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Encapsidated B is poised on an ordered RNA lattice

Joseph Che-Yen Wanga, David G. Nickensa, Thomas B. Lentzb, Daniel D. Loebb, and Adam Zlotnicka,1

aMolecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN 47405; and bMcArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706

Edited by Francis V. Chisari, The Scripps Research Institute, La Jolla, CA, and approved June 19, 2014 (received for review November 14, 2013) Assembly of a virus (HBV) virion begins with the required for subsequent reverse suggests that P and formation of an RNA-filled core composed of a symmetrical both ends of the RNA form a compact complex (22). Within the (built of core ), viral pregenomic RNA, and viral reverse immature core, tyrosine 63 of P protein’s terminal domain (TP) transcriptase. To generate the circular dsDNA of HBV, primes reverse transcription (17, 23). P protein remains co- reverse transcription requires multiple template switches within valently bound to the 5′ end of the nascent minus strand, al- the confines of the capsid. To date, most anti-HBV therapeutics though P will switch templates three times to complete dsDNA target this reverse transcription process. The detailed molecular synthesis (Fig. 1) (24, 25). Mature DNA-filled cores contain mechanisms of this crucial process are poorly understood because a complete minus strand with a covalently attached P protein and of the lack of structural information. We hypothesized that capsid, a partial plus strand. This DNA product is the relaxed circular RNA, and viral reverse transcriptase would need a precise geo- DNA genome of infectious virions. DNA-filled cores are either metric organization to accomplish reverse transcription. Here we transported to the nucleus or acquire an envelope and leave the present the asymmetric structure of authentic RNA-filled cores, cell (26). determined to 14.5-Å resolution from cryo-EM data. Capsid and We hypothesized that for successful reverse transcription, the RNA are concentric. On the interior of the RNA, we see a distinct 5′ and 3′ ends of the pgRNA, the P protein, and the capsid must donut-like density, assigned to viral reverse transcriptase, which form a uniform quaternary structure. To test our hypothesis, we pins the viral pregenomic RNA to the capsid inner surface. The have determined a single particle image reconstruction of authentic observation of a unique ordered structure inside the core suggests RNA-filled cores. We have introduced asymmetric reconstruction that assembly and the first steps of reverse transcription follow to obtain the unique structure of P inside the icosahedral capsid a single, determinate pathway and strongly suggests that all sub- and identified a putative P protein by its donut-like density, sequent steps in DNA synthesis do as well. which was consistent with structures of polymerase superfamily members, including HIV RT and HCV polymerase. The P protein asymmetric reconstruction | HBV intermediate was located at a unique site within the core, touching pgRNA where the pgRNA also contacted the inner surface of the core, near an icosahedral threefold axis. These ordered, asymmetric fea-

epatitis B virus (HBV) is one of the most serious BIOCHEMISTRY Hpathogens: 350 million people suffer from chronic HBV, tures suggest that assembly and the first steps of reverse transcrip- and 600,000 die from it annually. Its 3,200-base pair genome tion follow a single, determinate pathway. marks it as one of the smallest pathogens (1). HBV is an envel- Results and Discussion oped double-stranded DNA virus that is reverse transcribed from a RNA pregenome (pgRNA). To date, most anti-HBV therapeu- We reengineered an HBV expression system to isolate the uni- tics target polymerase (P) (2). However, the structural interplay formity of RNA-filled cores (27). The modified system has three between the components of the reverse transcription complex has key features: P protein was mutated (Y63F) so that it packages not been described. In an infected cell, HBV cores are the metabolic compart- Significance ments for reverse transcription; DNA-filled cores go on to be- come virions. The basic building block of the