Inhibition of hepatitis B virus gene expression and replication by endoribonuclease-prepared siRNA

Abstract

Endoribonuclease-prepared siRNA (esiRNA) is an alternative tool to chemical synthetic siRNA for gene silencing. Since esiRNAs are directed against long target sequences, the genetic variations in the target sequences will have little influence on their effectiveness. The ability of esiRNAs to inhibit hepatitis B virus (HBV) gene expression and replication was tested. EsiRNAs targeting the coding region of HBV surface antigen (HBsAg) and the nucleocapsid (HBcAg) inhibited specifically the expression of HBsAg and HBcAg when cotransfected with the respective expression plasmids. Both esiRNAs reduced the HBV transcripts and replication intermediates in transient transfected cells and cells with HBV genomes integrated stably. Compared with synthetic siRNA, esiRNA targeting HBsAg was less effective than the selected synthetic siRNA in terms of the inhibition of HBV gene expression and replication. However, while the ability of synthetic siRNAs for specific gene silencing was impaired strongly by the nucleotide substitutions within the target sequences. The efficiency of gene silencing by esiRNAs was not influenced by sequence variation. The transfection of esiRNA did not induce interferon-stimulated genes (ISGs) like STAT1 and ISG15, indicating the absence of off-target effects. In general, esiRNAs strongly inhibited HBV gene expression and replication and may have an advantage against HBV strains which are variable genetically.

Regulation of the blood-testis barrier by coxsackievirus and adenovirus receptor

“…2B, a and b). Furthermore, the expression of two interferon-stimulated genes (ISG), such as oligoadenylate synthetase 1 (OAS1) and signal transducer and activator of transcription 1 (STAT1) that were known to be upregulated due to off-target effects when cells were transfected by RNAi vectors for gene knockdown (1,34,43), were found to be unaffected with S16 served as a loading control in this semi-quantitative RT-PCR experiment (Fig. 3, A and B).…”

Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells

“…5B) without any apparent off-target effects since two interferon stimulated genes (ISG), including oligoadenylate synthetase 1 (OAS1) and signal transducer and activator of transcription 1 (STAT1), remained relatively steady (Fig. 5A), which were known to be up-regulated non-specifically when mammalian cells were transfected by siRNA duplexes with off-target effects5253. Interestingly, CRB3 KD led to a significant down-regulation of formin 1 (Fig.…”

Role of Toll-like receptor 2 in the immune response against hepadnaviral infection

“…The PI3k/Akt pathway inhibitor rapamycin was purchased from LC Laboratories (Woburn, MA). The siRNAs targeting HBV S and X region (Supplementary Table 1) [25], the validated siRNAs targeting TLR signaling adaptor proteins (IRAK1, IRAK4, TAK1, and TRAF6) (Supplementary Table 2) and an unrelated control siRNA (neg.siRNA) were purchased from Qiagen (Hilden, Germany).…”