Thursday, November 21, 2024
11/21/2024
PROJECT SUMMARY Coronaviruses (CoVs) are a large class of positive-strand RNA viruses that are capable of causing severe human disease and death, as is exemplified by the pandemic outbreak of SARS-CoV-2. The innate immune response to coronavirus infection includes a battle between poly-ADP-ribose polymerases (PARPs) and the coronavirus macrodomain (Mac1), which add and remove ADP-ribose from proteins, respectively. In the absence of Mac1 enzyme activity, CoVs replicate poorly in the face of the innate immune response and cause little to no disease in several animal models of infection, including SARS-CoV-2. These results demonstrate the power of PARP-mediated ADP-ribosylation to limit CoV-induced disease and support our central hypothesis that PARPs target host and viral proteins, and that the post-translational modification of these targets (MARylation) induces an antiviral state that limits virus replication. Despite the clear importance of PARP enzymes in driving the outcome of a CoV infection, a large gap in knowledge remains as to exactly how this battle plays out during CoV infections, most notably i) what PARPs are heavily involved in this battle; and ii) what cellular or viral proteins are ADP-ribosylated during infection. Functional redundancy, similar NAD+ binding sites, low protein abundance, and viral enzymes that reverse their effects have made it challenging to identify direct targets of individual PARPs during coronavirus infection. The objective of this proposal is to identify specific ADP-ribosylated targets of PARPs during a SARS-CoV-2 infection that will uncover novel mechanisms of virus restriction. This objective will be resolved with the following specific aims: 1) Identify PARPs proteins that are expressed during and impact SARS-CoV-2 infection, and 2) Identify viral and cellular PARP targets using chemical genetics and proximity labeling. This work is innovative because we will apply, for the first time, NAD+-based chemical proteomics, chemical genetics, and BioID proximity labeling to uncover the specific targets of of MARylating PARPs that impact SARS-CoV-2 infection. Furthermore, we have a unique tool, a virus that lacks the ability to counter PARP activity, to aid in our identification of physiologically relevant PARP target proteins. Our rationale is that identifying the targets of individual PARPs during SARS-CoV-2 infection will define novel mechanisms of virus restriction that will dramatically expand the landscape of ADP-ribosylation and how it can impact virus replication. Together, with our combined expertise in chemistry, PARP/ADP-ribose, and CoV biology, we are poised to address these challenges and make seminal discoveries describing novel targets of PARP-mediated ADP-ribosylation and how they can drive antiviral innate immune responses during SARS-CoV-2 infection. We anticipate identifying dozens of ADP-ribosylated proteins during infection, which will alter the landscape of how PTMs, outside of phosphorylation or ubiquitination, can impact the outcomes of virus infections, and will provide new avenues for antiviral therapy.
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