Zhigang Chen

APOBEC3 proteins (A3s) play an important role in host innate immunity against viruses and DNA mutations in cancer. A3s-induced mutations in both viral and human DNA genomes vary significantly from non-lethal mutations in viruses to localized hypermutations, such as kataegis in cancer. How A3s are regulated remains largely unknown. Since A3s exist in complexes and belong to the same family as APOBEC-1 (A1), which requires cofactors to be functional, we investigated the role of A1 cofactors and other A3 potentially associated hnRNPs on A3 mutational activity using hepatitis B virus (HBV) cellular replication as a model. We found that A1-associated cofactors and other hnRNPs were involved in A3 mutational activity regulation, and their regulatory effect was dependent on the strength of A3 association with hnRNPs with an order of A3C > 3G>3B and A1 cofactors > other hnRNPs. A1 cofactors had a strong protein interaction with A3 and significantly increased A3 mutational activity by co-expression. Endogenous gene expression knockdown by siRNA had the opposite decrease effect. Disruption of the protein interactions between A3 and hnRNPs through A3G and A3B mutagenesis decreased A3 mutational activity significantly, even to a level of near total loss, indicating that A1 cofactors and hnRNPs are required for A3 mutational activity. HBV genome-wide mutation analyses showed that A1 cofactors significantly increased A3C accessibility to HBV (−)DNA and A3C-induced mutational efficiency to generate kataegis-like hypermutation. These data demonstrate that A1 cofactors and hnRNPs are closely associated with A3s and may play an important regulatory role under physiological conditions. IMPORTANCE: As human host restriction factors, A3s play an important role against viral infections. A3s are also major mutagenic drivers of cancer. However, why A3-induced mutations vary significantly from non-lethal mutations in virus to localized hypermutations in cancer remains unknown. We found that A1 cofactor and other hnRNPs are not only associated with A3 complexes but also play important regulatory roles in A3-induced mutation activities. A1 cofactors like GRY-RBP significantly increased A3 accessibility and mutational efficiency to its single-strand DNA substrate during HBV reverse transcription to generate hypermutations. Disruption of the A3 protein association with hnRNPs by A3 mutagenesis diminished A3 mutational activity. This finding not only reveals a regulatory mechanism for A3-induced mutation but also indicates that A3-associated cellular factors can be a potential target for regulating A3-induced mutation for cancer therapeutics.

The class B scavenger receptor CD36 is known to bind and mediate the transport of lipid-related ligands and it functions as a pattern recognition receptor (PRR) for a variety of pathogens, including bacteria and viruses. In this study, we assessed CD36’s role as a PRR mediating pro-inflammatory effects of several known Danger-Associated Molecular Patterns (DAMPs) used either as a single preparation or as a combination of DAMPs in the form of total cell/skeletal muscle tissue lysates. Our data demonstrated that multiple DAMPs, including HMGB1, HSPs, histone H3, SAA, and oxPAPC, as well as cell/tissue lysate preparations, induced substantially higher (~7–10-fold) IL-8 cytokine responses in HEK293 cells overexpressing CD36 compared to control WT cells. At the same time, DAMP-induced secretion of IL-6 in bone marrow-derived macrophages (BMDM) from CD36−/− mice was markedly (~2–3 times) reduced, as compared to macrophages from normal mice. Synthetic amphipathic helical peptides (SAHPs), known CD36 ligands, efficiently blocked CD36-dependent inflammatory responses induced by both cell and tissue lysates, HMGB1 and histone H3 in CD36+ cells. IP injection of total cellular lysate preparation induced inflammatory responses that were assessed by the expression of liver and lung pro-inflammatory markers, including IL-6, TNF-α, CD68, and CXCL1, and was reduced by ~50% in CD36-deficient mice compared to normal mice. Our findings demonstrate that CD36 is a PRR contributing to the innate immune response via mediating DAMP-induced inflammatory signaling and highlight the importance of this receptor as a potential therapeutic target in DAMP-associated inflammatory conditions.