Truncated Active Human Matrix Metalloproteinase-8 Delivered by a Chimeric Adenovirus-Hepatitis B Virus Vector Ameliorates Rat Liver Cirrhosis
Figure 2
Trans-complementation rescues HBV vectors lacking endogenous gene products into infectious virions with enhanced transgene expression.
(A) Rescue of replication in intracellular nucleocapsids by helper plasmid pCH3142. HepG2 cells were cotransfected with equal amounts of pCH3142 and the indicated pCH-S or pCH-M5 plasmids. Transfection with the wild-type HBV expression plasmid pCH-9/3091 served as reference. DNA from cytoplasmic nucleocapsids was analyzed by Southern blotting using a 32P-labeled HBV-specific probe. The positions of the major replicative intermediates, i.e. relaxed circular (RC) DNA, double-stranded linear DNA (DS) and single-stranded DNA (SS) are indicated. (B) and (C) Enhanced transgene expression by HBV vectors devoid of endogenous gene products. HepG2 cells were transfected with either the first generation pCH-S-GFP plasmid, or the new pCH-M5-GFP vector and GFP expression at 48 h post transfection was monitored by fluorescence microscopy (B). Alternatively, HepG2 cells were cotransfected with the Renilla luciferase encoding plasmids pCH-S-Luc or pCH-M5-Luc plus a plasmid encoding firefly luciferase (pGL3-control). At 48 h post transfection, Renilla luciferase activity from the HBV vector was then normalized to firefly luciferase activity in the same cells (C). (D) Rescue of infectious HBV vector particle formation by wild-type HBV. HepG2 cells were cotransfected with the vector pCH-M5-GFP and, instead of the helper plasmid pCH3142 as in (A), with the wild-type HBV expression plasmid pCH-9/3091. Viral particles from the supernatant were then tested for infectivity on differentiated HepaRG cells, using a nominal moi of 5,000 vge/cell. GFP expression indicating the presence of infectious vector particles was assessed by fluorescence microscopy 6 days post infection (panel “Fluorescence”). An overlay with the brightfield image of the same field is shown in panel “Merged”.