Involvement of the Host DNA-Repair Enzyme TDP2 in Formation of The

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Involvement of the host DNA-repair enzyme TDP2 in PNAS PLUS formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

Christian Königera,b, Ida Wingerta, Moritz Marsmanna, Christine Röslera, Jürgen Becka,1, and Michael Nassala,1

aDepartment of Internal Medicine 2/Molecular Biology, University Hospital Freiburg, D-79106 Freiburg, Germany; and bFaculty of Biology, University of Freiburg, D-79104 Freiburg, Germany

Edited by Francis V. Chisari, The Scripps Research Institute, La Jolla, CA, and approved July 28, 2014 (received for review May 29, 2014) Hepatitis B virus (HBV), the causative agent of chronic hepatitis B nucleocapsids via P protein’s interaction with the 5′-proximal and prototypic hepadnavirus, is a small DNA virus that replicates e RNA stem-loop (Fig. 1A). A Tyr residue in the terminal protein by protein-primed reverse transcription. The product is a 3-kb (TP) domain of P protein acts as acceptor for the first dNTP of relaxed circular DNA (RC-DNA) in which one strand is linked to ashorte-templated oligonucleotide. This “protein-priming” re- the viral polymerase (P protein) through a tyrosyl–DNA phospho- action, which for DHBV can be reconstituted completely in vitro diester bond. Upon infection, the incoming RC-DNA is converted (7–10), establishes a 5′-phosphotyrosyl linkage between P pro- into covalently closed circular (ccc) DNA, which serves as a viral tein and DNA which is maintained during the formation of persistence reservoir that is refractory to current anti-HBV treat- complete minus-strand DNA (Fig. 1B), RNA-primed synthesis ments. The mechanism of cccDNA formation is unknown, but the of usually incomplete plus-strand DNA, and circularization into release of P protein is one mandatory step. Structural similarities RC-DNA via a short terminal redundancy (r) in the minus between RC-DNA and cellular topoisomerase–DNA adducts and strand. Hence the formation of cccDNA from RC-DNA requires their known repair by tyrosyl-DNA-phosphodiesterase (TDP) 1 or release of P protein and RNA, removal of r, completion of TDP2 suggested that HBV may usurp these enzymes for its own the plus strand, and eventually ligation (Fig. 1C). Ligation must purpose. Here we demonstrate that human and chicken TDP2, but

occur with single-nucleotide precision because each nucleo- MICROBIOLOGY – only the yeast ortholog of TDP1, can specifically cleave the Tyr tide has a coding function in at least one ORF. Notably, be- DNA bond in virus-adapted model substrates and release P protein cause cccDNA cannot be replicated semiconservatively (11), from authentic HBV and duck HBV (DHBV) RC-DNA in vitro, with- each single cccDNA molecule must have undergone this con- out prior proteolysis of the large P proteins. Consistent with TPD2’s version process. having a physiological role in cccDNA formation, RNAi-mediated To begin to decipher the conversion of RC-DNA to cccDNA, TDP2 depletion in human cells significantly slowed the conversion we focus here on the release of P protein from the minus-strand of RC-DNA to cccDNA. Ectopic TDP2 expression in the same cells DNA. Notably, chemically similar protein–DNA adducts occur restored faster conversion kinetics. These data strongly suggest that TDP2 is a first, although likely not the only, host DNA-repair naturally as abortive topoisomerase (TOP) cleavage complexes. factor involved in HBV cccDNA biogenesis. In addition to establish- TOPs relieve DNA supercoiling (12) by incising one (TOP1) or both (TOP2) strands via the usually transient formation of ing a functional link between hepadnaviruses and DNA repair, – ′ – ′ our results open new prospects for directly targeting HBV tyrosyl 3 DNA(TOP1)ortyrosyl5 DNA phosphodiester persistence. Significance hepatitis B virus persistence | TDP substrate specificity | virus–DNA repair interface Chronic hepatitis B virus (HBV) infection puts >250 million humans at risk for developing liver cirrhosis and liver cancer. ore than 250 million people worldwide are chronically Current therapies are not curative because they do not target Minfected with hepatitis B virus (HBV) (1) and are at a HBV´s persistence reservoir, the plasmid-like covalently closed highly increased risk for developing end-stage liver disease (2). circular DNA (cccDNA). RNA production from cccDNA initiates Current treatments with IFN-α or nucleos(t)ide analogs (NAs) the generation of progeny virus via protein-primed reverse are only partially effective (3, 4). Importantly, they do not transcription, yielding viral polymerase-linked relaxed-circular directly target the viral persistence reservoir, an episomal DNA (RC-DNA). Its conversion, upon infection, into cccDNA covalently closed circular (ccc) DNA form of the viral genome requires multiple poorly understood steps, including poly- that serves as template for all viral transcripts; hence a few merase removal. We found that the host enzyme tyrosyl-DNA- cccDNA molecules present in the liver can reactivate full viral phosphodiesterase 2 (TDP2), important for repair of cellular – replication. Eliminating infection thus will require the elimi- protein DNA adducts, performed this step in vitro and that nation of cccDNA. cccDNA is generated, upon infection, from TDP2 depletion impaired the conversion of RC-DNA to cccDNA the viral polymerase (P protein)-linked relaxed circular (RC) in cells. These data establish a functional link between HBV DNA present in incoming virions (Fig. 1A). The mechanism by and cellular DNA repair and pave the way for targeting which RC-DNA is converted to cccDNA is poorly understood, HBV persistence. but it must involve multiple steps (see below). Because the ∼3-kb Author contributions: C.K., J.B., and M.N. designed research; C.K., I.W., M.M., C.R., and J.B. genomes of HBV and the other hepadnaviruses [e.g., from ducks performed research; C.K., M.M., J.B., and M.N. analyzed data; and C.K., J.B., and M.N. (DHBV)] are too small to encode all the activities required for wrote the paper. this conversion, these activities must be provided by the host cell. The authors declare no conflict of interest. RC-DNA from all hepadnaviruses bears several molecular This article is a PNAS Direct Submission. peculiarities that result from its generation by protein-primed 1To whom correspondence may be addressed. Email: [email protected] reverse transcription (for reviews, see refs. 5 and 6). The pre- or [email protected]. genomic RNA (pgRNA) acts as mRNA for the capsid (core) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. proteinandPproteinandtheniscopackagedwithPinto 1073/pnas.1409986111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1409986111 PNAS Early Edition | 1of10 Downloaded by guest on September 26, 2021 Fig. 1. Study-relevant hepadnavirus features. (A) Replication cycle. Incoming P-protein–linked RC-DNA is converted into nuclear cccDNA from which viral RNAs are transcribed, exported, and translated. Via e, pgRNA is copackaged with P into new nucleocapsids and reverse transcribed into RC-DNA. Mature nucleocapsids can be secreted as virions or reenter the intracellular cycle. (B) Establishment of the 5′-phosphotyrosyl linkage. (Upper) P protein uses a Tyr residue in its TP domain to initiate synthesis of an e bulge-templated DNA oligo primer; its extension after translocation to 3′ DR1* yields minus-strand (−) DNA bearing a terminal redundancy (r). (Lower) HBV and DHBV primer and DR1* acceptor sequences are depicted. Subsequent RC-DNA formation is not depicted. DR, direct repeat; RH, RNase H domain of P protein; RT, reverse transcriptase domain of P protein. (C) P protein release is one of several obligatory steps in the conversion of RC-DNA to cccDNA. Before final ligation, all structural peculiarities of RC-DNA [i.e., linked P protein and r on minus-strand DNA, and RNA primer (red line) and the gap in plus-strand DNA] must be removed. (D) Similarity of abortive TOP1 and TOP2 cleavage complexes to P-protein–linked RC-DNA. TOP1 is bound to DNA via 3′- and TOP2 via 5′-phosphotyrosyl bonds. Major repair pathways include TDP1 for TOP1 and TDP2 for TOP2 adducts, but TDP2 may substitute for TDP1 and possibly vice versa. Alternative nucleolytic repair pathways are not indicated.

