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Virology 353 (2006) 443–450 www.elsevier.com/locate/yviro

The insertion domain of the duck core protein plays a role in nucleocapsid assembly ⁎ Haitao Guo, Carol E. Aldrich, Jeffry Saputelli, Chunxiao Xu, William S. Mason

Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA Received 12 April 2006; returned to author for revision 11 May 2006; accepted 6 June 2006 Available online 11 July 2006

Abstract

Synthesis of hepadnaviral DNA is dependent upon both the viral DNA polymerase and the viral core protein, the subunit of the nucleocapsids in which viral DNA synthesis takes place. In a study of natural isolates of duck (DHBV), we cloned full-length viral genomes from a puna teal. One of the clones failed to direct viral DNA replication in transfected cells, apparently as a result of a 3 nt inframe deletion of histidine 107 in the core protein. Histidine 107 is located in the center of a predicted helical region of the “insertion domain”, a stretch of 45 amino acids which appears to be at the tip of a spike on the surface of the nucleocapsid. The mutation was introduced into a well-characterized strain of DHBV for further analysis. Core protein accumulated in cells transfected with the mutant DHBV but was partially degraded, suggesting that it was unstable. Assembled nucleocapsids were not detected by gel electrophoresis. Interestingly, the mutant protein appeared to form chimeric nucleocapsids with wild-type core protein. The chimeric nucleocapsids supported viral DNA replication. These results suggest that the insertion domain of the spike may play a role either in assembly of stable nucleocapsids, possibly in formation of the dimer subunits, or in triggering nucleocapsid disintegration, required during initiation of new rounds of infection. © 2006 Elsevier Inc. All rights reserved.

Keywords: Duck hepatitis B virus; Nucleocapsid assembly; Insertion domain

Introduction Lanford, 1993; Hatton et al., 1992; Kock et al., 1998; Nassal, 1992; Yu and Summers, 1991). Hepadnaviruses are small enveloped DNA that At the 24 amino acid carboxy-terminus of the DHBV core replicate via reverse (Seeger and Mason, 2000). protein, there are four sites that may be phosphorylated (T239, The family is divided into two genera, Orthohepadnavirus S245, S257 and S259) (Yu and Summers, 1994b). Depho- and , with host specificity for mammals and sphorylation at these sites takes place as the DHBV birds, respectively. Reverse transcription takes place following nucleocapsids accumulate double-stranded DNA and is packaging of viral pregenomic RNA, polymerase and host- essentially complete in mature virions. This observation encoded chaperones (Hu and Seeger, 1996; Hu et al., 1997) implies that dephosphorylation is associated with nucleocapsid into icosahedral viral nucleocapsids, assembled from dimers of maturation. A maturation event is implied by the fact that the viral core protein (Zhou and Standring, 1992). The core virus is preferentially assembled from nucleocapsids contain- protein of avian hepadnaviruses is longer than that of HBV ing partially double-stranded DNA rather than single-stranded (262 aa versus 183) due to an extra 45 contiguous amino acids DNA or pregenomic RNA (Perlman et al., 2005; Pugh et al., (aa 86 to aa 130) (Bringas, 1997), defined as an insertion 1989; Rabe et al., 2003). The DHBV core protein is also domain, as well as additional amino acids in the arginine-rich phosphorylated at other amino acids. It has been suggested C-terminal, nucleic acid binding domain (Beames and that phosphorylation at threonine 174 triggers disintegration of the viral nucleocapsid and delivery of viral DNA into the nucleus (Barrasa et al., 2001). ⁎ Corresponding author. Fax: +1 215 728 3105. In contrast to the arginine-rich carboxy-terminus, the E-mail address: [email protected] (W.S. Mason). amino-terminus is involved in assembly of the nucleocapsid

