Asymmetric Modification of Hepatitis B Virus (HBV) Genomes by an Endogenous Cytidine Deaminase inside HBV Cores Informs a Model of Reverse Transcription

ABSTRACTCytidine deaminases inhibit replication of a broad range of DNA viruses by deaminating cytidines on single-stranded DNA (ssDNA) to generate uracil. While several lines of evidence have revealed hepatitis B virus (HBV) genome editing by deamination, it is still unclear which nucleic acid intermediate of HBV is modified. Hepatitis B virus has a relaxed circular double-stranded DNA (rcDNA) genome that is reverse transcribed within virus cores from a RNA template. The HBV genome also persists as covalently closed circular DNA (cccDNA) in the nucleus of an infected cell. In the present study, we found that in HBV-producing HepAD38 and HepG2.2.15 cell lines, endogenous cytidine deaminases edited 10 to 25% of HBV rcDNA genomes, asymmetrically with almost all mutations on the 5′ half of the minus strand. This region corresponds to the last half of the minus strand to be protected by plus-strand synthesis. Within this half of the genome, the number of mutations peaks in the middle. Overexpressed APOBEC3A and APOBEC3G could be packaged in HBV capsids but did not change the amount or distribution of mutations. We found no deamination on pregenomic RNA (pgRNA), indicating that an intact genome is encapsidated and deaminated during or after reverse transcription. The deamination pattern suggests a model of rcDNA synthesis in which pgRNA and then newly synthesized minus-sense single-stranded DNA are protected from deaminase by interaction with the virus capsid; during plus-strand synthesis, when enough dsDNA has been synthesized to displace the remaining minus strand from the capsid surface, the single-stranded DNA becomes deaminase sensitive.IMPORTANCEHost-induced mutation of the HBV genome by APOBEC proteins may be a path to clearing the virus. We examined cytidine-to-thymidine mutations in the genomes of HBV particles grown in the presence or absence of overexpressed APOBEC proteins. We found that genomes were subjected to deamination activity during reverse transcription, which takes place within the virus capsid. These observations provide a direct insight into the mechanics of reverse transcription, suggesting that newly synthesized dsDNA displaces ssDNA from the capsid walls, making the ssDNA accessible to deaminase activity.

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cis-Acting Sequences That Contribute to Synthesis of Minus-Strand DNA Are Not Conserved between Hepadnaviruses

Journal of Virology ◽

10.1128/jvi.01487-10 ◽

2010 ◽

Vol 84 (24) ◽

pp. 12824-12831 ◽

Cited By ~ 5

Author(s):

Megan L. Maguire ◽

Daniel D. Loeb

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Reverse Transcription ◽

Minus Strand ◽

Wild Type ◽

Dna Viruses ◽

Cis Acting ◽

Duck Hepatitis ◽

B Virus ◽

The Individual

ABSTRACT Hepadnaviruses are DNA viruses that are found in several mammalian and avian species. These viruses replicate their genome through reverse transcription of an RNA intermediate termed pregenomic RNA (pgRNA). pgRNA is reverse transcribed by the viral polymerase into a minus-strand DNA, followed by synthesis of the plus-strand DNA. There are multiple cis-acting sequences that contribute to the synthesis of minus-strand DNA for human hepatitis B virus (HBV). Less is known about the cis-acting sequences of avian hepadnaviruses that contribute to synthesis of minus-strand DNA. To identify cis-acting sequences of duck hepatitis B virus (DHBV) and heron hepatitis B virus (HHBV), we analyzed variants containing 200-nucleotide (nt) deletions. Most variants of DHBV synthesized minus-strand DNA to 50 to 100% of the wild-type (WT) level, while two variants synthesized less than 50%. For HHBV, most variants synthesized minus-strand DNA to less than 50% the WT level. These results differ from those for HBV, where most of the genome can be removed with little consequence. HBV contains a sequence, φ, that contributes to the synthesis of minus-strand DNA. It has been proposed that DHBV has an analogous sequence. We determined that the proposed φ sequence of DHBV does not contribute to the synthesis of minus-strand DNA. Finally, we found that the DR2 sequence present in all hepadnaviruses is important for synthesis of minus-strand DNA in both DHBV and HHBV but not in HBV. These differences in cis-acting sequences suggest that the individual hepadnaviruses have evolved differences in their mechanisms for synthesizing minus-strand DNA, more so than for other steps in replication.

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Evidence that the first strand-transfer reaction of duck hepatitis B virus reverse transcription requires the polymerase and that strand transfer is not needed for the switch of the polymerase to the elongation mode of DNA synthesis

Journal of General Virology ◽

10.1099/0022-1317-81-8-2059 ◽

2000 ◽

Vol 81 (8) ◽

pp. 2059-2065 ◽

Cited By ~ 6

Author(s):

Yunhao Gong ◽

Ermei Yao ◽

Melissa Stevens ◽

John E. Tavis

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Dna Synthesis ◽

Reverse Transcription ◽

Transfer Reaction ◽

Strand Transfer ◽

Minus Strand ◽

Duck Hepatitis B Virus ◽

Duck Hepatitis ◽

B Virus

Deletion of amino acids 79–88 in the duck hepatitis B virus reverse transcriptase had minimal effects on polymerase activities prior to the minus-strand DNA transfer reaction, yet it greatly diminished strand transfer and subsequent DNA synthesis. This mutation also reduced reverse transcription on exogenous RNA templates. The reaction on exogenous RNAs employed the phosphonoformic acid (PFA)-sensitive elongation mode of DNA synthesis rather than the PFA-resistant priming mode, despite the independence of DNA synthesis in this assay from the priming and minus-strand transfer reactions. These data provide experimental evidence that the polymerase is involved directly in the minus-strand transfer reaction and that the switch of the polymerase from the early PFA-resistant mode of DNA synthesis to the later PFA-sensitive elongation mode does not require the strand-transfer reaction.