capsid, the protein (HBV) is a double-stranded DNA virus that shell of the core, is a dimer of the183-residue core protein (Cp). packages a single-stranded RNA pregenome (pgRNA). The lin- Cp is highly conserved among different genotypes (3). The N- ear pgRNA is reverse transcribed to a gapped circular dsDNA terminal 149 amino acids of the Cp form the assembly domain, within the confines of the virus capsid. We hypothesized that which can self-assemble into an icosahedral capsid (4). In vitro a specific capsid-RNA-reverse transcriptase structure would be and in vivo, Cp dimers self-assemble mainly into T = 4 particles required to accomplish this task. In this article, we report the (120 dimers), with a small fraction of smaller 90-dimer T = 3 structure of the authentic pgRNA-filled HBV core as determined particles (5, 6). are dynamic: they are held together by by cryo-EM and asymmetric 3D reconstruction. The observed very weak Cp–Cp interactions and are subject to breathing ordered structure suggests the assembly process and the first modes involving partial unfolding of Cp (7, 8). The 34-amino steps of reverse transcription follow a single, determinate acid C-terminal domain (CTD), rich in arginine residues, is es- pathway. sential for packaging of the pgRNA (9). The CTD is subject to phosphorylation, which is required for RNA packaging and plays Author contributions: J.C.-Y.W., D.G.N., D.D.L., and A.Z. designed research; J.C.-Y.W. and D.G.N. performed research; J.C.-Y.W., D.G.N., T.B.L., and D.D.L. contributed new reagents/ a role in reorganizing packaged nucleic acid and the solution analytic tools; J.C.-Y.W., D.G.N., and A.Z. analyzed data; and J.C.-Y.W., D.G.N., D.D.L., and behavior of the core (10–16). A.Z. wrote the paper. Synthesis of the gapped, circular DNA genome of mature Conflict of interest statement: A.Z. has an interest in a biotechnology startup. HBV from the linear pgRNA template takes place inside the This article is a PNAS Direct Submission. core, as it resides in the host cytoplasm (17) (Fig. 1). Core for- Data deposition: The cryo-EM structure reported in this paper has been deposited in the mation begins with P protein binding to a stem loop, e, near the EMDataBank database (accession no. EMD-2509). 5′ end of the pgRNA (18). This complex is packaged by phos- 1To whom correspondence should be addressed. Email: [email protected]. – phorylated Cp to yield the immature, RNA-filled core (15, 19 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 21). Complementarity between the 5′ and 3′ ends of the RNA 1073/pnas.1321424111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1321424111 PNAS | August 5, 2014 | vol. 111 | no. 31 | 11329–11334 Downloaded by guest on September 26, 2021 Fig. 1. Reverse transcription in HBV. (A) Plus-sense pgRNA (green) is transcribed from covalently closed circular DNA (cccDNA) (red: plus-strand; black: minus- strand). Direct repeats 1 and 2 (DR1, DR2) e, ϕ, and ω are cis-acting sequences involved in genome replication. (B) Initiation of minus-strand DNA synthesis and the first template switch. Cp packages a complex of P protein (blue circle) bound to the encapsidation signal, e. P primes and synthesizes the first 3 of minus-strand DNA, using e as template. The P-minus strand complex switches template to the 3′ DR1 acceptor site. Interaction of e with ϕ and ω facilitate switching. (C and D) Minus-strand DNA elongation proceeds with concomitant pgRNA degradation. (E) Minus-strand elongation proceeds to the 5′ end of pgRNA. RNase H leaves a 17-nt RNA from the 5′ end of pgRNA. This nascent primer disassociates from the 3′ end of the minus strand and anneals to the complementary DR2c site. (F) Plus-strand synthesis proceeds to the 5′ end of the minus-strand template. (G) Circularization and the third template switch are accomplished when the 3′ end of plus-strand switches to the 3′ end of minus-strand DNA. (H and I) Elongation and completion of plus-strand DNA synthesis yields a relaxed-circular DNA genome. Cores containing relaxed-circular DNA may be transported to the nucleus or acquire an envelope and leave the cell. Steps B–I occur in the confines of the capsid. We have determined the structure of the intermediate in B.