(TOP2) bonds. Occasionally, or induced by near-by lesions or the “unlinkase” activity that can remove the covalently linked topoisomerase poisons (13), the back-reaction fails, leaving the VPg from poliovirus RNA (31). enzyme covalently trapped on the broken DNA (Fig. 1D). To Given the structural similarity of hepadnaviral P-protein– avoid the potentially fatal consequences of this and other kinds linked RC-DNA to TOP–DNA adducts and the increasingly of DNA damage, all cells are equipped with a sophisticated DNA acknowledged fact that all viruses with a nuclear DNA phase repair machinery comprising a large network of damage sensors, must cope with cellular DNA repair (32), we reasoned that signal transducers, and repair effectors (14, 15). Their concerted TDP1 and/or TDP2 might be able to release P protein from action usually arrests the cell cycle to allow time for repair or RC-DNA and thus catalyze one crucial step in cccDNA forma- induces cell death if repair fails. Because of the fundamental tion. As shown below, huTDP2 and chTDP2 but only yTDP1 ′ importance of genome integrity, many, if not all, repair pathways could cleave specifically hepadnavirus-adapted Tyr-5 -DNA are safeguarded by alternative systems, as is TOP cleavage model substrates, covalent complexes of DHBV P protein car- complex repair. For TOP1 adducts, one pathway involves tyrosyl- rying the short DNA oligonucleotide primer and, importantly, DNA-phosphodiesterase 1 (TDP1; Fig. 2A), an ∼600-aa member authentic RC-DNA from DHBV and HBV nucleocapsids. Last, of the phospholipase D superfamily (16) long known to cleave stable shRNA knockdown of TDP2 in human hepatoma cells significantly impaired the conversion of RC-DNA to cccDNA. 3′-phosphotyrosyl bonds (17); however, yeast TDP1 (yTDP1) (18), Together these data strongly suggest that TDP2, but not TDP1, and possibly human TDP1 (huTDP1) (19), also may possess is a first host-cell DNA-repair factor involved in hepadnaviral tyrosyl-5′-DNA phosphodiesterase activity (but see also ref. 20). cccDNA biogenesis. Alternatively, trapped TOP1 can be removed nucleolytically (21, 22). For repair of the 5′-phosphotyrosyl–linked TOP2 Results adducts, only nucleolytic mechanisms were considered (23, 24) Different Phosphodiesterase Activity Profiles of TDP1 and TDP2 from “ until a protein known as TRAF and TNF receptor-associated Different Organisms on Synthetic Tyr-5′-DNA and Tyr-3′-DNA Substrates. ” “ ” protein (TTRAP) or ETS1-associated protein II (EAPII) (25) The principal requirements in the conversion of RC-DNA to was found to have such an activity (26); that protein has been cccDNA are the same for HBV and DHBV. Because chicken as renamed “TDP2” (Fig. 2B). The preference of TDP2 for well as human hepatoma cells support efficient DHBV cccDNA 5′ phosphotyrosyl bonds is consistent with recent X-ray data formation, we investigated, in addition to huTDP1 and huTDP2 (27–29), but TDP2 also is active on 3′-substrates (20, 26, 30). Al- [National Center for Biotechnology Information (NCBI) reference though TDP2 likely is the only enzyme of its kind (30), complete sequences: NP_001008744.1 and NP_057698.2, respectively], their knockout of TDP2 in chicken DT40 cells and in mice was not chicken orthologs (NCBI reference sequences: XP_421313.1 and lethal (20), possibly reflecting the power of redundancy in DNA NP_001263313.1, respectively). We included yTDP1 (NCBI refer- repair. Notably, TDP2 recently also has been found to represent ence sequence: NP_009782.3) because of its reported 5′-substrate

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1409986111 Königer et al. Downloaded by guest on September 26, 2021 pendence of TDP2 activity (26, 34). Accordingly, these assays PNAS PLUS + were performed in the presence of 5 mM of Mg2 .Both huTDP2 and chTDP2, but not their mutant versions, exerted high activity on the 5′ substrate and a well-detectable but lower activity on the 3′ substrate. Hence huTDP2 and chTDP2 but only yTDP1 (and possibly chTDP1) possessed the proper enzymatic activity for the release of the 5′-phosphotyrosyl–linked P protein from RC-DNA.

Vertebrate TDP2 and Yeast TDP1 Can Cleave the Covalent P Protein– Oligonucleotide Bond Formed by in Vitro Protein Priming. Next we reconstituted the 5′-phosphotyrosyl bond between P protein and minus-strand DNA using in vitro protein priming with recombinant DHBV P protein and e RNA plus cellular chap- erones, ATP as the energy source, and dGTP, dTTP, and dATP (10). Using [α32P]-dGTP, the 5′ residue of the DHBV oligonucleotide primer GTA or GTAA (Fig. 1B, Lower), and hence the phosphotyrosyl bond to P protein, will be radioactively labeled (Fig. 4A). TDP cleavage should reduce P-protein labeling and release the 32P-labeled oligonucleotide (35). The priming products were incubated with the different TDP enzymes, and radiolabeled protein products were resolved by SDS/PAGE and visualized by autoradiography and/or phos- Fig. 2. General structures of TDP1 and TDP2. (A) TDP1 contains two con- phorimaging (Fig. 4 B–D). Wild-type yTDP1, but not its inactive served HKN motifs; their sequences are shown for yTDP1, huTDP1, and mutant, reduced the 32P P-protein signal by about 60% com- chTDP1. (B) TDP2 contains conserved LQEV and GDXN motifs; their sequences for huTDP2 and chTDP2 are indicated. Numbers refer to amino pared with the mock-treated control; this reduction was pre- acid positions. Catalytically important residues, for the chicken enzymes vented by vanadate, a known phosphodiesterase inhibitor (Fig.

inferred by sequence homology, mutated to Ala are listed on the right. In 4B). No loss of protein label was seen with chTDP1 and huTDP1 MICROBIOLOGY preliminary model substrate assays, all four TDP2 mutants exerted no activity, (Fig. 4C). In contrast, both vertebrate TDP2 enzymes, but not hence only those shown in bold face were used in further experiments. their inactive variants, also reduced P-protein labeling by about 50% (Fig. 4D). Loss of protein labeling correlated strictly with the detection activity (18). The generation of bacterial and mammalian ex- of distinct radiolabeled oligonucleotides by electrophoresis in pression vectors for these proteins, plus inactive variants with high-percentage polyacrylamide -7 M urea gels, with yTDP1 and mutations of key catalytic residues (hereafter collectively termed both vertebrate TDP2s, but not their mutants, generating very “ ” mut ) (Fig. 2), is described in SI Materials and Methods. All five similar patterns (Fig. 4E, Left). The strongest signals appeared at proteins and their mutants were well expressed in Escherichia the 4- and 5-nt marker positions, with weaker signals at the coli and could be purified with substantial yield and purity in 3- and 6- to 7-nt marker positions. Some products thus were longer a monomeric state (Fig. S1). than the tri- or tetranucleotide GTA(A) that productively primes To validate their enzymatic activities, we adapted an assay minus-strand extension in vivo. To address concerns that the reported for TDP1 activity on 3′ phosphotyrosyl bonds (33) to released products did not originate from e-templated primers, obtain synthetic substrates in which methylumbelliferon (MU), dATP was omitted from the priming reaction; this omission led a Tyr mimic, is attached via a phosphodiester bond to either the to the appearance of a product at the expected GT dinucleotide 5′ or the 3′ end of a 14-mer DNA oligo (MUP-DNA); its se- position, but some extended products obviously containing only quence corresponded to the 5′ end of DHBV minus-strand DNA dG and dT were formed. However, replacement of dTTP by its to which P protein is linked (Fig. 3A). Cleavage of the phos- dideoxy derivative ddTTP resulted exclusively in one product photyrosyl bond releases fluorescent MU from nonfluorescent with slightly faster mobility than the dinucleotide marker (likely MUP-DNA and can thus be monitored in real time using a fluo- because of the modified ribose part), as expected from efficient rescence reader. The most pertinent progress curves obtained using e-templated ddT incorporation at the second primer position 0.5 μM MUP-DNA substrate and a 100-nM protein concentra- (Fig. 4E, Center) and subsequent chain termination. Further- tion are shown in Fig. 3 B–G; data on bivalent metal ion de- more, using e RNA with an extended 16-nt bulge (eb16) as a pendence are provided in Fig. S2. priming template, yTDP1 generated cleavage products extending All three wild-type TDP1 orthologs, but not their mutant up to about 13 nt in length (Fig. 4E, Right). Hence the TDP- versions, caused a fast rise in fluorescence with the 3′ substrate released oligonucleotides are derived, at least in part, from au- (Fig. 3 B–D), independently of bivalent metal ions (Fig. S2A). In thentic protein-priming events. Potential reasons for the seeming contrast, activities on the 5′ substrate differed strongly. yTDP1 discrepancies between in vitro and in vivo priming are given was highly active (Fig. 3B), huTDP1 was inactive (Fig. 3C), and in Discussion. Besides illustrating the usefulness of vertebrate chTDP1 produced a slight increase in fluorescence (Fig. 3D) that TDP2s and yTDP1 for further investigation of hepadnaviral was more evident at higher enzyme concentrations and longer protein priming, the data showed that these enzymes were active incubation times (Fig. 3E). Under the same conditions, no in- on the authentic P protein–oligonucleotide phosphotyrosyl bond. crease occurred with chTDP1mut and wild-type huTDP1, indicating a low but specific 5′-substrate activity of the chicken but HuTDP2, chTDP2, and yTDP1 Can Cleave P Protein Specifically from not the human enzyme (Fig. 3E). Together, these data confirmed Authentic DHBV and HBV RC-DNA. As the closest in vitro approxi- the Tyr-3′-DNA phosphodiesterase activity of all three TDP1 mation to a possible in vivo role in cccDNA biogenesis, we asked orthologs and the proposed Tyr-5′-DNA activity of yTDP1 (18) whether the TDPs were able to release P protein from authentic but not huTDP1. viral RC-DNA as present in cell-derived nucleocapsids. The TDP2gaveadifferentpicture(Fig.3F and G). Initial experi- challenge was to make the packaged RC-DNA accessible to ments (Fig. S2B) confirmed the strict bivalent metal ion de- exogenous enzymes under conditions that maintain a soluble P

