Abstract Hepatitis B virus (HBV) and hepatitis C virus (HCV) have both distinct and shared pathways to mediate development of virus-associated hepatocellular carcinoma (HCC). It has been postulated that viral encoded proteins such as HCV polyprotein or HBV X protein (HBx) induce epigenetic changes that can lead to HCC during chronic infection. 5-Hydroxymethylcytosine (5hmC) is a recently identified cytosine variant and an intermediate in enzyme-catalyzed active DNA demethylation. 5hmC is also considered a stable epigenetic mark during embryo development and tumorigenesis. Herein, we developed a liquid chromatography-mass spectrometry (LC-MS) based approach to assess global genomic 5hmC levels in tissues using several transgenic (Tg) mouse models to determine epigenetic changes induced by HBV and HCV viral proteins and role in pathogenesis of HCC. A 2-to 3-fold reduction in genomic 5hmC was observed in liver samples from both Mdr2-knockout (Mdr2-KO) mice (a model of chronic inflammation-mediated HCC) and Mdr2-KO/HCV-Tg mice (a model carrying Mdr2-KO and HCV polyprotein). Interestingly, a similar fold reduction in 5hmC was detected in (Mdr2+/-)/HCV-Tg mice, which express HCV polyprotein, but do not develop HCC. In parallel, 5hmC levels were assessed in HBx-Tg mice with high-frequency of HCC. However, reduction of 5hmC was not detectable in liver samples from HBx-Tg mice as late as 14 months of age. These results suggest that HCV polyprotein may accelerate inflammation-associated hepato-carcinogenesis in the Mdr2-KO/HCV-Tg mice. Therefore, we speculate that the HCV proteins may directly induce deconstruction of genomic 5hmC to promote HCC development. In contrast, our results in HBx-Tg mice suggest that HBx may employ a different epigenetic mechanism on 5hmC from that of HCV polyprotein. Notably, a significant change in genomic 5hmC was observed early at the pre-cancerous stages of HCC development in Mdr2-KO mice. This finding implies that, during chronic inflammation, a precipitous drop in 5hmC emerges prior to tumor formation. Therefore, 5hmC may provide an important diagnostic marker for early detection of HCC in persons with HCV chronic infection. Citation Format: Yong Jiang, Ting Nie, James Kohler, Leda Bassit, Daniel Goldenberg, Mark Feitelson, Raymond F. Schinazi. Epigenetic effects of viral proteins on global levels of 5-hydroxymethylcytosine in HBV- and HCV-associated hepatocellular carcinoma in mice. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B38.