1 Extensive mutagenesis of the conserved box E motif in duck ...

1

Extensive mutagenesis of the conserved box E motif in duck hepatitis B virus 1

P protein reveals multiple functions in replication and a common structure with 2

the primer grip in HIV-1 reverse transcriptase 3

4

Yong-Xiang Wang1,2, Cheng Luo3, Dan Zhao3, Jürgen Beck1, Michael Nassal1,# 5

6 1University Hospital Freiburg, Internal Medicine II/Molecular Biology, Hugstetter 7

Strasse 55, Freiburg D-79106, Germany 8 2Key Laboratory of Medical Molecular Virology, Institute of Medical Microbiology, 9

Shanghai Medical College, Fudan University, Shanghai 200032, China 10 3State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, 11

Chinese Academy of Sciences, Shanghai 201203, China 12

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15 #Corresponding author´s mailing address: 16

Prof. Dr. Michael Nassal 17

University Hospital Freiburg 18

Internal Medicine II / Molecular Biology 19

Hugstetter Strasse 55 20

Freiburg D-79106, Germany 21

Phone and FAX: +49 761 270 35070; Email: [email protected] 22

23

Running title: DNA primer grip in DHBV reverse transcriptase 24

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Ms Information: 28

Word count Abstract: 239 words 29

Main text: 8841 words (excluding references and legends) 30

Figures 8 31

Supplementary Information: Supplementary Figures S1-S6; 32

plus Figs. Sa and Sb for reviewer inspection only 33

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.00011-12 JVI Accepts, published online ahead of print on 18 April 2012

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Abstract 36

Hepadnaviruses, including the pathogenic hepatitis B virus (HBV), replicate their 37

small DNA genomes through protein-primed reverse transcription, mediated by the 38

Terminal Protein (TP) domain in their P proteins and an RNA stem-loop, ε, on the 39

pregenomic (pg) RNA. No direct structural data are available for P proteins but their 40

reverse transcriptase (RT) domains contain motifs that are conserved in all RTs (box 41

A to box G), implying a similar architecture; however, experimental support for this 42

notion is limited. Exploiting assays available for duck HBV (DHBV) but not HBV P 43

protein, we assessed the functional consequences of numerous mutations in box E 44

which in human immunodeficiency virus 1 (HIV-1) RT forms the DNA primer grip. 45

This substructure coordinates primer 3´ end positioning and RT subdomain 46

movements during the polymerization cycle, and is a prime target for non-nucleosidic 47

RT inhibitors (NNRTIs) of HIV-1 RT. Box E was indeed critical for DHBV replication, 48

with the mutations affecting folding, ε RNA interaction and polymerase activity of P 49

protein in a similar position- and amino acid side-chain dependent fashion as in HIV-50

1 RT. Structural similarity to HIV-1 RT was underlined by molecular modeling and 51

confirmed by the replication activity of chimeric P proteins carrying box E, or even 52

box C to box E, from HIV-1 RT. Hence box E in DHBV and likely in HBV P protein 53

forms a primer grip-like structure that may provide a new target for anti-HBV NNRTIs. 54

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INTRODUCTION 57

Hepadnaviruses are small hepatotropic DNA viruses that infect humans and select 58

mammals and birds. Hepatitis B virus (HBV), one of the most relevant viral 59

pathogens of humans (20), is their prototypic member. All hepadnavirus replicate 60

their ~3.0 kb genomes by chaperone-assisted protein-primed reverse transcription 61

(10), executed by their P proteins. These are unusual reverse transcriptases (RTs) 62

which, beyond the common RNA-dependent and DNA-dependent DNA polymerase 63

and RNase H (RH) domains, contain a unique Terminal Protein (TP) domain at their 64

N terminus (Fig. 1A). To initiate reverse transcription, the phenolic OH-group of a 65

specific Tyr-residue in TP fills the role that conventionally is taken by the 3´ hydroxyl 66

end of a nucleic acid primer (30). 67

P proteins are translated from a greater-than-genome-length transcript, the 68

pregenomic (pg) RNA which also acts as mRNA for the viral core protein. The 69

interaction of P protein with an RNA stem-loop, ε, on the pgRNA is crucial for viral 70

replication; it triggers co-encapsidation of pgRNA and P protein into newly forming 71

nucleocapsids, and synthesis of a short DNA oligonucleotide which is templated by 72

the bulge in ε and via its 5´ terminal nucleotide (nt) becomes covalently attached to 73

the Tyr-residue in TP ("protein-priming"). Upon transfer to a 3´ proximal acceptor site 74

on pgRNA the oligonucleotide is extended into full-length (-)-strand DNA, and the 75

pgRNA template is concurrently degraded by P protein´s RH activity. Some 15 to 18 76

residues from the RNA 5´ end are spared and serve, upon another template switch, 77

as primer for (+) strand DNA. The final product is a capsid-borne relaxed circular 78

(RC-) DNA which still carries P protein covalently bound to the 5´ end of the (-) strand 79

(reviewed in reference (10)). 80

Duck HBV (DHBV) has as yet provided the deepest insights into the mechanism of 81

hepadnaviral replication. Beyond providing a feasible in vivo infection system (41, 42) 82


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DHBV is the only hepadnavirus for which replication initiation has successfully been 83

reconstituted in vitro. DHBV P protein in vitro translated in reticulocyte lysate (RRL) 84

(51), or expressed as fusion protein in E. coli and supplemented with RRL or purified 85

chaperones (Hsc70 and Hsp40, with further stimulation by Hsp90 and Hop (46)) 86

displays authentic protein priming activity when supplied with the cognate DHBV ε 87

(Dε) RNA and dNTPs, as manifested by the covalent labeling of the protein if α32P-88

labeled dNTPs are used (8, 25, 46). A simpler system exploits severely truncated P 89

proteins (miniPs) lacking part of TP, the spacer, the RH domain and the C terminal 90

part of the RT domain which exert chaperone-independent priming activity (7, 8, 12, 91

52). In any such in vitro assays, HBV P protein shows at most specific binding to its 92

cognate ε RNA (19, 24) but no enzymatic activity. 93

The structural correlates to the diverse activities of hepadnaviral P proteins are not 94

well defined because it has been impossible to generate sufficient amounts of 95

homogeneous P protein for direct analyses. Due to its importance as a drug target 96

(all five currently approved chemotherapeutics for hepatitis B are nucleos(t)idic RT 97

inhibitors (NRTIs); (31)) several models for the RT domain of HBV P protein have 98

been calculated using HIV-1 RT as template (5, 16, 17, 50). The rationale is the 99

presence in the RT domains of all P proteins of short motifs (boxes A to E, plus F (or 100

box II) and G (or box I); see Fig. 1A) that are universally conserved in RTs; for easier 101

comparison of different HBV isolates, a unified numbering system accounts 102

exclusively for the RT/DNA polymerase domain (47, 61). In retroviral RTs, the 103

conserved boxes constitute distinct structure elements that together form the catalytic 104

core. Experimental support for the modeled HBV P protein structures comes mainly 105

from the alike location of mutations conferring resistance to nucleoside analogs in 106

and around the conserved boxes as in HIV-1 RT (61), and from cell culture studies in 107

which mutations, e.g. in the YMDD motif (36) of the catalytic center (in box C) and in 108


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the proposed helix clamp motif (see Supplementary Fig. S1) downstream of box E 109

(53-55, 60), reduced or abolished viral replication. However, the limited experimental 110

repertoire applicable to HBV P protein´s function does often not allow to distinguish 111

whether such inhibitory mutations act specifically or simply induce global P protein 112

misfolding. 113

DHBV P protein shares about 30% sequence identity as well as susceptibility to 114

several NRTIs with its HBV counterpart but offers broader experimental options. We 115

have therefore previously begun a more systematic analysis of the functional 116

consequences of mutations in the conserved boxes. For instance, replacement of 117

F451 (in box A, corresponding to rtF88 in HBV and homologous to Y115 in HIV-1 RT) 118

rendered DHBV P protein capable of using ribonucleotide triphosphates as 119

substrates, demonstrating the involvement of box A in dNTP vs. NTP discrimination 120

as in HIV-1 RT (11). Here we focus on box E, the most C proximal motif. Based on 121

various crystal structures of HIV-1 RT - nucleic acid complexes it has been termed 122

the "DNA primer grip" (48). The sequence forms a hairpin (β12-β13) that acts as a 123

connecting hub for all other subdomains (fingers, palm, and thumb); its loop also 124

contacts the second last residue from the primer 3´ end (mostly via M230 and G231) 125

and thus helps positioning the 3´ terminal OH group close to the catalytic site for 126

nucleophilic attack on the incoming dNTP (Fig. 1C). Moreover, the primer grip forms 127

part of a binding pocket for non-nucleosidic HIV-1 RT inhibitors (NNRTIs) which 128

cause misalignment of important components at the polymerase active site and/or 129

prevent the dynamic inter-subdomain movements required during polymerization 130

(reviewed in references (38, 43)). Proper geometry can also be disturbed by 131

mutations in the primer grip, explaining the multiple phenotypes reported for such 132

mutants, including impacts on polymerase activity, primer/template utilization (21, 29) 133


