Developmental Regulation of Hepatitis B Virus Biosynthesis by Hepatocyte Nuclear Factor 4a Lie Li 1 , Claudia E. Oropeza 1 , Bruno Sainz, Jr. 2 , Susan L. Uprichard 1,2 , Frank J. Gonzalez 3 , Alan McLachlan 1 * 1 Department of Microbiology and Immunology College of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America, 2 Department of Medicine, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America, 3 Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America Abstract The host cellular factors that promote persistent viral infections in vivo are, in general, poorly understood. Utilizing the hepatitis B virus (HBV) transgenic mouse model of chronic infection, we demonstrate that the nuclear receptor, hepatocyte nuclear factor 4a (HNF4a, NR2A1), is essential for viral biosynthesis in the liver. The dependency of HBV transcription on HNF4a links viral biosynthesis and persistence to a developmentally regulated transcription factor essential for host viability. Citation: Li L, Oropeza CE, Sainz B Jr., Uprichard SL, Gonzalez FJ, et al. (2009) Developmental Regulation of Hepatitis B Virus Biosynthesis by Hepatocyte Nuclear Factor 4a. PLoS ONE 4(5): e5489. doi:10.1371/journal.pone.0005489 Editor: Donald E. Ganem, University of California San Francisco, United States of America Received March 24, 2009; Accepted April 15, 2009; Published May 8, 2009 Copyright: ß 2009 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Public Health Service grants AI30070 from the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mclach@uic.edu Introduction Persistent viral infections require the coexistence of pathogen and host for extended periods of time without the host resolving the infection or the virus killing the host. Human immunodefi- ciency virus, herpes simplex viruses, papillomaviruses and hepatitis B and C virus have evolved a variety of strategies to persistently infect man [1–4]. HBV chronically infects approximately 400 million people worldwide resulting in about a million deaths per year from liver cirrhosis and hepatocellular carcinoma [5,6]. Most HBV infections worldwide occur in neonates at or around the time of birth and usually result in persistent infections [6]. Although the immunological immaturity of the neonate is presumed to contribute to these chronic HBV infections, a precise understand- ing of the molecular events governing HBV persistence during development is lacking. HBV replicates its genomic DNA by reverse transcription of a pregenomic 3.5kb RNA which is transcribed from covalently closed circular 3.2kb viral genomic DNA in the nucleus of infected hepatocytes [7]. Thus transcriptional regulation plays a central role in controlling viral replication levels [8] and represents a potential antiviral target which has not, to date, been exploited clinically. In cell culture, the binding of hepatocyte nuclear factor 4a (HNF4a) or retinoid X receptor a (RXRa) plus peroxisome proliferator-activated receptor a (PPARa) to the nucleocapsid promoter regulatory elements governs the level of synthesis of this critical HBV pregenomic 3.5kb RNA template [9]. In this study, we demonstrate that the level of HBV transcription and replication throughout early postnatal develop- ment correlates with the level of liver HNF4a expression in the HBV transgenic mouse model of chronic HBV infection. The conditional depletion of HNF4a in the liver results in the loss of HBV transcription and replication indicating that this nuclear receptor is a major determinant of viral biosynthesis in vivo. These observations indicate that viral transcription, biosynthesis and antigen expression will increase progressively after infection at birth, possibly contributing to persistent infection. Additionally, the essential nature of the HNF4a transcription factor for host viability [10–12] may limit the hosts’ ability to resolve infection and increase the probability of viral persistence. Results Characterization of the conditional liver-specific HNF4a deficient HBV transgenic mouse during early postnatal development Using the HBV transgenic mouse (lineage 1.3.32) model of chronic infection [13], we previously demonstrated that PPARa did not affect the level of viral biosynthesis under normal physiological conditions but did mediate enhanced viral transcrip- tion and replication in response to peroxisome proliferators [14]. To determine the role of the transcription factor HNF4a in HBV biosynthesis, we bred HBV transgenic mice with mice carrying a floxed HNF4a (HNF4a fl/fl ) allele [10] and albumin Cre recombi- nase (lineage B6.Cg-Tg(Alb-cre)21Mgn/J, Jackson Laboratory) transgene (AlbCre) [15] to generate HBVAlbCreHNF4a fl/fl transgenic mice. The presence of the AlbCre transgene in the HNF4a fl/fl mice results in the postnatal liver-specific loss of exons 4 and 5 of the HNF4a gene [10]. From the initial crosses generating HBVAlbCreHNF4a fl/fl transgenic mice, it was apparent that some of the pups were not growing at the same rate as their littermates (Figure 1A and B). Indeed, these pups were approximately 50% the weight of their littermates between age 1 and 2 weeks (Figure 1B) and failed to survive past 16 days. Genotyping of these mice demonstrated that all the pups with reduced growth were Cre positive (sCre(+); small, PLoS ONE | www.plosone.org 1 May 2009 | Volume 4 | Issue 5 | e5489