The initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, ) on the pregenomic RNA (pgRNA) by the viral reverse transcriptase (RT). RT-interaction in the duck hepatitis B virus (DHBV) was recently shown to require the molecular chaperone complex, the heat shock protein 90 (Hsp90). However, the requirement for RT-interaction in the human HBV has remained unknown due to the inability to obtain a purified RT protein active in specific binding. We now report that Hsp90 is also required for HBV RT-interaction. Inhibition of Hsp90 led to diminished HBV pgRNA packaging into nucleocapsids in cells, which depends on RTinteraction. Furthermore, using truncated HBV RT proteins purified from bacteria and five purified Hsp90 chaperone factors, we have developed an in vitro RT-binding assay. Our results demonstrate that Hsp90, in a dynamic process that was dependent on ATP hydrolysis, facilitated RT-interaction in HBV, as in DHBV. Specific binding required sequences from both the amino-terminal terminal protein and the carboxyterminal RT domain. Only the cognate HBV , but not the DHBV , could bind the HBV RT proteins. Furthermore, the internal bulge, but not the apical loop, of was required for RT binding. The establishment of a defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT-interaction and chaperone activation.Hepatitis B virus (HBV) infection is a major global public health problem with over 300 million chronically infected patients worldwide (34). Patients with chronic HBV infection carry a great risk of developing severe liver diseases, including cirrhosis and liver cancer, which result in a million mortalities annually (4, 10). HBV is a member of the Hepadnaviridae family, a group of small hepatotropic DNA viruses that also includes related animal viruses, such as the duck hepatitis B virus (DHBV) and the woodchuck hepatitis virus. All hepadnaviruses carry a small (ca. 3.2 kb), relaxed circular, partially double-stranded DNA genome and replicate this DNA genome through an RNA intermediate, the pregenomic RNA (pgRNA), by reverse transcription (52). The reverse transcription pathway employed by hepadnaviruses is similar to, yet distinct from, that used by retroviruses (for reviews, see references 49 and 50).All hepadnaviruses encode a novel, multifunctional reverse transcriptase (RT). Like its retroviral counterparts, the hepadnavirus RT catalyzes RNA-and DNA-dependent DNA polymerization and has an intrinsic RNase H activity (12,43,56). Reflecting this functional conservation, the central catalytic RT domain and carboxy (C)-terminal RNase H domain of the hepadnavirus RT are homologous to the corresponding domains of retroviral RTs. However, all hepadnavirus RTs share an amino (N)-terminal domain, called the terminal protein (TP) (2,12,43). The TP domain is absent from retroviral RTs. Sequence database searches indicate tha...

Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, ) on the pregenomic RNA template by the viral reverse transcriptase (RT) and is primed by the RT itself (protein priming). We have previously shown that the RT-interaction and protein priming require the cellular heat shock protein, Hsp90. However, additional host factors required for these reactions remained to be identified. We now report that five cellular chaperone proteins, all known cofactors of Hsp90, were sufficient to reconstitute a duck hepatitis B virus RT active in binding and protein priming in vitro. Four proteins, Hsp90, Hsp70, Hsp40, and Hop, were required for reconstitution of RT activity, and the fifth protein, p23, further enhanced the kinetics of reconstitution. RT activation by the chaperone proteins is a dynamic process dependent on ATP hydrolysis and the Hsp90 ATPase activity. Thus, our results have defined a minimal complement of host factors necessary and sufficient for RT activation. Furthermore, this defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT activation and chaperone functions. Reverse transcription in hepadnaviruses (hepatitis B viruses[HBVs]) is carried out by a virally encoded reverse transcriptase (RT), which is unique among all known RTs in several aspects (39, 43). The RT initiates DNA synthesis de novo using a specific tyrosine residue located within its unique N-terminal domain as a protein primer (27,46,48,51; for a recent review, see reference 19). This protein-priming reaction requires interaction between the RT and a specific RNA signal, termed ε, located on the viral pregenomic RNA (pgRNA) (the template for reverse transcription) (33, 47). The ε RNA forms a conserved stem-loop structure with an internal bulge, which is used as the specific template for protein priming (and thus, the origin of reverse transcription) (29,45,47; for a recent review, see reference 21). In addition, ε serves as the RNA packaging signal (16, 24) and, through its interaction with the RT, directs the selective encapsidation of both the pgRNA and the RT into viral nucleocapsids (1, 33). Therefore, the specific interaction between the RT and ε triggers two essential early steps in hepadnavirus assembly and replication, i.e., the proteinprimed initiation of reverse transcription and the assembly of replication-competent nucleocapsids.Using the duck hepatitis B virus (DHBV) RT as a model system, we have recently found that the RT requires the assistance of host cell factors in order to carry out specific ε binding and protein-priming functions (20,22). One such cellular factor is the 90-kDa heat shock protein (Hsp90). Hsp90 associates with the DHBV RT and is required for RT-ε interaction and protein priming in vitro and for pgRNA packaging and DNA synthesis in vivo. Furthermore, the chaperone is specifically incorporated into viral nucleocapsids via associatio...