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and dNTP binding (57), fidelity of DNA synthesis (14, 22, 56), and (via the thumb 134

domain that connects to the RH domain) also on RNase H activity (21, 34). 135

Characteristic residues for box E in retroviral RTs and in P proteins from diverse 136

hosts (Fig. 1B) are an aromatic residue at position +3, a hydrophobic residue at 137

position +4, and a GY dipeptide at positions +5 and +6, as exemplified by DHBV P 138

protein residues 556 to 563. Moreover, the box E motif (rt247-254 in the unified HBV 139

RT numbering system) is almost invariably conserved in the >3000 sequences in the 140

HBV database (http://hivdb.stanford.edu/HBV/DB/cgi-141

bin/MutPrevByGenotypeRxHBV.cgi). More directly, box E residue Y561 in DHBV P 142

protein must be close to the active site because this residue can replace Y96, though 143

less efficiently and only in truncated DHBV miniP, in the in vitro protein-priming 144

reaction (7). 145

Therefore, we used DHBV P protein as a model to reveal the potential presence in 146

hepadnaviral P proteins of an actual primer grip element that is functionally and 147

structurally related to that in HIV-1 RT. To this end we extensively mutagenized the 148

box E motif and subjected the variant P proteins to numerous functional assays, from 149

replication in transfected cells to in vitro Dε RNA binding. Together with HIV-1 RT 150

homology-based modeling and the functionality of chimeric P proteins carrying box E, 151

or even the sequence encompassing the catalytic YMDD motif in box C to box E, 152

from HIV-1 RT, these data demonstrate a common catalytic core architecture of the 153

RT domains of hepadnaviral P proteins and retroviral RTs, in particular the formation 154

of a primer-grip like structure by the box E residues in P proteins. Because the primer 155

grip in HIV-1 RT is the target for multiple approved and experimental NNRTIs (13, 156

18), the presence of a primer grip equivalent in P proteins suggests that related 157

compounds might be developed into a new class of HBV inhibitors. 158

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MATERIALS AND METHODS 160

161

Cell culture and transfection 162

LMH cells were cultured in IMDM (Invitrogen) supplemented with 10% fetal bovine 163

serum, 100 U/ml penicillin G and 100 μg/ml streptomycin, 0.2 mM L-glutamine, at 164

37°C in a humidified atmosphere of 5% CO2. Cells were transfected using TransIT-165

LT1 reagent (Mirus) as recommended by the manufacturer. 166

Compounds 167

Lamivudine (LAM) and phosphonoformic acid (PFA) were purchased from Sigma-168

Aldrich, MG132 from Axxora Platform Biochemicals. 169

Plasmid constructs 170

All variant DHBV expression vectors were based on plasmid pCD16 which harbors a 171

1.1-fold DHBV16 genome (GenBank accession no.: K01834) under control of the 172

CMV-IE promoter (33), followed by a T7 promoter in reverse orientation. Mutations 173

were introduced by standard mutagenic PCR. In the priming-defective variant 174

pCD16-Y96D the codon for Y96 was changed from TAT to GAT; in the replication-175

defective variant pCD16-YMHA the codons for the two essential Asp residues in the 176

YMDD motif were changed to codons for His and Ala (15). In pCD16-P-null the P 177

ORF was prematurely terminated by replacing the codon for Y96 by TGA. Box E 178

mutations (aa 556-563 in the P ORF) introduced into pCD16 or pCD16-Y96D 179

included I556A (ATA>GCT), R557A (AGA>GCT), F558A (TTC>GCT), F558L 180

(TTC>CTC), F558W (TTC>TGG), L559A (CTC>GCT), L559M (CTC>ATG), L559I 181

(CTC>ATC), G560A (GGT>GCT), Y561A (TAC>GCT), Q562A (CAG>GCT), and 182

I563A (ATT>GCT). Expression vectors for wt and mutant DHBV P proteins with an N 183

terminal 3xFLAG tag were based on plasmid pcDNA3.1/Hygro(-) (Invitrogen). For the 184

chimeric P proteins (chimera 1 and 2), the DHBV sequences encoding P protein aa 185


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556-563 and 505-563, respectively, were replaced by fragments encoding aa 227-186

234 and 177-234 from HIV-1 RT (Genbank accession no.: AAK08484.2). Vectors for 187

in vitro transcription and translation of DHBV P protein were based on plasmid 188

pT7AMVpol16H6 (6) which encodes a His6-tag between P protein aa 2 and 3. pAAV-189

Dcore contains the DHBV core ORF between the Cla I and Xho I sites in pAAV-MCS 190

(Stratagene). All HBV vectors were based on plasmid pCH-9/3091, encoding a 191

genotype D, subtype ayw wt HBV genome (32). All plasmid constructs were 192

confirmed by DNA sequencing. 193

Extraction of capsid-associated viral DNA from transfected cells and Southern 194

blotting 195

Cells were lysed with NP40 lysis buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 50 196

mM NaCl, and 0.5% Nonidet P-40) and incubated with 10 mM MgAc2, 100 units/ml 197

DNaseI and 100 μg/ml RNaseA to remove free nucleic acids. Extraction and 198

detection by Southern blotting of intracellular capsid-associated viral DNA was 199

conducteded as described (54). Densitometry was performed using MultiGauge V2.2 200

software (Fujifilm). 201

RNA encapsidation assay 202

RNA encapsidation efficiency was determined as reported (54). In brief, transfected 203

cells were lysed with NP40 lysis buffer. Lysates were centrifuged at 12,000 g for 2 204

min, and 40 μl supernatants were electrophoresed on a native agarose gel and 205

transferred onto a nitrocellulose membrane using TNE buffer (10 mM Tris–HCl, pH 206

7.5, 1 mM EDTA, and 50 mM NaCl). Capsids were detected using anti-DHBV core 207

monoclonal antibody (mAb) 2B9-4F8 (49), followed by peroxidase-conjugated anti-208

mouse secondary antibody (Jackson Immunoresearch Laboratories) and ECL 209

reagent (GE Healthcare). For detection of encapsidated viral RNA, the same 210

membrane was subsequently treated with 0.2 M NaOH/1.5 M NaCl for 30 s followed 211


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by neutralization with 0.2 M Tris-HCl, pH 7.4/1.5 M NaCl for 5 min. RNAs were then 212

detected using a 32P-labeled antisense riboprobe. 213

Western blotting 214

SDS/PAGE and Western blotting were conducted following standard procedures. 215

Primary antibodies were anti-FLAG mAb M2, or mAbs against tubulin, actin, or lamin 216

(all Sigma); against GFP (Roche); and mAb9 against DHBV P protein (58). For 217

detection, the blots were incubated with peroxidase conjugated secondary 218

antibodies, followed by ECL+ reagent (GE Healthcare). Chemiluminescent signals 219

were recorded on X-ray film or using a Fuji LAS 3000 instrument. 220

Endogenous polymerase assay 221

For enrichment of cytoplasmic nucleocapsids, NP40 lysates were treated with 10 mM 222

Mg acetate, 100 units/ml DNase I, and 100 μg/ml RNaseA and then loaded onto a 223

cushion solution (NP40 lysis buffer plus 20% sucrose). After centrifugation in a 224

TLS55 rotor at 55,000 rpm for 90 min at 20°C the pellets were suspended in capsid 225

buffer (20 mM Tris-HCl, pH7.5, 50 mM NaCl, 1 mM EDTA, 0.01% Triton X-100, 0.1 226

% NP40, and 0.05% β-mercaptoethanol) and then incubated overnight at 4°C. The 227

suspension was briefly sonicated and centrifuged at 15,000 g for 5 min to remove 228

insoluble material. Cleared supernatants were treated with 15 U micrococcal 229

nuclease and 5 mM CaCl2, and then incubated with reaction buffer containing 50 mM 230

Tris–HCl, pH 7.5, 75 mM NH4Cl, 1 mM EDTA, 10 mM EGTA, 20 mM MgCl2, 0.1% 231

(v/v) β-mercaptoethanol, 0.5% (v/v) Nonidet P-40, 0.4 mM dATP, 0.4 mM dCTP, 0.4 232

mM dGTP, and 10 μCi [-32P]dTTP (3000 Ci/mmol) at 37°C for various times. 233

Labeled viral DNA was released by addition of 20 mM EDTA, 0.5% SDS, and 0.5 234

μg/μl proteinase K and incubation at 50°C for 2 h. After extraction by 235

phenol/chloroform and precipitation by ethanol, labeled DNAs were separated on 1 % 236

agarose gels in 0.5×TBE buffer. Radioactive signals on the dried gels were recorded 237


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using a Typhoon imager and quantitated using ImageQuant software (both GE 238

Healthcare). 239

DHBV P protein trans-complementation 240

One μg pCD16-P-null plasmid DNA was mixed with either 1 μg pCDNA-3×FLAG-241

Dpol or its derivative plasmids and then transfected into LMH cells. Four days later, 242

capsid-associated viral DNA was extracted and determined by Southern blotting. 243

In vitro transcription 244

Dε RNA as template for in vitro priming was obtained from Cla I linearized pDε1 (6) 245

using the Ampliscribe T7 High Yield Transcription Kit (Epicentre Biotechnologies). 32P 246

labeled Dε RNA and antisense riboprobes were generated using the Riboprobe 247

system-T7 (Promega) from Cla I linearized pDε1 and Nhe I linearized pCD16 248

plasmid, respectively. 249

Dε RNA binding assay 250

This assay was performed as previously described (6). In brief, using 251

pT7AMVpol16H6 plasmid as template, His6-tagged P proteins were in vitro 252

translated in the presence of [35S]-Met using the TNT T7 quick coupled 253

transcription/translation system (Promega). Three μl 32P labeled Dε RNA (approx. 254