Königer et al. PNAS Early Edition | 3of10 Downloaded by guest on September 26, 2021 Fig. 3. Differential activities of TDP1 and TDP2 from different species on 5′- and 3′-phosphotyrosyl model substrates. (A) Assay principle. MU as a Tyr mimic was coupled to a 5′-or3′-phosphorylated 14-mer oligonucleotide representing the P-protein–linked 5′ end of DHBV minus-strand DNA (MUP-DNA). Specific cleavage of the nonfluorescent MUP-DNA substrate releases fluorescent MU. (B–G) MU release activity of the indicated TDP proteins. Fluorescence development over time was recorded in a microplate fluorescence reader. Because of the bivalent metal ion dependence of TDP2, but not TDP1 (Fig. S2), TDP2 reactions were performed in the presence of 5 mM Mg2+. In all panels except E, 100 nM protein (filled symbols for wild-type, open symbols for the respective mutants) was reacted with 0.5 μM substrate (indicated by circles for the 3′ substrate and by triangles for the 5′ substrate). In E, protein concentrations were increased to 500 nM, and the observation time was extended to 5.5 h. Substrate conversions are expressed as the percent of the maximal fluorescence signal (in relative fluorescence units, rfu) obtained when the wild-type enzyme reaction with the optimal substrate reached a plateau. Error bars representSD (n = 3). Formal progress curve fitting, serving only illustrative, not analytical purposes, was done by nonlinear regression using GraphPad Prism 5.

protein–DNA complex and allow TDP activity. For DHBV (but DNA 5′ end after treatment with PK only and after treatment not HBV) nucleocapsids we confirmed the previously reported with PK plus TDP2. PK-treated DNA still contains at least the destabilization by incubation at >500 mM NaCl (36); under these DNA-linked Tyr residue, whereas TDP cleavage should result in conditions all TDP enzymes retained substantial activity (Fig. S3A). a free 5′ phosphoryl end. λ 5′→3′ exonuclease is highly active on We therefore incubated cytoplasmic nucleocapsids from DStet5 dsDNA with phosphorylated 5′ ends but is much less active on cells, a chicken LMH cell-derived (TetOFF) line stably trans- ssDNA. Because the 5′ end of plus-strand DNA is at least par- − formed with an envelope-deficient (env ) DHBV genome (37), tially protected by the RNA primer, selective digestion of minus- at 500 mM NaCl with yTDP1, huTDP2, and chTDP2 or their strand DNA in RC-DNA and dsL-DNA should lead to the inactive mutants, or for control with proteinase K (PK) which accumulation of single-stranded plus strands. In contrast, degrades most of the P protein. Without such treatment, which is 5′ aminoacylated minus-strand DNA would be resistant (Fig. applied routinely for Southern blot analyses in HBV research, 6A). The impact of λ exonuclease then was analyzed by Southern the protein-linked viral DNA partitions into the organic phase blotting using strand-specific riboprobes (Fig. 6B). Without upon phenol extraction (38) and is not detected. Release of P huTDP2 treatment, at most a slight increase in single-stranded protein by TDP cleavage should result in a signal increase similar minus-strand and plus-strand DNA was detectable, the latter to that seen with proteolysis (Fig. 5A). possibly arising from the fraction of naturally deproteinized viral Incubation of DHBV RC-DNA with yTDP1, but not with its DNA. In contrast, huTDP2 treatment caused a massive loss of inactive mutant, indeed caused a strong increase in Southern blot double-stranded forms in favor of single-stranded plus-strands. signal as compared with the mock-treated (no PK, no TDP) Hence, as expected, huTDP2 generated minus-strand DNA ends control (Fig. 5B); the weak signals observed in the control rep- from the specific cleavage of the tyrosyl-DNA phosphodiester resent a fraction of already “deproteinized” DNA, which routinely bond connecting P protein and DNA. accounts for 10% of the total viral DNA (39). Importantly, signal enhancement similar to that of PK also was achieved by huTDP2 TDP2 Knockdown Impairs the Formation of Hepadnaviral cccDNA in and chTDP2, but not by their inactive variants (Fig. 5C). Cells. If the biochemically demonstrated ability of vertebrate HBV nucleocapsids obtained from induced cultures of TDP2 and yTDP1 to release P protein from RC-DNA is physi- TetOFF wild-type HBV HepG2.117 cells (40) were refractory ologically relevant, reducing TDP2 expression should negatively to high-salt treatment, but freezing in the presence of 0.01% affect the conversion of RC-DNA to cccDNA in cells. Pre- (wt/vol) ethidium bromide (EB) made the RC-DNA and double- requisites for dependable detection of such an effect were the stranded linear (dsL) DNA amenable to digestion by micrococcal efficient, verifiable silencing of cellular TDP2 and sufficiently nuclease (Fig. S3B), possibly via their increased space requirements high basal levels of cccDNA so that a potential reduction could upon EB intercalation; in line with this notion, ssDNA remained be detected unambiguously by Southern blotting. In addition, be- protected (Fig. S3B). Incubation of HBV nucleocapsids destabilized cause cccDNA, once formed, appears to be highly stable (see ref. in this way with huTDP2 and chTDP2, but not their mutants, 41 and references therein), interfering with conversion should have caused an increase in Southern blot-detectable RC-DNA and the largest impact during the phase of maximal cccDNA pro- dsL-DNA similar to that caused by PK (Fig. 5D), indicating that duction, i.e., upon synchronized induction of RC-DNA formation. the vertebrate TDP2s, as well as yTDP1, can cleave P protein Because of equivocal immunoblot results with a commercial from authentic DHBV and HBV RC-DNA. anti-TDP2 antibody (Sigma AV33010) and similar problems To demonstrate formally the specific cleavage of the phos- reported for antibodies from other sources (42), we first used the photyrosyl bond, we assessed the nature of the minus-strand recombinant huTDP2 and chTDP2 proteins to obtain custom

