Site‐Specific Engineering of Chemical Functionalities on the Surface of Live Hepatitis D Virus

Abstract

The genetic code expansion strategy, the recently emerged pyrrolysine (Pyl)-based system in particular, has become a generally applicable method for site-specific incorporation of unnatural amino acids (UAAs) into a protein of interest in bacteria, yeast, mammalian cells, and even in animals. However, this technique has yet to be applied to intact and live viruses, which is largely due to the fragile nature as well as the complicated assembly process of many human viruses. To address this challenge, we here coupled the genetic-code expansion strategy with an engineered virus assembly process in human hepatocytes to site-specifically introduce unnatural chemical groups onto virus surface proteins by using hepatitis D virus (HDV) as a model system. HDV has infectedmore than 15 million people worldwide, and currently there are no drugs clinically available against this virus. HDV is a satellite virus of human hepatitis B virus (HBV), which has infected two billion people and among them about 240 million are currently chronically infected. Both HBV and HDV share the same envelope proteins for infection of hepatocytes. Study of HBV and HDV infection has long been hampered by the lack of efficient and easily accessible in vitro infection system. Recently, a bile acid transporter predominantly expressed in liver, sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for HDV and HBV. The NTCP complemented human hepatoma cell line HepG2 provided a feasible in vitro infection system for studying HBV and HDV infection. However, the lack of methods to selectively label, monitor, and/or manipulate an intact virus under living conditions still restricts investigations into molecular details of the infection. Many problems are due to the distinct topological features of the critical viral proteins, as well as complex virus assembly processes. For example, HDV has developed a tightly regulated assembly process to produce infectious viral particles in human hepatocytes: the HDV RNAs were first encapsulated with delta antigens and then packaged with three HBV envelope proteins, namely large (L), middle (M), and small (S) proteins, to produce the intact viral particle before being secreted to the extracellular space (Supporting Information, Figure S1). The resulting HDV, with a diameter of 36 nm, is one of the smallest animal viruses known to date. It is therefore exceedingly difficult to chemically label this tiny virus with delicate structures under living conditions. Furthermore, the virus surface envelope proteins contain many chemically active amino acids (for example, cysteine and lysine) that are essential for virus entry in host cells. Conjugation or modification of these natural residues will severely compromise viral infectivity. A noninvasive strategy for manipulation of living viral particles without impairment of their viability and infectivity is thus highly desired. Bioorthogonal reactions have revolutionized our ability to label and manipulate various biomolecules and even whole cells and organisms under living conditions. As a critical step for applying such chemistry for virus labeling, several approaches have been reported for installation of bioorthogonal handles, typically in the form of UAAs into proteins from sub-viral-like particle (SVP) or intact virus. For instance, site-specific or residue-specific incorporation of UAAs bearing an azide or an alkyne moiety into SVP has been demonstrated in bacterial cells by several laboratories. These methods allow the conjugation of SVPs with various fluorescent dyes or therapeutic reagents for biomedical or biomaterial applications. However, SVPs are non-infectious and not suitable for investigating virus infection mechanisms. Indeed, SVPs produced from prokaryotic cells lack posttranslational modifications, particularly on their surface envelope proteins, and therefore differ from the native SVPs. Although attempts have been made to extend some of these methods for virus production in mammalian cells, such strategies typically require the metabolic replacement of a specific type of amino acid permissive only to simple groups (for example, azide and ketone) from the entire virus proteome, which may disrupt the virion assembly process or permute the vulnerable virion structure, resulting in compromised viral infectivity. Taken together, a general approach for precise labeling and manipulation of intact [*] S.-X. Lin, M.-Y. Yang, Prof. Dr. P. R. Chen Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center and Peking-Tsinghua Center for Life Sciences Peking University, Beijing 100871 (China) E-mail: pengchen@pku.edu.cn H. Yan Graduate program in School of Life Sciences Peking University, Beijing (China) H. Yan, L. Li, B. Peng, S. Chen, Prof. Dr. W.-H. Li National Institute of Biological Sciences, Beijing (China) E-mail: liwenhui@nibs.ac.cn [] These authors contributed equally to this work.