Hepatitis B virus p25 precore protein accumulates in Xenopus oocytes as an untranslocated phosphoprotein with an uncleaved signal peptide

Yang, S Q; Walter, M.R.; Standring, David N.

doi:10.1128/jvi.66.1.37-45.1992

Cited by 21 publications

(13 citation statements)

References 38 publications

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“…reported that the 3a protein of SARS‐CoV might undergo specific proteolytic processing [14]. Studies have also shown that the multiple cleavages of the precore protein of the hepatitis B virus resulted in different sizes of smaller proteins which could be translocated from the cytosol to other compartments of the cell [15]. SARS 8b may possibly employ similar mechanism as the 3a or the precore protein and the smaller band might be a processed form of the protein.…”

Section: Resultsmentioning

confidence: 99%

Expression and functional characterization of the putative protein 8b of the severe acute respiratory syndrome‐associated coronavirus

et al. 2006

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SARS 8b is one of the putative accessory proteins of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) with unknown functions. In this study, the cellular localization and activity of this estimated 9.6 kDa protein were examined. Confocal microscopy results indicated that SARS 8b is localized in both nucleus and cytoplasm of mammalian cells. Functional study revealed that overexpression of SARS 8b induced DNA synthesis. Coexpression of SARS 8b and SARS 6, a previously characterized SARS-CoV accessory protein, did not elicit synergistic effects on DNA synthesis.

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Section: Resultsmentioning

confidence: 99%

Expression and functional characterization of the putative protein 8b of the severe acute respiratory syndrome‐associated coronavirus

et al. 2006

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“…Oocytes rival cell-free systems in terms of experimental flexibility, making it simple to vary parameters such as the p21.5 concentration or to introduce additional viral components and other molecular reagents, yet oocytes also possess most of the properties anticipated for cultured animal cells. Thus, oocytes generate appropriately posttranslationally modified proteins, and p21.5 is modified by phosphorylation within the protamine region in oocytes (28), as it is in virions (1, 10) and animal cells (24); such modifications can be important for viral assembly, as has been shown for polyomavirus (8). Perhaps more important, oocytes possess subcellular compartments such as the nucleus and the secretory organelles which allow studies on the trafficking properties of viral proteins (25,26,30); these compartments may also be important for specific stages of animal virus assembly.…”

Section: Discussionmentioning

confidence: 99%

Characterization of hepatitis B virus capsid particle assembly in Xenopus oocytes

Zhou

Yang

Standring

1992

J Virol

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Little is known about the assembly of the 28-nm nucleocapsid or core particle of hepatitis B virus. Here we show that this assembly process can be reconstituted in Xenopus oocytes injected with a synthetic mRNA encoding the hepatitis B virus capsid protein (p21.5). Injected oocytes produce both a nonparticulate p21.5 species (free p21.5) and capsid particles. We describe rapid and simple methods for fractionating these species on a small scale either with step gradients of 10 to 60%o (wt/vol) sucrose or by centrifugation to pellet the particles, and we characterize the oocyte core particles. Free p21.5 exhibits chemical and physical properties distinctly different from those of particles. Free p21.5 is partially cleaved by proteinase K, whereas core particles are almost completely resistant to cleavage. This suggests that the carboxyl-terminal protamine region, the main target for proteases within p21.5, is exposed in free p21.5 but faces the interior of the p21.5 core particle. Finally, pulse-chase experiments demonstrated that free p21.5 can be chased almost quantitatively into core particles, establishing that free p21.5 is fully competent to form particles and represents an assembly intermediate on the pathway for core particle formation. However, core particle assembly appears very dependent on p21.5 concentration and is rapidly compromised if the p21.5 concentration is lowered. The advantages of oocytes for studying assembly are discussed.

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“…The first 149 residues of the 185-residue p21.5 capsid protein form a domain which is very resistant to proteolytic attack (36) and directs assembly of the 28-nm capsid shell (5, 13). The final 36 residues form an Arg-rich region that bears a striking similarity to protamines and mediates the interaction with nucleic acid detected by either an RNA encapsidation assay (5) or a Southwestern (DNA-protein) blot assay (13,24).…”

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confidence: 99%

RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication

Hatton

Zhou

Standring

1992

J Virol

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192

102

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The hepatitis B virus capsid or core protein (p21.5) binds nucleic acid through a carboxy-terminal protamine region that contains nucleic acid-binding motifs organized into four repeats (I to MV). Using carboxy-terminally truncated proteins expressed in Escherichia coli, we detected both RNAand DNA-binding activities within the repeats. RNA-binding and packaging activity, assessed by resolving purified E. coli capsids on agarose gels and disclosing their RNA content with ethidium bromide, required only the proximal repeat I (RRRDRGRS). Strikingly, a mutant in which four Arg residues replaced repeat I was competent to package RNA, demonstrating that Arg residues drive RNA binding. In contrast, probing immobilized core proteins with 32P-nucleic acid revealed an activity which (i) required more of the protamine region (repeats I and II), (ii) appeared to bind DNA better than RNA, and (iii) was apparently modulated by phosphorylation in p21.5 derived from Xenopus oocytes. Deletion analysis suggested that this activity may depend on an SPXX-type DNA-binding motif in repeat II. Similar motifs found in repeats III and IV may also function to bind DNA. On * Corresponding author. p21.5 appear to be specifically configured for a role in HBV replication. MATERIALS AND METHODS Reagents. Homogeneous preparations of recombinant capsids from Escherichia coli and from Saccharomyces cerevisiae were kindly provided by Chiron, Emeryville, Calif., and Merck, West Point, Pa., respectively. All chemical and biochemical reagents were of fine reagent grade. Restriction enzymes and T4 DNA ligase were obtained from Boehringer Mannheim; radiochemicals were obtained from NEN. Oligonucleotides were synthesized on a Milligen Biosearch 8700 synthesizer. DNA sequencing was performed with an Applied Biosystems 373A DNA sequencer. Standard protocols were used for molecular biology procedures. Construction of mutants and expression plasmids. HBV DNA of subtype adw (34) was used for all constructs. For expression in E. coli, the 185-amino-acid p21.5 coding region was mobilized by the polymerase chain reaction from plasmid 64-C (29) to the expression vector pAR3040 (31). The 5'

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Hepatitis B virus p25 precore protein accumulates in Xenopus oocytes as an untranslocated phosphoprotein with an uncleaved signal peptide

Cited by 21 publications

References 38 publications

Expression and functional characterization of the putative protein 8b of the severe acute respiratory syndrome‐associated coronavirus

Expression and functional characterization of the putative protein 8b of the severe acute respiratory syndrome‐associated coronavirus

Characterization of hepatitis B virus capsid particle assembly in Xenopus oocytes

RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication

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