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1409986111 Königer et al. Downloaded by guest on September 26, 2021 when transfected into Huh7 cells, reduced huTDP2 levels by up PNAS PLUS to 90% (SI Materials and Methods and Fig. S5A). As a model of inducible, human cell-based conversion of RC-DNA to cccDNA, − we exploited the fact that env DHBV generates drastically more cccDNA (39) than does HBV even in human hepatoma cells and − established a stable DHBVenv Huh7 TetOFF cell line. With- drawal of tetracycline (Tet)-initiated virus replication and RC- DNA formation, as expected and in contrast to analogous HBV lines (40), was accompanied by easily detectable accumulation of cccDNA from day 9 to day 18 postinduction (Fig. S6). However, using transient siRNA transfection, we were unable to maintain substantially decreased TDP2 levels for more than 3 d (Fig. S5B), and repeated siRNA transfections were poorly tolerated by the cells. We therefore used stable shRNA-mediated knockdown as alternative. Screening of four anti-huTDP2 and anti-chTDP2 shRNAs predicted by i-Score (44) and transiently expressed from a nonselectable vector (45) identified one particularly efficient target site each (hu526 and ch660; Fig. S7 A and B). Inciden- tally, a selectable vector for a hu526-like shRNA (termed “siTTRAP_T1”) plus one for an shRNA not validated in our experiments (siTTRAP_T4) had been used successfully to knock down TTRAP (i.e., TDP2) in neuronal cells (46). Using these plasmids (kindly provided by S. Gustincich, International School of Advanced Studies, Trieste, Italy), we selected several HepG2 clones with clearly reduced TDP2 levels as compared with naive HepG2 cells (Fig. 7A); clone T4#6, showing an 80–90% re-

duction in TDP2, was used for most subsequent experiments. MICROBIOLOGY Next, T4#6 cells and naive HepG2 cells were transiently transfected with a cytomegalovirus (CMV) promoter-driven expres- − sion vector for DHBV env ; cells were harvested at days 2, 3, and 4 posttransfection, and nuclear viral DNAs were analyzed by Southern blotting. To account for potential variations in transfection efficiency and/or sample loading, the ratio of cccDNA to RC-DNA in the same lane was used to assess the efficiency of the conversion of RC-DNA to cccDNA. As already evident by visual inspection (Fig. 7B), TDP2 depletion markedly slowed the Fig. 4. Both vertebrate TDP2s but only yTDP1 can cleave the P protein–DNA primer complex. (A) Assay principle. Recombinant DHBV P protein was ac- conversion kinetics, with the cccDNA:RC-DNA ratio in the tivated in vitro to bind e RNA, then incubated with [α32P]-dGTP plus dTTP shRNA line being about half that in naive HepG2 cells at day 2 plus dATP to initiate synthesis of the P-protein–linked primer. If P protein is and about two-thirds at day 3 posttransfection; at day 4, the a substrate for TDP, radiolabeling of P protein should be reduced by release difference had leveled off and did not change further. Compa- of the labeled primer. (B–D) Impact on the radiolabeled P protein. Equal rable results in six independent experiments (Fig. 7C) confirmed aliquots of the priming reaction were incubated with the indicated wild-type the significance of the difference between naive and TDP2- (wt) or mutant (mut) proteins or in buffer only (mock); in B, one reaction was depleted cells. Delayed cccDNA formation was not caused by performed in the presence of vanadate (VAN). Reactions were separated by a general effect of the TDP2-knockdown on the cells, because SDS/PAGE, and labeled P protein (plus smaller degradation products) was they had doubling times [1.048 d; 95% confidence interval (CI) visualized by autoradiography. The top bands were quantified by phos- ± 1.019–1.079 d] virtually identical to those of naive HepG2 cells phorimaging. Mean values SD are indicated at the bottom of each panel. – (E–G) Detection of released oligonucleotides. After incubation with the in- (1.052 d; 95% CI 1.019 1.087 d). Furthermore, delayed conver- dicated TDP proteins, labeled nucleic acids were separated by electropho- sion of RC-DNA to cccDNA also was seen in an siTTRAP_T1 resis in 20% polyacrylamide-7 M urea gels. M, 5′-32P–labeled marker DNA shRNA-derived cell clone in which the shRNA targets a different oligos of the indicated sequences and lengths. Bands marked with asterisks site on the huTP2 mRNA. Finally, cotransfection into T4#6 cells − and double asterisks occurred in all reactions and are nonspecific. dGMP was of the DHBVenv vector plus a huTDP2 expression vector, and 32 identified by apyrase treatment of [α P]-dGTP. In E, priming was performed more so a vector encoding a huTDP2 mRNA carrying silent with all three nucleotides required for the authentic primer. In F,[α32P]-dGTP e e mutations in the shRNA target site (SI Materials and Methods), was supplemented with only dTTP or ddTTP. In G, a variant RNA ( b16) increased the cccDNA:RC-RNA ratio as compared with cells with a 16-nt bulge (5′ C in the bulge replaced by 5′ U2GU8) was used as priming template. cotransfected with empty vector, thus corroborating the TDP2- specificity of the effect (Fig. 7D). Altogether, these in-cell experiments established that reducing TDP2 by 80–90% mark- rabbit antibodies (Biogenes) which detected the two TDP2 edly decelerated the conversion of RC-DNA to cccDNA, as proteins with high specificity and sensitivity (SI Materials and expected if TDP2 is involved in the removal of P protein from RC-DNA as one of the mandatory steps in cccDNA formation. Methods and Fig. S4). Notably, in all anti-huTDP2 immunoblots two bands were visible, one at ∼51 kDa and one at ∼37 kDa that Discussion was more pronounced in Huh7 than in HepG2 cells; similar The central role of cccDNA in enabling productive infection by observations have been made recently in other cell lines 31, 43). and persistence of hepadnaviruses is long established, but the The smaller product likely represents an N-terminally shortened mechanism by which cccDNA is formed from the incoming but enzymatically active form (Discussion). Next, we confirmed protein-bound RC-DNA genome remained elusive. Our in vitro that one of four commercial anti-huTDP2 siRNAs (Qiagen), si 9, data firmly establish that two distant vertebrate orthologs of

Königer et al. PNAS Early Edition | 5of10 Downloaded by guest on September 26, 2021 reported (18), only yTDP1 was highly active on the 5′ substrate (Fig. 3). An obvious difference between yTDP1 and the vertebrate enzymes is the shorter N-terminal domain of yTDP1 (Fig. 2). However, deletion of the first 39 or 149 residues from recombinant huTDP1 did not unleash detectable activity in the 5′ substrate (48). The low but apparently specific 5′ activity of chTDP1 but not huTDP1 (Fig. 3E) indicates that conclusions obtained in avian systems, e.g., DT40 cells, may not apply strictly to mammalian cells. Taken together, however, these data made a role for TDP1 in HBV cccDNA formation unlikely. A remaining caveat is that in vivo TDP1 substrate specificity may be modu- lated by posttranslational modifications or by protein-interaction partners such as XRCC1 and PARP1 (49). For huTDP2 and chTDP2, the assay confirmed a strictly bivalent metal ion-dependent 5′-aswellasalower3′-phosphotyrosyl cleavage activity (Fig. 3 F and G), in accord with recent reports (20, 26, 35). Given the interest in TDP2 inhibitors for cancer therapy (13), we note that our fluorescent TDP2 assay should be easily adaptable to high-throughput applications with potential advantages over assays using low-affinity chromogenic substrates (42, 50). All TDPs active on the 5′-model substrate also were able to Fig. 5. Vertebrate TDP2 and yTDP1 release P protein from authentic RC- cleave the covalent P protein–oligonucleotide complex formed DNA. (A) Assay principle. P-protein–linked viral DNA partitions into phenol during in vitro priming. Less than complete loss of label from the unless the bulk of the protein is digested by PK or is released by TDP. Only protein (Fig. 4A) might indicate that a fraction of the P-protein the water-soluble DNA is detected by Southern blotting. (B–D) The amount of water-soluble viral DNA is increased upon incubation with wild-type but molecules were present as larger aggregates in which the scissile not mutant TDP proteins. Cell-derived DHBV or HBV nucleocapsids were permeabilized (SI Materials and Methods and Fig. S3) and incubated with the indicated TDP proteins, with PK, or with buffer only (mock). After phenol extraction, DNAs were detected by Southern blotting using 32P-labeled DHBV or HBV DNA probes. The low signals in the mock reactions reflect a small fraction of RC-DNA that naturally lacks intact P protein. Numbers below panels C and D indicate mean RC-DNA signal intensities ± SD (n = 3) relative to intensities after PK treatment, which were set to 100%.

TDP2, but only the yeast ortholog of TDP1, have the proper specific enzymatic activity to release P protein from DHBV and HBV RC-DNA. Furthermore, TDP2 depletion significantly decelerated the conversion of RC-DNA to cccDNA in cells; this observation is consistent with this DNA repair enzyme catalyzing an obligate step in the process. Given the biochemical data, this step is P protein release from the RC-DNA. Although we are only beginning to decipher the complete pathway from RC-DNA to cccDNA, our study provides the first experimental evidence, to our knowledge, that hepadnaviruses interact functionally with the cellular DNA repair machinery; given the multiple modifications RC-DNA must undergo for conversion into cccDNA, it is likely that this interaction is much more extensive.