8×105 cpm/μl) was added to translated products. The mixture was incubated at 30°C 255

for 30 min to allow the formation of P protein - Dε RNA complexes. Complexes were 256

immobilized on Ni-NTA agarose beads (Qiagen) in 300 μl binding buffer (0.1 M 257

sodium phosphate pH 7.5, 150 mM NaCl, 0.1% NP40, 20 mM imidazole, 100 μg/ml 258

yeast total RNA). After shaking for 2 h at 4 °C, the beads were washed twice 259

individually with 1 ml binding buffer and 1 ml TMK buffer (50 mM Tris-HCl, pH 7.5, 40 260

mM KCl, 10 mM MgCl2, 100 μg/ml yeast total RNA). Bound complexes were released 261

by addition of 2×SDS loading buffer (100 mM Tris-HCl, pH6.8, 4% (w/v) SDS, 20% 262


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(v/v) glycerin, 2% (w/v) β-mercaptoethanol, and 0.2% (w/v) bromophenol blue) 263

containing 100 mM EDTA and subsequently denatured at 100°C for 5 min. 35S-P 264

protein and 32P-labeled RNA were analyzed by SDS-PAGE, followed by 265

phosphorimaging. 266

In vitro protein priming assay 267

In vitro protein priming was performed as described (11). In brief, wild-type (wt) and 268

mutant P proteins were in vitro translated in the presence of 35S-Met. Five µl of the 269

translation mixture products were analyzed by SDS–PAGE to control translation 270

efficiency. For priming, 10 µl translated products were mixed with 0.6 µM Dε RNA 271

and 10 µCi of [-32P]dGTP (3000 Ci/mmol, Perkin-Elmer) in a 15 µl volume containing 272

10 mM Tris-HCl, pH 7.5, 2 mM MnCl2, and 6 mM MgCl2. The mixture was incubated 273

at 30°C for 1 h and analyzed by SDS-PAGE. After drying, the gels were covered with 274

or without 3 layers of Inkjet films and exposed to a phosphorimage plate to 275

separately monitor 32P and 35S signals. 276

Molecular modeling 277

Molecular models of the RT domains of WT DHBV P protein and chimeras 1 and 2 278

were constructed similarly as reported for HBV P protein (17, 50). BLASTP (1) was 279

used to search for homologs; multiple sequence alignment was performed using 280

ClustalW from EBI Tools (http://www.ebi.ac.uk/). Based on the results, chain A from 281

the crystal structure (PDB ID: 1RTD) of the HIV-1 RT and DNA complex (28) was 282

used as template to model the 3D structures of RT domains using Insight II software 283

(Accelrys Inc., San Diego, CA, USA). Energy minimizations were performed using 284

molecular mechanics in the SYBYL software package (Version 6.8, Tripos 285

Associates, St Louis, MO), with the Kollman-all force field. All protein atoms were 286

assigned the Kollman-all-atom charges and the energy convergence gradient value 287

of the simulation systems was set to 0.005 kcal x mol-1 x Å-1. Geometrical 288


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reasonability of modeled structures was tested by procheck analysis 289

(http://nihserver.mbi.ucla.edu/SAVS/). DNA and dTTP were superimposed to the RT 290

structures in Pymol (http://www.pymol.sourceforge.net), followed by energy 291

minimization so as to remove unreasonable contacts. Graphic representations were 292

generated using DS ViewerPro 5.0 (Accelrys). Distances between atoms were 293

calculated using 3D-Mol Viewer (a module of Vector NTI 8.0, InforMax, Inc.). 294

Drug sensitivity assays 295

LMH cells were transfected with equal amounts of plasmids pCD16, pCD16-F558A, 296

or pCD16-L559A. Five hours later, different concentrations of LAM or PFA were 297

added to the media. Four days later, cells were lysed and intracellular capsid-298

associated viral DNA was determined by Southern blotting. All experiments were 299

performed five times. IC50 values for LAM and PFA were calculated by sigmoidal 300

curve fitting as implemented in Origin 8.0 software (OriginLab, Northampton, MA). 301

Statistical significance was evaluated by one-way ANOVA using Origin 8.0. 302

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RESULTS 304

The box E motif in DHBV P protein is crucial for viral replication 305

Box E in DHBV P protein comprises residues I556 to I563 (Fig. 1B). To address a 306

potential role in viral replication, all 8 residues were individually changed to alanines. 307

All mutations were transferred into the DHBV expression vector pCD16 which 308

harbors a 1.1-fold wt DHBV16 genome under control of the CMV-IE 309

enhancer/promoter and produces intact virions upon transfection into the chicken 310

hepatoma cell line LMH (33). Viral DNAs from cytoplasmic nucleocapsids of cells 311

transfected with the mutant genomes and the wt DHBV16 vector as reference were 312

analyzed by Southern blotting (Fig. 2A). The amounts in the respective cytoplasmic 313

lysates of viral core protein and P protein, and of cellular β-actin, were assessed by 314

Western blotting (Fig. 2A). For quantitation, all signals were first normalized to equal 315

loading using the β-actin signals. Then the signals for viral DNA (RC-DNA plus some 316

double-stranded linear DL-DNA which is regularly formed as a by-product of reverse 317

transcription) were correlated with the amounts of core protein in the same sample. 318

Mean replication levels and standard deviations (from 5 to 7 experiments for the Ala-319

scanning mutants, and 3 experiments for the other variants) relative to wt DHBV set 320

at 100% are indicated at the bottom of Fig. 2A; a graphical representation of inter-321

experiment variation is shown in Supplementary Fig. S6. Accordingly, mutant Q562A 322

replicated with wt-like efficiency, and mutants I556A, R557A and G560A displayed 323

less than 50% reductions; the lowest replication levels were seen for variants F558A, 324

L559A, Y561A and I563A. Hence at the latter four positions (+3, +4, +6, +8 in box E) 325

replacing the bulky hydrophobic side-chains by the small methyl groups of Ala had 326

the strongest negative impact on P protein functionality. This position-specific pattern 327

of relative functional importance in box E strongly resembles that reported for Ala-328

scanning mutations in the HIV-1 RT primer grip (21), with mutations W229A, M230A 329


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and Y232A but not the flanking residues producing the strongest impairments in DNA 330

polymerase activity. 331

We also analyzed four mutants with more conservative exchanges, namely F558W 332

and F558L, and L559M and L559I. In all RTs the analogous position to DHBV P 333

protein F558 (residue +3 in box E) harbors an aromatic residue (F, Y, or W) but 334

invariably F in hepadnaviral P proteins. Residue +4 (L559 in DHBV) is L in all avian, 335

and M in the mammalian hepadnaviruses and HIV-1 RT; an I residue here is 336

uncommon but may be selected in HBV (rtM250I) in response to entecavir treatment 337

(50). 338

The F558W exchange had little impact (Fig. 2A) whereas the F558L exchange 339

reduced replication similarly strongly as the F588A mutation. Hence an aromatic 340

residue at the +3 position of box E in P protein appears as important as it is in the 341

HIV-1 primer grip. Both the L559M and L559I mutations were much better tolerated 342

than the L559A exchange. Hence the +4 position of box E exerts a strong preference 343

for a large hydrophobic side-chain, again in line with the preference for L and M at 344

this position in the primer grip of retroviral RTs (Fig. 1B). 345

346

Differential effects of box E mutations on pgRNA packaging 347

Because formation of capsid-borne RC-DNA is a multi-step process, multiple reasons 348

for the pronounced negative impact on viral replication of some of the box E 349

mutations could be envisaged. Reverse transcription is preceded by co-350

encapsidation of P protein and pgRNA, which depends on P protein binding to ε and 351

the availability of core protein. We did not see major differences in core protein (Fig. 352

2A); potential effects on transcript levels (see Fig. 3A) or pgRNA functionality were 353

excluded in subsequent experiments by using a trans-complementation format (see 354


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below). Hence either the amounts, or the properties of the mutant P proteins were 355

responsible for poor replication. 356

To address pgRNA encapsidation competence, we used native agarose gel 357

electrophoresis (NAGE) of intact capsids to compare their relative pgRNA contents; 358

in this assay, capsids can be detected by immunoblotting, and encapsidated viral 359

RNA by molecular hybridization with a 32P labeled probe. To prevent a loss of pgRNA 360

due to conversion into DNA we analyzed the box E mutants in pCD16 vectors 361

encoding a P protein lacking the protein-priming Tyr residue in TP (Y96D) which 362

prevents reverse transcription but not pgRNA encapsidation. Another packaging-363

proficient but catalytically inactivated variant with altered YMDD motif (YMHA), and a 364

vector encoding DHBV core protein (pAAV-Dcore) as the sole viral component 365

served as additional controls. 366

As shown in Fig. 2B, cytoplasmic lysates of cells transfected with all viral vectors 367

produced similar amounts of capsids (the low capsid levels from the YMHA variant 368

were specific for this particular sample); enhanced capsid production from the pAAV-369

