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Jianming Hu

Publications and source records attributed to Jianming Hu.

13 recordsLinked to original sources
PubMed2006

Regulation of hepadnavirus reverse transcription by dynamic nucleocapsid phosphorylation.

Reverse transcription, an essential step in the life cycle of all retroelements, is a complex, multistep process whose regulation is not yet clearly understood. We have recently shown that reverse transcription in the pararetrovirus duck hepatitis B virus is associated with complete dephosphorylation of the viral core protein, which forms the nucleocapsid wherein reverse transcription takes place. Here we present a genetic study of the role of this dynamic nucleocapsid phosphorylation in regulating viral reverse transcription. Detailed analyses of the reverse transcription products synthesized within nucleocapsids composed of core phosphorylation site mutants revealed that alanine substitutions, mimicking the nonphosphorylated state, completely blocked reverse transcription at a very early stage. In contrast, aspartate substitutions, mimicking the phosphorylated state, allowed complete first-strand DNA synthesis but were severely defective in accumulating mature double-stranded DNA. The latter defect was due to a combination of mutant nucleocapsid instability during maturation and a block in mature second-strand DNA synthesis. Thus, the reversible phosphorylation of the nucleocapsids regulates the ordered progression of reverse transcription.

Amino Acid Sequence↗
PubMed2006

Aberration measurement of projection optics in lithographic tools by use of an alternating phase-shifting mask.

As a critical dimension shrinks, the degradation in image quality caused by wavefront aberrations of projection optics in lithographic tools becomes a serious problem. It is necessary to establish a technique for a fast and accurate in situ aberration measurement. We introduce what we believe to be a novel technique for characterizing the aberrations of projection optics by using an alternating phase-shifting mask. The even aberrations, such as spherical aberration and astigmatism, and the odd aberrations, such as coma, are extracted from focus shifts and image displacements of the phase-shifted pattern, respectively. The focus shifts and the image displacements are measured by a transmission image sensor. The simulation results show that, compared with the accuracy of the previous straightforward measurement technique, the accuracy of the coma measurement increases by more than 30% and the accuracy of the spherical-aberration measurement increases by approximately 20%.

Journal Article↗
PubMed2006

Hepatitis B virus reverse transcriptase and epsilon RNA sequences required for specific interaction in vitro.

Initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, epsilon) on the pregenomic RNA by the viral reverse transcriptase (RT). Using an in vitro reconstitution system whereby the cellular heat shock protein 90 chaperone system activates recombinant HBV RT for specific epsilon binding, we have defined the protein and RNA sequences required for specific HBV RT-epsilon interaction in vitro. Our results indicated that approximately 150 amino acid residues from the terminal protein domain and 230 from the RT domain were necessary and sufficient for epsilon binding. With respect to the epsilon RNA sequence, its internal bulge and, in particular, the first nucleotide (C) of the bulge were specifically required for RT binding. Sequences from the upper portion of the lower stem and the lower portion of the upper stem also contributed to RT binding, as did the base pairing of the upper portion and the single unpaired U residue of the upper stem. Surprisingly, the apical loop of epsilon, known to be required for RNA packaging, was entirely dispensable for RT binding. A comparison of the requirements for in vitro RT-epsilon interaction with those for in vivo pregenomic RNA (pgRNA) packaging clearly indicated that RT-epsilon interaction was necessary but not sufficient for pgRNA packaging. In addition, our results suggest that recognition of some epsilon sequences by the RT may be required specifically for viral DNA synthesis.

Electrophoretic Mobility Shift Assay↗
PubMed2005

Reverse transcription-associated dephosphorylation of hepadnavirus nucleocapsids.

