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Seminars in VIROLOGY 8, 205–211 (1997) Article No. VI970123

RNA Signals That Control DNA Replication in Hepadnaviruses Jianming Hu and Christoph Seeger1 Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

The small 3.2-kb-long DNA genomes of hepadnaviruses are replicated by reverse transcription of an RNA intermediate. This RNA ‘‘pregenome’’ contains important signals that control critical steps of replication that include RNA packaging, initiation of reverse transcription, and elongation of minus strand DNA. Transcribed with terminally redundant ends, from a covalently closed circular DNA, pregenomic RNA also contains signals that regulate the conditional use of a polyadenylation site. To function as a pregenome the transcript must not enter the splicing pathway and therefore bears signals that permit splicing-independent egress from the nucleus and transport to ribosomes where it exerts its other role as a messenger RNA for the synthesis of capsid and polymerase polypeptides. Translation, in turn, demands signals that maintain the stoichiometry of about 200 capsid proteins per molecule of polymerase synthesized. r 1997 Academic Press

Key Words: hepadnavirus; Hsp90; protein priming; reverse transcription; RNA packaging.

Synthesis of the hepadnavirus pregenome occurs in the nucleus of the infected hepatocyte from a covalently closed circular (ccc) DNA, which is the processed viral genome (for a recent review, see 1). The result is a terminally redundant 3.5-kb-long RNA that is transported into the cytoplasm as an unspliced messenger RNA. It first acts as a template for the synthesis of the capsid protein and the polymerase, a reverse transcriptase (RT), and subsequently succumbs to sequestration into capsids to serve as a template for reverse transcription to synthesize the first (minus) strand DNA and as a substrate for RNase H. The 58 end of the RNA, generated by RNase H, then acts as a primer for second (plus) strand DNA synthesis and becomes, together with the RT, a part of the viral genome.

RNA SIGNALS FOR RNA SYNTHESIS The synthesis of unspliced, terminally redundant RNAs from a circular DNA template poses two obvious problems: 38 end formation and nuclear export. To synthesize the terminally redundant pregenomic RNA 1To whom correspondence should be addressed. Fax: (215) 7283616. E-mail: [email protected].

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(pgRNA) the polyadenylation site is bypassed once and used only the second time to form the 38 end (Fig. 1). The polyadenylation signals of the mammalian hepadnaviruses differ from the canonical AAUAAA motif at the first position where they carry a U instead of an A residue. This variation renders the signal per se inefficient and requires additional signals to compensate for the loss of function (2–4). These activating sequences, called processing signals (PS), are located within a ca. 400-nucleotide-long segment upstream of the polyadenylation site (Fig. 1). As a result, they are not contained in toto at the 58 end of pgRNA, which is part of the reason why the 58 polyadenylation signal is not recognized. In addition, the proximity of the 58 polyadenylation site to the cap site suppresses its usage, as has been observed in certain retroviruses (5). This may, in fact, be the major reason for transcriptional readthrough in avian hepadnaviruses indigenous to domestic ducks (duck hepatitis B virus, DHBV) and gray herons (heron hepatitis B virus, HHBV) where the consensus AAUAAA polyadenylation motif is present, approximately 250 nucleotides downstream of the cap site. Exactly how cap proximity suppresses the polyadenylation signal remains an enigma. An RNA element located near the 58 end of the pregenome of DHBV is required for efficient expres-

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FIG. 1. Summary of cis-acting signals on pgRNAs of mammalian and avian hepadnaviruses. (A) Mammalian hepadnaviruses. The terminal redundancy (R) harbors the RNA packaging signal («) and the polyadenylation site (shaded oval). t denotes a region upstream of the 38 terminal redundancy which serves as an acceptor site for the nascent minus strands (see Fig. 3 for detail). Also indicated are the processing signal (PS), required for 38 end formation, and the posttranscriptional regulatory element (PRE), required for nuclear export of unspliced viral RNAs. In addition, the core protein and polymerase open reading frames (ORF) are indicated. For simplicity, the surface and X ORFs are omitted (see 1). The nucleotide positions (with the cap site defined as nucleotide 1) of the various signals are indicated. (B) Avian hepadnaviruses. The RNA packaging signal «, the polyadenylation signal and core, and polymerase ORFs are as described above for the mammalian viruses. Pac 2 denotes a second region of pgRNA required for RNA packaging in DHBV. In addition, the positive effector of transcription (pet), overlapping «, is required for efficient transcriptional elongation of pgRNA across a downstream attenuator site (see text for details).