To facilitate investigations of replication and host cell interactions in the hepadnavirus system, we have developed cell lines permitting the conditional replication of duck hepatitis B virus (DHBV). With the help of this system, we devised conditions for core particle isolation that preserve replicase activity, which was not found in previous preparations. Investigations of the stability of viral DNA intermediates indicated that both encapsidated DNA and covalently closed circular DNA (cccDNA) were turned over independently of cell division. Moreover, we showed that alpha interferon reduced the accumulation of RNA-containing viral particles. The availability of a synchronized replication system will permit the biochemical analysis of individual steps of the viral replication cycle, including the mechanism and regulation of cccDNA formation.Hepadnaviruses are small DNA viruses that contain a relaxed circular DNA (rcDNA) genome with modified 5Ј ends. The 5Ј end of minus strand DNA is covalently attached to the viral reverse transcriptase (RT), whereas the 5Ј end of the plus strand is linked to an 18-nucleotide-long capped RNA oligomer. After infection, the rcDNA is converted into covalently closed circular DNA (cccDNA), which is the template for the transcription of at least three viral RNA species. The longest transcript, termed pregenomic RNA (pgRNA

The cellular chaperone Hsp90 has been shown to associate with the reverse transcriptase (RT) of the duck hepatitis B virus and is required for RT functions. However, the molecular basis for the specific interaction between the RT and Hsp90 remains unknown. Comparison of protein compositional properties suggests that the RT is highly related to the protein kinase c-Raf, which interacts with Hsp90 via the cochaperone p50 (CDC37). We tested whether the RT, like c-Raf, is specifically recognized by p50. Immunoprecipitation and pulldown assays showed that p50 or p50␦C, a p50 mutant defective in Hsp90 binding, could interact specifically with the RT both in vitro and in vivo, indicating that p50 can bind the RT independently of Hsp90. Furthermore, purified p50 and p50␦C interacted directly with purified RT. The importance of p50-RT interaction for RT functions was underscored by 1) inhibition of proteinprimed initiation of reverse transcription by p50␦C in vitro and 2) stimulation of viral DNA replication and RNA packaging by p50 and their inhibition by p50␦C in transfected cells. These results suggest that p50 can function as a cellular cofactor for the hepadnavirus RT by mediating the interaction between the RT and Hsp90.Reverse transcription in hepadnaviruses (hepatitis B viruses) is carried out by a novel virally encoded reverse transcriptase (RT) 1 (1, 2). The RT is able to initiate DNA synthesis de novo using a specific tyrosine residue located within its N-terminal domain (the terminal protein (TP)) as a protein primer (Refs. 3-6; for a recent review, see Ref. 7). This protein priming reaction requires the interaction between the RT and a specific RNA signal (termed ⑀) located on the viral pregenomic RNA (pgRNA; the template for reverse transcription) (8, 9). The ⑀ RNA is used as a specific template for protein priming (and thus, the origin of reverse transcription) (Refs. 9 -11; for a recent review, see Ref. 12). In addition, ⑀ serves as the RNA packaging signal (13, 14) and directs, through its interaction with the RT, the selective encapsidation of both the pgRNA and the RT into viral nucleocapsids (8,15). Therefore, the specific interaction between the RT and ⑀ triggers two essential early steps in hepadnavirus assembly and replication, i.e. the protein-primed initiation of reverse transcription and the assembly of replication-competent nucleocapsids.Using the duck hepatitis B virus (DHBV) as a model system, we have recently found that the RT requires the assistance of host cell factors to carry out specific ⑀ binding and protein priming functions (16,17). One such cellular factor is the 90-kDa heat shock protein (Hsp90). Hsp90 associates with the DHBV RT and is required for RT-⑀ interaction and protein priming in vitro and for pgRNA packaging and DNA synthesis in vivo. Hsp90 is thought to facilitate the functions of a specific subset of cellular proteins (the target or substrate proteins) by helping to establish and maintain certain poised but labile conformations of these target proteins through a dynamic...

