Suppression of Host Antiviral Responses by a SARS-CoV-2 Histone Mimetic

PROJECT SUMMARY/ABSTRACT Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS- CoV-2) and has resulted in more than 1.4 million deaths globally. In contrast to other highly pathogenic influenza viruses, SARS-CoV-2 infection is characterized by low levels of type I interferons and over production of pro- inflammatory cytokines in patients. While concerted efforts have been made to understand the pathogenesis of this virus, how SARS-CoV-2 is able to evade the innate immune response is unclear. The central hypothesis of this proposal is that the envelope (E) protein of the virus interacts with BRD4 in a bromodomain-dependent manner to disrupt the induction of host innate inflammatory and antiviral responses. This hypothesis is supported by the recent proteomic study identifying this interaction and the presence of a histone H3-like motif containing two lysine residues in the E protein. This model is also strongly supported by previous studies demonstrating BRD4 as an important transcriptional coactivator of interferon and inflammatory genes during viral infection. The central hypothesis will be tested in two specific aims: 1) To define the role of bromodomains in the interaction between SARS-CoV-2 E protein and BRD4. The working hypothesis is that the lysine residues of the E protein are acetylated and mediate interaction with the bromodomains of BRD4, acting as a histone mimetic. I will test this model with label-free mass spectrometry to identify acetylated residues in E protein and immunoprecipitation assays with domain constructs to characterize the interaction. 2) To determine the functional relevance of the SARS-CoV-2 E protein and BRD4 interaction on the viral replication. The working hypothesis is that the E protein acts as a histone mimetic to sequester BRD4 from chromatin thereby disrupting the transcription of pro- inflammatory and interferon genes. I will use CRISPR/Cas9-mediated BRD4 knockout cell lines and BRD4 reconstitution studies in infected cells to test this model. My analysis will also focus on the transcriptional activation of canonical pro-inflammatory and interferon genes along with the chromatin occupancy of BRD4 in the presence of the E protein through RT-qPCR and ChIP-qPCR studies. I expect my proposed studies to inform our fundamental understanding of coronavirus pathogenesis and provide novel therapeutic targets to combat SARS-CoV-2 infection and immune evasion.