Dcore plasmid appears to be an intrinsic feature of this vector (MN, unpublished 370

data). Visual inspection of the RNA blot (Fig. 2B) already indicated clear reductions in 371

encapsidated pgRNA for variants Y561A and I563A. As expected, no signal was 372

produced by the pAAV-Dcore vector. For semiquantitation, all RNA signals 373

(determined by phosphorimaging) were normalized to the capsid signals from the 374

same sample (by densitometry of the immunoblot signals), which confirmed about 375

equal encapsidation efficiencies for all other variants; the slightly enhanced pgRNA 376

levels per capsid for some variants were not significant. Hence none of the 377

mutations, save Y561A and I563A, affected the mutant P proteins´ ability to interact 378

with ε and core protein. Their reduced replication activities (Fig. 2A) therefore likely 379

resulted from impaired DNA polymerase activity. 380


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Notably, low pgRNA encapsidation by variants Y561A and I563A correlated with 381

reduced amounts of detectable P protein (Fig. 2A, panel Dpol), suggesting these 382

mutations affected P protein solubility and/or stability. The immunoblot samples had 383

been obtained from the soluble fraction of cell lysates produced using NP40 384

detergent which selectively solubilizes the plasma membrane. To distinguish 385

between reduced production, increased proteolytic turnover, or insolubility we directly 386

compared the P protein contents in the soluble and the insoluble fractions (containing 387

nuclei and insoluble aggregates) of the NP40 lysates; in addition, the cells were 388

treated, or not, with the proteasome inhibitor MG132 to reduce potential proteasomal 389

degradation (Fig. 3B). DHBV core protein, and tubulin or lamin B (for the soluble and 390

insoluble NP40 fractions, respectively) were assessed for reference. Consistent with 391

the data shown in Fig. 2A, much less of the mutant P proteins than of the wt protein 392

was detectable in the soluble NP40 fraction, whereas the reverse was true for the 393

insoluble fraction. Proteasome inhibition for 24 h, starting on day 3 post transfection, 394

caused no major change in this distribution, except that the overall core protein levels 395

appeared reduced compared to the non-treated cells; similar results were obtained 396

after 7 h of MG132 treatment (not shown). We have not investigated whether this 397

reflects a specific effect on the core protein or is due to the known cytotoxic effects of 398

prolonged MG132 treatment. Clearly, however, P protein variants Y561A and I563A 399

displayed reduced solubility regardless of the presence or absence of MG132, in line 400

with a negative impact of these mutations on protein folding. 401

402

Provision of P proteins in trans excludes that altered properties of box E 403

sequence-modified pgRNAs reduce replication 404

Though not significantly altering pgRNA steady-state levels (Fig. 3A) the nucleotide 405

exchanges required for the box E mutations could still have affected the quality of the 406


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modified pgRNAs as encapsidation and replication templates, thereby contributing to 407

the reduced replication of variants F558A, L559A, Y561A, and I563A (Fig. 2A). To 408

exclude such effects, we trans-complemented a DHBV plasmid defective for P 409

protein translation (pCD16-P-null) with P protein expression vectors encoding 410

3xFLAG-tagged versions of wt and the four mutant P proteins. Hence regardless of 411

the specific P protein, always the pgRNA from the pCD16-P-null vector would be 412

packaged and reverse transcribed. Cytoplasmic lysates of the cotransfected cells 413

were then analyzed by Southern blotting and by NAGE. The wt P protein vector 414

clearly rescued viral replication (Fig. 3C); the lower level of viral DNA compared to 415

the parental pCD16 vector (about 20%) probably reflects the preferential cis-416

encapsidation of pgRNA molecules from which P protein is translated (4). Importantly, 417

all four mutant P proteins generated much weaker signals (5-10% of those seen with 418

wt P protein vector); these data were confirmed by the equally low amounts of viral 419

DNA per capsid seen in the NAGE analysis (Fig. 3D). Hence the reduced replication 420

levels were attributable to the mutant P proteins, not to altered properties of the 421

packaged pgRNA. 422

423

Reduced replication efficiency of box E mutants F558A and L559A is due to 424

decreased polymerase activity 425

To directly confirm an impact of the F558A and L559A mutations on polymerase 426

activity, we monitored the kinetics of recovery of viral DNA formation after treatment 427

with the pyrophosphate analog phosphonoformic acid (PFA). PFA traps HIV-1 RT in 428

the pretranslocated state (39) where the dNTP binding pocket is still occupied by the 429

primer 3´ end, and thus prevents access of the next incoming dNTP. PFA also 430

inhibits P protein´s polymerase activity but not pgRNA encapsidation and protein-431

priming (26). Cells were transfected with pCD16 vectors encoding the two variants, 432


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or wt DHBV; 5 h post transfection, the cells were split and subjected to four different 433

treatments (Fig. 4A). Group 1 was cultured without PFA for another 96 h to serve as 434

untreated control. The treatment groups received 0.4 mM PFA for 72 h; in group 2 435

treatment was continued until harvest at 96 h post transfection; group 3 was cultured 436

for another 7 h, and group 4 for another 24 h in the absence of PFA. Then viral DNA 437

in cytoplasmic nucleocapsids was analyzed by Southern blotting (Fig. 4B). Untreated 438

cells showed similar relative amounts of viral DNA as before (Fig. 2), with variant 439

F558A reaching about 20% and variant L559A about 40% the level of wt virus. 440

Permanent PFA treatment reduced DNA levels to nearly undetectable amounts; 441

notably, the signals for the two mutants were slightly stronger than for wt DHBV, 442

suggesting a low level of resistance to the drug, as confirmed below. In cells allowed 443

to recover for 7 h, signals for wt DHBV already exceeded those for both mutants (Fig. 444

4B, group 3), and after 24 h, a similar ratio in the DNA signals of wt DHBV vs. F558A 445

vs. L559A was restored as in the untreated cells. A graphic representation of the 446

results is shown in Fig. 4C. As both variants encapsidated wt-like amounts of pgRNA 447

(Fig. 2B), the reduced levels of viral DNA must result from reduced polymerase 448

activity. 449

This conclusion was corroborated using the endogenous polymerase reaction in 450

which P protein present in isolated nucleocapsids extends immature replicative 451

intermediates in vitro when provided with dNTPs; employing at least one α-32P 452

labeled dNTP, the extended products can directly be visualized by autoradiography. 453

Here, we subjected nucleocapsids from cells kept for 96 h in the presence of 0.4 mM 454

PFA to this procedure, and analyzed the levels of newly formed radiolabeled DNA 1 455

h, 3 h, 5 h, and 16 h after dNTP addition (Supplementary Fig. S2). As in cells (Fig. 4), 456

de novo synthesis of 32P labeled viral DNA in the mutant nucleocapsids proceeded 5- 457


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to 10-times slower and less efficiently than in wt nucleocapsids, corroborating a direct 458

impact on polymerase activity. 459

To detect potentially strand-specific impairments in DNA synthesis activity, we 460

analyzed viral DNAs from cells transfected with pCD16 vectors for variants F558A 461

and L559A by Southern blotting using strand-specific probes (data not shown). The 462

levels of immature minus-strands were similarly affected as the corresponding 463

mature strands, and no accumulation of single-stranded minus-strands (as expected 464

from a defect in plus-strand synthesis) was observed. Though not excluding minor 465

impacts, these data gave no hints for strand-specific differences. 466

Box E in DHBV P protein is important for protein priming and ε RNA binding 467

For in vitro priming, DHBV P protein is provided with Dε RNA as template plus 32P 468

labeled dNTPs; successful formation of the tyrosyl-DNA phosphodiester bond results 469

in 32P labeling of the protein. The simplest, chaperone-independent system exploits 470

severely truncated miniP proteins that lack, inter alia, the C terminal region which in 471

HIV-1 RT forms the thumb (Supplementary Fig. S1) such that the region 472

encompassing box E is not stably folded (7). Here, we therefore used full-length P 473

protein in vitro translated in RRL to assess the impact of box E mutations on protein-474

priming, using α32P-dGTP as the only exogenously added dNTP; dGMP, specified by 475

the C residue at the last position of the Dε bulge, constitutes the natural first nt that is 476

added to the protein. 477

The 35S labeled in vitro translation products of variants F558A, L559A, Y561A, and 478

I563A plus wt P protein as a positive and variant Y96D as a negative control were 479

first subjected to centrifugation (at 16,000 g for 20 min) to remove insoluble 480

molecules. SDS-PAGE followed by autoradiography showed similar amounts of P 481

protein in all supernatants, including for variants Y561A and I563A (Fig. 5A, panel 482

35S). When subjected to priming conditions, all reactions except those containing no 483


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P protein or the priming-deficient Y96D variant generated specific signals, however of 484

markedly differing intensity (Fig. 5A, panel 32P). 485

The signal from variant L559A was about 70% as intense as that from the wt protein, 486

followed by Y561A and F558A (30% to 40% of wt); the signal for variant I563A was 487

barely above background. These results correlated largely with the replication assays 488

shown in Fig. 2A, except that the well priming variant L559A poorly supported 489

replication (see below for a likely explanation). For variants F558W, F558L, L559M 490

and L559I (Fig. 5A, right panel) high priming activity was seen when F558 was 491

replaced by an aromatic but not an aliphatic residue, and L559 was tolerant against 492

substitution with other large hydrophobic (I, M) residues, as in the replication assays. 493

This indicated that many of the box E mutations exert their effects already during 494

initiation of reverse transcription. 495

Finally, we directly assessed the Dε RNA binding capacity of variants F558A, L559A, 496