The reverse transcriptase (RT) encoded by hepadnaviruses (hepatitis B viruses) is a multifunctional protein critical for several aspects of viral assembly and replication. Reverse transcription is triggered by the specific interaction between the RT and an RNA signal located on the viral pregenomic RNA, termed , and is initiated through a novel protein priming mechanism whereby the RT itself serves as a protein primer and serves as the obligatory template. Using the RT from duck hepatitis B virus as a model, we previously demonstrated that RT-interaction and protein priming require the assistance of a host cell chaperone complex, heat shock protein 90 (Hsp90) and its cochaperones, which associates with the RT and facilitates the folding of the RT into an active conformation. We now report that extensive truncation removing the entire C-terminal RNase H domain and part of the central RT domain could relieve this dependence on Hsp90 for RT folding such that the truncated RT variants could function in interaction and protein priming independently of Hsp90. The presence of certain nonionic or zwitterionic detergent was sufficient to establish and maintain the truncated RT proteins in an active, albeit labile, state. Furthermore, we were able to refold an RT truncation variant de novo after complete denaturation. In contrast, the full-length RT and also RT variants with less-extensive Cterminal truncations required Hsp90 for activation. Surprisingly, the presence of detergent plus some yet-tobe-identified cytoplasmic factor(s) led to a dramatic suppression of the RT activities. These results have important implications for RT folding and conformational maturation, Hsp90 chaperone function, and potential inhibition of RT functions by host cell factors. Reverse transcription in hepadnaviruses (hepatitis B viruses[HBVs]) is carried out by a novel virally encoded reverse transcriptase (RT) (27,30). The RT has the unique ability to initiate DNA synthesis de novo, using itself as a protein primer (18,19,35,39,43; for a review, see reference 9). This protein priming reaction requires the specific interaction between the RT and a short RNA signal, termed ε, located at the 5Ј end of the viral pregenomic RNA (pgRNA; the template for reverse transcription) (22,36). The product of protein priming is a three-to four-nucleotide DNA oligomer, representing the 5Ј end of the viral minus-strand DNA, covalently attached to the RT via an invariant tyrosine residue located at its N-terminal domain (19,21,32,34; for a review, see reference 11).The ability of the hepadnavirus RT to carry out specific RNA recognition and protein priming is reflected in its structural organization, which displays both similarities to, as well as differences from, conventional RTs encoded by retroviruses and other retroelements (4, 9, 23). As mentioned above, the N-terminal domain (the so-called terminal protein [TP]) bears the invariant primer tyrosine residue; the TP domain is conserved among all hepadnaviruses but absent from any other known RTs. The centr...

