Relative Abundance of Integrant-Derived Viral RNAs in Infected Tissues Harvested from Chronic Hepatitis B Virus Carriers

Abstract

Five matching sets of nonmalignant liver tissues and hepatocellular carcinoma (HCC) samples from individuals chronically infected with hepatitis B virus (HBV) were examined. The HBV genomic sequences were determined by using overlapping PCR amplicons covering the entire viral genome. Four pairs of tissues were infected with HBV genotype C, while one pair was infected with HBV genotype B. HBV replication markers were found in all tissues. In the majority of HCC samples, the levels of pregenomic/precore RNA (pgRNA) and covalently closed circular DNA (cccDNA) were lower than those in liver tissue counterparts. Regardless of the presence of HBV replication markers, (i) integrant-derived HBV RNAs (id-RNAs) were found in all tissues by reverse transcription-PCR (RT-PCR) analysis and were considerably abundant or predominant in 6/10 tissue samples (2 liver and 4 HCC samples), (ii) RNAs that were polyadenylated using the cryptic HBV polyadenylation signal and therefore could be produced by HBV replication or derived from integrated HBV DNA were found in 5/10 samples (3 liver and 2 HCC samples) and were considerably abundant species in 3/10 tissues (2 livers and 1 HCC), and (iii) cccDNA-transcribed RNAs polyadenylated near position 1931 were not abundant in 7/10 tissues (2 liver and 5 HCC samples) and were predominant in only two liver samples. Subsequent RNA sequencing analysis of selected liver/HCC samples also showed relative abundance of id-RNAs in most of the examined tissues. Our findings suggesting that id-RNAs could represent a significant source of HBV envelope proteins, which is independent of viral replication, are discussed in the context of the possible contribution of id-RNAs to the HBV life cycle. The relative abundance of integrant-derived HBV RNAs (id-RNAs) in chronically infected tissues suggest that id-RNAs coding for the envelope proteins may facilitate the production of a considerable fraction of surface antigens (HBsAg) in infected cells bearing HBV integrants. If the same cells support HBV replication, then a significant fraction of assembled HBV virions could bear id-RNA-derived HBsAg as a major component of their envelopes. Therefore, the infectivity of these HBV virions and their ability to facilitate virus cell-to-cell spread could be determined mainly by the properties of id-RNA-derived envelope proteins and not by the properties of replication-derived HBsAg. These interpretations suggest that id-RNAs may play a role in the maintenance of chronic HBV infection and therefore contribute to the HBV life cycle. Furthermore, the production of HBsAg from id-RNAs independently of viral replication may explain at least in part why treatment with interferon or nucleos(t)ides in most cases fails to achieve a loss of serum HBsAg.

Will we need novel combinations to cure HBV infection?

“…HBsAg can be transcribed from ccc/DNA but in HBeAg negative patients the probable predominant source of HBsAg is from RNAs transcribed from integrant HBV DNA . This source of HBsAg is relatively inaccessible without cell loss.…”

Pregenomic RNA: How to assist the management of chronic hepatitis B?

“…Integrated HBV DNA lacks the ability to transcribe pgRNA but produces novel viral‐host fusion RNA, which may have pathological functions. The relative abundance of integrant‐derived HBV RNAs (id‐RNAs) in chronically infected tissues suggest that id‐RNAs coding for the envelope proteins may facilitate the production of a considerable fraction of HBsAg in infected cells bearing HBV integrants . In patients treated with NA, HBsAg may still be high despite low transcriptional activity of cccDNA because integrated HBV DNA can maintain HBsAg production .…”

Commentary to: Relative Abundance of Integrant-Derived Viral RNAs in Infected Tissues Harvested from Chronic Hepatitis B Virus Carriers by Freitas N, et al. 2018

“…Since integrated HBV DNA cannot serve as the source of the progeny virus, it has been commonly assumed that the HBV life cycle is independent of HBV DNA integrants. Using liver and matching hepatocellular carcinoma (HCC) tissues harvested from individuals that were chronically infected with HBV and that were not treated with anti-HBV drugs, our recent study demonstrated that RNA transcripts arising from integrated HBV DNA (i.e., HBV integrant-derived RNAs or id-RNAs) were abundantly or even predominantly present, when compared to viral RNAs transcribed from the episomal, covalentlyclosed circular viral DNA (cccDNA) (i.e., replication-derived or rd-RNAs) [4]. The observed relative abundance of id-RNAs suggested that these RNA molecules could serve as a considerable source of the HBV envelope proteins (or surface antigen, HBsAg) that is independent of the HBV genome replication.…”

“…The data further suggested that id-RNAs/id-HBsAg therefore can possibly influence the maintenance of the chronic state of HBV infection. In addition, the study proposed to revise the current model of the HBV life cycle by incorporating the potential contributions of id-RNAs and id-HBsAg as described above [4]. Moreover, it became apparent that the inability of current anti-HBV drugs to achieve loss of serum HBsAg in the vast majority of treated patients can be likely explained by significant amounts of id-HBsAg that are translated from id-RNAs independently of HBV replication [4].…”

“…In addition, the study proposed to revise the current model of the HBV life cycle by incorporating the potential contributions of id-RNAs and id-HBsAg as described above [4]. Moreover, it became apparent that the inability of current anti-HBV drugs to achieve loss of serum HBsAg in the vast majority of treated patients can be likely explained by significant amounts of id-HBsAg that are translated from id-RNAs independently of HBV replication [4]. Consequently, it is reasonable to suggest that the abundant presence of id-RNAs/id-HBsAg during chronic HBV infection may also have important implications for HBVrelated liver pathogenesis (including carcinogenesis), co-or superinfection with Hepatitis Delta Virus (HDV) [4], and innate and adaptive immunity ( Figure 1).…”