Nuclear dengue virus NS5 antagonizes expression of PAF1-dependent immune response genes

Abstract

Dengue virus (DENV) disruption of the innate immune response is critical to establish infection. DENV non-structural protein 5 (NS5) plays a central role in this disruption, such as antagonism of STAT2. We recently found that DENV serotype 2 (DENV2) NS5 interacts with Polymerase associated factor 1 complex (PAF1C). The primary members of PAF1C are PAF1, LEO1, CTR9 and CDC73. This nuclear complex is an emerging player in the immune response. It promotes the expression of many genes, including genes related to the antiviral, antimicrobial and inflammatory responses, through close association with the chromatin of these genes. Our previous work demonstrated that NS5 antagonizes PAF1C recruitment to immune response genes. However, it remains unknown if NS5 antagonism of PAF1C is complementary to its antagonism of STAT2. Here, we show that knockout of PAF1 enhances DENV2 infectious virion production. By comparing gene expression profiles in PAF1 and STAT2 knockout cells, we find that PAF1 is necessary to express immune response genes that are STAT2-independent. Finally, we mapped the viral determinants for the NS5-PAF1C protein interaction. We found that NS5 nuclear localization and the C-terminal region of the methyltransferase domain are required for its interaction with PAF1C. Mutation of these regions rescued the expression of PAF1-dependent immune response genes that are antagonized by NS5. In sum, our results support a role for PAF1C in restricting DENV2 replication that NS5 antagonizes through its protein interaction with PAF1C.

Transcriptomic profiling implicates PAF1 in both active and repressive immune regulatory networks

“…The diminished expression of cGAS and PYCARD/ASC across all stimuli (Fig. 2B), for example, mirrors our group’s past work in finding cGAS expression to be reduced following PAF1 knockout in poly I:C-stimulated cells (14). Considering both cGAS and ASC are involved in dsDNA-mediated immune activation and exhibit cross-regulation (50), it is particularly interesting to see their shared downregulation.…”

“…This way, we could differentiate a core set of genes correlated to PAF1 while preserving any nuance for a particular stress response (Table 1). Based on our work using a dsRNA mimic in PAF1 KO cells, we hypothesized that genes both upstream and downstream of type I interferon (IFN-I) signaling were PAF1-dependent (14). PAF1 KO and non-targeting control (ncgRNA) cells were previously developed in A549 lung epithelial cells, which have been used extensively for the study of the innate immune response (20)(21)(22).…”

“…PAF1 KO and non-targeting control (ncgRNA) cells were previously developed in A549 lung epithelial cells, which have been used extensively for the study of the innate immune response [21–23]. We have shown PAF1 Rescue cells recapitulate PAF1-dependent gene expression [14]. Therefore, we opted to use ncgRNA cells as a universal control that has the potential to enable comparisons across multiple gene knockouts in the future.…”

“…Cells. ncgRNA and PAF1 KO cell lines were generated by CRISPR/Cas9 in A549 lung epithelial cells as previously described (14). All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco ThermoFisher) with 10 % fetal bovine serum (FBS, Gibco ThermoFisher) at 37ºC.…”

“…PAF1C regulation of gene expression impacts the ability of cells to mount an antimicrobial response. Specifically, PAF1 depletion has been shown to disrupt interferon-related signaling and lead to increases in viral replication for both dengue virus and influenza A virus (IAV) (13)(14)(15). Similarly, past studies have identified PAF1 and various complex members to be HIV-1 restriction factors (16)(17)(18).…”