Biochemical Evidence for a Role of Vertebrate TDP2 but Not TDP1 in P Protein Release from Hepadnaviral RC-DNA. Cleavage of the P protein–RC-DNA linkage requires a 5′-phosphotyrosyl bond– specific activity. Hence TDP2 was a prime candidate (26, 27), but Fig. 6. TDP2-mediated release of P protein from RC-DNA is caused by potential species-specific differences (18) and partly ambiguous specific phosphotyrosyl bond cleavage. (A) Assay principle. Viral DNA with 5′-phosphorylated minus-strand ends (TDP2 pathway) but not literature data (19) did not exclude TDP1 a priori. Further- 5′-aminoacylated minus-strand ends (PK pathway) is an efficient substrate more, the peculiarities of the hepadnaviral RC-DNA struc- for 5′→3′ λ exonuclease; plus-strand 5′ ends are protected by the RNA ture, including the large size of P protein (around 90 kDa), primer. Specific phosphotyrosyl bond cleavage should result in the accu- could affect whether one enzyme or the other is capable of mulation of free plus-strands. (B) TDP2-treated viral DNA is sensitive to λ specific P-protein release. exonuclease. Viral DNA from cell-derived DHBV nucleocapsids was treated The fluorescence-based MUP-DNA assay allowed the 5′ and with PK only or with PK and subsequently with huTDP2. Thereafter, DNAs 3′ activities of numerous enzyme preparations to be compared were incubated with λ exonuclease, and the products were detected by – – simultaneously. Although deriving enzyme kinetic parameters Southern blotting, using either a minus-strand specific (Left) or plus-strand from progress curves is complex (47), this straightforward anal- specific (Right) riboprobe. A marked increase in single-stranded plus-strand DNA was seen only upon TDP2 treatment. All lanes within each panel are ysis excluded the possibility that inactivity on one substrate was derived from the same exposure of the same blot, but the order of lanes has caused by general inactivity, and it revealed relative quantitative been rearranged (indicated by the dotted separation lines) to facilitate differences. All three TDP1 orthologs exerted high, bivalent a direct comparison of the impact of the nuclease on TDP2-treated versus metal ion–independent activity on the 3′ substrate, but, as not TDP2-treated DNA. See text for further details.

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1409986111 Königer et al. Downloaded by guest on September 26, 2021 (27, 54). A plausible explanation is the exposed position of the PNAS PLUS hepadnaviral phosphotyrosyl bond on the TP domain, which must be flexible enough to slide the pertinent Tyr residue into P protein’s active site during priming. Finally, the most compelling in vitro evidence for TDP2 as a potential cccDNA-relevant host factor comes from the ability of huTDP2 and chTDP2 to release P protein from authentic DHBV and HBV RC-DNA, as indicated by a similarly strong increase in water-soluble (i.e., non–protein-linked) viral DNA as that achieved by deliberate proteolysis (Fig. 5). That yeast TDP1 had the same capability is unlikely to be relevant for hepadnaviral infection. Why nucleocapsid-borne DHBV DNA was easily exposed by high-salt treatment but HBV DNA was not (Fig. S3) remains to be determined; although the two core proteins differ substantially in length and sequence, the capsids share a common architecture (55, 56). However, freezing in the presence of EB provided a novel way of permeabilizing HBV capsids. The preferential sensitivity of dsDNA containing HBV nucleocapsids and the comparable impact on DHBV Fig. 7. Stable TDP2 knockdown in HepG2 cells significantly delays the nucleocapsids support the view that EB, sufficiently small to conversion of RC-DNA to cccDNA. (A) Reduced huTDP2 levels in stable TDP2- knockdown HepG2 cell clones. TDP2 levels (bands marked with white diffuse into capsids (57), intercalates into the packaged DNA, asterisks) in naive HepG2 cells versus several HepG2 clones selected after increases its space requirements, and thus destabilizes the capsid transfection with a vector encoding shRNA si_TTRAP_4 were monitored by shell from within. immunoblotting using rabbit anti-huTDP2 antiserum #6152 (SI Materials and Methods and Fig. S4). The black asterisk indicates a nonspecific signal. (B) In-Cell Evidence for TDP2 Involvement in Hepadnaviral cccDNA TDP2 depletion slows the conversion of RC-DNA to cccDNA. Nuclear viral Formation. Given the multistep nature of the RC-DNA–to– DNAs from naive or TDP2-depleted HepG2 cells transfected with the − cccDNA conversion (Fig. 1C), blocking any of the intermediate DHBVenv vector harvested at the indicated days posttransfection were steps by inactivating the responsible factor(s) should prevent the MICROBIOLOGY analyzed by Southern blotting. RC-DNA and cccDNA signals were quantified accumulation of cccDNA. Consistently, in HepG2 cell clones by phosphorimaging. For each time point, the ratio of cccDNA to RC-DNA in – the knockdown cells was compared with that in the naive cells (set at 100%); stably transfected with the anti huTDP2-shRNA vector and the resulting relative ratios ± SD indicated at the bottom are derived from C. endurably expressing 80–90% lower TDP2 levels than the pa- (C) Statistical significance. Individual relative cccDNA:RC-DNA ratios in TDP2 rental cells, the conversion of RC-DNA to cccDNA was slowed knockdown vs. naive HepG2 cells on days 2, 3, and 4 posttransfection from significantly (Fig. 7C); eventually, the relative and total amounts six independent experiments are shown as scatter plots. The horizontal bar of the two DNA forms stabilized at similar levels in both cell represents the median. P values were derived by a paired two-tailed t test. backgrounds. Comparable results in knockdown cells with a dif- (D) Ectopic TDP2 expression restores faster conversion of RC-DNA to cccDNA. TDP2-knockdown cells were cotransfected with the DHBVenv− vector plus ferent target site on the TDP2 mRNA and the recovery of faster pcDNA3.1 expression vectors for wild-type huTDP2 (wt), a variant with mu- conversion rates upon cotransfection with vectors for ectopic tated shRNA target site (sh-res), or empty vector (Ø). RC-DNA and cccDNA huTDP2 expression (Fig. 7D) made off-target effects unlikely. were analyzed on day 3 posttransfection as in B. Relative cccDNA:RC-DNA Furthermore, TDP2 depletion had no negative impact on cell ratios in cells transfected with theTDP2 expression vector and in cells viability or duplication rate. transfected with empty vector (set at 100%), ± SD, are indicated at the bottom (n = 3).

bond was sterically shielded; the notorious problems in keeping recombinant P protein soluble (9, 51) support this view. Cleavage specificity was demonstrated directly by the detectability of labeled oligonucleotides upon incubation with the wild-type but not the mutant TDP enzymes (Fig. 4 E–G). Their wider length distribution (up to six or seven residues) (Fig. 1B) compared with the 3 or 4 nt expected from genetic analyses might indicate that in vitro priming is less specific per se, although our data suggest that the authentic e bulge template is used in vitro also. Because mutational impairment of primer transfer in cells facilitates repetitive copying of the same bulge template nucleotides (52), Fig. 8. Summary model and clinical implications. At normal levels, TDP2 such polymerase slipping also may occur in vitro. Furthermore, efficiently releases P protein from RC-DNA (thick black arrow), and P-pro- e tein–free RC-DNA is processed further by other factors of the host DNA- -independent terminal transferase activity recently reported for repair machinery to yield cccDNA. P-protein release is not necessarily the first HBV P protein expressed in mammalian cells (53) might con- step in the pathway. In TDP2-depleted cells (smaller gray arrow), P-protein tribute to the longer-than-expected priming products. Alterna- release becomes rate limiting, feeding less P-protein–free RC-DNA per time tively, oversized primers also may form in vivo but escape into the processing pathway and slowing the accumulation of cccDNA. detection by assays that rely on prior transfer to DR1* (Fig. 1B). Eventual formation of cccDNA levels similar to those in normal cells could be Of practical importance, yTDP1 and the vertebrate TDP2s lend caused by residual TDP2 or, alternatively, by nucleolytic pathways. Clinically themselves as tools to clarify these issues further. used NAs inhibit reverse transcription of cccDNA-derived pgRNA but not cccDNA formation and pgRNA production. Inhibition of TDP2 or other More relevant here, TDP2 (and yTDP1) could accept the – cccDNA-relevant host factors should provide a strategy for targeting the entire P protein oligonucleotide complex as substrate, without HBV persistence reservoir directly. A critical unresolved issue is whether the prior proteasome-mediated proteolysis which appears required reported longevity of cccDNA holds on the individual-molecule level or reflects for efficient TDP-based repair of topoisomerase–DNA adducts turnover and replenishment via intracellular amplification or infection.