Y561A and I563A; wt P protein and variant Y96D served as positive and a reaction 497

containing no P as negative control. The in vitro translated 35S-labeled His-tagged 498

proteins were incubated with 32P labeled Dε RNA, the proteins were immobilized to 499

Ni-NTA beads (6), and protein and RNA remaining on the beads after extensive 500

washing were analyzed by SDS-PAGE and autoradiography (Fig. 5B; the complete 501

autoradiograms are shown in Supplementary Fig. S3). For quantitation, the RNA 502

signals were normalized to the full-length P protein 35S signals in the same lane. 503

Expectedly, wt P protein and the Y96D variant bound similar amounts of Dε RNA, 504

and so did variant F558A. Clearly less RNA was bound by variants Y561A and I563A 505

which had also shown low (Y561A) and very low (I563A) priming activities (Fig. 5A). 506

Most notably, variant L559A reproducibly retained about twice as much RNA as the 507

wt protein, indicating an enhanced affinity for Dε RNA. Nonetheless, it produced a 508


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weaker priming signal (Fig. 5A), indicating a reduced capacity to use the bound RNA 509

as template. 510

511

Box E, and even box C to box E, from HIV-1 RT can functionally replace the 512

corresponding sequences in DHBV P protein 513

Together, the results described above suggested that box E in DHBV P protein is of 514

similar importance for functionality as the primer grip in HIV-1 RT, implying a similar 515

structure. To substantiate this interpretation, we replaced the box E motif in P protein 516

(residues I556 to I563) by the primer grip sequence from HIV-1 RT (F227 to L234; 517

chimera 1) in which the GY dipeptide is the only strictly identical feature (Fig. 1B). 518

More daringly, in chimera 2 the entire ~60 aa sequence encompassing the active site 519

YMDD motif in box C to box E is derived from the HIV-1 enzyme (P protein N505 to 520

to I563 replaced by HIV-1 RT D177 to L237; see Fig. 1A). Functional activity would 521

only be expected if both sequences, despite their divergent primary sequences (15 522

identical plus 11 similar residues within 59 residues total), could adopt similar 3D 523

structures. To avoid impacts on pgRNA by the numerous nucleotide exchanges, 524

functionality was assessed in the trans-complementation format. Co-transfection of 525

pCD16-P-null with the wt P protein expression vector and the empty pCDNA plasmid 526

served as controls. As shown in Fig. 6, both chimeric P proteins supported formation 527

of the same pattern of RC-DNA plus DL-DNA as trans-complemented wt P protein, 528

albeit with lower efficiency (around 30% of wt). This was partly due to reduced levels 529

of soluble chimeric P proteins (Fig. 6, panels α-FLAG and α-DPol), but additional 530

contributions of the numerous mutations to reduced polymerase activity are likely. 531

Most importantly, however, replacement by the short and even the long HIV-1 RT 532

sequence still rendered the chimeric proteins capable of fulfilling all P protein 533

functions, including pgRNA encapsidation and proper protein-primed reverse 534


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transcription. Compared to the strong negative effects that even some single box E 535

mutations exerted on DHBV P protein function, this high tolerance towards multiple 536

mutations derived from the homologous HIV-1 RT sequence strongly supports the 537

formation of similar 3D structures by both protein sequences. 538

539

Molecular modeling is consistent with a primer grip-like structure of box E in 540

DHBV P protein 541

The modeled structures of the RT domain of HBV P protein have been derived using 542

HIV-1 RT as template (5, 16, 17). Here we took a similar approach for DHBV P 543

protein and its two chimeric derivatives, as described in Materials and Methods. In 544

brief, P protein residues 382-657, and the corresponding regions of chimera 1 and 2, 545

were modeled on residues 53-314 from chain A of the crystal structure of HIV-1 RT 546

complexed with DNA and dTTP (pdb: 1RTD; (28)); that structure displays the typical 547

right-hand architecture with palm, fingers and thumb (Fig. 7A). Then, DNA and dTTP 548

were superimposed on the energy-minimized model structures. 549

The DHBV sequence adopted an overall similar architecture as HIV-1 RT (Fig. 7B), 550

and the box E residues formed a hairpin structure (Fig. 7F) as in the HIV-1 RT primer 551

grip (Fig. 7E) around which the subdomains, including the YMDD active site motif, 552

and the 3´ proximal primer strand residues organize. Residues L559 and G560 were 553

situated at the tip of the primer grip hairpin (Fig. 7F), like HIV-1 RT residues M230 554

and G231 (residues +4 and +5; Fig. 7E), and thus similarly able to contact the 555

penultimate primer residue and properly position the terminal 3’-OH relative to the 556

incoming dNTP. These interactions would be perturbed by replacing either residue by 557

Ala. F558, at the +3 position, contacted Y511 in the YMDD motif (highlighted in 558

purple in Fig. 7E-H), an overall similar though not identical arrangement as that 559

between HIV-1 RT residues W229/M230 (in the primer grip) and Y183 (in the YMDD 560


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motif); there, M230 rather than W229 is closest to Y183. Replacement of F558 by 561

smaller residues would weaken the interaction with the YMDD motif, consistent with 562

the reduced replication capacity of the F558A variant, and possibly affect resistance 563

to NAs (a modest hypersensitivity towards LAM was indeed observed; see below). 564

The sidechains of Y561 and I563 displayed extensive hydrophobic interactions with 565

residues of the palm and thumb subdomains (L432, I570, F609; and L432, I435, I556, 566

I570; see Supplementary Fig. S4) which would be not be maintained by the smaller 567

Ala side-chain; the implied impact on overall folding is in line with partial misfolding 568

and the observed insolubility. The +7 position (Q562 in DHBV P) shows substantial 569

variability between different RTs (Fig. 1B) and thus the negligible impact of its 570

replacement by Ala in DHBV P protein is consistent with a non-essential role in 571

folding and activity. 572

Modeling of the two chimeric DHBV P protein RT domains resulted in a similar 573

overall architecture, with some alterations in the thumb domain (Fig. 7C-D). The 574

primer grip hairpin was also formed yet the contact to Y511 in the YMDD motif was 575

largely mediated by M559 (position +4) rather than W558 (Fig. 7G-H), very similar to 576

HIV-1 RT. Together, these and other (7) modeling studies make a common structure 577

of the box E motifs in DHBV P protein and HIV-1 RT highly plausible. 578

579

F558A and L559A in DHBV P protein inversely affect sensitivity to lamivudine 580

and PFA 581

As a final, independent line of evidence supporting such similarity we analyzed the 582

impact of the box E mutations F558A and L559A in DHBV P protein on sensitivity 583

against lamivudine and PFA. In HIV-1 RT, the primer grip does not directly contact 584

the incoming dNTP but due to its scaffolding function, mutations can indirectly affect 585

susceptibility to nucleoside analogs. For instance, the box E mutation F227A 586


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rendered the enzyme 8-fold hypersensitive to azidothymidine (AZT), and various 587

HIV-1 RT mutants selected for resistance to PFA increased sensitivity to AZT (23, 588

44). Both DHBV P protein mutations induced an about 3-fold, statistically significant 589

resistance against PFA (mean IC50 = 189 µM and 143 µM vs. 55 µM for wt DHBV), 590

whereas sensitivity against lamivudine was about 2-fold enhanced (IC50 = 3.4 and 2.5 591

µM vs. 6.8 µM for wt DHBV; see Supplementary Fig. S5 for representative titration 592

experiments). For HBV, a similar cross-talk between box E and the dNTP binding 593

pocket in HBV P protein is suggested by changes in sensitivity towards lamivudine 594

and entecavir reported for mutations of the box E +4 residue rtM250 (3, 50); for 595

instance, rtM250V (in the absence of other mutations) rendered the HBV enzyme 596

seven-fold hypersensitive to LAM (50). Hence these data are again consistent with a 597

common structural role for the box E motif in hepadnaviral P proteins and the DNA 598

primer grip in HIV-1 RT. 599

600

Similar impact on replication of box E mutations in human HBV as in DHBV P 601

protein 602

DHBV and HBV are more closely related to each other than is either of them to HIV-1. 603

Hence if box E in DHBV P protein can adopt a primer grip-like structure this should 604

also hold for the HBV protein, as also implied by the published HBV RT models (5, 605

16, 17, 50). Mutations of box E In HBV P protein should then have similar functional 606

consequences as for DHBV. We therefore replaced residues F584, M585 (rtM250), 607

Y587 and I589 in HBV P protein (box E positions +3, +4, +6, and +8, corresponding 608

to F558, L559, Y561 and L563 in DHBV) by Ala and compared the replication 609

competence and pgRNA encapsidation capacity of the corresponding virus variants 610

versus wt HBV upon transfection into the human heptatoma cell line Huh7 (Fig. 8A). 611

Indeed, all mutations reduced replication and RNA encapsidation efficiency, without 612


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detectably influencing capsid levels (Fig. 8B,C). Relative reductions were similar to 613

DHBV for the +6 position, more pronounced for the +8 position, and less pronounced 614

for the +3 and +4 positions than in DHBV (F558 and L559); a similarly minor effect of 615

the rtM250A mutation at +3 has also been seen in the context of a different HBV 616

genotype where the corresponding residue is M598 (3). Apart from the limitations in 617

accurate quantitation of Southern blots and immunoblots (see Supplementary Fig. S6 618

for the DHBV mutants) the gradual differences of mutational impact on HBV and 619

