A core viral protein binds host nucleosomes to sequester immune danger signals

Abstract

Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses1. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important role in innate immune responses2. Viral encoded core basic proteins compact viral genomes but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones3. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles4,5, it is unknown whether protein VII impacts cellular chromatin. Our observation that protein VII alters cellular chromatin led us to hypothesize that this impacts antiviral responses during adenovirus infection. We found that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in chromatin of members of the high-mobility group protein B family (HMGB1, HMGB2, and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses6,7. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.

Differential Salt Fractionation of Nuclei to Analyze Chromatin-associated Proteins from Cultured Mammalian Cells

“…In addition, proteomics can be performed to identify changes to global chromatin composition under different conditions. Further details concerning mass spectrometry of salt fractions can be found in (Avgousti et al, 2016). …”

“…We are specifically interested in how virus infection alters the composition of factors associated with the cellular chromatin. Nuclear replicating viruses, such as adenovirus, herpes simplex virus, and Epstein-Barr virus, dramatically alter the appearance of the host chromatin during infection (Avgousti et al , 2016; Lam et al ., 2010; Simpson-Holley et al , 2005; Chiu et al ., 2013). We hypothesized that these changes in appearance are partly due to differences in protein composition of host chromatin.…”

“…In this protocol we isolate nuclei, digest the DNA down to mono-nucleosome length, and then wash the nuclei with increasing concentrations of salt, collecting each fraction for analysis by Western blotting. We recently used this protocol to elucidate changes to cellular chromatin during infection with adenovirus (Avgousti et al , 2016). We now present this protocol as a general approach to monitor association of proteins with chromatin under a wide range of perturbing conditions.…”

Time-resolved Global and Chromatin Proteomics during Herpes Simplex Virus Type 1 (HSV-1) Infection

“…Western blot (WB) analysis was carried out using standard methods. Salt fractionation of nuclei and preparation of salt fractions for nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) analysis was performed as described previously (25). nLC-MS/MS and data analysis settings were the same as the proteomic analysis described in the next paragraph.…”

Adenovirus sensing by the immune system

“…Additionally, Ad protein VII in the nucleus binds and sequesters high-mobility-group B protein 1 (HMGB1) that functions as an immune danger signal if released from the cell. However, the mechanism of sensing the Ad infection and release of HMBG1 is unknown [33]. …”