Plasmacytoid dendritic cells (pDCs) are a dendritic cell subset specialized to rapidly secrete copious amounts of Type I Interferon (IFN-I), a group of innate mediators that play key roles in antiviral immune defense and autoimmune diseases. Loss of pDC-derived IFN-I during chronic viral infection enhances susceptibility to secondary infection while excessive pDC IFN-I production contributes to autoimmune pathology, demonstrating the need for further investigation into the mechanisms of IFN- I regulation in pDCs. Through comparing gene expression profiles of pDCs and conventional (c) DCs, we found that Fyn, a member of the src-family kinases, and CD28, a prototypic T cell co-stimulatory receptor, were highly and selectively expressed in pDCs. Fyn acted as a positive regulator of pDC IFN-I and inflammatory cytokine production upon toll-like receptor (TLR) stimulation. In contrast, CD28 acted as a negative regulator of pDC IFN-I production but did not affect production of inflammatory cytokines or maturation. Our data suggests that Fyn and CD28 may play important roles in the regulation of pDC cytokine response during pathogenic challenge. These pathways may have evolved to fine-tune the magnitude of innate responses and to coordinate them with the adaptive immune response. Future studies will determine the mechanisms by which CD28 and Fyn regulate pDC cytokine production, including potential cross-talk with the Phosphoinositide 3-Kinase and TLR signaling pathways

The N 6 -methyladenosine (m 6 A) RNA-modification pathway substantially affects the outcome of viral infection. Studies now show that m 6 A modification of transcripts encoding type I interferons limits the duration of anti-viral signaling.

Transcription of herpesviral late genes is stimulated after the onset of viral DNA replication but otherwise restricted. Late gene expression in gammaherpesviruses requires the coordination of six early viral proteins, termed viral transactivation factors (vTFs). Here, we mapped the organization of this protein complex for Kaposi's sarcoma-associated herpesvirus. Disruption of this complex via point mutation of the interaction interface between the open reading frame 24 (ORF24) and ORF34 vTFs ablated both late gene expression and viral replication.

Methylation at the N6 position of adenosine (m6A) is a highly prevalent and reversible modification within eukaryotic mRNAs that has been linked to many stages of RNA processing and fate. Recent studies suggest that m6A deposition and proteins involved in the m6A pathway play a diverse set of roles in either restricting or modulating the lifecycles of select viruses. Here, we report that m6A levels are significantly increased in cells infected with the oncogenic human DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV). Transcriptome-wide m6A-sequencing of the KSHV-positive renal carcinoma cell line iSLK.219 during lytic reactivation revealed the presence of m6A across multiple kinetic classes of viral transcripts, and a concomitant decrease in m6A levels across much of the host transcriptome. However, we found that depletion of the m6A machinery had differential pro- and anti-viral impacts on viral gene expression depending on the cell-type analyzed. In iSLK.219 and iSLK.BAC16 cells the pathway functioned in a pro-viral manner, as depletion of the m6A writer METTL3 and the reader YTHDF2 significantly impaired virion production. In iSLK.219 cells the defect was linked to their roles in the post-transcriptional accumulation of the major viral lytic transactivator ORF50, which is m6A modified. In contrast, although the ORF50 mRNA was also m6A modified in KSHV infected B cells, ORF50 protein expression was instead increased upon depletion of METTL3, or, to a lesser extent, YTHDF2. These results highlight that the m6A pathway is centrally involved in regulating KSHV gene expression, and underscore how the outcome of this dynamically regulated modification can vary significantly between cell types.

Recognition and elimination of tumor cells by the immune system is crucial for limiting tumor growth. Natural killer (NK) cells become activated when the receptor NKG2D is engaged by ligands that are frequently upregulated in primary tumors and on cancer cell lines. However, the molecular mechanisms driving NKG2D ligand expression on tumor cells are not well defined. Using a forward genetic screen in a tumor-derived human cell line, we identified several novel factors supporting expression of the NKG2D ligand ULBP1. Our results show stepwise contributions of independent pathways working at multiple stages of ULBP1 biogenesis. Deeper investigation of selected hits from the screen showed that the transcription factor ATF4 drives ULBP1 gene expression in cancer cell lines, while the RNA-binding protein RBM4 supports ULBP1 expression by suppressing a novel alternatively spliced isoform of ULBP1 mRNA. These findings offer insight into the stress pathways that alert the immune system to danger.