DHBV likely reflect the substantial sequence divergence between the two proteins. 620

However, compared with the results reported for +3 mutations other than M598A (3) 621

the trend for DHBV L559 mutations was very similar in that larger aliphatic amino 622

acids were better tolerated than smaller ones. Hence together the data are fully 623

compatible with a primer grip-like structure of the box E residues in HBV and DHBV 624

P protein. 625

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DISCUSSION 627

Despite the importance of HBV as a pathogen structural information on hepadnaviral 628

replication is limited to homology-based models of the DNA polymerase (RT) domain 629

of HBV P protein (5, 16, 17, 50). While consistent with NRTI resistance data, HBV 630

provides few experimental opportunities to directly validate the structural predictions. 631

Using the related DHBV P protein as a model, here we extended our previous 632

functional analysis (11) to the conserved box E which in HIV-1 RT forms the DNA 633

primer grip. All our experimental data plus molecular modeling strongly suggest that 634

box E in DHBV as well as HBV P protein adopts a similar structure as the primer grip 635

in HIV-1 RT, with a similarly central role in coordinating the interactions between RT 636

subdomains and the primer 3´ end. Most compellingly, this is indicated by the 637

replication competence of chimeric DHBV P proteins carrying box E, or the entire 638

sequence from box C to box E, from HIV-1 RT. Our data provide new insights into 639

the replication mechanism of hepadnaviruses that should also be useful for the 640

design of new non-nucleosidic anti-HBV compounds. 641

Specific versus global effects of box E mutations in DHBV P protein - evidence 642

for a primer grip 643

Most box E mutations in DHBV P protein, unless to chemically similar residues, 644

reduced overall replication efficiency (Fig. 2A); such negative effects could be 645

specific or simply reflect global misfolding. For distinction, we tested the impact of the 646

mutations on P protein solubility and stability, and we subjected the variants to 647

multiple functional assays, reflecting the consecutive steps of hepadnaviral reverse 648

transcription. 649

The soluble fraction of the NP40 lysates should contain P protein encapsidated in 650

cytoplasmic nucleocapsids plus free, nonaggregated molecules; molecules that were 651

aggregated and/or associated with nuclei, large cellular complexes or debris should 652


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accumulate in the insoluble fraction. In transfected cells, the fraction of non-653

encapsidated P protein exceeds that of encapsidated molecules, and most of those 654

apparently precipitate (59). We did not differentiate between capsid-borne and free 655

soluble P protein yet clearly, variants Y561 A and I563A produced much less soluble 656

protein than wt P protein and the other variants (Fig. 2A, 3B), suggesting a folding 657

defect. In all viral RTs the corresponding primer grip positions +6 and +8 are 658

occupied by an aromatic and a large hydrophobic residue (Fig. 1B) that undergo 659

extensive hydrophobic interactions with the palm and thumb subdomains, and the 660

same was predicted in our modeled DHBV RT domain (Supplementary Fig. S4). The 661

lack of these interactions in the Y561A and I563A mutants could therefore easily 662

promote misfolding and insolubility; notably, an L234A exchange (equivalent to I563A) 663

in HIV-1 RT also affects proper folding (29). In turn, the lack of soluble P protein 664

largely explains the reduced replication capacity of these two variants. Insolubility in 665

the RRL in vitro translation system was less pronounced, perhaps because only 666

freshly translated protein is analyzed. However, the low level of Dε RNA binding and 667

in vitro priming displayed by the two variants (Fig. 5) suggests that even the RRL-668

soluble molecules are functionally impaired. In sum, an impact of the Y561A and 669

I563A on folding is compatible with the predicted primer grip structure but more 670

specific conclusions can be drawn from the other variants which did not display 671

obvious folding problems. 672

Implications for the hepadnaviral replication mechanism 673

While protein-priming is unique to hepadnaviruses, the subsequent chain elongation 674

reactions must proceed as with other reverse transcriptases. Mutations in the primer 675

grip of HIV-1 RT, depending on their nature and relative position, can severely impair 676

polymerase activity (14, 21, 22, 29, 34, 56, 57). Due to the central position of the 677

primer grip, such impairment can occur by various mechanisms; these may be 678


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summarized as misalignments of the components involved in catalysis, and/or 679

hindrance of the dynamic structure changes ("ratcheting") that accompany cycling of 680

the enzyme through successive polymerization steps (43). A comprehensive 681

mechanistic analysis was beyond the scope of this study but the data still allow for 682

several conclusions of relevance for the hepadnaviral replication mechanism. 683

Box E variants other than Y561A and I563A displayed low replication phenotypes 684

despite the absence of obvious solubility or pgRNA packaging deficits (Fig. 2). We 685

first confirmed a direct effect on P protein´s polymerase activity by following the 686

kinetics of DNA synthesis after releasing a block by PFA. Both in intact cells (Fig. 4) 687

as well as in isolated nucleocapsids (Supplementary Fig. S2) variants F558A and 688

L559A generated less DNA from the encapsidated pgRNA than the wt protein, and 689

they did so with slower kinetics. In HIV-1 RT, the corresponding box E positions +3 690

and +4, W229 and M230, and the universally conserved G at +5 form the loop of the 691

β12-β13 primer grip hairpin and mediate the contacts to the Y residue in the YMDD 692

motif and the -2 residue in the primer (Fig. 1C). In our modeled DHBV P protein RT 693

domain, F558 and L559 are located at equivalent positions (Fig. 7F). Tolerance of 694

polymerase activity against replacement by the HIV-1 specific W and M residues 695

(F558W and L559M) but not by Ala with its small side-chain (F558A and L559A) is 696

fully consistent with this model. Similarly, replacement of the universally conserved 697

glycine at position +5 by a more bulky amino acid would affect the interaction with the 698

second last primer residue and proper positioning of the primer 3´ end. Indeed, the 699

G560A mutant displayed a modestly reduced replication competence despite 700

maintained pgRNA encapsidation efficiency (Fig. 2). Thus all these data are in line 701

with box E of P protein forming a primer grip structure with a homologous function 702

during polymerization as in other RTs. 703


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The perhaps most compelling evidence for a common catalytic core structure comes 704

from the ability of the two chimeric P proteins to support DHBV replication, with 705

formation of the same replication products as by the genuine DHBV P protein (Fig. 6). 706

Hence despite replacement of box E, or even the entire sequence from box C 707

containing the catalytic YMDD motif to box E, by the divergent sequence from the 708

HIV-1 enzyme the chimeric proteins were able to specifically package pgRNA, and to 709

initiate and complete reverse transcription, including RNase H-mediated pgRNA 710

degradation. Thus also the unique ε RNA and TP-dependent steps of hepadnaviral 711

replication proceeded normally, though with decreased efficiency. Notably, the 712

reduction in overall replication level was not more pronounced than that caused by 713

the single F558A and L559A mutations in the genuine P protein context. 714

Together with the importance of the YMDD motif in P proteins and our previous 715

demonstration that DHBV P protein F451 in box A fulfills an analogous role in dNTP 716

vs. NTP discrimination as Y115 in HIV-1 RT, our new data firmly establish a common 717

catalytic core structure in the RT domains of P proteins and retroviral reverse 718

transcriptases; in turn, this strongly supports the plausibility of the modeled P protein 719

RT domain structures. 720

A role for the primer grip in protein-priming 721

Regarding the periphery of the RT domain, reliability of the predicted P protein 722

structures is limited by the low sequence similarity to the HIV-1 RT template outside 723

the conserved boxes. For lack of known homologues, homology-modeling is not at all 724

applicable to the hepadnavirus TP domain; direct structural analyses of P proteins, 725

including TP, therefore remain a highly desirable goal. 726

Still, our in vitro priming data with the box E variants also provide new insights into 727

hepadnaviral replication initiation. Priming as well as pgRNA encapsidation depend 728

on P protein binding to the ε RNA stem-loop. Many of the box E variants supported 729


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similarly efficient pgRNA encapsidation as the wt enzyme, suggesting the absence of 730

global misfolding, yet they less efficiently converted the RNA into DNA (Fig. 2). This 731

can in part be attributed to less efficient DNA elongation (Fig. 4, Supplementary Fig. 732

S2) but the in vitro priming data suggest that box E mutations can also affect TP-733

dependent protein-priming. Most evident was the strict correlation between in vitro 734

priming activity and overall replication capacity of most variants. Interpretation of the 735

Y561A and I563A data is not straightforward due to the solubility issues related with 736

these mutations (see above). However, replacements of F558 by Ala and Leu but not 737

Trp reduced both activities; priming, like replication, was more tolerant towards 738

mutations L559I and L559M than towards L559A, and variant F558A performed more 739

poorly than variant L559A in either assay (Fig. 5 vs. Fig. 2A). Hence addition of the 740

first nt to the protein-priming Tyr residue in TP apparently has very similar 741

requirements as the subsequent strand elongations. This is in accord with previous 742

data: Active site mutations such as YMHD or YMHA prevent polymerization as well 743

as priming, and as in elongation, addition of the first nt to TP occurs in a templated 744

fashion (7). Hence the templating nt in the ε RNA bulge must be properly positioned 745

so as to base-pair with the bound dNTP, and the priming tyrosyl-OH group must 746

occupy the same position in the catalytic center as the 3´ end of a nucleic acid primer 747