sion of this RNA species (6, 7). The activity of this so-called positive effector of transcription, or pet (Fig. 1), is shown to be position and orientation dependent and to function in cis. Pet appears to act mainly by enhancing transcriptional elongation across a downstream negative element, a transcriptional attenuator or pause site. However, the details of this mechanism remain to be determined. Once generated, the pregenome must be transported across the nuclear membrane to assemble into a polysome and act as a messenger RNA for the synthesis of capsid proteins and polymerase. Its nature as an intermediate for viral genome replication precludes splicing of pgRNA as that would result in the permanent loss of genetic information. Although, theoretically, a small deletion of one of the terminally redundant ends could be tolerated, this has not been observed. However, since splicing and export of mRNAs in eukaryotic cells are generally coupled events, pgRNA may have to enter specialized path-

ways for export to the cytoplasm, which depend on one or more RNA signals. An RNA element located near the 38 end of pgRNA displays the profile of such a signal. It was first noticed in experiments aimed at the expression of the viral surface protein in mammalian cell cultures. Deletion of a region spanning 360 nucleotides downstream of the coding region reduced expression approximately 10-fold (8). Subsequent studies by several other groups led to the identification of a 400to 500-nucleotide-long region downstream of the surface gene that affected the accumulation of viral RNA in the cytoplasm of transfected cells (9–11). This socalled posttranscriptional regulatory element (PRE, Fig. 1) could act by facilitating the export of the unspliced viral RNAs to the cytoplasm, perhaps similar to the rev-responsive element in human immunodeficiency virus (HIV) (12). However, unlike in HIV, the protein substrates, if any, have yet to be discovered. Viral proteins do not appear to have a role in this process.

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RNA SIGNALS FOR TRANSLATION Whereas translation of the capsid protein occurs from an AUG located at the 58 end of pgRNA, translation of the polymerase gene occurs from an internal AUG that is located within the capsid gene. Structural and biochemical analyses of core particles indicated that one or two polymerase polypeptides are present in capsids composed of 180 or 240 monomeric subunits (13, 14). To maintain this ratio, one would expect that the polymerase AUG is used with an efficiency of approximately 0.5% compared to the core AUG. Based on the suboptimal context of the polymerase initiation AUG and the presence of many upstream AUGs in the core gene, internal ribosomal entry was favored over a leaky scanning mechanism for polymerase expression (15, 16). However, genetic experiments aimed at the identification of a signal in the vicinity of the polymerase AUG that could facilitate internal entry of ribosomes have not been successful. Also, expression of the poliovirus protease 2A, which prevents cap dependent translation, appeared to inhibit internal initiation from the polymerase initiation site, suggesting that this event occurs in a cap-dependent manner (16a). Consistent with this view is the observation that interference with initiation of translation at the core AUG by insertion of an RNA sequence that can form a stable hairpin or by annealing of an antisense RNA upstream of the core AUG also curtails polymerase expression. Thus, in spite of the earlier considerations, ribosomal leaky scanning appears to be the most likely mechanism for the internal initiation of polymerase translation.

RNA SIGNALS FOR RNA PACKAGING Once it has directed the synthesis of the polymerase, pgRNA is converted from a messenger RNA to a template for reverse transcription. Genetic experiments revealed that the formation of a ribonucleoprotein (RNP) complex between the polymerase and an RNA signal, termed «, withdraws the messenger from the ribosome and commits it to the pathway of viral assembly (17–19). « is located within the terminally redundant ends of pgRNA (20) but only the 58 copy of « is required for RNA packaging and, so far, no function has been attributed to the 38 copy. While in mammalian hepadnaviruses « is sufficient for RNA packaging, in avian hepadnaviruses a second signal located approximately 1000 nucleotides downstream from « is required (Fig. 1, pac 2; Refs. 21, 22). Besides its role in viral assembly, the interaction between « and the RT also plays a critical role for the priming of

