FIG. 7.

Overexpression of the antisense NESI RNA blocked the nuclear export of HDV genomic RNA and assembly of the viral RNA into virus-like particles mediated by HDAg-L. HepG2 cells were cotransfected with plasmid pSVD2 expressing a dimeric HDV RNA and plasmids encoding the small form of HBsAg (pECE-C-ES) and HDAg-L (pECE-d-BE) in the presence of the control plasmid pcDNA3.1 (Control) or the effector plasmid pcDNA3.1-AS-NESI (NESI antisense) that expresses an antisense RNA of NESI. Four days posttransfection, both RNA and protein lysates were prepared from the transfected cells and from the viral pellets collected from theculture media. A DIG-labeled trimeric HDV antigenomic RNA transcribed in vitro from plasmid pD3 was used as a probe to perform Northern blot analysis (A). Protein G-purified rabbit antiserum specific for HDAgs and goat polyclonal antibodies specific to HBsAg were used to perform Western blot analysis as indicated (B). Following a partition, RNAs were isolated from both nuclear and cytoplasmic fractions of the transfected HepG2 cells and subjected to RT-PCR analysis to detect HDV genomic RNA (C, upper panel) and β-actin mRNA (C, lower panel). M, 100-bp DNA ladder. N, nuclear fraction; C, cytoplasmic fraction.