(see Fig. 9 in reference (7)). By analogy, the primer grip might be involved in Tyr-748

positioning by contacting nearby TP residues. Alternatively, the primer grip mutations 749

could more indirectly distort the geometry of the initiation complex because, just as 750

during elongation, all components must be properly aligned. 751

Information on how the ε RNA interacts with P protein during priming is limited. The 752

regions around the bulge (which serves as template) and the apical loop are 753

particularly important for P protein binding (7, 40, 41) and, once the RNA is bound, 754

unfolding of the upper stem is critical for its template function (9). On P protein, one 755


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RNA binding site appears to comprise the conserved T3 motif in the C terminal TP 756

region (2, 45) and the so-called RT-1 motif (2) in the N terminal RT domain part 757

(residues 381-416), far apart in primary sequence from box E. Most notable in this 758

light is the enhanced Dε RNA binding yet reduced priming activity observed for 759

variant L559A (Fig. 5), a phenotype previously observed with mutations in the RNA 760

rather than in the protein (27). Though only two-fold the effects were statistically 761

significant, indicating that the primer grip contributes to ε RNA binding as well as its 762

utilization as template. Two, not mutually exclusive, options are that structural 763

changes in the mutant protein favor non-productive interactions with the RNA, e.g. by 764

impeding its rearrangement into the template-active conformation, or that the RNA 765

can rearrange but distortions in the catalytic core of the protein reduce its efficient 766

utilization as template. Nuclease accessibility experiments comparing the Dε RNA 767

bound to the mutant versus wt P protein (9) may help to distinguish between these 768

possibilities. 769

The primer grip in P protein as antiviral target? 770

As for HIV-1 RT we found that box E mutations in DHBV P protein had modest, 771

differential impacts on sensitivity against the nucleoside analog lamivudine and the 772

pyrophosphate analog PFA (Supplementary Fig. S5), supporting crosstalk between 773

box E and the dNTP binding pocket as in the HIV-1 enzyme. Such crosstalk is also 774

implied for HBV P protein where mutations of residue rtM250 (the equivalent box E 775

residue to L559) can differentially affect resistance against entecavir and lamivudine 776

(3, 50). While independently corroborating a similar architecture as in HIV-1 RT, a 777

practically more important implication of the presence of a primer grip in DHBV and 778

most likely also in HBV P protein is that it may provide a new target for non-779

nucleosidic RT inhibitors. NNRTIs are important components of highly active anti-780

retroviral therapy (HAART) against HIV-1 that distort the geometry of the catalytic 781


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core, including by dislodging the primer grip (43). In HIV-1 RT the primer grip forms 782

one sidewall of the NNRTI binding pocket, together with residues immediately 783

upstream of box A, the box C β9-β10 hairpin presenting the YMDD motif and some 784

others. Our data predict that hepadnaviral P proteins contain a similar pocket that 785

may provide a valuable new target for HBV antivirals. Given that most NNRTIs 786

against HIV-1 RT are not active against HIV-2 RT (37) it is unlikely that the already 787

established anti-HIV-1 drugs will be active against the more distantly related HBV P 788

protein. However, by screening of compound libraries that maintain the principal HIV-789

1 NNRTI butterfly-like shape (13, 35) and by advanced modeling NNRTIs with activity 790

against HBV should be identifiable. Because all current anti-HBV therapies beyond 791

type I interferon rely exclusively on nucleos(t)ide analogs, such NNRTIs with their 792

different mechanism-of-action would be a most desired alternative for, or 793

complement to, the limited drug repertoire available against chronic hepatitis B. 794

795 796

797


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ACKNOWLEDGEMENTS 798

This work was supported in part by the Shanghai Eduction and Development 799

Foundation (grant KBF101038); the National Natural Science Foundation of China 800

(grant 30800048); and the Deutsche Forschungsgemeinschaft (grant DFG Na154/7-801

3). YXW is indebted to the Alexander-von-Humboldt Foundation for a post-doctoral 802

fellowship. We thank John E. Tavis for providing the anti-DHBV P protein antibody 803

mAb9. 804

805


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57. Wohrl BM, Krebs R, Thrall SH, Le Grice SF, Scheidig AJ, Goody RS. 1997. Kinetic 967 analysis of four HIV-1 reverse transcriptase enzymes mutated in the primer grip region of p66. 968 Implications for DNA synthesis and dimerization. J Biol Chem. 272:17581-17587. 969

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980 981


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FIGURE LEGENDS 982 983 Fig. 1. Relationship of hepadnaviral P proteins to other reverse transcriptases. 984

(A) Domain structure, relative positions and primary sequence of the 985

conserved motifs box A to box E in P proteins versus HIV-1 RT. Beyond the RT 986

(DNA polymerase) and RNase H (RH) domains found in all RTs, P proteins contain 987

an extra Terminal Protein (TP) domain, in which a specific Tyr-residue (Y96 for 988

DHBV, Y63 for HBV) acts as acceptor for the first nt of (-)-strand DNA. Numbers are 989

amino acid (aa) positions for DHBV P protein. Of the conserved boxes, box E 990

constitutes the primer grip in HIV-1 RT. The alignment (as by Clustal W) comprises 991

the sequences in HIV-1 RT, and DHBV and HBV P protein from box A to E; the 992

position of the first aa is given in each line; the fourth line (rt no.) refers to HBV P 993

positions in the unified RT numbering system (47). For HIV-1 RT, residues 994

contributing the palm and finger subdomains are shown in green and blue lettering; β 995

strands β9/β10 (box C with the YMDD motif) and β12/β13 (box E with the primer grip) 996

are highlighted by yellow boxes. HIV-1 RT sequences transplanted into DHBV P 997

protein are indicated as chimera 2 and chim 1. For an extended version of the 998

alignment see Supplementary Fig. S1. (B) Conservation of box E. Alignment of box 999

E from HIV-1 RT and Moloney Murine Leukemia Virus (MuLV) RT with the 1000

corresponding regions in the P proteins of various hepadnaviruses (WMHBV, woolly 1001

monkey HBV; WHV, woodchuck hepatitis virus; GSHV, ground squirrel hepatitis 1002

virus; HHBV, heron hepatitis B virus). Residues forming the loop between β12 and 1003

β13 in the HIV-1 RT primer grip are boxed. For comparison, box E positions have 1004

been numbered from +1 to +8. (C) The primer grip as a central hub in HIV-1 RT. 1005

The primer grip coordinates the relative positions of the palm, finger and thumb 1006

subdomains; its loop interacts with the active site YMDD motif and with the second 1007


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last residue of the primer, thereby properly positioning the primer 3´ end towards the 1008

incoming dNTP (red pentangle); green spheres, bivalent metal ions. 1009

1010

Fig. 2. Distinct impact of box E mutations on DHBV overall replication and 1011

pgRNA encapsidation. (A) Relative replication levels. LMH cells were transfected 1012

with the wt DHBV expression vector pCD16 and its indicated derivatives, then viral 1013

DNAs from cytoplasmic nucleocapsids were analyzed by Southern blotting. RC, 1014

relaxed circular DNA; DL, double-stranded linear DNA; M, marker for DL-DNA. 1015

Aliquots from the same cell lysates were analyzed by immunoblotting for β-actin and 1016

DHBV core protein (Dcore) to normalize for loading and transfection efficiency. 1017

Presence of soluble DHBV P protein was monitored by using an anti-DHBV P protein 1018

antibody (Dpol). DNA signal intensities were determined by phosphorimaging and, 1019

after normalization, used to calculate replication efficiencies relative to pCD16 set at 1020

100%. Mean values and SD are from 5 to 7 experiments (Ala-scanning mutants) and 1021

three experiments (non-Ala mutants), respectively. A graphical representation of 1022

inter-experiment variation is shown in Supplementary Fig. S6. (B) Relative pgRNA 1023

encapsidation efficiencies. LMH cells were transfected with the protein-priming 1024

deficient but pgRNA encapsidation-proficient DHBV vector pCD16-Y96D or its 1025

indicated box E derivatives. Vectors for a polymerase-defective DHBV genome 1026

(pCD16-YMHA) and for DHBV core protein only (pAAV-Dcore) served as controls. 1027

Aliquots from cytoplasmic lysates were subjected to native agarose gel 1028

electrophoresis; capsids were detected by immunoblotting and capsid-borne viral 1029

RNA by molecular hybridization using a 32P-labeled DHBV-specific riboprobe. RNA 1030

signals were quantified by phosphorimaging and normalized to the capsid signals 1031

from the same sample to derive relative encapsidation efficiencies. Mean values ± 1032

SD are based on four independent experiments. 1033


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1034

Fig. 3. Box E nt exchanges reduce replication on the P protein level, not the 1035

pgRNA level. (A) Lack of a significant impact of box E mutations on steady-1036

state levels of viral RNAs. LMH cells were cotransfected with the wt DHBV 1037

expression vector pCD16 or its indicated derivatives plus a constant amount of a 1038

CMV promoter based GFP expression vector. Mock, cells transfected with the GFP 1039

vector only. Cytoplasmic RNAs were analyzed by Northern blotting using probes 1040

specific for DHBV (top panel), GAPDH (center panel), or GFP (bottom panel). 1041

Relative viral RNA levels were calculated by normalization of the pgRNA plus preS/S 1042