reverse transcription. Investigations of the determinants on RNA and protein that control RNA packaging have been performed in tissue culture cells where packaged pgRNA can be distinguished from cytoplasmic RNA by its resistance to treatment with ribonuclease. Efforts to reconstitute this reaction with purified components in vitro have not been successful. However, results reported by our laboratory showed that enzymatically active reverse transcriptase of DHBV can be expressed in reticulocyte lysates, which has provided a means for biochemical and genetic studies of the polymerase–« interaction (23, 24). As will be discussed below, these efforts led to the findings that « is not simply a packaging signal but acts as a template for reverse transcription in a, so far, unique protein priming reaction and, furthermore, that this event depends on the presence of host factors belonging to the heat shock 90 protein (Hsp90) complex. « bears two inverted repeats that can form a stemloop structure. In HBV, the proposed structure features a lower and an upper stem of 13 and 11 nucleotides in length, an apical loop and an internal bulge, both of which span six nucleotides (Fig. 2; Ref. 20). In addition, the upper stem bears a single bulged U residue. Evidence for the proposed structure in solution has been obtained by RNase mapping and chemical probing experiments (25, 26). In addition, extensive mutagenesis studies have, in general, confirmed the importance of the « structure in viral RNA packaging and DNA synthesis (25–30). The nature of the cis-acting regions of « for efficient pgRNA packaging can be summarized as follows. First, the lower stem and the internal bulge appear to play mainly a structural role, the sequence of which can be altered without significant effect on RNA packaging. Second, the specific sequences on the lower right side of the upper stem is required. Third, sequences of the apical loop, in contrast to the internal bulge, contribute critically, in a sequence specific manner, to RNA packaging. The availability of an enzymatically active polymerase of DHBV, expressed in a cell-free system, afforded the opportunity to examine directly the determinants of « that dictate its specific interaction with the polymerase (23, 24). It has been demonstrated that the DHBV RT, expressed in vitro, forms a stable RNP complex with « (24, 28). As expected, these in vitro RNA-binding studies have confirmed that the specific interaction between the RT and « is a prerequisite for RNA packaging and DNA synthesis. Thus, the requirements of « for efficient binding to the RT, in general, mimic those already described above for pgRNA packaging except for the apical loop, whose specific sequences appear to be critical for RNA packaging but not for RT binding (28).

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FIG. 2. Proposed structures for the RNA packaging signal «. The stem-loop structures for the HBV, DHBV, and HHBV « RNA, as proposed by Junker-Niepmann et al. (20), are shown. Note the much shorter upper stem and much larger apical loop (shaded region) in the HHBV «, compared to HBV or DHBV.

The precise structure of « as determined by NMR or X-ray crystallography is not available. Interestingly, the stem-loop structure proposed for the DHBV and, in particular, HHBV « (Fig. 2; Refs. 24, 31), based on phylogenetic and functional conservation to their mammalian counterparts, is not the most energetically favored. However, recent enzymatic and chemical probing studies have largely confirmed the proposed structures (31). These results suggest that additional, not-yet-recognized secondary and/or tertiary interactions may stabilize the proposed structure. In addition, the upper stem of the HHBV « is much shorter and leaves a much larger apical loop, compared to DHBV. In spite of these apparent structural differences, the HHBV « can efficiently bind to the DHBV polymerase (24, 31). These and other observations suggest that only the lower stem and bulge region of « are critical for the initial binding to the RT. Upon binding to the RT, both the DHBV and the HHBV « may adopt a new structure that is similar for both RNAs, i.e., by an induced-fit mechanism common to RNA–protein interactions. In addition, the requirement for the specific sequences of the apical loop in RNA packaging, but not RT binding, has led to the hypothesis that in addition to the viral polymerase, host proteins may also bind to the apical loop in a sequence specific manner and play an essential role in viral RNA packaging (28). Genetic studies showed that a structurally intact polymerase is required for RNA packaging (17–19). However, the enzymatic activities of the RT are not

required. Similar observations were made with the in vitro system where the RT–« interaction can be studied independently of RNA packaging (24, 28). Differences between RNA packaging and RT–« binding became apparent with C-terminal deletions of the RT which abolish RNA packaging but seem not to affect « binding. The analyses of more than 50 variants of the DHBV polymerase with single amino acid changes point to two regions with conserved arginine residues that are critical for polymerase–« interaction (32). These map to the N-terminal (terminal protein, TP) and the central RT domains, consistent with previous observations that both domains are required for « binding and RNA packaging. All these results support a model whereby the polymerase polypeptide acts as a scaffold for the assembly of nucleocapsids, with residues from the TP and RT domains contacting « and, perhaps, the C-terminal domain interacting with the core proteins, either directly or indirectly. Efforts to biochemically identify the amino acid residues on the polymerase that make direct contact with « have been hampered by the presence of cellular factors that form a large complex with the polymerase, as will be described below.

RNA SIGNALS FOR DNA SYNTHESIS Experiments with in vitro synthesized DHBV RT revealed that sequences within the internal bulge of « serve as the template for initiation of reverse transcrip-

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tion. This reaction leads to the synthesis of a three- to four-nucleotide-long DNA strand (Fig. 3; Refs. 27, 30, 33, 34). Most notably, this reaction is primed by a tyrosine residue on the reverse transcriptase and not, as expected, by RNA (23, 35). Different from other known systems where proteins prime DNA synthesis, in hepadnaviruses the protein priming and the polymerase activities reside on the same polypeptide (36, 37). As a consequence, the polymerase remains attached to the 58 end of minus strand until it is removed by an unknown process following infection, which leads to the formation of ccc DNA. To continue DNA synthesis following the protein priming reaction, the polymerase–DNA complex is translocated to the 38 end of pgRNA, where the nascent minus DNA strand anneals to complementary sequences at a specific acceptor site. Mutagenesis studies have demonstrated that the short homology between the donor and acceptor sites is necessary but not sufficient to specify the site of minus strand translocation (38). The precise nature of these additional RNA signals which specify the acceptor site selection is not known at present. Also unclear is the mechanism whereby minus strand DNA synthesis