pgRNA but cannot prime DNA synthesis because of the re- prediction of our previously stated hypothesis is that P protein, placement of the reactive tyrosine (28); a 4- sub- probably present as a single copy (30), should be evident at stitution eliminated the primary splice acceptor site in the HBV the lower resolution, even in an averaged map, as a weak but pgRNA (the majority of pgRNA produced from this mutant is discrete density. The key to identifying the correct orientation for full length) (29); and of HBV surface was inactivated, eliminating a potentially complicating set of paths for immature virions. We validated that particles purified from our expression system had hallmarks of authentic HBV cores. SDS/ PAGE showed a single dominant protein (Fig. 2 and Fig. S1), identified as Cp by Western blot; proteins other than Cp were detectable by silver stain. One expects 240 copies of Cp and only one copy of P per capsid. In this fraction, the pgRNA:capsid molar ratio was 1.00 ± 0.14. Cryo-EM of the highly purified samples showed the expected 36-nm-diameter T = 4 HBV capsids (Fig. 2D). The small number (∼5%) of 32-nm T = 3 particles were not examined further. When images were translationally averaged, an inner ring of RNA was evident that was stronger than the outer protein density (Fig. 2D, Inset), similar to pgRNA-filled capsids assem- bled in vitro (13). A de novo-built icosahedrally symmetrized image reconstruction of 13,575 capsids, calculated to 10.2 Å resolution, showed a typical T = 4 configuration of the HBV particle (Fig. 3A). A similar result was also obtained when using low-pass filtered atomic structure (PDB ID code 1QGT) as an initial model (Fig. S2). The inner layer of density had charac- teristics of RNA seen in capsids of unphosphorylated and Fig. 2. Purified cores are uniform. (A) Size exclusion chromatography, phopshomimic Cp (13), and RNA was particularly strong be- the last step in purification, shows a single major peak. (B) The peak is neath fivefold and quasi-sixfold arrays of Cp. However, the lu- dominated by Cp, confirmed by immunoblot (Fig. S1). (C) The ratios of men of the symmetrized capsid had weak density not found in in 260 nm to 280 nm absorbance and pgRNA to capsid indicate cores have B C uniform RNA content. These are shown as squares and triangles, re- vitro assembled particles (Fig. 3 and ) (13). spectively, in A.(D) Cryo-EM shows mainly 36 nm T = 4 particles, with The major challenge to identifying unique features in an ico- a small number of 32 nm T = 3 particles. A translational average of T = 4 sahedral complex is that the symmetrical protein shell leads to particles (inset) confirmed the presence of an inner concentric layer, misalignments that average and obscure asymmetric density. A assigned as RNA.

11330 | www.pnas.org/cgi/doi/10.1073/pnas.1321424111 Wang et al. Downloaded by guest on September 26, 2021 Fig. 3. Symmetric and asymmetric reconstructions of HBV. (A) The exterior of a 10-Å resolution reconstruction, viewed down a twofold axis, shows T = 4 symmetry. Symmetry axes are highlighted by symbols. (B) The central section of the reconstruction in A shows an RNA layer and weak internal density, suggesting the presence of asymmetric features. (C) An isosurface of the symmetrized interior shows density thought to result from averaging of asymmetric features. (D) The exterior of the asymmetric reconstruction is very similar to A.(E) A central section of the asymmetric reconstruction shows internal asymmetric features (red arrow). (F) A histogram of the correlation between images and the asymmetric reconstruction. The mean value of the cross-correlation calculated from the “best orientations” is 2.3 σ above the average cross-correlation calculated from the symmetry-related orientations.

asymmetric reconstruction was to choose a feature to use as a fi- S6). RNA density around fivefold and sixfold vertices correlates ducial marker, which was a trial-and-error process. We finally with the position of the RNA-binding CTD of Cp (Fig. S7), proceeded with a lower-resolution search model that started with a stretch of 34 amino acids that includes 17 arginines (13). How- a clump of density located near the icosahedral threefold axis (see ever, the connectivity between vertices is not regular (Fig. 4C), Fig. S3 and Materials and Methods for details). This volume was set suggesting a preferred organization for the RNA.