0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2006.06.004 444 H. Guo et al. / Virology 353 (2006) 443–450 shell. When the arginine-rich region is deleted, nucleocapsids the H107 deletion, for disassembly of nucleocapsids (e.g., are still assembled, but packaging of viral RNA does not take when wt virus enters the nuclear basket; Rabe et al., 2003). place (Kock et al., 1998; Yang et al., 1994). The structure of Interestingly, core protein with the H107 deletion appeared to the HBV nucleocapsid has been derived by cryoEM and, at form viable, chimeric nucleocapsids when co-expressed with higher resolution, by X-ray crystallography (Bottcher et al., wtCore protein. 1997; Conway et al., 1997; Wynne et al., 1999). The building block is a core protein homodimer (Zhou and Standring, Results 1992). The regions from amino acids 50–73 and 79–110 of the monomer fold into α helices that fold back on each other HisDHBV is replication defective in LMH cells to form a hairpin with a short loop between amino acid 73 and 79. Interactions between the first helix of each monomer DHBV DNA replication intermediates were not detected in stabilize the homodimer. The resulting four helix structure LMH cells transfected with DHBV DNA containing the histi- forms a spike projecting from the nucleocapsid shell, with the dine 107 deletion mutation in the core protein (HisDHBV). loop regions at the tip of the spike. Large insertions near the Replication was rescued by trans-complementation with wild- tip of the spike of HBV, at amino acid 80, typically do not type DHBV core protein (wtCore), indicating that the mu- block nucleocapsid assembly, while mutations in the alpha tation was not trans-dominant (Figs. 1, 2A). It has been helices forming the sides of the spike can block assembly reported that viral DNA synthesis may destabilize nucleocap- (Beames and Lanford, 1995; Brown et al., 1991; Newman et sids assembled from mutated core proteins (Kock et al., 1998). al., 2003). Early studies suggested that the tip of the spike We therefore investigated the possibility that the DNA within interacted with proteins during virus formation nucleocapsids formed by mutant core protein was destroyed because a peptide that bound to the tip blocked virion by the DNaseI digestion used to eliminate plasmid DNA prior formation (Bottcher et al., 1998). However, a later genetic to extraction of viral DNA from nucleocapsids. Extractions analysis suggested that amino acids involved in interactions were performed without DNaseI treatment, and samples were between the nucleocapsid and envelope proteins and critical incubated with DpnI to digest the input plasmid DNA, which for virion formation mapped to the base of the spike (Ponsel is methylated. The results were the same as with DNaseI and Bruss, 2003). Thus, the function of the distal portion of treatment (Fig. 2A). the spikes remains unclear. To determine if the defect in DNA synthesis might be due to Relative to HBV, DHBV contains a 45 amino acid insertion a failure to package pregenomic RNA into nucleocapsids, which, based on amino acid alignments (Bringas, 1997), northern blot analysis was carried out on total cellular RNA and would be located at a position homologous to the tip of the on RNA isolated from viral nucleocapsids present in transfected spike of the HBV nucleocapsid. Low resolution cryoEM of cells. As shown in Fig. 2B, packaging of pregenomic RNA was DHBV nucleocapsids is consistent with the placement of the not detected in cells transfected with HisDHBV. This defect insertion domain in the spikes (Kenney et al., 1995). Model could be rescued by co-transfection with a plasmid expressing building using the crystal structure of the HBV nucleocapsid the wtDHBV core protein. The slightly smaller size of the as a template suggests a location at the tip of the spike, pgRNA within nucleocapsids than the core/pol mRNAs has forming the loop of a stem loop structure with α helices as the been previously reported and attributed to digestion of the 3′ stem (Thermet et al., 2004). In agreement with the idea that end by ribonucleases during nucleocapsid isolation (Ostrow and the insertion domain is exposed at the tip of the spike, amino Loeb, 2004). The smear of signal below the pregenome acids 99–112 in the insertion domain, with a predicted α presumably reflects partial degradation of pgRNA by the viral helical structure (Bringas, 1997; Thermet et al., 2004), are RNaseH during elongation of minus strand DNA by reverse recognized by antisera from chronically infected ducks transcription. (Thermet et al., 2004). The function of the DHBV insertion The accumulation of mutant core protein in transfected domain is unknown. cells was assayed by immunoblotting. Partial degradation was In a study of natural isolates of avian hepadnaviruses, we evident in the HisCore transfected cells (Fig. 3), suggesting cloned from the puna teal a virus mutant that was defective in that the mutant protein may be less stable than the wt, perhaps viral DNA synthesis as the result of a histidine deletion (aa due to a failure to assemble into dimers or nucleocapsids. The 107) in the insertion domain of the core protein (Guo et al., presence of multiple species of mutant protein that, like the 2005). In order to characterize this mutation further, we wild-type core protein, migrates slower than the major band introduced it into the genome of a laboratory strain of DHBV, suggested that the mutant core protein is phosphorylated DHBV-16 (Mandart et al., 1984), and demonstrated that the (Pugh et al., 1989). defect in DNA synthesis was due to a failure to form nucleocapsids. Substitutions of H107 that conserved the ability The mutant core protein forms functional chimeric to form an α helix had no effect on nucleocapsid formation, nucleocapsids when co-expressed with wtCore protein while a proline substitution, which would disrupt the α helix, blocked nucleocapsid assembly. Thus, the insertion domain of To see if the mutant core protein could assemble into the DHBV spike either provides information essential for nucleocapsids, lysates of the transfected cells were subjected formation of nucleocapsids or encodes a signal, activated by to capsid gel electrophoresis in agarose. The nucleocapsids H. Guo et al. / Virology 353 (2006) 443–450 445

