Engineering Hepadnaviruses as Reporter-Expressing Vectors: Recent Progress and Future Perspectives

Abstract

The Hepadnaviridae family of small, enveloped DNA viruses are characterized by a strict host range and hepatocyte tropism. The prototype hepatitis B virus (HBV) is a major human pathogen and constitutes a public health problem, especially in high-incidence areas. Reporter-expressing recombinant viruses are powerful tools in both studies of basic virology and development of antiviral therapeutics. In addition, the highly restricted tropism of HBV for human hepatocytes makes it an ideal tool for hepatocyte-targeting in vivo applications such as liver-specific gene delivery. However, compact genome organization and complex replication mechanisms of hepadnaviruses have made it difficult to engineer replication-competent recombinant viruses that express biologically-relevant cargo genes. This review analyzes difficulties associated with recombinant hepadnavirus vector development, summarizes and compares the progress made in this field both historically and recently, and discusses future perspectives regarding both vector design and application.

A single hepatitis B virus genome with a reporter allows the entire viral life cycle to be monitored in primary human hepatocytes

“…Many studies have attempted to create reporter‐expressing recombinant HBV because such viruses are powerful tools for basic virology research and the development of antiviral medicines. [ 6 ] Replication and packaging of the HBV genome are highly restricted by its size. Although 399–720 bp can be inserted into the HBV genome while retaining replication and packaging, replication capacity and infectivity are severely impaired by such insertions.…”

“…Although various methods have been developed to produce recombinant HBV, attempts to create replication‐ and infection‐competent recombinant HBV have been unsuccessful. [ 6 ]…”

“…Although various methods have been developed to produce recombinant HBV, attempts to create replicationand infection-competent recombinant HBV have been unsuccessful. [6] To overcome the difficulties associated with inserting a foreign gene into the HBV genome without severely affecting its life cycle, in the present study we applied nano-luciferase (NanoLuc) binary technology (NanoBiT). [7] NanoBiT is a split reporter consisting of two subunits: high-affinity NanoBiT (HiBiT) and large NanoBiT.…”

DNA Engineering and Hepatitis B Virus Replication

“…Insertion of differently sized exogenous genes, including HIV Tat, ZeoR, fluorescent proteins (GFP, EGFP, hrGFP, RFP, DsRed), Renilla luciferase (Rluc), Guassia luciferase (Gluc), NanoLuc luciferase (Nluc), and blasticidin S deaminase (BSD), into HBV genome has allowed for the construction of associated recombinant viruses ( Kimura et al, 1994 ; Chaisomchit et al, 1997 ; Protzer et al, 1999 ; Yoo et al, 2002 ; Untergasser and Protzer, 2004 ; Liu et al, 2009 , 2013 ; Hong et al, 2013 ; Nishitsuji et al, 2015 , 2018 ). However, some of these efforts were unsuccessful ( Bai et al, 2016 ), and no systematic analysis of HBV’s engineering strategy has been done.…”