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 part 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 affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find 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 the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the 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 signalling.
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We thank members of the Weitzman laboratory for insightful discussions and input, especially R. Dilley and B. Simpson for generating reagents. We also thank R. Panetierri and C. Koziol-White for providing precision-cut lung slices. We are grateful to D. Curiel for sharing recombinant protein-VII–GFP vectors and L. Gerace for anti-protein-VII antibodies. We thank the Penn Vector Core for assistance in purifying recombinant vectors, the Penn CDB Microscopy Core for imaging and FRAP assistance, and the CHOP Pathology core for immunostaining of mouse lungs. We thank members of the Black, Garcia and Worthen laboratories for technical help. We thank C. Bassing, I. Brodsky, J. Henao-Mejia, R. Kohli, C. Lopez, A. Resnick, S. Shin, K. Spindler and J. Weitzman for advice and critical reading of the manuscript. D.C.A. was supported in part by T32 CA115299 and F32 GM112414. N.J.P. was supported in part by T32 NS007180. N.S. was supported in part by funding from the American Cancer Society. Research was supported by grants from the National Institutes of Health (CA097093 to M.D.W., AI102577 and CA122677 to P.H., AI118891 and GM110174 to B.A.G., and GM082989 to B.E.B.), the Institute for Immunology of the University of Pennsylvania, and funds from the Children’s Hospital of Philadelphia (M.D.W.).
Extended data figures
Summary of post-translational modifications found on histones upon expression of protein VII.
Total list of proteins significantly changed upon protein VII expression identified by mass spectrometry analysis of high salt fractions. Table includes the log2 fold change of MaxQuant-derived iBAQ values obtained for protein VII-HA induced and uninduced highest salt fractions (600mM). Proteins with homoscedastic two-tailed t-test p-value smaller than 0.05 were considered as significantly altered between the two tested conditions. N/A defines not assigned t-test p-values; this is either due to the presence of the protein in only one condition, or if in one condition the protein was quantified in only one replicate. The number of peptides used for quantification was also highlighted. Commonly occurring contaminants, such as human keratins or trypsin, were removed from the final list.
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International Immunopharmacology (2018)