to the density level of the protein shell, and the rest of equivalent The innermost layer of density in this reconstruction was un- BIOCHEMISTRY copies were masked out. The resulting asymmetric model was ambiguously asymmetric (Fig. 3E and 4B) and was evident in the compared with each image to identify the best of the 60 icosa- reference-free 2D class averages (Fig. S8A). A number of distinct hedrally equivalent orientations previously determined for each masses were observed, all clustered in one hemisphere. In image during calculation of the symmetrical structure. The best a control experiment, the internal density disappeared when the orientation for all particles had an average cross-correlation of model was used to refine orientations of in vitro-reassembled 2.1 σ above the mean calculated for all icosahedrally equivalent pgRNA-filled Cp183 capsids (lacking P and host factors; orientations. When the internal clump of density was moved to Fig. S9). The strength of their electron density was similar to different locations to simulate alternative initial asymmetric models that of the RNA and of the capsid; therefore, they were not likely as control experiments, the average cross-correlation of the “best to represent low-occupancy proteins. The largest mass was an orientation” in all cases dropped to ∼1.9 σ (Fig. S4). In these elliptical donut that was ∼75 Å in diameter at its widest. The structures, internal density was noisy and recapitulated input donut touched the RNA layer, where the RNA layer also con- features, suggesting the wrong starting models or orientations. tacted a Cp dimer near an icosahedral threefold axis of sym- After 50 iterations of refinement, the resulting 14.5-Å resolution metry. Although colored red, gold, and gray in the figure, at this map had a symmetric exterior and internal density that was resolution it is not possible to specifically state exactly where one clearly asymmetric (Fig. 3 D and E and Fig. S5). For our final component ends and another begins. Nevertheless, the Fourier model of 11,727 images, the average cross-correlation of shell correlation at the radii corresponding to the donut density the “best orientation” was improved to 2.3 σ (Fig. 3F), a corre- (60–90 Å) indicated a resolution of 15.4 Å, which is only slightly lation between the asymmetric model and image significantly worse than the resolution estimated for the whole structure above the level expected for 60 random orientations. The (Fig. S8B). model we identified as the basis of our reconstructions led to The well-resolved portion of the donut was consistent with the a structure with unique features and was significantly better curved shape of polymerases: the polymerase domains of HIV than the alternatives. reverse transcriptase and HCV RNA-dependent RNA poly- The exterior of the asymmetric reconstruction resembled merase fit neatly into density (32, 33). HBV P protein is mo- symmetrized T = 4 structures (Figs. 3D and 4A and Fig. S4A). nomeric but larger, at 95 kDa, than most reverse transcriptases. There were no systematic differences between a molecular cap- The extra mass is localized to the N-terminal TP and spacer sid model [1QGT (31)] and our asymmetric density (Fig. S6B domains. These and the C-terminal RNaseH would occupy and Movie S1). Diameters measured from equivalent points a larger fraction of the donut. We caution that this identification confirm the visually apparent symmetry. Thus, small differences is only tentative. Interestingly, when the polymerase domain of in spike shape reflect the low noise level of this asymmetric the p66 subunit of HIV RT is fit to the donut, the RNaseH structure. domain hangs out of density. Efforts to work with P from HBV In the asymmetric reconstruction, the pgRNA layer of the core and from the related duck virus have shown that the protein has symmetric and asymmetric character (Fig. 4 B and C and Fig. in solution requires chaperones for solubility. However, the

Wang et al. PNAS | August 5, 2014 | vol. 111 | no. 31 | 11331 Downloaded by guest on September 26, 2021 The most important conclusion we draw from this study is that the asymmetric interior of the pgRNA-containing HBV core is uniform from particle to particle. RNA and non-RNA density was well-ordered. Therefore, we suggest that 5′ and 3′ ends of the RNA, critical for reverse transcription, are structurally identical from capsid to capsid. This may be accomplished during assembly if the P-pgRNA complex acts to nucleate assembly, accumulating the first few subunits of the growing capsid, which direct sub- sequent growth and symmetry. It is not possible to unambiguously model a single path for the whole RNA, consistent with obser- vation that with large deletions, which retained 5′ and 3′ ends, were still able to support reverse transcription (43). The density internal to the RNA shows more order than the RNA itself. Reverse transcription (Fig. 1) would be impossibly complex if the ends of the nucleic acid had random locations; if the ends can conform to one structure, as if the capsid were a molecular jig, the template switches are much easier to envision. Because of the organization of the RNA and the presence of the P complex inside the RNA shell, minimally interacting with the capsid, we propose that the P complex is