arrests following the polymerization of three to four nucleotides during the protein priming reaction. It is conceivable that the last two nucleotides of the bulge may be inaccessible to the polymerase active site due to structural features of the lower stem and/or of the bulge itself or to tight binding by the polymerase (29). Interestingly, a similar problem is encountered by telomerase, which also copy only a small portion of its RNA template into DNA (39). An important consequence of the protein priming reaction is that the «–RT interaction is transient and that conformational changes have to promote the transition from the protein priming reaction to the elongation step. In particular, the bulge of «, which occupies the active site of the RT must be displaced by sequences at the 38 end of pgRNA, which provide the template for subsequent elongation of minus strands. Increased protease resistance has provided the first evidence for a conformational change of the RT following RNP formation with « in vitro (40). However, nothing is known about the structural dynamics that is likely to occur during the minus strand transfer reaction. Investigations of these events are hampered, in part, by the low efficiency of the transfer reaction in the

FIG. 3. The protein priming reaction in hepadnaviruses. Shown schematically are the « stem loop at the 58 end of pgRNA and the polymerase with its TP and RT domains. Initiation of reverse transcription in triggered by the formation of an RNP complex between « and the polymerase. A tyrosine residue located at the TP domain acts as the primer for minus DNA synthesis (36, 37). After the RT copies three or four nucleotides from the « bulge, producing a short DNA oligomer which remains attached to the RT via the primer tyrosine residue, a template switch occurs whereby the nascent minus DNA–RT complex is translocated to the 38 end of pgRNA. The nascent DNA strand then anneals to the homologous sequences at the acceptor site (t) to permit elongation of the minus DNA strand.

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reticulocyte lysate. It is possible that this process depends on the formation of nucleocapsids, the assembly of which depends on core subunit concentrations that are difficult to obtain in the lysates. Biochemical analyses of the requirements for RNP formation between the RT and «, using the cell-free system that expresses a functional DHBV RT, have led to the discovery that cellular factors, in particular, a multicomponent chaperone complex consisting of Hsp90 and its cochaperones, are required for the polymerase to interact with « (41, 42). By using specific monoclonal antibodies and pharmacological inhibitors, it has been demonstrated that the Hsp90 complex is functionally required for RNP formation between the RT and «, and thus, for protein priming in vitro and RNA packaging and DNA replication in vivo. The chaperone complex is physically associated with the RT and appears to act by establishing and maintaining a specific conformation of the RT that is competent for « binding. The action of this chaperone complex in facilitating the RT–« interaction, as has been observed with other target proteins such as the steroid receptors, is highly dynamic; it requires energy in the form of ATP hydrolysis and another chaperone and a known ATPase, the heat shock protein 70. Furthermore, the chaperone complex appears to remain associated with the RT following assembly and is incorporated into nucleocapsids. Whether it plays additional roles in the viral replication cycle remains to be determined.

PERSPECTIVES The presence of multiple cis-acting sequences on the hepadnavirus pgRNA is a reflection of its many roles in viral replication and the intricate mechanisms of regulating its genesis and function. Save for the «–RT interaction, only limited information is available about the nature of other RNA-binding proteins that interact with the known cis-acting sequences on pgRNA. Nevertheless, the identification and characterization of the cis-acting sequences on the viral RNAs have opened up the possibility of identifying the putative transacting proteins, viral or cellular, that specifically bind to these RNA sequences. Recently, the identification of a 65-kDa nuclear protein (p65) that specifically binds to the terminally redundant region of HBV pgRNA has been reported (43). Although its role in viral replication remains to be determined, preliminary results showed that mutations of this region that abolish p65 binding dramatically inhibited viral DNA replication, suggesting that p65 may indeed play an essential role

in viral DNA synthesis. Efforts to isolate specific cellular factors binding to the PRE have also identified two nuclear proteins, 30 and 45 kDa, which bind to multiple elements within the PRE (44). Mutagenesis of the PRE elements indicated that binding of these cellular proteins to the PRE and its mutant derivatives correlated with the RNA export function of the PRE, suggesting that these proteins may mediate the activities of the signal. Future efforts to further identify and characterize these host proteins binding to the viral RNA signals will not only provide important insights into the molecular mechanisms of hepadnavirus replication but may also reveal novel mechanisms of RNA–protein interactions in general.

ACKNOWLEDGMENTS We thank William Mason for comments to the manuscript. This work was supported by grants from the National Institutes of Health and the Commonwealth of Pennsylvania. J.H. is a recipient of an NRSA Postdoctoral Training Grant (CA09035-20).

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