Fig. 1. Schematic representation of the plasmids and mutants used in this study. Numbers refer to nucleotides in the DHBV genome (Mandart et al., 1984). (1) wtDHBV: also known as pCMVDHBV (Condreay et al., 1990), is a plasmid in which transcription of the viral pregenome is under control of a CMV IE promoter. (2) HisDHBV: a CAC (nt 2965–2967) deletion removed H107 from the insertion domain. (3) wtCore: a DHBV core expression plasmid in which wtDHBV core protein is expressed under the control of a CMV IE promoter. (4) HisCore: vector in which a DHBV core protein with a deletion of histidine 107 is expressed under the control of a CMV IE promoter. were transferred to nitrocellulose membranes and detected by Histidine substitution immunostaining of the viral core protein. As shown in Fig. 4, neither HisDHBV nor HisCore produced stable viral nuc- To determine if substituting other amino acid at position leocapsids. When wtCore was co-transfected with HisDHBV 107 of the core protein affected nucleocapsid assembly, we or HisCore, nucleocapsids were detected. We inferred that replaced histidine (aa 107) in the plasmid wtCore with these are chimeric since a higher electrophoretic mobility was glutamate (E), tyrosine (Y), or asparagine (N), which is detected than for nucleocapsids assembled from wtCore by present at the homologous location in the closely related core itself. A mixing experiment also suggested that the electro- protein of the snow goose hepatitis B virus (SGHBV), ross phoretic difference was intrinsic to the nucleocapsids (Fig. goose hepatitis B virus (RGHBV) and stork hepatitis B virus 5F). In particular, the mobility of wild-type nucleocapsids was (STHBV), respectively. These three substitutions did not not increased when a cell lysate containing these was inhibit viral DNA synthesis. Replacement of histidine 107 mixed with a lysate containing chimeric nucleocapsids. with glutamine (Q) and methionine (M), which represent Chimeric nucleocapsids appeared to be functional for viral typical polar and hydrophobic amino acid, respectively, also DNA synthesis since viral DNA containing nucleocapsids did not affect the ability of core protein to assemble into also had a higher electrophoretic mobility (Fig. 5). nucleocapsids. Substitution with a proline, which disrupts To obtain further evidence for assembly of chimeric alpha helices, blocked nucleocapsid assembly (Fig. 7) and, nucleocapsids, we examined the effect on nucleocapsid like core protein with the H107 deletion, core protein with this electrophoretic mobility of varying the ratio of the wt and substitution appeared to form chimeric nucleocapsids with the mutant core protein expressed in transfected cells. LMH cells wild-type protein (not shown). This observation suggests that were transfected with varying ratios of HisDHBV/wtCore and integrity of the alpha helix in the insertion domain of the wtDHBV/HisCore. Again, core protein in nucleocapsids was DHBV core protein may be essential for nucleocapsid detected by western blot analysis (Fig. 5A). Viral DNA within assembly or that disruption may be a part of a trigger for nucleocapsids was detected by probing the blots with nucleocapsid disassembly. radiolabeled DHBV DNA. An increase in the ratio of HisCore to wtDHBV correlated with an increase in the mobility of Discussion DNA containing nucleocapsids (Figs. 5A–B). Southern blot analysis of viral DNA extracted from the nucleocapsids Deletion of histidine 107 in the insertion domain of the revealed a typical pattern (Mason et al., 1982) of viral DNA DHBV core protein prevented formation of stable nucleocap- replication intermediates (Fig. 5C). Thus, the results implied sids and viral DNA synthesis, which takes place in that chimeric nucleocapsids supported typical viral DNA nucleocapsids. Histidine 107 is predicted to be near the synthesis. cccDNA synthesis also took place (Fig. 5D). middle of a short alpha helix located in the center of the Virus production was also measured and found to be insertion domain, which is thought to be at the tip of the proportional to intracellular levels of viral DNA, suggesting external spike (Bringas, 1997; Kenney et al., 1995; Thermet et that chimeric nucleocapsids were able to form virions (Fig. 5E). al., 2004). An external location is consistent with antibody Infectivity of virus formed in cells transfected with wtDHBV or binding studies (Thermet et al., 2004). Histidine 107 is not with varying ratios of wtDHBV/HisCore was assessed by highly conserved in DHBV isolates, and its replacement with infection of primary duck hepatocytes. Virus assembled in cells amino acids that are present at the homologous position in containing predominantly chimeric nucleocapsids (Fig. 5A) other avihepadnaviruses did not affect viral nucleocapsid was able to infect primary duck hepatocyte cultures (Fig. 6). assembly. Substitution with proline, a residue that would 446 H. Guo et al. / Virology 353 (2006) 443–450