mobile, processively traveling along the viral nucleic acid, similar to a train on a track. [With current data, we cannot preclude a reovirus-like (44) model of P function, where P is static. However, the train track model allows the ends of the genome to be organized and does not require detaching large sequences of nucleic acid from the very basic capsid surface so they can be threaded through a replication machine.] In its travels, the P complex would also interact with Cp, so that bound kinases and phosphatases could sequentially modify Cp phosphorylation, a progressive change observed during duck HBV replication (11, 12). Indeed, the capsid has been im- Fig. 4. An asymmetric reconstruction of authentic RNA-filled HBV. (A) The plicated as an active participant in reverse transcription (43, 45). exterior view is tilted so that the viewer is faced by the threefold vertex associated with the putative P protein density. Unlike the other threefolds in Following the nucleic acid track, the P complex would return to the the asymmetric or the symmetric reconstructions, this one is blocked. (B)In same location to perform the template switches required the cut-away view, the particle has been rotated 90° so the P-associated for dsDNA synthesis. This “train on a track” model requires threefold is pointed up. The assembly domain (gray) is fit well by Cp dimers a nonoverlapping Hamiltonian path proposed to be general to and remains nearly symmetric. A donut-shaped density (red) has tentatively ssRNA (46, 47); such paths also imply a deterministic been assigned as the P protein. Additional internal (blue) density is attrib- capsid assembly path. utable to unidentified proteins and misaligned capsid. (C) A projection of the RNA shell showing it is not symmetrical but does have some features Materials and Methods common to many, but not all, icosahedral facets. The P-associated dimer is identified by the dimer (upper left). The strongest RNA density is actually in Molecular Clones and Plasmids. Molecular clones of HBV used in this work the adjacent facet (upper row, second from left). (D and E) Polymerase have been deposited in the GenBank database (accession no. V01460, subtype domains from HIV (PDB ID code 1RTD) and HCV (PDB ID code 3MWV), rep- ayw, genotype D). The plasmid 1159 is a derivative of the HBV expression resenting the superfamily, fit well into the red density. plasmid TL7 (27), which contains the 7xTetO-CMVp-HBV sequence and the oriP sequence of the Epstein Barr virus. The plasmid TL7 includes a series of base substitutions that result in stop codons that prevent expression of viral putative P density within the capsid lacks this extra bulk, sug- envelope proteins without altering the overlapping polymerase gene. These gesting they are shed when the single P protein of the virion is mutations block the major route of virus excretion from the cell, leading to bound within the RNA-filled core. Other density in the capsid an intracellular accumulation of pgRNA-filled particles. To construct 1159 interior was not connected directly to the RNA, but most had from TL7, mutations were introduced that create a Y63F change in HBV Pol that eliminates polymerase priming activity and that allow accumulation of weak connections with each other. This additional density, which pgRNA-filled cores. A 4-nucleotide substitution within the splice acceptor is at lower resolution (Fig. S8), is likely to include signal from site at nt 485 in the HBV sequence (cagg→gtcc at positions 484–487) was misaligned particles as well as a mixture of host-derived proteins. introduced. This mutation has been shown to eliminate the majority of Although studies are currently underway to determine the pro- spliced pgRNA products, increasing the percentage of full-length pgRNA teome of our HBV samples, complementary studies have iden- filled cores (29). tified possible candidates. For example, Hsp90 plays an essential role in initiation of viral DNA synthesis and RNA packaging (34, Cell Cultures and Transfections. Human hepatoma cell line Huh7-H1 was used 35) and Hsp70 and Hsp40 facilitate transitions of the polymerase for production of pgRNA-filled cores and has been demonstrated to support (34, 36, 37). Proteins that have been associated with HBV core high levels of HBV replication (27). Cells were maintained in DMEM/F12 μ phosphorylation include cyclin-dependent kinase 2, serine- media supplemented with 5% FBS and 600 g/mL G418. Cell cultures were transfected using the calcium phosphate precipitation method described arginine protein kinase 2, and protein kinase C (38–40). Pre- previously (48). For production of pgRNA-filled cores, 107 cells were seeded sumably there is also a phosphatase involved in the capsid de- into 100-mm plates and grown to 70–80% confluence. Media was removed phosphorylation (11). Some host proteins, such as the DEAD and replaced by DMEM/F12 supplemented with 5% (vol/vol) FBS without box RNA helicase DDX3X (41) and APOBEC3C (42), may be G418 0–4 h before transfection. Cells were transfected with 32 μg plasmid host restriction factors that limit HBV and are only 1159 and 1 μg plasmid 1929. Medium [DMEM/F12 plus 5% (vol/vol) FBS] was present in a subset of particles. replaced at 16–20 h after transfection and was changed every 24 h for 4 d.