Königer et al. PNAS Early Edition | 7of10 Downloaded by guest on September 26, 2021 Combined with the biochemical data, a straightforward inter- in circulating virions by inhibiting the reformation of RC-DNA pretation, summarized in Fig. 8, is that in the TDP2-depleted (3), but transcription from cccDNA continues (Fig. 8). Com- cells TDP2-dependent release of P protein from RC-DNA becomes pletely purging cccDNA from a patient´s liver by this approach rate limiting. Once the relative cccDNA:RC-DNA levels are equal could take >50 y (62). Silencing the transcriptional activity of those in naive HepG2 cells, the same unknown mechanism that cccDNA (63) may suffer the same limitations. Our data suggest limits further expansion of the cccDNA pool takes effect. These inactivation of TDP2, or any other host DNA-repair factor in- data strongly suggest that TDP2 is a host DNA-repair factor volved in the conversion of RC-DNA to cccDNA, as a means of usurped by hepadnaviruses to generate cccDNA, which they interrupting the formation of the HBV persistence reservoir need to establish and maintain infection. (Fig. 8). Targeting host factors has become a valid option in drug However, the data leave room for more complex scenarios. development (64), and TDP2 inhibitors already are in de- First, the pleiotropic functions of TDP2 (25) could affect the velopment to enhance the efficacy of TOP poisons in cancer conversion of RC-DNA to cccDNA indirectly; we consider this therapy (13). However, several issues must be resolved before possibility unlikely, because RC-DNA is a proven substrate the conversion of RC-DNA to cccDNA can be considered a valid for TDP2’s tyrosyl-DNA phosphodiesterase activity. A second target for chronic hepatitis B treatment. One is the redundancy issue is whether alternative nucleolytic pathways such as those in DNA repair; if the redundancy is fully exploitable by HBV, safeguarding cells from TDP2 failure (58) could step in when inhibiting a single repair factor would have little impact. Con- TDP2 is limiting (Fig. 8). Slow formation of cccDNA in our versely, if the virus were restricted to just one of several repair TDP2-knockdown cells could be caused by residual TDP2 but pathways, redundancy should minimize adverse effects on the also could reflect a contribution by alternative pathway(s); one cell. A second key issue is whether the longevity of cccDNA (ref. candidate would be the MRE11/RAD50/NBS (MRN) complex, 41 and references therein) holds on the single cccDNA molecule whichisimplicatedinbothTOP1-andTOP2-DNAadduct level or instead results from replenishment by low-level intra- resolution (23) and (detrimentally to the virus) in removing the cellular amplification or infection (Fig. 8) because suppression of TP from the double-stranded adenovirus genome (32). To dis- reverse transcription is not absolute. Without any turnover, tinguish among these possibilities, we attempted to knock out blocking the conversion of RC-DNA to cccDNA would be re- TDP2 in Huh7 cells, using transcription activator-like effector stricted to prophylactic applications; if turnover occurs, such a nucleases targeting the region immediately downstream of the blockade could be turned into a relevant complement to current initiator ATG specified by the TDP2 mRNA (RefSeq accession therapies. As recently reported, high doses of IFN-α or, more no. NM_016614.2). However, in corresponding cell clones only efficiently, activation of the lymphotoxin-β receptor can induce the larger of the two anti-TDP2 reactive bands (Figs. S4B, S5 cccDNA degradation experimentally (65), but about one third of B and C, and S6A) was deleted, as is consistent with the smaller the cccDNA remained refractory, available for later ream- form representing an N-terminally truncated protein. In line plification. Hence further investigation into how cccDNA is with the absence of catalytic residues in the deleted region formed appears highly warranted. (Fig. 2B), a corresponding recombinant protein was active in the MUP-DNA assay. Hence the knockout cells were not func- Materials and Methods tionally TDP2 deficient. Cell Culture and Transfection. Huh7, HepG2, and LMH cells were maintained in In any case, the multiple structural peculiarities of RC-DNA DMEM F12 (PAA Laboratories) supplemented with 10% FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C and 5% CO2. Media for the (Fig. 1C) indicate that additional host DNA-repair factors are − involved in cccDNA conversion (Fig. 8). Examples are structure- TetOFF Dstet5 DHBVenv (37) and HBV HepG2.117 lines (40) contained in μ μ specific endonucleases (59) involved in the removal of the ter- addition 200 g/mL G418 and 80 g/mL hygromycin to maintain the in- minal redundancy from minus-strand DNA or the RNA primer tegrated Tet-transactivator (tTA) and Tet-responsive element (TRE) promoter-controlled virus expression cassettes. Expression was repressed by from plus-strand DNA, or the involvement of host DNA 1 μg/mL of doxycycline (DOX) and was induced by its withdrawal. Trans- ligases in catalyzing the final step of cccDNA formation. Other fections of plasmid DNA and siRNA were performed using TransIT-LT1 such factors may be identified only by more comprehensive (Mirus) and HiPerFect transfection reagent (Qiagen), respectively, as rec- approaches, such as RNAi-based screening. Even small RNAs ommended by the manufacturers. recently have been directly linked to DNA repair (60). Hence − it is more than likely the interaction between hepadnaviruses Expression Vectors. The DHBVenv vector (39) and plasmid pET23b-yTDP1 and cellular DNA repair involves more than TDP2 alone. (18), kindly provided by J. Nitiss (University of Illinois at Chicago, Chicago), have been described previously. Coding sequences for huTDP1 (NM_001008744.1) Virological Implications. Although the release of P protein from and huTDP2 (NM_016614.2) were amplified from sequence-verified cDNA RC-DNA is evidently beneficial to hepadnaviruses, TDP’sability clones (Source Bioscience); those for chTDP1 (XM_421313.4) and chTDP2 to clip off the short oligonucleotide generated during protein (NM_001276384.1) were from an LMH cell cDNA library. PCR primers provided terminal restriction sites for cloning into the prokaryotic expression priming (Fig. 4) also poses the threat of premature termination vector pET30a(+) or the eukaryotic vector pcDNA3.1, as detailed in SI of replication, especially because TDP2 is not a strictly nuclear Materials and Methods. Enzymatically inactivated mutants (Fig. 2) were protein (25). The typical hepadnavirus feature of restricting the obtained by conventional mutagenic PCR. bulk of reverse transcription to the inside of intact cytoplasmic nucleocapsids which then deliver the RC-DNA to the nuclear Recombinant TDP Expression. Wild-type and mutant TDP proteins were pore for release into the nucleoplasm (61) appears to be an expressed in E. coli BL21-CodonPlus cells (Stratagene) and purified by se- appropriate strategy for avoiding such untimely encounters. If quential immobilized metal ion affinity chromatography and size-exclusion so, the most dangerous period would be the period immediately chromatography, as detailed in SI Materials and Methods. Representative after P protein has bound to e, triggering both pgRNA encap- purifications are shown in Fig. S1. sidation and replication initiation (5, 6). The order of these events is unknown, but initiating assembly before reverse tran- Fluorescent TDP Activity Assay. MUP-DNA substrates were obtained from – a commercial service (IBA GmbH) by carbodiimid-mediated condensation of scription would protect the replication initiation complex from 4-MU to a 3′-phosphorylated (33) or a 5′-phosphorylated (this work) DNA inappropriate TDP2 activity. oligo of the sequence 5′-GTAATTCTTAAGTTG. Cleavage assays (total vol- ume, 200 μL) were performed in 96-well flat-bottom plates in MUP-DNA Medical Implications. The persistence of cccDNA under current buffer [50 mM Tris·HCl (pH 8.0), 80 mM KCl, 2 mM EDTA, 1 mM DTT, anti-HBV treatments is the key obstacle for eventually curative 40 μg/mL BSA, 10% DMSO] and 0.5 μM MUP-DNA, usually plus 100 nM TDP. therapies. The most potent NAs achieve several-log reductions For TDP2, the buffer was supplemented with 5 mM Mg2+ (Fig. S2). Time-resolved