Fig. 3. Immunoblot analysis of intracellular core protein expression in transfected LMH cells. LHM cells were transfected with 1.8 μg of HisCore or wtCore (Fig. 1). Total protein recovered from one fiftieth of a 60-mm- diameter dish at 5 days post-transfection was loaded in each lane for SDS- polyacrylamide gel electrophoresis. Following transfer to a nitrocellulose filter, core protein was detected with rabbit antiserum raised to purified DHBV nucleocapsids. β-actin, detected with mouse anti-chicken actin, was assayed as a loading control.

into mature and functional nucleocapsids. Additional experi- ments (Zhou and Standring, 1992) will be necessary to determine if the mutant protein is functional for homodimer formation and/or if mutant and wild-type core protein are able Fig. 2. DNA replication in cells transfected with wild-type and mutant DHBV. to assemble into chimeric dimers. LMH cells were transfected with 2.5 μg of wtDHBV or HisDHBV and 1.8 μg In addition to its apparent influence on nucleocapsid of wtCore, as indicated. (A) Five days later, nucleocapsids were isolated and assembly, there remains the question of whether the insertion DNA replication intermediates were extracted. One quarter of each sample domain has a role in the functions of the assembled was subjected to Southern blot analysis. The positions of the relaxed circular nucleocapsid. For HBV, interactions with the viral envelope (RC), double-stranded linear (DSL) and single-stranded (SS) DNAs are indicated. DpnI digestions were carried out prior to loading of the gels to have been mapped near the base of the spike (Ponsel and Bruss, digest input plasmid DNA. Fragments from the DpnI-digested plasmids are 2003) and not, as might have been considered a priori, to the tip. indicated. The blot was probed with a radiolabeled DNA representing the However, a firmly established feature of DHBV infection, but as complete viral genome. (B) Total RNA and RNA packaged into nucleocapsids were extracted at 5 days post-transfection and analyzed by northern blotting. For total RNA analysis, each lane contained 10 μg of RNA. For nucleocapsid RNA analysis, each lane contains the amount of encapsidated RNA in one quarter of the nucleocapsids prepared from a single 60 mm tissue culture dish. The blot was probed with a genome-length, plus strand specific, DHBV riboprobe. disrupt the alpha helix, blocked nucleocapsid assembly. Thus, the insertion domain, unlike insertions at the tip of the HBV spike, plays an essential structural role in accumulation of nucleocapsids. A particularly puzzling feature of the current results, when compared to studies of insertions at the tip of the HBV spike, Fig. 4. Capsid gel assay for nucleocapsid production by HisCore and is that the H107 deletion prevented nucleocapsid formation. A HisDHBV. LMH cells were transfected with 2.5 μg of wtDHBV or HisDHBV μ simple interpretation of the data is that wt and mutant are able and 1.8 g of wtCore or HisCore, as indicated. At 5 days post-transfection, nucleocapsids were isolated and analyzed by electrophoresis through a non- to form chimeric dimers that are then functional for denaturing agarose gel followed by transfer to a nitrocellulose membrane. nucleocapsid formation. That is, that the insertion domain is Core protein was detected by western blotting using rabbit antiserum raised to essential for dimer formation but not for assembly of dimers DHBV nucleocapsids. H. Guo et al. / Virology 353 (2006) 443–450 447