11332 | www.pnas.org/cgi/doi/10.1073/pnas.1321424111 Wang et al. Downloaded by guest on September 26, 2021 Purification of HBV Particles. Four days after transfection cells were washed [7.5 μL2× IQ SYBR Green Supermix (Bio-Rad), 3 pmoles RevC1 primer, and with 10 mL ice-cold Hepes-buffered saline (HBS) plus EGTA buffer (2 mM 3 pmoles ForC1rep primer (5′-TTTCCATGGCTGCTAGGCTGTGC-3′)]. The PCR Hepes at pH 7.5, 150 mM NaCl, and 0.5 mM EGTA) and frozen at −80 °C. Plates program was 95 °C for 4 min, followed by 40 cycles of 95 °C for 20 s, 54 °C for were thawed and cells were treated with 1 mL lysis buffer [50 mM Hepes at 20 s, and 72 °C for 30 s, followed by 4 °C until retrieved. Concentration of pH 7.5, 100 mM NaCl, 0.25% Nonidet P-40 (wt/vol), 0.5 mM EDTA, and one unknown samples was determined by comparison of Ct values with the in Complete Mini EDTA-free protease inhibitor tablet (Roche) per 10 mL lysis vitro pgRNA generated standard curve. solution]. Cells were scraped, collected, and vortexed several times. Lysate Core protein was measured using the native particle blot Western assay × was clarified by centrifugation at 10,000 g for 1 h to pellet cell debris. described earlier, with the following modifications: Primary Dako core an- Clarified lysate was loaded onto a 40%/50%/60% (wt/vol) sucrose step tibody was used at a 1:1,000 dilution. Standards for the Western blots were × gradient in 25 89 mm Ultra-Clear centrifuge tubes (Beckman Coulter). Each Cp183 capsids prepared in Escherichia coli (49). sucrose solution was prepared in CLPm buffer [50 mM Hepes at pH 7.5, 100 mM NaCl, 50 mM L-arginine, 50 mM L-glutamate, 1 mM EDTA, 2 mM DTT, Cryo-EM. Four microliters HBV samples were absorbed on a glow-discharged 0.05% Nonidet P-40 (wt/vol), and 1% Trehalose and one Complete EDTA- continuous carbon film for 25 s at 4 °C under 100% humidity. The samples free protease inhibitor tablet (Roche) per 50 mL sucrose solution]. Sucrose were blotted and vitrified in a liquid ethane bath cooled by liquid nitrogen, gradients were centrifuged for 16 h at 110,000 × g in a SW 32Ti rotor using a FEI Vitrobot (FEI Mark IV). The frozen hydrated specimen was (Beckman Coulter). One-milliliter fractions were collected and stored at 4 °C. transferred into a JEOL 3200FS electron microscope operated at an accel- Sucrose gradient fractions with cores were identified by a native-particle- eration voltage of 300 kV with an in-column energy filter, using a slit width blot Western assay. Samples were resolved using a 1% agarose gel run at 80 V of 20 eV, and kept at the low temperature (<−176 °C). Digital images were and 300 mA in 1× Tris-acetate-EDTA buffer for 75 min. Gels and filter papers recorded on a Gatan UltraScan 4000 CCD camera under low-dose condition were equilibrated in 1× Tris-NaCl-EDTA (TNE) buffer (10 mM Tris at pH 8.0, − (≤20 e /Å2) and at a nominal magnification of 40,000× (corresponding to 150 mM NaCl, 1 mM EDTA) for at least 15 min. Immobilon-P 0.45-μm pore a pixel size of 2.94 Å), with a defocus level ranging from 0.73–4.21 μm. size transfer membranes (Millipore) were treated with methanol and then equilibrated in TNE. Capsids were transferred to this membrane by capillary action, and then membranes were air dried and blocked by incubation at Image Processing. A total of 16,970 particles were semimanually selected, room temperature for 1 h in 1× phosphate-buffered saline with 0.05% using e2boxer.py. The defocus levels were estimated by CTFFIND3 and Tween 20 (PBST) buffer supplemented with 5% (wt/vol) skim milk powder. ROBEM. The contrast transfer function of the microscope was partially cor- Capsid particles were detected by incubation with a polyclonal rabbit HBV rected by flipping the phase of the particles, using either applyctf for 2D core antibody (Dako) at a 1:2,000 dilution for 1 h in 1× PBST buffer. Mem- classification or AUTO3DEM (50), during data processing. For 2D classifica- branes were washed three times for 15 min with 1× PBST and then in- tion, all particles were aligned and classified using EMAN (version 1.9) (51). cubated for 1 h at room temperature with a Protein A-HPR conjugate For the 3D reconstruction of a symmetrized particle, an ab initio model was (Thermo/Pierce) at a 1:5,000 dilution in 1× PBST buffer. Membranes were first generated and followed by well-established refinement processing, washed three times for 15 min with 1× PBST and once for 5 min in PBS to using icosahedral averaging in AUTO3DEM. The refinement continues until remove Tween. Washed membranes were treated with Pierce ECL Western no further improvement on the 3D reconstruction is observed. To generate