. However, although LGTV NS5 residues 355 to 735 are sufficient to inhibit IFN signaling equally as well as the full-length protein, the analogous truncation of WNV-NY99 or TBEV NS5 did not function efficiently as antagonists (data not shown). While we did not finely map the antagonism domains in these two proteins, only expression constructs corresponding to residues 1 to 735 retained resistance to IFN in both cases. This is consistent with previous mapping studies of JEV NS5 (23
) and with the requirement for sequences in the MTase domain of TBEV NS5 for optimal inhibition (59
). Thus, additional features of some NS5 molecules may also contribute to suppression.
The relationship between NS5 function and virulence of the corresponding virus was not observed for the tick-borne flaviviruses. NS5 from attenuated LGTV and pathogenic TBEV both exhibited the same high degree of pY-STAT1 suppression (Fig. ). Of course, flaviviruses encode factors other than NS5 that contribute to pathogenicity. The E protein, for example, is particularly important in flavivirus virulence since it mediates virus binding to cellular receptors and entry to the host cell (reviewed in reference 32
). The presence of specific glycosylation sites in E is associated with WNV virulence (3
), and the WNV E protein can suppress innate immune responses to double-stranded RNA, a phenomenon dependent on E glycosylation status (1
). The E protein has recently been demonstrated to affect sensitivity of JEV to host IFN responses since a mutation in E that reduced replication efficiency also reduced the capacity to antagonize IFN-mediated JAK-STAT signaling (22
). Hence, while NS5 function in IFN resistance is likely required for virus replication and pathogenesis, it is not the only candidate for defining flavivirus virulence.
The accumulated data presented here and previously (2
) suggest that NS5 is the most potent of the flavivirus-encoded IFN antagonists in mammalian cells. However, NS4B also antagonizes responses, a function that is dependent on the 2K signal sequence derived from NS4A, and is enhanced in the presence of the other small hydrophobic NS proteins, NS2A and NS4A (37
). During flavivirus replication, these three proteins are involved in endoplasmic reticulum membrane proliferation, membrane anchoring of the viral replication complex, and RNA replication (28
). In the case of WNV and probably all flaviviruses, membrane rearrangement is concomitant with redistribution of cellular cholesterol to sites of viral replication (30
). The resulting loss of cholesterol-rich lipid rafts in the plasma membrane is associated with reduced IFN-mediated JAK-STAT signal transduction (30
). Thus, it is highly possible that the functions of NS4A, NS4B, and the intervening 2K signal sequence in membrane rearrangement contribute to their IFN antagonism. However, this does not readily explain why 2KNS4B from JEV can suppress STAT1 phosphorylation at levels far greater than other 2KNS4B molecules, for example, from TBEV (Fig. ), unless their roles differ in membrane alteration and potentially cholesterol metabolism, which seems unlikely. Thus, a more specific mechanism of NS4B-mediated IFN antagonism may exist.
The use of multiple proteins to suppress IFN-mediated JAK-STAT signaling, as well as using one relatively conserved protein to target this pathway using different mechanisms, is not unique to the flaviviruses. The best described examples of this are the paramyxoviruses, a large family of negative-stranded RNA viruses that includes several important human pathogens such as measles virus, mumps virus, and NiV. The V protein from mumps virus targets both STAT1 and STAT3 for proteasomal degradation (41
) whereas the simian virus 5 V protein degrades only STAT1 (10
), and the type II human parainfluenza virus V protein degrades only STAT2 (43
). The NiV P gene encodes four proteins, P, V, W, and C, all capable of functioning in IFN antagonism (21
). NiV V and P proteins sequester STAT1 and STAT2 in the cytoplasm in high-molecular-weight complexes (49
), whereas the W protein, which shares a common N terminus with P and V, sequesters unphosphorylated STAT1 in the nucleus (52
). As has been speculated for NiV (52
), encoding multiple IFN antagonists may be associated with the high virulence of some flaviviruses or contribute to their broad host range (both invertebrate and vertebrate) by overcoming IFN responses from multiple species.
The most outstanding question raised by the current study, given the clear effect of the S653F mutation on NS5-mediated IFN antagonism, is what is its role in WNV virulence? We are currently addressing this question in the mouse model. Interestingly, in a comparison of sequences from WNV strains of high and low virulence in humans, the virulent SPU116/89 strain (WNV lineage II, isolated from a fatal infection in South Africa) had a number of variable residues in NS5 (7
). Four out of five of these (residues 623, 635, 641, and 643) map within the same pocket on NS5 as residue 653 (data not shown). Thus, we speculate that this virus may have an increased capacity to suppress IFN responses compared to its closely related but less virulent South African strains. A greater understanding of the precise roles of specific residues required for IFN antagonism by WNV NS5 will shed light on their role in virulence and could be exploited in the development of live attenuated vaccines or antiviral therapeutics.