Reverse transcription in hepadnaviruses is primed by the viral reverse transcriptase (RT) (protein priming) and requires the specific interaction between the RT and a viral RNA signal termed , which bears the specific template sequence for protein priming. The product of protein priming is a short oligodeoxynucleotide which represents the 5 end of the viral minus-strand DNA and is covalently attached to the RT. We have now identified truncated RT variants from the duck hepatitis B virus that were fully active in the initial step of protein priming, i.e., the covalent attachment of the first nucleotide to the protein (RT deoxynucleotidylation), but defective in any subsequent DNA polymerization. A short sequence in the RT domain was localized that was dispensable for RT deoxynucleotidylation but essential for the subsequent DNA polymerization. These results have thus revealed two distinct stages of protein priming, i.e., the initial attachment of the first nucleotide to the RT (RT deoxynucleotidylation or initiation of protein priming) and the subsequent DNA synthesis (polymerization) to complete protein priming, with the second step entailing additional RT sequences. Two models are proposed to explain the observed differential sequence requirement for the two distinct stages of the protein priming reaction.Reverse transcription in hepadnaviruses (hepatitis B viruses, [HBVs]) is carried out by a unique virus-encoded reverse transcriptase (RT) (26, 28). The RT is able to initiate DNA synthesis de novo, using the RT itself as a protein primer (19,32,34,35; for a review, see reference 12). This protein priming reaction requires the specific interaction between the RT and a short RNA signal, termed ε, located at the 5Ј end of the viral pregenomic RNA (pgRNA) (the template for reverse transcription) (23, 33). The unique ability of the hepadnavirus RT to carry out specific RNA recognition and protein priming is reflected in its structural organization, which is both similar to, and distinct from, that of conventional RTs (6,12,24). The N-terminal TP (so-called terminal protein) domain is conserved among all hepadnaviruses but absent from all other known RTs encoded by retroviruses or other retroelements. In contrast, the central RT domain and the C-terminal RNase H domain share sequence homologies with conventional RTs. A highly variable spacer or tether domain appears to link the TP and RT domains.It is now well established that both the N-terminal TP and central RT domains are required for the RT to bind to ε and to carry out protein priming. Furthermore, protein priming requires additional sequences from the RT domain that are dispensable for ε binding (11,23,33). These additional amino acid sequences from the RT domain are presumably required for some aspects of viral DNA synthesis during protein priming, as follows. Using an internal bulge located on the ε stemloop structure as a specific template and an invariant tyrosine residue within the TP domain as a protein primer, the RT synthesizes, de novo, a 3-to 4-nucl...

Rabies remains a globally significant zoonotic disease, but rabies control is achievable under certain circumstances.Canine rabies has been eliminated from the U.S.; however, approximately 55,000 humans die annually worldwide from the disease. In the U.S., economic losses continue to be substantial and the risk to humans and domestic animals has not been eliminated. As an example of the complexity of rabies management, we describe a local rabies control program and efforts to restore Cape Cod, MA to terrestrial rabies-free status, after a 2004 oral rabies vaccination (ORV) barrier breach following 10 years of rabies-free status. The emergence of raccoon rabies in southeastern New England in 1992 prompted the U.S. Centers for Disease Control and Prevention, the Tufts Cummings School of Veterinary Medicine, and the Massachusetts Department of Public Health to begin an ORV program to reduce the occurrence of carnivore rabies in an area directly adjacent to the Cape Cod Canal. In 2001, USDA APHIS Wildlife Services began full-time collaboration on the Cape Cod Oral Rabies Vaccination Program (CCORVP) as part of national wildlife rabies control efforts. The primary objective of the CCORVP was to use ORV in tandem with the physical barrier created by the Canal to prevent the spread of rabies to peninsular Cape Cod, a heavily-populated tourist destination southeast of Boston. After an increase in rabies cases within the traditional Cape Cod ORV zone, ORV bait distribution efforts were modified to reduce the risk of rabies spread onto the Cape. In spite of these modifications, raccoon rabies was detected for the first time on peninsular Cape Cod in March 2004. A trap-vaccinate-release campaign, removal of suspect raccoons and skunks, and expanded ORV efforts were unsuccessful in preventing the spread of the virus. Rabies surveillance became the priority of the Cape Cod Rabies Task Force. In 2006, rabies was finally detected at the eastern extremity of the peninsula. In this paper, we summarize ORV efforts, explore possible causes for the spread of raccoon rabies onto the Cape, summarize several small-scale Cape Cod rabies research projects, and suggest a 5-year plan for future Cape Cod rabies controls efforts.

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