Blotting Substrate according to the manufacturer’s recommendations the search model for asymmetric structure determination, we used a lower- (Thermo/Pierce). resolution model from the refinement of the icosahedrally symmetric re- Selected sucrose gradient fractions were pooled and loaded onto a 200-mL construction. The model was first rendered at low contour level to reveal the Sephacryl 300-high resolution column equilibrated at 4 °C with CLPm buffer internal density (Fig. S3A). This model was used to create an initial asym- without protease inhibitor. Fractions with high A260/A280 ratios were metric model by selecting one unit of internal density and then masking shown by Western blotting to contain the highest concentration of cores. symmetry-related masses using the segmentation tool in UCSF Chimera. The These were loaded onto a Superose6 column (GE Healthcare) equilibrated capsid and RNA layers were also segmented into a new volume. Because the BIOCHEMISTRY with 1× CLPm buffer. Samples were run at 0.5 mL/min, and fractions with internal density was much weaker than the capsid density and because of high A260/A280 ratios were collected. Superose6 fractions with the highest icosahedral averaging, we manually increased the weight to the average core concentration samples were pooled and concentrated with a Nanosep level of capsid. The adjusted internal density was then combined with the 100K Omega spin concentrators at 1500 × g (Pall Life Sciences). Concen- capsid/RNA volume to create a new model to search the 60-symmetry equiv- trated samples were stored at 4 °C. alent positions from previously obtained origins and orientations (Fig. S3B). The particles with the greatest agreement (the highest cross-correlation or the pgRNA/Capsid Ratio Measurement. To determine the ratio of pgRNA/Capsid, “best orientation”) to the first search model were used to generate a new pgRNA was isolated from pgRNA-filled core preparations at each stage of asymmetric model. Because the first search model had asymmetric density as purification. To each sample, an equal volume of 2× ProK digestion buffer (50 a fiducial marker, the resulting map had better-defined asymmetric features. mM Tris at pH 8.0, 300 mM NaCl, 4 mM EDTA, 1% (wt/vol) SDS, Proteinase K We again segmented the internal density of the resulting reconstruction, 22 ng/μL) was added, and samples were incubated for 1 h at 37 °C. Samples based on the location from the icosahedral reconstruction. This volume had were then extracted with an equal volume of TRIzol and precipitated ions its density scaled and was combined with the capsid/RNA layer from the according to the manufacturer’s protocol. Samples were resuspended in icosahedrally averaged model to create the second search model (Fig. S2C), 10 μL nuclease-free water and stored at −20 °C. which was compared with the 60 equivalent orientations for each image, pgRNA for use as a standard was prepared by in vitro transcription from determined from the icosahedral reconstruction. The resulting asymmetric plasmid 1135 (9). Standard pgRNA concentrations were determined by ab- model (Fig. S4D) was further refined without imposing symmetry using – sorbance to prepare standards from 3 0.003 fmoles per reverse transcription AUTO3DEM. A total of 11,727 particle projections from 513 micrographs quantitative PCR (RT-PCR) reaction. were used to generate the final 3D reconstruction at 14.5 Å, determined by The concentration of pgRNA was determined by RT-qPCR. To copy pgRNA the 0.5 Fourier shell correlation criterion. The electron density map was vi- μ to a cDNA, 2.5 L RNA sample was annealed to 2 pmoles oligonucleotide sualized using ROBEM and UCSF Chimera (52). Segmentation and fitting were ′ ′ ′ RevC1 (5 -CCGGCAGATGAGAAGGCACAGAC-3 ) complementary to the 3 both performed using Chimera. region of the pgRNA. Annealing was performed in 6 μL at 65 °C for 5 min, followed by 5 min on ice. Reverse transcription was performed in SuperScript μ × ACKNOWLEDGMENTS. We are indebted to the Electron Microscopy Center III (Invitrogen) reaction buffer (6 L of annealing reaction, 1 reaction and the Indiana Molecular Biology Institute, both at Indiana University. This buffer, 5 mM DTT, 750 μM dNTPs, 1 mM MgCl2, 60 U SuperScript III reverse work was supported by National Institutes of Health Grants R01-AI077688 transcriptase) in 20-μL reaction volumes at 52 °C for 4 h. A 2.5-μL aliquot of and R01-AI067417 (to A.Z.) and P01-CA022443 and R01-AI060018 (to D.D.L.); each reverse transcription reaction was added to 12.5-μL qPCR reaction mix T.B.L. was supported by T32 CA009135.

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