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1409986111 Königer et al. Downloaded by guest on September 26, 2021 fluorescence was monitored in a Genios Pro Microplate Reader (Tecan) at 37 °C Nuclear Viral DNA. Transfected cells were lysed in Nonidet P-40 lysis buffer, PNAS PLUS using a 355-nm excitation filter and a 460-nm emission filter. and the nuclei were separated by low-speed centrifugation as previously described (39). Nuclear DNAs were prepared using the QiaAmp Blood Mini P Protein–Oligonucleotide Primer Cleavage Assays. In vitro priming was per- Kit (Qiagen) according to the manufacturer´s tissue protocol for subsequent formed as previously described (9, 10) using recombinant GrpE-fused DHBV P Southern blotting. protein and Hsc70 and Hsp40; in vitro transcribed DHBV e RNA or a mutant containing an extended bulge region (e b16) served as templates. Primer Southern Blotting. Southern blotting was performed as previously described 32 synthesis was induced by adding [α32P]-dGTP (3,000 Ci/mMol; Perkin-Elmer) (39). P-labeled DNA probes were obtained by random priming using and various combinations of other dNTPs. Equal aliquots of the reactions cloned full-length DHBV or HBV genomes as templates. Strand-specific were incubated with the respective TDP proteins at 1.5-μM concentration at riboprobes were obtained by in vitro transcription from a pBluescript plasmid containing the complete DHBV genome between T7 and T3 RNA 37 °C for 30 min; thereafter, one-half of each sample was incubated with polymerase promoters. SDS sample buffer for SDS/PAGE and detection of 32P-labeled P protein. The other half was incubated with formamide gel buffer (95% formamide, Validation of TDP2 Knockdown Efficiency. Rabbit polyclonal anti-TDP2 antisera 20 mM EDTA) for detection of clipped-off 32P-labeled oligonucleotides were obtained from a commercial service (Biogenes) using recombinant by urea-PAGE [7M urea, 20% acrylamide-bisacrylamide (19:1), 0.5× Tris- huTDP2 and chTDP2 as immunogens. Specificity and sensitivity of TDP2 borate-EDTA]. Radiolabeled products were visualized by autoradiography detection were determined as detailed in Fig. S4. Using these antisera, or phosphorimaging (Typhoon FLA 7000, GE Healthcare) and quantified TDP2 levels in Huh7 cells transiently transfected with four commercial using ImageQuant software. huTDP2-specific and two control siRNAs (Qiagen) were evaluated as detailed in Fig. S5. Efficient target sites for shRNAs on huTDP2 and RC-DNA Susceptibility for TDP. Conditions for exposing packaged viral DNAs chTDP2 mRNA were identified analogously after transient transfection were established by incubating cytoplasmic nucleocapsids from induced with human H1 promoter-driven shRNA vectors (45), as detailed in cultures of Dstet5 or HepG2.117 cells under various conditions and assessing Fig. S7. their resistance or sensitivity toward micrococcal nuclease, as detailed in SI Materials and Methods and Fig. S3. The TDP-mediated release of P protein Stable Cell Lines. A Tet-regulatable DHBVenv−-expressing Huh7 TetOFF cell from RC-DNA was assessed by incubation with 50 nM wild-type or mu- clone was obtained by transfection of the tTA-containing line Huh7.TA61 − tant TDP2 or 30 nM yTDP1 for 1 h at 37 °C, followed by phenol extraction (40) with a pTRE2hyg–based (Clontech) vector producing DHBVenv pgRNA and ethanol precipitation of the water-soluble DNA and subsequent under the control of a Tet-responsive promoter and selection with Southern blotting. hygromycin. RC-DNA and cccDNA induction by DOX withdrawal are shown in Fig. S6. Stably huTDP2-silenced HepG2 cell clones were obtained by

Susceptibility of huTDP2-Treated RC-DNA for λ 5′→3′ Exonuclease. Cytoplasmic transfection of pSuperior.neo+GFP–based vectors (Oligoengine) encoding MICROBIOLOGY DHBV nucleocapsids from Dstet5 cells were treated with 0.5 mg/mL PK for 1 h the shRNAs siTTRAP_1 and siTTRAP_4 (46), enrichment of transfected cells by + at 37 °C. After phenol extraction and ethanol precipitation, DNA from the sorting for GFP , and G418 selection. + aqueous phase was incubated in Mg2 -supplemented MUP-DNA buffer with 25 nM huTDP2 for 2 h at 37 °C. After a second round of phenol extraction ACKNOWLEDGMENTS. We thank Dr. S. Gustincich for selectable pSuperior vectors encoding shRNAs siTTRAP_1 and siTTRAP_4; Dr. J. Nitiss for vector and ethanol precipitation, the DNA was dissolved in 10 μL water plus 1 μL pET23b-yTDP1; and Prof. Dr. Hubert E. Blum for his continuous support. This × λ λ 10 -exonuclease-buffer and was treated with 2.5 U exonuclease (New work was supported by the Deutsche Forschungsgemeinschaft Grant Na154/ England Biolabs) for 2 h at 37 °C. Products were analyzed by Southern 12-1,2 within the Collaborative Research Unit 1202 (Persistence of Hepato- blotting using minus-strand– or plus-strand–specific DHBV riboprobes. tropic Viruses).

1. Ott JJ, Stevens GA, Groeger J, Wiersma ST (2012) Global epidemiology of hepatitis 17. Yang SW, et al. (1996) A eukaryotic enzyme that can disjoin dead-end covalent B virus infection: New estimates of age-specific HBsAg seroprevalence and endemic- complexes between DNA and type I topoisomerases. Proc Natl Acad Sci USA 93(21): ity. Vaccine 30(12):2212–2219. 11534–11539. 2. Ganem D, Prince AM (2004) Hepatitis B virus infection—natural history and clinical 18. Nitiss KC, Malik M, He X, White SW, Nitiss JL (2006) Tyrosyl-DNA phosphodiesterase consequences. N Engl J Med 350(11):1118–1129. (Tdp1) participates in the repair of Top2-mediated DNA damage. Proc Natl Acad Sci 3. Zoulim F, Locarnini S (2009) Hepatitis B virus resistance to nucleos(t)ide analogues. USA 103(24):8953–8958. Gastroenterology 137(5):1593–1608 e1591-1592. 19. Murai J, et al. (2012) Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs DNA damage 4. Scaglione SJ, Lok AS (2012) Effectiveness of hepatitis B treatment in clinical practice. induced by topoisomerases I and II and base alkylation in vertebrate cells. J Biol Chem – Gastroenterology 142(6):1360–1368 e1361. 287(16):12848 12857. 5. Beck J, Nassal M (2007) Hepatitis B virus replication. World J Gastroenterol 13(1): 20. Zeng Z, et al. (2012) TDP2 promotes repair of topoisomerase I-mediated DNA damage – 48–64. in the absence of TDP1. Nucleic Acids Res 40(17):8371 8380. 6. Nassal M (2008) Hepatitis B viruses: Reverse transcription a different way. Virus Res 21. Pommier Y, et al. (2006) Repair of topoisomerase I-mediated DNA damage. Prog – 134(1-2):235–249. Nucleic Acid Res Mol Biol 81:179 229. 7. Hu J, Anselmo D (2000) In vitro reconstitution of a functional duck hepatitis B virus 22. Maede Y, et al. (2014) Differential and common DNA repair pathways for topoisomerase I- and II-targeted drugs in a genetic DT40 repair cell screen panel. Mol reverse transcriptase: Posttranslational activation by Hsp90. JVirol74(24):11447–11455. Cancer Ther 13(1):214–220. 8. Beck J, Nassal M (2001) Reconstitution of a functional duck hepatitis B virus replica- 23. Hartsuiker E, Neale MJ, Carr AM (2009) Distinct requirements for the Rad32(Mre11) tion initiation complex from separate reverse transcriptase domains expressed in nuclease and Ctp1(CtIP) in the removal of covalently bound topoisomerase I and II Escherichia coli. J Virol 75(16):7410–7419. from DNA. Mol Cell 33(1):117–123. 9. Beck J, Nassal M (2003) Efficient Hsp90-independent in vitro activation by Hsc70 and 24. Nakamura K, et al. (2010) Collaborative action of Brca1 and CtIP in elimination of Hsp40 of duck hepatitis B virus reverse transcriptase, an assumed Hsp90 client protein. covalent modifications from double-strand breaks to facilitate subsequent break re- J Biol Chem 278(38):36128–36138. pair. PLoS Genet 6(1):e1000828. 10. Stahl M, Retzlaff M, Nassal M, Beck J (2007) Chaperone activation of the hepadnaviral 25. Li C, Sun SY, Khuri FR, Li R (2011) Pleiotropic functions of EAPII/TTRAP/TDP2: Cancer reverse transcriptase for template RNA binding is established by the Hsp70 and development, chemoresistance and beyond. Cell Cycle 10(19):3274–3283. – stimulated by the Hsp90 system. Nucleic Acids Res 35(18):6124 6136. 26. Cortes Ledesma F, El Khamisy SF, Zuma MC, Osborn K, Caldecott KW (2009) A human 11. Tuttleman JS, Pourcel C, Summers J (1986) Formation of the pool of covalently closed 5′-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA dam- – circular viral DNA in hepadnavirus-infected cells. Cell 47(3):451 460. age. Nature 461(7264):674–678. 12. Chen SH, Chan NL, Hsieh TS (2013) New mechanistic and functional insights into DNA 27. Caldecott KW (2012) Tyrosyl DNA phosphodiesterase 2, an enzyme fit for purpose. – topoisomerases. Annu Rev Biochem 82:139 170. Nat Struct Mol Biol 19(12):1212–1213. 13. Pommier Y (2013) Drugging topoisomerases: Lessons and challenges. ACS Chem Biol 28. Schellenberg MJ, et al. (2012) Mechanism of repair of 5′-topoisomerase II-DNA ad- 8(1):82–95. ducts by mammalian tyrosyl-DNA phosphodiesterase 2. Nat Struct Mol Biol 19(12): 14. Sancar A, Lindsey-Boltz LA, Unsal-Kaçmaz K, Linn S (2004) Molecular mechanisms of 1363–1371. mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 73: 29. Shi K, et al. (2012) Structural basis for recognition of 5′-phosphotyrosine adducts by 39–85. Tdp2. Nat Struct Mol Biol 19(12):1372–1377. 15. Deriano L, Roth DB (2013) Modernizing the nonhomologous end-joining repertoire: 30. Zeng Z, Cortés-Ledesma F, El Khamisy SF, Caldecott KW (2011) TDP2/TTRAP is the Alternative and classical NHEJ share the stage. Annu Rev Genet 47:433–455. major 5′-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for 16. Huang SN, Pommier Y, Marchand C (2011) Tyrosyl-DNA Phosphodiesterase 1 (Tdp1) cellular resistance to topoisomerase II-induced DNA damage. J Biol Chem 286(1): inhibitors. Expert Opin Ther Pat 21(9):1285–1292. 403–409.