RNA signals in one lane to the GAPDH (loading control) and GFP (transfection 1043

efficiency control) signals in the same lane, relative to the pCD16 wt signals set at 1044

100%. The range of relative signal intensities derived from 2 independent 1045

experiments is indicated. (B) Low levels of soluble P protein variants Y561A and 1046

I563A are largely due to insolubility rather than proteasomal degradation. LMH 1047

cells were transfected pCD16 or its Y561A and I563A dervatives. Three days post 1048

transfection, cells were split and cultured for another 24 h in the absence or presence 1049

of the proteasome inhibitor MG132. After cell lysis using NP40 detergent, the soluble 1050

and insoluble fractions were separately analyzed by immunoblotting for DHBV P 1051

protein (Dpol), DHBV core protein (Dcore), and cellular tubulin (soluble) and lamin 1052

(insoluble), respectively. Note the strong Dpol signals for both variants in the 1053

insoluble fraction and their near absence in the soluble fraction; MG132 treatment did 1054

not change this distribution. (C,D) Poor replication rescue by box E mutated P 1055

proteins in trans. LMH cells were co-transfected with a DHBV vector deficient for P 1056

protein production (pCD16-P-null, providing in each reaction the same pgRNA as 1057

encapsidation substrate) plus the indicated pCDNA-based expression vectors for 1058

3xFLAG-tagged versions of the indicated P proteins, or plus empty pCDNA plasmid. 1059


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Cells transfected with pCD16 served as control. In (C), intracellular capsid-borne viral 1060

DNAs were analyzed by Southern blotting and quantified by phosphorimaging. 1061

Replication efficiencies, relative to pCD16-P-null complementation by the wt P 1062

protein expression vector set at 100%, were calculated from three independent 1063

experiments. In (D), capsids and encapsidated DNA in aliquots from the same 1064

samples as in (C) were subjected to NAGE. Capsids were monitored by 1065

immunoblotting, and encapsidated viral DNA by molecular hybridization with a 32P-1066

labeled probe. 1067

1068

Fig. 4. Box E mutations F558A and L559A affect P protein´s DNA polymerase 1069

activity. (A) Treatment schemes for groups 1 to 4. Cells were transfected with 1070

pCD16 or its F558A and L559A variants. Five h post transfection, cell received fresh 1071

media without PFA (group 1) or with 0.4 mM PFA (groups 2-4); 72 h later, PFA was 1072

removed for groups 3 and 4, and the cells were grown for another 7 h (group 3) or 24 1073

h (group 4) in the absence of PFA. (B) Viral DNA production. Viral DNAs in 1074

cytoplasmic nucleocapsids were analyzed by Southern blotting and quantitated by 1075

phosphorimaging. Values were normalized to the amount of DHBV core protein 1076

present in the same sample. Permanent PFA treatment reduced the DNA signals by 1077

50- to 200-fold (group 2). Releasing the PFA block caused a partial recovery in DNA 1078

levels at 7 h (group 3), and a more complete recovery at 24 h post PFA removal 1079

(group 4). (C) Graphic evaluation of DNA levels upon different treatments, 1080

relative to the untreated group 1 set at 100%. Comparable results were obtained 1081

upon monitoring de novo DNA synthesis in isolated nucleocapsids using the 1082

endogenous polymerase reaction (see Supplementary Fig. S2). 1083

1084


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Fig. 5. Differential impact of box E mutations on in vitro priming and Dε RNA 1085

binding of DHBV P protein. (A) In vitro priming. Wt P protein, the protein-priming 1086

defective but Dε RNA binding-proficient Y96D mutant, and the indicated box E 1087

variants were in vitro translated in rabbit reticulocyte lysate in the presence of 35S-1088

Met; a reaction programmed for luciferase (Luc) translation and non-programmed 1089

lysate (No P) served as controls. For priming, the reactions were supplemented with 1090

Dε RNA and [α-32P]-dGTP in priming buffer as detailed in Materials and Methods. 1091

Primed, 32P-labeled P proteins were detected by autoradiography after SDS-PAGE. 1092

The amounts of soluble P protein present in each reaction were determined by SDS-1093

PAGE and 35S autoradiography of aliquots withdrawn from each translation reaction 1094

immediately before adjustment to priming conditions. Relative priming activities were 1095

assessed by normalizing the 32P signals to the 35S P protein signals, with those for wt 1096

P protein set at 100%. Mean values and SD are derived from three independent 1097

experiments. (B) Dε RNA binding. His6-tagged P proteins were in vitro translated in 1098

the presence of 35S-Met, then mixed with 32P-labeled Dε RNA; one aliquot each was 1099

directly analyzed by SDS-PAGE and autoradiography for protein and RNA (panel 1100

input). From the rest of each mixture, P protein and bound RNA were captured on Ni-1101

NTA beads. Protein and RNA remaining bound after extensive washing were 1102

analyzed by SDS-PAGE/autoradiography as in (A). Dε RNA binding activity was 1103

assessed by normalizing the 32P RNA signals to the 35S-P protein signal in the same 1104

lane. Mean values, relative to those produced by wt P protein set at 100%, and SD 1105

were derived from three independent experiments. Note the significantly enhanced 1106

RNA binding by variant L559A. The complete autoradiograms are show in 1107

Supplementary Figure S3. 1108

1109


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Fig. 6. Replacement of box E, or box C to E, by the corresponding HIV-1 RT 1110

elements yields functional P proteins. Cells were co-transfected with pCD16-P-1111

null plus pCDNA vectors encoding 3xFLAG-tagged wt DHBV P protein or its chimera 1112

1 and 2 derivatives (Fig. 1A). Co-transfection with empty pCDNA vector and 1113

transfection with the wt DHBV expression plasmid pCD16 served as controls. In 1114

chimera 1, box E residues 556-563 are replaced by HIV-1 RT residues 227-234; in 1115

chimera 2, residues 505-563 (box C to E) are replaced by HIV-1 RT residues 177-1116

234. Viral DNA from cytoplasmic nucleocapsids was analyzed by Southern blotting, 1117

levels of DHBV core protein and tubulin were monitored by Western blotting. 1118

Presence of soluble P proteins was addressed using anti-FLAG and anti-DHBV P 1119

protein antibodies. Replication capacities relative to trans-complemented wt P protein 1120

were calculated from the DNA signal intensities after normalization to tubulin and 1121

core protein levels in the same sample; mean ± SD are from three independent 1122

experiments. 1123

1124

Fig. 7. Molecular modeling supports formation of a primer grip-like structure by 1125

box E in DHBV P protein. (A-D) Overall structures of the RT domains plus bound 1126

template/primer duplex (in blue) and dTTP (in red) from HIV-1 RT (pdb: 1RTD), and 1127

in the modeled structures of wt DHBV P protein (Dpol) and its chimeric derivatives in 1128

which box E (chimera 1) or box C to box E (chimera 2) were replaced by the 1129

corresponding HIV-1 RT elements. The primer grip hairpin is shown in yellow, 1130

template and primer strands in blue, and bound dTTP in red. The red squares 1131

indicate the parts of the structures that are enlarged in panels E to H. (E-H) Close-up 1132

views of the interactions between the active site YMDD motif (magenta; with Y183 in 1133

HIV-1RT and Y511 in P protein shown as stick models) and the primer grip loop; 1134

residue numbering in the primer grip is as in Fig. 1B. As in the HIV-1 enzyme, primer 1135


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grip residues +3, +4, +5 are in close proximity to the penultimate primer residue and 1136

the Y residue in the YMDD motif; however, the major interaction with the latter is with 1137

the +3 residue F558 rather than with the +4 residue as in HIV-RT (M230); in the 1138

chimeric proteins, a more HIV-1 RT-like arrangement appears to be restored. 1139

1140

Fig. 8. Box E mutations in HBV P protein generate similar replication 1141

phenotypes as in DHBV P protein. (A) Southern blot of HBV DNA from 1142

cytoplasmic nucleocapsids. Human Huh7 cells were transfected with the wild-type 1143

HBV expression vector pCH-9/3091 (pCH-wt) or derivatives carrying the indicated P 1144

protein mutations. For easier comparison, the box E positions of the mutated 1145

residues are indicated on the top; homologous residues in HBV vs. DHBV are 1146

F584/F558; M585 (rtM250)/L559; Y587/Y561; and I589/I563. Relative replication 1147

efficiencies were calculated from the DNA signals relative to the amounts of HBV 1148

core protein (Hcore) and tubulin present in the same samples; mean ± SD are from 1149

three independent experiments. (B) Relative viral DNA contents of cytoplasmic 1150

nucleocapsids. Equal aliquots from the cytoplasmic lysates were analyzed by 1151

NAGE, followed by immunodetection of HBV capsids and molecular hybridization of 1152

packaged DNA with a 32P-labeled HBV probe. Relative DNA content per capsid was 1153

calculated from the intensity of the DNA signals normalized to the capsid signals from 1154

the same sample. (C) Relative viral RNA contents of cytoplasmic nucleocapsids. 1155

Huh7 cells transfected with the same vectors as in (A) were cultured in the presence 1156

of 250 µM LAM to prevent conversion of packaged pgRNA into DNA. Aliquots from 1157

cytoplasmic lysates were subjected to NAGE and processed as in (B). RNA contents 1158

per capsid relative to wild-type vector transfected cells (100%) are derived from three 1159

experiments. 1160

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