Characterization of hepatitis B virus capsid particle assembly in Xenopus oocytes

Zhou, Sen; Yang, S Q; Standring, David N.

doi:10.1128/jvi.66.5.3086-3092.1992

Cited by 36 publications

(36 citation statements)

References 29 publications

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“…Furthermore, this 65kd protein was shown to be more resistant to proteolytic digestion than prion protein in the same homogenate. This proteolytic resistance is most consistent with the presence of tight capsid-nucleic acid complexes, as only bound but not free capsid proteins of hepatitis B (one of the retroviridae) are similarly resistant to proteolysisl", 28 We also analyzed the characteristics of this putative gag protein in 2-D protein blots of our 120S preparations. A protein of the same size showed an acidic charge (pI of -5.3) as would be expected for the IAP gag protein.…”

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confidence: 67%

Endogenous viral complexes with long RNA cosediment with the agent of Creutzfeldt-Jakob Disease

1994

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A class of viruslike agents that induces Creutzfeldt-Jakob Disease (CJD) and scrapie remains undefined at the molecular level. Several investigators believe this infectious agent is constituted by a single host protein or 'prion', and have emphasized data that would seem to exclude the presence of any viral nucleic acids. However, more rigorous evaluations in scrapie have shown reasonably abundant nucleic acids. Additionally, in highly purified 120S CJD preparations that have been treated with nucleases, RNAs as long as 6,000 bases have been detected. Few nucleic acids have been characterized in either scrapie or CJD, but previous cloning experiments delineated relatively short LTR regions of the endogenous IAP retrovirus in 120S CJD preparations. We therefore used specific primers encompassing the entire IAP genome to test for the presence of long viral RNAs, and here show approximately 5,000 contiguous bases of the IAP RNA genome can be recovered from reasonable amounts of starting brain. The 3' env region of IAP is comparably truncated in CJD and normal preparations, and we find no evidence for IAP transduction of CJD-specific sequences. Because IAP cores can coencapsidate unrelated sequences, and are unusually resistant to physical and chemical treatments, it was relevant to find if cosedimenting cognate proteins of the IAP core, such as gag, could be detected. The predicted approximately 65 kd acidic gag protein, showing appropriate antigenic and nucleic acid binding features, was apparent in both one and 2-D Western blots. This data strongly indicates specific viral complexes cofractionate with the CJD agent. Interestingly, these nuclease resistant IAPs do not appear to be in morphologically recognizable 'R' particles. This cosedimenting viral assembly therefore provides a paradigm for non-particulate CJD complexes in infectious preparations. In developing strategies to identify a CJD specific sequence, cosedimenting IAPs can be used to assess the quality, length and recovery of RNAs extracted from highly resistant viral complexes.

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confidence: 67%

Endogenous viral complexes with long RNA cosediment with the agent of Creutzfeldt-Jakob Disease

1994

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“…ն denotes a heterogeneous population of nucleotide sequences at the given position which may code for S, T, or C. tein in hepatocytes of HBV infected patients is in a particulate form was unexpected, in view of some earlier in vitro studies. It was reported that the assembly of core particles is strongly dependent on the concentration of core protein and occurs mainly in the cytoplasm [Zhou et al, 1991[Zhou et al, , 1992Seifer et al, 1993]. Our data suggest that the concentration of HBc in the nucleus of infected hepatocytes from chronic HBV carriers is higher than in Xenopus oocyte nuclei and sufficient to drive most of the core protein into the assembled form.…”

Section: Discussionmentioning

confidence: 52%

Differentiation of core gene products of the hepatitis B virus in infected liver tissue using monoclonal antibodies

et al. 1997

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Hepatitis B virus (HBV) core gene translational products were localised previously in the cytoplasm and/or in the nuclei of infected cells. We investigated in naturally infected human hepatocytes whether this variation in the subcellular expression is due to differences in the presence of assembled core particles and other core gene derived proteins, the expression of HBeAg and the processing of liver tissue. By immunostaining of liver specimens infected with HBeAg-positive and HBeAg-minus variants of HBV, using monoclonal antibodies specific for assembled core particles and for various epitopes on denatured core protein, it was shown that virtually all immunoreactive core gene products are assembled into core particles. The latter are present both in the nuclei and in the cytoplasm of hepatocytes, independent of the infecting virus strain. A marked reduction or absence of immunoreactivity, observed with some monoclonal antibodies, was shown to result from nucleotide sequence variations within or close to the corresponding epitope. These results demonstrate that immunoreactive products, derived from the HBV core gene, in the nuclei and cytoplasm of human hepatocytes represent assembled core particles and that monoclonal antibodies with known recognition sites can reveal region-specific core gene variation of the infecting HBV population.

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“…The core protein has been shown in a variety of exogenous systems (8,13,17,23,25,40) to spontaneously form capsids morphologically resembling WT capsids found in cells permissive for HBV replication in which all HBV proteins are produced. Core assembly is cooperative, proceeding as a sufficient concentration is reached (18,32,41). Core assembly in an in vitro translation system has been shown to be chaperonin dependent at low protein concentrations (18), suggesting possible differences in assembly between high-level expression systems and permissive cells.…”

Section: Discussionmentioning

confidence: 99%

“…Assembly of core protein in Huh7 cells. Since core protein is expressed at a much lower level in mammalian cells than in insect cells and capsid formation is sensitive to the core protein concentration (18,32,41), it was necessary to confirm whether mutant core proteins can form high-molecular-weight assemblies in Huh7 cells. Metabolically labeled WT and mutant core proteins from transfected Huh7 cell lysates were assayed by pelleting through 30% sucrose onto a 70% sucrose cushion.…”

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Insertions within the hepatitis B virus capsid protein influence capsid formation and RNA encapsidation

Beames

Lanford

1995

J Virol

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Hepatitis B virus (HBV) capsid proteins, termed core proteins, with two-to four-amino-acid insertions were assessed for capsid formation, RNA encapsidation, and the ability to support reverse transcription of the pregenome by the polymerase molecule. Velocity sedimentation analysis of insect cell-expressed recombinant core proteins revealed that only two of the nine insertion mutant proteins formed capsids with the tight banding patterns of wild-type capsids. The remaining mutant core proteins were spread over the gradients, suggesting aggregate formation, or at the top of the gradients, suggesting lack of stable capsid formation. The mutant capsid proteins were coexpressed in Huh7 cells with an HBV genome lacking a functional core gene to test for trans complementation of HBV replication. Three of the mutant core proteins formed capsids containing HBV RNA, but only two of these contained reverse-transcribed HBV DNA. While the core protein has shown resiliency in capsid formation following insertion of foreign residues into the major B-cell epitope, several of the small insertions severely reduced the efficiency of capsid formation and inhibited capsid function.

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Characterization of hepatitis B virus capsid particle assembly in Xenopus oocytes

Cited by 36 publications

References 29 publications

Endogenous viral complexes with long RNA cosediment with the agent of Creutzfeldt-Jakob Disease

Endogenous viral complexes with long RNA cosediment with the agent of Creutzfeldt-Jakob Disease

Differentiation of core gene products of the hepatitis B virus in infected liver tissue using monoclonal antibodies

Insertions within the hepatitis B virus capsid protein influence capsid formation and RNA encapsidation

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