Antisense RNA complementary to hepatitis B virus specifically inhibits viral replication  Jasper Zu Putlitz*, Stefan Wieland‡, Hubert E. Blum‡, Jack R. Wands*  Gastroenterology  Volume 115, Issue 3, Pages 702-713 (September 1998) DOI: 10.1016/S0016-5085(98)70150-7 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Map of the HBV genome and location of antisense and sense RNAs used in the study. HBV has a 3.2-kb partially double-stranded DNA genome (central circle) from which four major classes of transcripts (circular, thin arrows) are synthesized. The 3.5-kb RNA contains coding regions (wide circular arrows) for the nucleocapsid (C) and the reverse transcriptase (pol). A subclass of this transcript with a slightly longer 5' end codes for the precore (pre-C) protein that, after processing, is secreted as HBeAg. The 2.4-kb RNA encompasses the preS1 open reading frame, which encodes for the large surface protein. The 2.1-kb RNA encompasses the preS2 and S open reading frames, which encode for the middle and small surface proteins, respectively. The smallest transcript (≈0.8 kb) codes for the X protein. The outermost arrows represent antisense RNAs AS 1, 2, 3, 4, and 5 (counterclockwise) as well as their sense RNA counterparts (clockwise). The table shows exact positions and lengths of antisense and sense RNAs on the genome of HBV, subtype adw2.16 ϵ, 5'-encapsidation signal on pregenomic RNA. Gastroenterology 1998 115, 702-713DOI: (10.1016/S0016-5085(98)70150-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Inhibition of HBV replication by antisense RNA. Southern blot analysis of nucleocapsid-associated replication products of HBV after cotransfection of a replication-competent HBV-HTD with antisense or sense RNA expression constructs. (A) No impact of antisense RNAs AS 1 (lane 1), AS 2 (lane 5), AS 3 (lane 9), and their sense counterparts (S 1, lane 2; S 2, lane 6; and S 3, lane 10) on HBV replication (respective wild-type replication levels are shown in lanes 3, 7, and 11) is observed. (B) A 60% inhibitory effect of antisense RNA AS 4 (lane 1) on HBV replication is detected compared with the wild-type replication level (lane 2). A 75% inhibitory effect of antisense RNA AS 5 (lane 4) but not sense RNA S 5 (lane 5) on HBV replication (compare with the respective wild-type level in lane 6) is observed. These experiments were repeated four times with similar results. NC, negative controls (mock-transfected HuH-7 HCC cells); DS, position of double-stranded linear HBV DNA. Gastroenterology 1998 115, 702-713DOI: (10.1016/S0016-5085(98)70150-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Inhibition of HBV replication by antisense RNA. Southern blot analysis of nucleocapsid-associated replication products of HBV after cotransfection of a replication-competent HBV-HTD with antisense or sense RNA expression constructs. (A) No impact of antisense RNAs AS 1 (lane 1), AS 2 (lane 5), AS 3 (lane 9), and their sense counterparts (S 1, lane 2; S 2, lane 6; and S 3, lane 10) on HBV replication (respective wild-type replication levels are shown in lanes 3, 7, and 11) is observed. (B) A 60% inhibitory effect of antisense RNA AS 4 (lane 1) on HBV replication is detected compared with the wild-type replication level (lane 2). A 75% inhibitory effect of antisense RNA AS 5 (lane 4) but not sense RNA S 5 (lane 5) on HBV replication (compare with the respective wild-type level in lane 6) is observed. These experiments were repeated four times with similar results. NC, negative controls (mock-transfected HuH-7 HCC cells); DS, position of double-stranded linear HBV DNA. Gastroenterology 1998 115, 702-713DOI: (10.1016/S0016-5085(98)70150-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of antisense RNAs on HBsAg (left) and HBeAg (right) secretion. Cell culture supernatants after cotransfection of a replication-competent HBV-HTD with antisense or sense RNA expression constructs were assayed by radioimmunoassay for HBsAg or HBeAg. AS 4 and AS 5 substantially inhibit HBsAg and HBeAg secretion, whereas the sense RNA control S 5 does not. No effect on HBsAg secretion is noted for antisense RNAs AS 1, AS 2, and AS 3 as well as their sense RNA counterparts. Gastroenterology 1998 115, 702-713DOI: (10.1016/S0016-5085(98)70150-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of antisense RNA species on DHBV replication and effect of transcriptionally silent antisense constructs on HBV replication. (A) Southern blot analysis of nucleocapsid-associated replication products of DHBV after cotransfection of a replication-competent DHBV-HTD with antisense or sense RNA expression constructs directed against HBV into LMH chicken HCC cells. The expression levels of HBV antisense RNAs in LMH cells were comparable with levels observed in HuH-7 cells (data not shown). No inhibitory effect of HBV antisense RNA AS 4 (lane 1) on DHBV replication is observed compared with the wild-type replication level (lane 2). No inhibitory effect of HBV antisense RNA AS 5 (lane 4) or sense RNA S 5 (lane 5) is detected compared with the wild-type replication level (lane 6). (B) Transcriptionally silent antisense constructs AS/S4 (lane 1) and AS/S5 (lane 4) do not inhibit HBV replication (respective wild-type replication levels are shown in lane 2 and lane 5). These experiments