Application of allele-specific RNAi in hepatitis B virus lamivudine resistance

[Guest guest]

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2893.2011.01483.x/abstract

Application of allele-specific RNAi in hepatitis B virus lamivudine resistance

X. Teng1,2,3,†, J.-Y. Liu4,†, D. Li1,2,3,†, Y. Fang1,2,3, X.-Y. Wang5,

Y.-X. Ma1,2,3, S.-J. Chen1,2,3, Y.-X. Zhao1,2,3, W.-Z. Xu1,2,3, H.-X. Gu1,2,3

Article first published online: 7 JUL 2011

DOI: 10.1111/j.1365-2893.2011.01483.x

© 2011 Blackwell Publishing Ltd

Issue

Journal of Viral Hepatitis

Early View (Online Version of Record published before inclusion in an issue)

Summary.  Understanding the consequences of mutation in the

tyrosine–methionine–aspartate–aspartate (YMDD) motif of hepatitis B virus

(HBV) genome on replication is critical for treating chronic hepatitis B with

lamivudine. Allele-specific gene silencing by RNAi (allele-specific RNAi:

ASP-RNAi) is an advanced application of RNAi techniques. Use of this strategy as

a means for specifically inhibiting an allele expression of interest suggested

that it can specifically suppress the expression of alleles causing disease

without inhibiting the expression of corresponding wild-type alleles. However,

no studies have used ASP-RNAi to address the issue of HBV lamivudine resistance.

In this study, we applied ASP-RNAi into two long-term eukaryotic cell lines of

full-length HBV containing either lamivudine-resistant mutants (HBV-YIDD) or

wild type (HBV–WT) which we generated in previously. The designed siRNAs were

also used in this eukaryotic expression system together with lamivudine. ELISA

and real-time PCR were performed to monitor virus-specific protein synthesis and

viral DNA replication. The results showed that the base substitutions conferring

marked ASP-RNAi appeared to be largely present in positions 1, 3, 6, 11, 12, 15

and 19 of the sense strand of siRNAs which were different from the most

sensitive positions of this application in eukaryotes. In addition,

siRNA–lamivudine combinations did not possess the prominent anti-HBV activity

we expected because of some unknown mechanisms. These findings recapitulated

many of the features of ASP-RNAi in hepadnaviruses which provided a new insight

into the development of a potent strategy against HBV drug resistance.

[Guest guest]

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2893.2011.01483.x/abstract

Application of allele-specific RNAi in hepatitis B virus lamivudine resistance

X. Teng1,2,3,†, J.-Y. Liu4,†, D. Li1,2,3,†, Y. Fang1,2,3, X.-Y. Wang5,

Y.-X. Ma1,2,3, S.-J. Chen1,2,3, Y.-X. Zhao1,2,3, W.-Z. Xu1,2,3, H.-X. Gu1,2,3

Article first published online: 7 JUL 2011

DOI: 10.1111/j.1365-2893.2011.01483.x

© 2011 Blackwell Publishing Ltd

Issue

Journal of Viral Hepatitis

Early View (Online Version of Record published before inclusion in an issue)

Summary.  Understanding the consequences of mutation in the

tyrosine–methionine–aspartate–aspartate (YMDD) motif of hepatitis B virus

(HBV) genome on replication is critical for treating chronic hepatitis B with

lamivudine. Allele-specific gene silencing by RNAi (allele-specific RNAi:

ASP-RNAi) is an advanced application of RNAi techniques. Use of this strategy as

a means for specifically inhibiting an allele expression of interest suggested

that it can specifically suppress the expression of alleles causing disease

without inhibiting the expression of corresponding wild-type alleles. However,

no studies have used ASP-RNAi to address the issue of HBV lamivudine resistance.

In this study, we applied ASP-RNAi into two long-term eukaryotic cell lines of

full-length HBV containing either lamivudine-resistant mutants (HBV-YIDD) or

wild type (HBV–WT) which we generated in previously. The designed siRNAs were

also used in this eukaryotic expression system together with lamivudine. ELISA

and real-time PCR were performed to monitor virus-specific protein synthesis and

viral DNA replication. The results showed that the base substitutions conferring

marked ASP-RNAi appeared to be largely present in positions 1, 3, 6, 11, 12, 15

and 19 of the sense strand of siRNAs which were different from the most

sensitive positions of this application in eukaryotes. In addition,

siRNA–lamivudine combinations did not possess the prominent anti-HBV activity

we expected because of some unknown mechanisms. These findings recapitulated

many of the features of ASP-RNAi in hepadnaviruses which provided a new insight

into the development of a potent strategy against HBV drug resistance.