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.

Keywords: DHBV; HBV; gene delivery; recombinant virus; viral vector.

Figure 1

Schematic representation of genome organization of HBV and DHBV and major recombinant (D)HBV vector designs. Terminally-redundant wild-type genomes are shown to reflect the circularity and 1.0 copy of each genome is marked out beginning at the start codon of preC(pC)/C ORF. ORFs are represented by boxes and ORFs destroyed in recombinant vectors are depicted using dotted lines. Promoters (Cp, Sp1, Sp2, and Xp) and enhancers (En, EnI, EnII) are represented by arrows pointing in the direction of transcription. Cis elements required for replication and encapsidation are also depicted. ε, encapsidation signal. A(n), polyadenylation signal. DR, direct repeats. (A) Wild-type DHBV genome and recombinant DHBV design: I, cargo gene replaces S ORF in-frame and destroys the overlapping P ORF. (B) wild-type HBV genome and recombinant HBV designs: I, cargo gene is inserted in-frame with P ORF within spacer region, upstream of preS1 ORF. P ORF may or may not be terminated depending on cargo. II, cargo gene is inserted in a deletion in polymerase spacer in-frame with preS1/preS2 ORF, without terminating the overlapping P ORF. II’, derivative of II. The cargo gene replaces the central part of prematurely-terminated C ORF, followed by IRES upstream of P ORF with a maximized deletion in the spacer region that retains polymerase activity but destroys preS1/preS2 ORF. III, the cargo gene replaces central part of C ORF and may or may not be expressly fused to the remaining N-terminal of preC/C, depending on the design. IV, IRES units are introduced to separate de-overlapped C and P ORFs. No viral ORF is obliterated. V, the cargo gene replaces S ORF in-frame and destroys the overlapping P ORF. VI, the cargo gene replaces the central and C-terminal of C ORF, destroying the overlapping P ORF. Arrows indicate insertion sites. Vector designs with obliterated C and/or P ORFs require trans-complementation of the obliterated proteins for genome replication. Vector designs with obliterated envelope ORFs require trans-complementation of envelope proteins for production of progeny virus.