were repeated three times with similar results. NC, negative controls (mock-transfected HuH-7 HCC cells); DS, position of double-stranded linear DHBV- (A) or HBV- (B) DNA. Gastroenterology 1998 115, 702-713DOI: (10.1016/S0016-5085(98)70150-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) Detection of HBV antisense and sense transcripts. Northern blot analysis of total cellular RNA and detection of antisense and sense transcripts 3 days after transfection of respective expression constructs. Transcripts corresponding to AS 1 (lane 1), S 1 (lane 2), AS 2 (lane 3), S 2 (lane 4), AS 3 (lane 5), S 3 (lane 6), AS 4 (lane 7), AS 5 (lane 9), and S 5 (lane 10) are expressed at high levels in transfected cells. Note that the sense control RNA S 4 is not detectable. These experiments were repeated several times with similar results. Numbers below the lanes refer to the nt length of HBV sequences present in the transcripts. (B) Effect of antisense RNA on HBV transcript levels. Northern blot analysis of total cellular RNA and detection of HBV-associated as well as antisense- and sense-transcripts 3 days after cotransfection of a replication-competent HBV-HTD with antisense or sense RNA expression constructs. The signal at 3.5 kb corresponds to terminally redundant HBV transcripts of which a subpopulation is encapsidated into the nucleocapsid; signals at 2.4/2.1 kb correspond to the preS1 and preS2/S transcripts, respectively. Coexpression of antisense RNAs AS 4 (lane 1) and AS 5 (lane 3) as well as sense RNA S 5 (lane 4) with HBV transcripts is observed. In the presence of antisense or sense RNAs, there is no significant reduction of the level of either the 3.5-kb or the 2.4/2.1-kb HBV transcripts detectable (compare lane 1 with lane 2; lanes 3 and 4 with lane 5). These studies were repeated three times with similar results. NC, negative control (mock-transfected HuH-7 HCC cells). Gastroenterology 1998 115, 702-713DOI: (10.1016/S0016-5085(98)70150-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) Detection of HBV antisense and sense transcripts. Northern blot analysis of total cellular RNA and detection of antisense and sense transcripts 3 days after transfection of respective expression constructs. Transcripts corresponding to AS 1 (lane 1), S 1 (lane 2), AS 2 (lane 3), S 2 (lane 4), AS 3 (lane 5), S 3 (lane 6), AS 4 (lane 7), AS 5 (lane 9), and S 5 (lane 10) are expressed at high levels in transfected cells. Note that the sense control RNA S 4 is not detectable. These experiments were repeated several times with similar results. Numbers below the lanes refer to the nt length of HBV sequences present in the transcripts. (B) Effect of antisense RNA on HBV transcript levels. Northern blot analysis of total cellular RNA and detection of HBV-associated as well as antisense- and sense-transcripts 3 days after cotransfection of a replication-competent HBV-HTD with antisense or sense RNA expression constructs. The signal at 3.5 kb corresponds to terminally redundant HBV transcripts of which a subpopulation is encapsidated into the nucleocapsid; signals at 2.4/2.1 kb correspond to the preS1 and preS2/S transcripts, respectively. Coexpression of antisense RNAs AS 4 (lane 1) and AS 5 (lane 3) as well as sense RNA S 5 (lane 4) with HBV transcripts is observed. In the presence of antisense or sense RNAs, there is no significant reduction of the level of either the 3.5-kb or the 2.4/2.1-kb HBV transcripts detectable (compare lane 1 with lane 2; lanes 3 and 4 with lane 5). These studies were repeated three times with similar results. NC, negative control (mock-transfected HuH-7 HCC cells). Gastroenterology 1998 115, 702-713DOI: (10.1016/S0016-5085(98)70150-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Antisense RNA AS 5 reduces the levels of encapsidated pregenomic RNA. Cotransfection of a replication-competent HBV-HTD with antisense RNA expression constructs AS 4 or AS 5 and control sense RNA S 5 into HuH-7 HCC cells. Nucleocapsids were isolated by ultracentrifugation through a sucrose cushion. (A) Primer extension analysis of encapsidated, pregenomic RNA from nucleocapsids. Relative amounts of encapsidated RNA were calculated using densitometric data after transfection efficiencies (shown at the bottom) were determined with an HGH assay as described in Materials and Methods. Note that encapsidated pregenomic RNA becomes undetectable (*) when antisense RNA AS 5 is present (lane 2), whereas the presence of antisense RNA AS 4 (lane 1, 110% of wild-type) does not reduce the amount of pregenomic RNA. A slight reduction (81% of the wild-type level) is noted in the presence of control sense RNA S 5 (lane 3). (B) Parallel quantification of nucleocapsids (p21) by immunoblot with an HBcAg-specific antiserum. Relative amounts of nucleocapsids were calculated using densitometric data after determination of transfection efficiencies with the HGH assay. No reductions in the level of nucleocapsids are observed for antisense RNAs AS 4 (lane 1) and AS 5 (lane 2) as well as for control sense RNA S 5 (lane 3) compared with the wild-type level (lane 4). These experiments were performed three times with similar results. Gastroenterology 1998 115, 702-713DOI: (10.1016/S0016-5085(98)70150-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions