Replication-Competent Infectious Hepatitis B Virus Vectors Carrying Substantially Sized Transgenes by Redesigned Viral Polymerase Translation
Figure 2
Genomic organization and transcripts of HBV and recombinant HBV vectors.
The parental plasmid pCH-9/3093 is based on a 1.05× HBV genome (thick black line, HBV nucleotides 3093 to 3182-1 plus 3182/1 to 84) in which the CMV promoter replaces the endogenous HBV core promoter (transparent grey arrowhead close to the 3′ end) that drives pgRNA transcription on the authentic, circular DNA genome. Numbers are nt coordinates relative to the core ORF start [75]; the Eco RI site at position 1280 is the reference point in an alternative numbering system [76]. ORFs are indicated by boxes: pC, precore; C, core; pS1/2 and S, preS1, preS2, and S domains of the envelope proteins, respectively; X, HBx; TP, RT, and RH, terminal protein, reverse transcriptase, and RNase H domains of Pol (P); hrGFP, humanized Renilla GFP. RNAs (bottom) are indicated as sinusoidal lines. Authentic pgRNA is about 3.5 kb in length, encodes core protein and Pol, and carries the 5′ proximal encapsidation signal ε which mediates, via Pol binding, specific encapsidation of the pgRNA and initiation of reverse transcription. The subgenomic 2.4 kb (preS1) and 2.1 kb (preS2/S) transcripts for the L, and the M and S envelope proteins and the 0.8 kb HBx RNA are generated from internal promoters located in the Pol ORF. The recombinant genomes are expected to generate size-increased pgRNAs (symbolized by the inserted dashed sinusoidal line) plus unmodified subgenomic RNAs.