Abstract
Persistence of hepatitis B virus (HBV) infection requires covalently closed circular (ccc)DNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc) DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV) in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process.
Highlights
More than 350 million people suffer from chronic hepatitis B virus (HBV) infection
To identify encapsidated DNAs, we incubated the cytoplasmic extracts with micrococcal nuclease (MN) which digests free nucleic acids but not those protected inside capsids
One option for the seeming discrepancy to efficient cccDNA formation during HBV infection in vivo is that cellular activities promoting the conversion are limited in these cell lines, another that inhibitory factors are overexpressed; this seems unlikely, though, because HBV cccDNA accumulation was not boosted in the avian LMH cells
Summary
More than 350 million people suffer from chronic HBV infection. Chronic hepatitis B frequently progresses to liver cirrhosis and hepatocellular carcinoma, a leading cause of cancerrelated morbidity and mortality worldwide [1,2].HBV is a small enveloped hepatotropic DNA virus which replicates by reverse transcription of an RNA intermediate, the pregenomic (pg) RNA (for review: [3,4]), to yield encapsidated, partially double-stranded rcDNA to which the viral polymerase is covalently bound [5]. Episomal cccDNA acts as template for all viral transcripts These include the subgenomic RNAs encoding the surface proteins, and the pgRNA that serves as mRNA for the polymerase protein and the capsid, or core, protein. DNA-containing capsids are enveloped by surface proteins and cellular lipids and secreted as virions They are redirected to the nucleus to increase cccDNA copy number by a mechanism termed intracellular recycling; many estimates for cccDNA copies per infected hepatocyte are in the range of 5 to 50 [6]. This amplification prevents loss during cell division of the cccDNA which can not be replicated semiconservatively [7]. CccDNA formation and recycling are central to establish and maintain persistent infection, and they limit the efficacy of antiviral nucleot(s)ides in the treatment of chronic hepatitis B, as these do not directly target cccDNA (for review: [8])
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