doi:10.1128/JVI.01970-16. protein are present constitutively and enhance innate immunity. During ZIKV infection or Jujuboside A NS5 expression, we found that ZIKV NS5 evicts PML from STAT2 NBs, forming NS5/STAT2 NBs that dramatically reduce PML expression in hBMECs and inhibit the transcription of interferon-stimulated genes (ISG). Expressing the ZIKV NS5 SUMO site mutant (K252R) resulted in NS5/STAT2/PML NBs that failed to degrade PML, reduce STAT2 expression, or inhibit ISG induction. Additionally, the K252 SUMOylation site and NS5 nuclear localization were required for ZIKV NS5 Rabbit Polyclonal to DDX50 to regulate hBMEC cell cycle transcriptional responses. Our data reveal NS5 SUMO Jujuboside A motifs as novel NB coordinating factors that distinguish flavivirus NS5 proteins. These findings establish SUMOylation of ZIKV NS5 as critical in the regulation of antiviral ISG and cell cycle responses that permit ZIKV to persistently infect hBMECs. IMPORTANCE ZIKV is a unique neurovirulent flavivirus that persistently infects human brain microvascular endothelial cells (hBMECs), the primary barrier that restricts viral access to neuronal compartments. Here, we demonstrate that flavivirus-specific SIM and SUMO sites determine the assembly of NS5 proteins into discrete nuclear bodies (NBs). We found that NS5 SIM sites are required for NS5 nuclear localization and that SUMO sites regulate NS5 NB complex constituents, assembly, and function. We reveal that ZIKV NS5 SUMO sites direct NS5 binding to STAT2, disrupt the formation of antiviral PML-STAT2 NBs, Jujuboside A and direct PML degradation. ZIKV NS5 Jujuboside A SUMO sites also transcriptionally regulate cell cycle and ISG responses that permit ZIKV to persistently infect hBMECs. Our findings demonstrate the function of SUMO sites in ZIKV NS5 NB formation and their importance in regulating nuclear responses that permit ZIKV to persistently infect hBMECs and thereby gain access to neurons. test. Experiments were performed at least 3 times with similar results. ZIKV NS5 SUMO K252 is required for regulating IFN-induced ISGs. As NS5 was previously reported to restrict STAT2-directed ISG responses (13), we evaluated NS5 mutant regulation of IFN-directed ISG induction. Cells expressing WT NS5 from ZIKV or DENV suppressed IFN- induction of IFIT1, OAS2, MX1, and ISG15 by 60- to 1 1,250-fold (Fig. 9). In contrast, NS5 NLS and K252R Jujuboside A SUMO mutants failed to inhibit ISG induction (Fig. 9). These findings indicate that the NS5-K252 SUMO motif and NLS are required to regulate IFN-directed ISG responses in hBMECs. Open in a separate window FIG 9 ZIKV NS5 K252 is required for regulating IFN induced ISGs. Lentivirus-transduced hBMECs expressing ZIKV NS5 WT, K252R, NLSmut, DENV3 NS5 WT, or GFP were induced with IFN-, total RNA was purified, and changes in ISG induction (OAS2, MX1, IFIT1, and ISG15) were analyzed by qRT-PCR at 24 h. Asterisks indicate statistical significance (*, test. Experiments were performed at least 3 times with similar results. Prior studies found that in HEK293T or A549 cells, ZIKV NS5 binds STAT2, and that NS5 directs STAT2 ubiquitination and degradation in response to IFN- addition (39,C41). In hBMECs, STAT2 is constitutively present in the nucleus (Fig. 10A), and we found that STAT2 colocalizes with ZIKV NS5 nuclear speckles (WT or -K252R mutant) in the presence or absence of added IFN- (Fig. 10A and ?andB).B). However, in response to IFN- addition, we also observed a significant reduction in cytoplasmic and nuclear-localized STAT2 in hBMECs expressing ZIKV NS5 or NS5-K252R proteins (Fig. 10). Open in a separate window FIG 10 ZIKV NS5 speckles colocalized with STAT2 in the nucleus of hBMECs. Lentivirus-transduced hBMECs constitutively expressing ZIKV NS5 WT or K252R mutant proteins (A) or induced with IFN- (right) and hBMEC controls (B) were immunostained for NS5 using anti-FLAG antibody and for STAT2 and analyzed using confocal microscopy. Experiments were done in triplicate, repeated at least three times, and representative data are presented. Bars represent 10?m. Consistent with subcellular localization, expressing ZIKV NS5 in hBMECs resulted in.