The NS5 protein of both LGTV and JEV mediates multiple indispensable and distinct functions during replication. The RdRP and MTase domains are obligatory for RNA replication, whereas a third domain confers resistance to IFN via inhibition of JAK-STAT signal transduction (2
). The structure and function of the RdRP and MTase are relatively well characterized, although the domain responsible for JAK-STAT inhibition was largely unknown. In this work, we first identified the linear domain within LGTV NS5 responsible for JAK-STAT inhibition as residing between amino acid residues 355 and 735 (Fig. ). This domain overlaps precisely with conserved features of the RdRP, namely the finger region and the eight conserved RdRP motifs (Fig. ). The superimposition of the JAK-STAT inhibitory domain within the RdRP suggests an inextricable evolutionary relationship between RNA replication and IFN resistance.
By analyzing an extensive panel of mutants, we further refined the specific amino acids critical for the function of NS5 as a suppressor of IFN-mediated signaling. These residues clustered in two short, noncontiguous stretches of amino acids within the JAK-STAT inhibitory domain. The first of these, residues 374 to 380, resides within the RdRP finger domain, whereas residues 624 to 647 belong to the RdRP palm domain. Despite their significant separation on the linear NS5 sequence, the two amino acid groups localize adjacent to one another when modeled on the crystal structure of the WNV RdRP (17
) (Fig. ). Taken together, these results strongly suggest that the specific amino acids identified by mutagenesis contribute to a unique functional site on the polymerase responsible for the disruption of JAK-STAT signaling.
The IFN antagonist function of the RdRP did not appear to depend upon key motifs necessary for RdRP activity. Each of the eight conserved motifs within the Flaviviridae
RdRP contain residues involved in RNA replication, functioning in template binding (motif II), binding catalytic ions (motif VI, which contains the RdRP active site, GDD), and GTP or NTP binding (motifs I, III, IV, V, VII, and VIII) (5
). Conserved residues within each motif are obligatory for HCV RdRP activity (16
) or contribute to the GTP binding site of BVDV RdRP (5
). In the course of our studies, mutation of many conserved residues critical for GTP or NTP binding in motifs I (for LGTV NS5, M456 and K458), II (R473), IV (D540), V (T608 and N612), and VI (DD664/665) had no effect on NS5-mediated inhibition of JAK-STAT signaling (Fig. and ). Notably, the DD664/665AA site-directed double mutant of LGTV NS5 should be incapable of RdRP activity (9
), but this mutation had no effect on nuclear accumulation of pY-STAT1. Thus, while the IFN-antagonist function of NS5 will certainly be affected by structural alterations in the RdRP, neither the RdRP active site nor an intact GTP binding site are required. However, the RdRP activity of LGTV NS5 containing specific mutations in 374 to 380 and 624 to 647 must be tested to determine if polymerase activity and JAK-STAT inhibition are strictly independent.
The precise mechanism of JAK-STAT inhibition by NS5 is not known. In the current study, four of the five mutant expression constructs exhibiting a strong change in the phenotype of pY-STAT1 inhibition contained substitutions for charged residues (R376, D380, E626, and E628), which would be expected to reside on the surface of NS5. Hence, these residues are potential mediators of the protein-protein interactions involved in the suppression of JAK-STAT signal transduction. We previously identified an association between LGTV and IFN receptor complexes (2
) and are currently working to determine if this association is affected by key mutations in NS5. The finding that the C-terminal border of the LGTV NS5 JAK-STAT antagonist domain occurs near residue 735 is consistent with work by Lin et al. whereby the IFN antagonist function of JEV NS5 was not affected by C-terminal truncation to residue 762 but was compromised following further truncation to residue 667 (14
). However, in contrast to our results, N-terminal deletion of the first 84 or 167 residues of JEV NS5 abrogated its function as an IFN antagonist. Despite these differences between studies, it remains possible that the JAK-STAT inhibitory domains in LGTV and JEV are similarly located in the proteins. A simple explanation of the differences between studies is that the N-terminal deletion series we made in LGTV NS5 were more structurally favorable and retained the nascent properties of the protein.
Inhibition of JAK phosphorylation by JEV NS5 was associated with protein tyrosine phosphatase (PTP) activity (14
). Significant PTPs involved in the normal negative regulation of JAK-STAT signal transduction include Src homology 2 (SH2) domain containing tyrosine phosphatase 1 (SHP-1) and SHP-2, PTP1B/T-cell PTP, and CD45. Specific inhibition of PTP1B and CD45 did not restore the IFN antagonist activity of JEV NS5 (14
). SHP activation requires substrate recognition of phosphorylated tyrosine residues (21
). We mutated every tyrosine residue within LGTV NS5:355-735 by random or site-directed mutagenesis with no effect on IFN-stimulated nuclear accumulation of pY-STAT1. Hence, it is unlikely that phosphorylated tyrosines within NS5 serve as substrates to directly activate SHPs. However, indirect activation of these phosphatases by NS5 may occur, such as through the activation of additional kinases that positively regulate PTPs. Alternatively, noncanonical PTPs may also be invoked. Importantly, although JAK-STAT signaling is regulated via a series of tyrosine phosphorylation events, our results suggest that NS5's function as a JAK-STAT antagonist is not itself directly mediated by tyrosine phosphorylation.
Results from the LGTV/DEN4 chimeras suggested that LGTV-specific residues exist in the N terminus of the JAK-STAT inhibitory domain and are important for the suppression of pY-STAT1. We found two LGTV NS5 residues, R376 and D380, that were both critical for inhibition and different from the corresponding DEN4 sequence, thus potentially contributing to this virus-specific function of NS5. However, an examination of protein sequences from the broader group of vector-borne viruses showed that these two residues are similar between LGTV and DEN2 NS5 yet different between LGTV and JEV NS5 (Fig. ). Furthermore, the second cluster of LGTV NS5 residues critical for inhibiting JAK-STAT signaling is highly conserved among the vector-borne flaviviruses (Fig. ). Since our results do not identify unique amino acids that correlate with the viruses that utilize NS5 as their predominant JAK-STAT antagonist (14
), additional residues are likely to influence IFN antagonism. Furthermore, structural deviations between the various flavivirus RdRPs may also determine NS5's competence to suppress IFN responses. The recently determined atomic structure of RdRP domains from WNV and DEN (17
) can be used to predict, and thus more precisely define, residues involved in the suppression of IFN signaling. This work will provide further insight into the immune evasion strategies utilized by these highly pathogenic viruses. Identification of the specific RdRP residues responsible for the antagonism of IFN responses is an important first step in the development of therapeutics aimed at disrupting this critical aspect of virus pathogenesis.