Fig. 5. Analysis of the chimeric nucleocapsids. LMH cells were co-transfected with different ratios of wtDHBVand HisCore, or HisDHBVand wtCore, and harvested at 5 days post-transfection. In each lane, the plasmid marked with a 1 was a full-length virus expression vector, the plasmid marked with a number less than 1 was a core protein expression vector. The number 1 means the cells were transfected with 2.5 μg of a full-length construct and the indicated molar fraction of a core expression vector, with the exception of the 1:1 ratio, where 2.5 μg of wtDHBVand 1.8 μg of HisCore were used. The first two panels show the results of capsid gel analysis of the native nucleocapsids. Core protein was detected by western blotting analysis using a rabbit antiserum reactive to purified DHBV nucleocapsids (A). Following the western analysis, the viral DNA was denatured with NaOH and the filter was probed to detect viral DNAs (B). Viral DNA replication intermediates isolated from nucleocapsids preparations (C), cccDNA (D) and viral DNA in virions isolated from the culture fluids (E) are shown in the next three panels. Panel F shows the results of a mixing experiment in which lysates of cells transfected with wtDHBVand wtDHBV + HisCore (wt:mt) were analyzed directly, or after mixing in a 1:1 ratio, and nucleocapsids were detected by western analysis. The positions of the relaxed circular (RC), double-stranded linear (DSL), single-stranded (SS) and cccDNAs are indicated in the appropriate panels. yet to be shown for HBV or woodchuck hepatitis virus, is passage through the nuclear pore (Mabit et al., 2001; Rabe et al., negative regulation of cccDNA formation by the viral envelope 2003). proteins (Summers et al., 1990, 1991). Indeed, in the absence of viral envelope proteins, DHBV infection is cytocidal (Lenhoff Materials and methods et al., 1999). Thus, one scenario is that the interaction of the nucleocapsid and viral envelope proteins is different than for Plasmids and mutant constructions HBV and that the external domain of the DHBV core protein is involved in negative regulation of nucleocapsid trafficking to pCMVDHBV (Condreay et al., 1990) is a plasmid, herein the nuclear pore by viral envelope proteins, a regulatory denoted wtDHBV, in which expression of the pregenome of mechanism that has not so far been found with the mammalian the wild-type DHBV strain DHBV-16 is under control of a hepadnaviruses. A positive role in transport into the nucleus CMV-IE promoter. With this construct, precore protein is not seems precluded by evidence that a nuclear localization signal made. To make a DHBV core protein expression plasmid, not in the distal portion of the core protein regulates trafficking to including the precore region, we amplified a fragment the nucleus (Mabit et al., 2001). Another possible function of containing the DHBV core gene by PCR using primers 5′ the insertion domain in the assembled nucleocapsid, not ATTCTCGAGTAAATATGGATATCAATGCTTCTAGAGCC 3′ necessarily excluding the first, is that the insertion domain is and 5′ GCCGGATCCGTAATTTATTTCCTAGGCGAGGGAG- part of a trigger for nucleocapsid disintegration following ATC 3′. XhoI and BamHI restriction sites (underlined), placed 448 H. Guo et al. / Virology 353 (2006) 443–450

