Determining mechanisms of innate immune modulation by ADP-ribosylation

PROJECT SUMMARY The mammalian antiviral response is largely controlled by post-translational modifications (PTMs), the addition of small proteins, chemicals, lipids, or sugars to proteins to quickly alter their function. Phosphorylation and ubiquitination are well known PTMs that play key roles in the antiviral response. However, research has demonstrated that ADP-ribosylation, the addition of single (mono-ADP-ribose or MAR) or multiple (poly-ADP- ribose or PAR) ADP-ribose units onto proteins, is also critical, as it both negatively and positively regulates key proteins in these pathways. However, the mechanisms by which ADP-ribosylation controls the antiviral response are not fully understood. ADP-ribose is added to proteins by ADP-ribosyltransferases, better known as PARPs. On the other side of the reaction, macrodomains are proteins that both bind and erase MAR on protein substrates. All coronaviruses (CoVs) encode for a macrodomain (Mac1), indicating that CoVs are highly sensitive to MAR. Reverse genetic approaches that remove Mac1 activity from CoVs make for excellent model systems to study how MAR functions in the antiviral response, as Mac1 is the only CoV protein that counters MAR, and prior studies had determined by Mac1 was critical for the ability of CoVs to replicate and cause disease. The investigator’s long-term goal is to determine how ADP-ribosylation inhibits virus replication and enhances the innate immune response. Using Mac1 mutant CoVs and other model systems, the PI’s group has demonstrated that MAR independently represses both CoV transcription and protein production in the absence of Mac1, and that MAR promotes interferon (IFN) production following RNA virus infection. PARP12 and PARP14 were identified as PARPs that restrict Mac1 mutant virus replication, and PARP14 enhances IFN responses. Furthermore, a novel PARP14/PARP1 interaction has been identified and this interaction likely contributes to the ADP-ribose-dependent induction of IFN. However, many of the mechanistic details driving these phenotypes remain unknown. The major questions being addressed are: I) How does MAR restrict CoV transcription (1) and protein accumulation (2) and what host factors mediate this restriction? II) What are the molecular determinants of the PARP1/PARP14 interaction (3), and how do PARP1 and PARP14 together drive the IFN response (4)? These proposed studies will provide more mechanistic data regarding the functions of ADP- ribose during infection. The rationale for this research is that it and will uncover novel cellular proteins or processes that repress virus replication and promote IFN production. The work is innovative because it explores unique cellular targets of ADP-ribosylation that have not previously been implicated in a CoV infection. Finally, these projects are significant and relevant to the NIGMS mission because they will provide a more thorough understanding of how MAR impacts the anti-viral response that could lay the foundation for advances in the treatment of virus infections or other human diseases impacted by MAR.