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Induction of Antiviral Cytidine Deaminases Does Not Explain the Inhibition of Hepatitis B Virus Replication by Interferons

Journal of Virology ◽

10.1128/jvi.02489-06 ◽

2007 ◽

Vol 81 (19) ◽

pp. 10588-10596 ◽

Cited By ~ 40

Author(s):

Stéphanie Jost ◽

Priscilla Turelli ◽

Bastien Mangeat ◽

Ulrike Protzer ◽

Didier Trono

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Cytidine Deaminase ◽

Chronic Phase ◽

Inhibitory Effect ◽

Type I ◽

Cytidine Deaminases ◽

Virion Infectivity Factor ◽

B Virus ◽

Ifn Treatment

ABSTRACT Interferons (IFNs) play a major role in the control of hepatitis B virus (HBV), whether as endogenous cytokines limiting the spread of the virus during the acute phase of the infection or as drugs for the treatment of its chronic phase. However, the mechanism by which IFNs inhibit HBV replication has so far remained elusive. Here, we show that type I and II IFN treatment of human hepatocytes induces the production of APOBEC3G (A3G) and, to a lesser extent, that of APOBEC3F (A3F) and APOBEC3B (A3B) but not that of two other cytidine deaminases also endowed with anti-HBV activity, activation-induced cytidine deaminase (AID), and APOBEC1. Most importantly, we reveal that blocking A3B, A3F, and A3G by combining RNA interference and the virion infectivity factor (Vif) protein of human immunodeficiency virus does not abrogate the inhibitory effect of IFNs on HBV. We conclude that these cytidine deaminases are not essential effectors of IFN in its action against this pathogen.

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Inhibitory Effect of Penciclovir-Triphosphate on Duck Hepatitis B Virus Reverse Transcription

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029700800104 ◽

1997 ◽

Vol 8 (1) ◽

pp. 38-46 ◽

Cited By ~ 29

Author(s):

E Dannaoui ◽

C Trépo ◽

F Zoulim

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Reverse Transcription ◽

Inhibitory Effect ◽

In Vitro Translation ◽

Chain Elongation ◽

Minus Strand ◽

Duck Hepatitis B Virus ◽

Duck Hepatitis ◽

B Virus

The aim of this study was to investigate the mechanism of inhibition of hepatitis B virus replication by penciclovir-triphosphate, the active metabolite of famciclovir. A recently developed in vitro translation assay for the expression of an enzymatically active duck hepatitis B virus (DHBV) reverse transcriptase was used to assess the inhibitory activity of penciclovir-triphosphate (PCV-TP) in comparison with other guanosine analogue triphosphates. Acyclovir-triphosphate (ACV-TP), the chiral triphosphates of penciclovir (PCV), ( R)-PCV-TP and ( S)-PCV-TP, and carbocyclic 2′-deoxyguanosine-TP (CDG-TP) did inhibit reproducibly minus strand DNA synthesis to different extents. CDG-TP was the most potent inhibitor of dGTP incorporation. The inhibitory effect of these compounds against the incorporation of the first nucleotide of minus strand DNA, dGMP, was similar to that observed with DNA chain elongation. 2′,3′-dideoxyguanosine-TP (ddG-TP), ACV-TP and both ( R) and ( S)-PCV-TP inhibited the incorporation of the next nucleotides in the short DNA primer, whereas CDG-TP did not. These results demonstrate that PCV-TP inhibits hepadnavirus reverse transcription by inhibiting the synthesis of the short DNA primer. The data obtained with the inhibition of the enzymatic activity of the DHBV polymerase provides a new insight into the mechanism of action of penciclovir-triphosphate on HBV replication.

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Deamination-Independent Inhibition of Hepatitis B Virus Reverse Transcription by APOBEC3G

Journal of Virology ◽

10.1128/jvi.02510-06 ◽

2007 ◽

Vol 81 (9) ◽

pp. 4465-4472 ◽

Cited By ~ 103

Author(s):

David H. Nguyen ◽

Suryaram Gummuluru ◽

Jianming Hu

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Reverse Transcription ◽

Inhibitory Effect ◽

Minor Effect ◽

Minus Strand ◽

Rna Packaging ◽

Hbv Replication ◽

B Virus ◽

A Minor

ABSTRACT The APOBEC3 family of mammalian cytidine deaminases, including APOBEC3G (A3G), has been shown to function as innate antiviral factors against retroviruses and can also suppress the replication of the hepatitis B virus (HBV). The mechanism by which A3G inhibits HBV replication remains to be elucidated. In this study, we show that the inhibitory effect of APOBEC3 proteins on HBV replication was mainly at the DNA level, with only a minor effect on viral RNA packaging. The anti-HBV effect of A3G was independent of the DNA-editing function, and the mode of inhibition was not due to HBV DNA degradation. The editing-independent antiviral activity of A3G could target DNA-RNA hybrids as well as single-stranded DNA. Finally, we show that there was a preferential decrease in the accumulation of longer minus-strand DNA by A3G, compared to the shorter minus-strand DNA, and suggest that A3G exerts its inhibitory effect at very early stages during viral reverse transcription.

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