Assay of viral DNA from transfected cells and culture supernatants

Viral DNA replication intermediates were isolated from cytoplasmic nucleocapsids as described (Calvert and Summers, 1994), including digestion with DNaseI to eliminate input plasmid DNA before disruption of the viral nucleocapsids. In some cases, DNaseI digestion was omitted and DpnI was used to digest any remaining input plasmid DNA before sample loading. The detection of cccDNA in the nuclei of transfected cells was performed as described (Yu and Summers, 1994a). Viral DNA in secreted enveloped virus particles was extracted and purified from 400 μl of clarified culture fluids using a published method (Lenhoff and Summers, 1994). Viral DNAs were subjected to electrophoresis through a 1.5% agarose gel and analyzed by Fig. 6. Assay for the ability of virions with chimeric cores to infect primary 32 duck hepatocyte cultures. Virions were purified, as described in Materials and Southern blotting (Condreay et al., 1990) with P-TTP-labeled methods, from the culture fluids of the transfected LMH cells shown in Fig. DNA probes representing the entire DHBV genome. 5. Primary duck hepatocyte cultures were infected with 1.8 × 108 virions per 60 mm tissue culture dish. Viral DNA was isolated from the infected cells at Analysis of viral RNA 7 days post-infection and subjected to southern blot analysis. Each lane contained one quarter of the total DNA isolated from the hepatocytes in a single dish. Viral DNAs were detected with a DNA probe representing the Total cellular RNAwas extracted from transfected LMH cells entire viral genome. The positions of the relaxed circular (RC) and single- using the Micro-to-Midi Total RNA Purification System stranded (SS) DNAs are indicated. (Invitrogen), and encapsidated viral pgRNA was purified as described (Guo et al., 2003). Total RNA and encapsidated pgRNA were subjected to northern blotting and hybridized with outside the core coding regions, were introduced at the 5′ ends a 32P-UTP (800 Ci/mmol)-labeled full-length DHBV minus of the two primers for subsequent subcloning. The DHBV strand riboprobe. regions are in italics and start at nt 2645 and 419, respectively (Mandart et al., 1984). The PCR product was then inserted into SDS-PAGE and immunoblotting phCMV1 (Gene Therapy Systems, Inc.) between XhoI and BamHI in the MCS, generating the plasmid wtCore. Deletion of Transfected LMH cells were directly lysed and subjected to the triplet encoding histidine 107 of the core protein was carried SDS-PAGE as described (Prassolov et al., 2003). Viral core out with the virus and core expression plasmids using the Quick protein was detected by immunoblotting using rabbit anti- Change II Site-Directed Mutagenesis Kit (Stratagene), generat- DHBcAg serum (diluted 1:1000; Jilbert et al., 1992). Chicken β- ing plasmids HisDHBV and HisCore, respectively. actin was detected with a mouse anti-chicken actin monoclonal To generate the plasmids expressing core protein with antibody clone C4 purchased from Chemicon International. substitutions of the histidine at amino acid 107, the CAC sequence (nt 2965–2967) was changed to GAG, TAC, AAC, CAG, ATG or CCG, which changed the histidine to glutamate (CoreH107E), tyrosine (CoreH107Y), asparagine (Cor- eH107N), glutamine (CoreH107Q), methionine (CoreH107M) and Proline (CoreH107P), respectively.

Cells and transfections

The chicken hepatoma cell line LMH, which supports high levels of DHBV replication from transfected viral DNA (Condreay et al., 1990; Kawaguchi et al., 1987), was grown at 37 °C on 60 mm diameter tissue culture dishes in 1:1 Dulbecco's minimal essential medium:Ham's nutrient mixture F12 supplemented with 10 mM NaH2CO3 and 10% calf serum. Transfection of plasmid DNA was performed using Lipofecta- Fig. 7. Effect of substitutions of amino acid H107 on replication of DHBV DNA. mine 2000 reagent (Invitrogen) when cultures were 70–80% The indicated mutations were inserted into a core gene expression vector. LMH μ confluent. The medium was changed daily until harvest. The cells were transfected with 2.5 g of each of the indicated plasmids and harvested 4 days later. Viral DNA was prepared from nucleocapsids isolated monolayers were rinsed with PBS, and the medium was from the cytoplasm of transfected cells and subjected to Southern blot analysis, clarified to remove floating cells and cell debris. Both were as shown. The positions of the relaxed circular (RC), double-stranded linear stored at −80 °C until analyzed. (DSL) and single-stranded (SS) DNAs are indicated. H. Guo et al. / Virology 353 (2006) 443–450 449

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