Königer et al. PNAS Early Edition | 9of10 Downloaded by guest on September 26, 2021 31. Virgen-Slane R, et al. (2012) An RNA virus hijacks an incognito function of a DNA 48. Königer C (2014) Impact of cellular DNA repair factors on formation of the covalently repair enzyme. Proc Natl Acad Sci USA 109(36):14634–14639. closed circular (ccc) DNA of hepatitis B viruses. PhD thesis (University of Freiburg, 32. Weitzman MD, Lilley CE, Chaurushiya MS (2010) Genomes in conflict: Maintaining Freiburg, Germany). genome integrity during virus infection. Annu Rev Microbiol 64:61–81. 49. Das BB, et al. (2014) PARP1-TDP1 coupling for the repair of topoisomerase I-induced 33. Rideout MC, Raymond AC, Burgin AB, Jr (2004) Design and synthesis of fluorescent DNA damage. Nucleic Acids Res 42(7):4435–4449. substrates for human tyrosyl-DNA phosphodiesterase I. Nucleic Acids Res 32(15): 50. Adhikari S, et al. (2011) Development of a novel assay for human tyrosyl DNA 4657–4664. phosphodiesterase 2. Anal Biochem 416(1):112–116. 34. Gao R, Huang SY, Marchand C, Pommier Y (2012) Biochemical characterization of 51. Vörös J, et al. (2014) Large-scale production and structural and biophysical charac- – human tyrosyl-DNA phosphodiesterase 2 (TDP2/TTRAP): A Mg(2+)/Mn(2+)-dependent terizations of the human hepatitis B virus polymerase. J Virol 88(5):2584 2599. phosphodiesterase specific for the repair of topoisomerase cleavage complexes. J Biol 52. Nassal M, Rieger A (1996) A bulged region of the hepatitis B virus RNA encapsidation Chem 287(36):30842–30852. signal contains the replication origin for discontinuous first-strand DNA synthesis. – 35. Jones SA, Boregowda R, Spratt TE, Hu J (2012) In vitro epsilon RNA-dependent protein J Virol 70(5):2764 2773. priming activity of human hepatitis B virus polymerase. J Virol 86(9):5134–5150. 53. Jones SA, Hu J (2013) Protein-primed terminal transferase activity of hepatitis B virus – 36. Radziwill G, Zentgraf H, Schaller H, Bosch V (1988) The duck hepatitis B virus DNA polymerase. J Virol 87(5):2563 2576. 54. Interthal H, Champoux JJ (2011) Effects of DNA and protein size on substrate cleavage polymerase is tightly associated with the viral core structure and unable to switch to by human tyrosyl-DNA phosphodiesterase 1. Biochem J 436(3):559–566. an exogenous template. Virology 163(1):123–132. 55. Kenney JM, von Bonsdorff CH, Nassal M, Fuller SD (1995) Evolutionary conservation in 37. Guo JT, et al. (2003) Conditional replication of duck hepatitis B virus in hepatoma the hepatitis B virus core structure: Comparison of human and duck cores. Structure cells. J Virol 77(3):1885–1893. 3(10):1009–1019. 38. Gerlich WH, Robinson WS (1980) Hepatitis B virus contains protein attached to the 56. Nassal M, et al. (2007) A structural model for duck hepatitis B virus core protein de- 5′ terminus of its complete DNA strand. Cell 21(3):801–809. rived by extensive mutagenesis. J Virol 81(23):13218–13229. 39. Köck J, et al. (2010) Generation of covalently closed circular DNA of hepatitis B viruses 57. Birnbaum F, Nassal M (1990) Hepatitis B virus nucleocapsid assembly: Primary struc- via intracellular recycling is regulated in a virus specific manner. PLoS Pathog 6(9): ture requirements in the core protein. J Virol 64(7):3319–3330. e1001082. 58. Nitiss JL, Nitiss KC (2013) Tdp2: A means to fixing the ends. PLoS Genet 9(3):e1003370. 40. Sun D, Nassal M (2006) Stable HepG2- and Huh7-based human hepatoma cell lines for 59. Tsutakawa SE, Lafrance-Vanasse J, Tainer JA (2014) The cutting edges in DNA repair, – efficient regulated expression of infectious hepatitis B virus. J Hepatol 45(5):636 645. licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, 41. Reaiche-Miller GY, et al. (2013) Duck hepatitis B virus covalently closed circular DNA cut once. DNA Repair (Amst) 19:95–107. – appears to survive hepatocyte mitosis in the growing liver. Virology 446(1-2):357 364. 60. d’Adda di Fagagna F (2014) A direct role for small non-coding RNAs in DNA damage 42. Thomson G, et al. (2013) Generation of assays and antibodies to facilitate the study of response. Trends Cell Biol 24(3):171–178. ′ – human 5 -tyrosyl DNA phosphodiesterase. Anal Biochem 436(2):145 150. 61. Kann M, Schmitz A, Rabe B (2007) Intracellular transport of hepatitis B virus. World 43. Li C, et al. (2011) Oncogenic role of EAPII in lung cancer development and its acti- J Gastroenterol 13(1):39–47. – vation of the MAPK-ERK pathway. Oncogene 30(35):3802 3812. 62. Chevaliez S, Hézode C, Bahrami S, Grare M, Pawlotsky JM (2013) Long-term hepatitis 44. Ichihara M, et al. (2007) Thermodynamic instability of siRNA duplex is a prerequisite B surface antigen (HBsAg) kinetics during nucleoside/nucleotide analogue therapy: for dependable prediction of siRNA activities. Nucleic Acids Res 35(18):e123. Finite treatment duration unlikely. J Hepatol 58(4):676–683. 45. Sun D, Rösler C, Kidd-Ljunggren K, Nassal M (2010) Quantitative assessment of the 63. Levrero M, et al. (2009) Control of cccDNA function in hepatitis B virus infection. antiviral potencies of 21 shRNA vectors targeting conserved, including structured, J Hepatol 51(3):581–592. hepatitis B virus sites. J Hepatol 52(6):817–826. 64. Zeisel MB, Lupberger J, Fofana I, Baumert TF (2013) Host-targeting agents for pre- 46. Zucchelli S, et al. (2009) Aggresome-forming TTRAP mediates pro-apoptotic properties vention and treatment of chronic hepatitis C - perspectives and challenges. J Hepatol of Parkinson’s disease-associated DJ-1 missense mutations. Cell Death Differ 16(3):428–438. 58(2):375–384. 47. Nikolova N, Tenekedjiev K, Kolev K (2008) Uses and misuses of progress curve analysis 65. Lucifora J, et al. (2014) Specific and nonhepatotoxic degradation of nuclear hepatitis in enzyme kinetics. Cent Eur J Biol 3(4):345–350. B virus cccDNA. Science 343(6176):1221–1228.

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