Hepatitis B viruses are pararetroviruses that contain a partially dsDNA genome and replicate this DNA through an RNA intermediate (the pregenomic RNA, pgRNA) by reverse transcription. Viral assembly begins with the packaging of the pgRNA into nucleocapsids (NCs), with subsequent reverse transcription within NCs converting the pgRNA into the characteristic dsDNA genome. Only NCs containing this dsDNA (the so-called "mature" NCs) are enveloped by the viral envelope proteins and secreted as virions; "immature" NCs, i.e., those containing pgRNA or immature reverse transcription intermediates, are excluded from virion formation. This phenomenon is thought to be caused by the emergence of an intrinsic maturation signal only on the mature NCs. To define the maturation signal, we have devised a method to separate mature from immature duck hepatitis B virus NCs and have compared them to NCs derived from secreted virions. Detailed mass spectrometric analyses revealed that the core protein from immature NCs was phosphorylated on at least six sites, whereas the core protein from mature NCs and that from secreted virions was entirely dephosphorylated. These results, together with the known requirement of core phosphorylation for pgRNA packaging and DNA synthesis, suggest that the NC undergoes a dynamic change in phosphorylation state to fulfill its multiple roles at different stages of viral replication. Although phosphorylation of the NCs is required for efficient RNA packaging and DNA synthesis by the immature NCs, dephosphorylation of the mature NCs may trigger envelopment and secretion.

Amino Acid Sequence↗
PubMed2004

Requirement of heat shock protein 90 for human hepatitis B virus reverse transcriptase function.

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, epsilon) on the pregenomic RNA (pgRNA) by the viral reverse transcriptase (RT). RT-epsilon 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-epsilon interaction in the human HBV has remained unknown due to the inability to obtain a purified RT protein active in specific epsilon binding. We now report that Hsp90 is also required for HBV RT-epsilon interaction. Inhibition of Hsp90 led to diminished HBV pgRNA packaging into nucleocapsids in cells, which depends on RT-epsilon interaction. Furthermore, using truncated HBV RT proteins purified from bacteria and five purified Hsp90 chaperone factors, we have developed an in vitro RT-epsilon binding assay. Our results demonstrate that Hsp90, in a dynamic process that was dependent on ATP hydrolysis, facilitated RT-epsilon interaction in HBV, as in DHBV. Specific epsilon binding required sequences from both the amino-terminal terminal protein and the carboxy-terminal RT domain. Only the cognate HBV epsilon, but not the DHBV epsilon, could bind the HBV RT proteins. Furthermore, the internal bulge, but not the apical loop, of epsilon 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-epsilon interaction and chaperone activation.

DNA Replication↗
PubMed2003

Conditional replication of duck hepatitis B virus in hepatoma cells.

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.

Animals↗
PubMed2003

Duck hepatitis B virus virion secretion requires a double-stranded DNA genome.

Hepatitis B virus assembly begins with the packaging of an RNA pregenome into intracellular nucleocapsids, with subsequent reverse transcription within these nucleocapsids converting the RNA into a characteristic, partially double-stranded DNA, which, alone, is found in enveloped extracellular virions as the viral genome. Using a synchronized replication system for the duck hepatitis B virus, together with a stringent two-step assay for virion secretion, we demonstrate that this selective genome secretion results from an intrinsic secretion competence gained only by the nucleocapsids at the late stage of reverse transcription.

Animals↗
PubMed2003

Heat shock protein 90-independent activation of truncated hepadnavirus reverse transcriptase.

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 epsilon, and is initiated through a novel protein priming mechanism whereby the RT itself serves as a protein primer and epsilon serves as the obligatory template. Using the RT from duck hepatitis B virus as a model, we previously demonstrated that RT-epsilon interaction and protein priming require the assistance of a host cell chaperone complex, heat shock protein 90 (Hsp90) and its co-chaperones, 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 epsilon 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 C-terminal truncations required Hsp90 for activation. Surprisingly, the presence of detergent plus some yet-to-be-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.

Animals↗
PubMed2002

Role of p50/CDC37 in hepadnavirus assembly and replication.

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 pull-down assays showed that p50 or p50deltaC, 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 p50deltaC interacted directly with purified RT. The importance of p50-RT interaction for RT functions was underscored by 1) inhibition of protein-primed initiation of reverse transcription by p50deltaC in vitro and 2) stimulation of viral DNA replication and RNA packaging by p50 and their inhibition by p50deltaC 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.

Animals↗
PubMed2002

In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins.

Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, epsilon) 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-epsilon 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 epsilon 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.

Chaperonins↗
PubMed2002

Distinct requirement for two stages of protein-primed initiation of reverse transcription in hepadnaviruses.

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 epsilon, 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.

5' Untranslated Regions↗