Expression of hepatitis B virus core and precore antigens in insect cells and characterization of a core-associated kinase activity

Lanford, Robert E.; Notvall, Lena

doi:10.1016/0042-6822(90)90247-o

Cited by 57 publications

(46 citation statements)

References 44 publications

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“…Our results here indicate that neither the viral RT, pgRNA, nor any hepatocyte-specific host factor is required for kinase packaging and fit well with previous reports of endogenous kinase activity observed in recombinant HBV capsids made in insect cells and in empty core particles from infected livers (1,18,30). These results thus suggest that the core protein alone may be able to capture and package the host kinase(s).…”

Section: Discussionsupporting

confidence: 91%

“…The endogenous kinase activity seen in our studies and by others (1,18,25,30) could be due to a kinase that either cofractionates with the capsids in the sucrose gradient or is tightly associated with the exterior of the capsid. To test these possibilities, we performed proteinase K digestions of the capsid fractions followed by the endogenous kinase reaction.…”

Section: Resultssupporting

confidence: 63%

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Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids

Ludgate

Ning

Nguyen

et al. 2012

J Virol

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Phosphorylation of the hepadnavirus core protein C-terminal domain (CTD) is important for viral RNA packaging, reverse transcription, and subcellular localization. Hepadnavirus capsids also package a cellular kinase. The identity of the host kinase that phosphorylates the core CTD or gets packaged remains to be resolved. In particular, both the human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) core CTDs harbor several conserved serine/threonine-proline (S/T-P) sites whose phosphorylation state is known to regulate CTD functions. We report here that the endogenous kinase in the HBV capsids was blocked by chemical inhibitors of the cyclin-dependent kinases (CDKs), in particular, CDK2 inhibitors. The kinase phosphorylated the HBV CTD at the serine-proline (S-P) sites. Furthermore, we were able to detect CDK2 in purified HBV capsids by immunoblotting. Purified CDK2 phosphorylated the S/T-P sites of the HBV and DHBV CTD in vitro. Inhibitors of CDKs, of CDK2 in particular, decreased both HBV and DHBV CTD phosphorylation in vivo. Moreover, CDK2 inhibitors blocked DHBV CTD phosphorylation, specifically at the S/T-P sites, in a mammalian cell lysate. These results indicate that cellular CDK2 phosphorylates the functionally critical S/T-P sites of the hepadnavirus core CTD and is incorporated into viral capsids.T he human hepatitis B virus (HBV) continues to pose a significant health risk worldwide, causing more than one million deaths annually (52). Chronic HBV infection, estimated to affect 350 million people globally, dramatically elevates the risk for developing serious liver diseases, including cirrhosis and hepatocellular carcinoma. HBV is a member of the Hepadnaviridae family, which includes hepatotropic DNA viruses that consist of an enveloped icosahedral capsid enclosing an approximately 3-kb DNA genome in a partially double-stranded, relaxed circular (RC) form. These DNA viruses are also retroid viruses and encode a reverse transcriptase (RT) enzyme that converts a so-called pregenomic RNA (pgRNA) template to the RC DNA through reverse transcription within cytoplasmic capsids. Capsids are composed of multiple copies (180 or 240) of one virally encoded protein, the core or capsid protein (9,63,65,71).Phosphorylation of the hepadnavirus core protein is important for RNA packaging, DNA synthesis, and subcellular localization. The HBV core protein (HBc) contains three major serine-proline (S-P) phosphorylation sites in its C-terminal domain (CTD) (32). The duck hepatitis B virus (DHBV) core protein (DHBc) contains six known phosphorylation sites, four of which also have the serine/threonine-proline (S/T-P) motifs (43, 68). Mutational analyses indicate that phosphorylation of the core protein at these S/T-P sites is required for RNA packaging and DNA synthesis in HBV (29, 31). For DHBV, dynamic CTD phosphorylation at the S/T-P sites is required for complete DNA synthesis such that the S/T-P phosphorylation is needed for first-strand DNA synthesis and dephosphorylation is required for second-strand DNA s...

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Section: Discussionsupporting

confidence: 91%

Section: Resultssupporting

confidence: 63%

Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids

Ludgate

Ning

Nguyen

et al. 2012

J Virol

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“…The core protein spontaneously forms capsids in the absence of the pregenomic RNA or Pol when expressed in insect cells (23), bacteria (7), yeast (28), or Xenopus oocytes (47). The core protein contains numerous phosphorylation sites, with a cluster of sites located at the carboxy terminus.…”

mentioning

confidence: 99%

Interaction between Hepatitis B Virus Core Protein and Reverse Transcriptase

Lott

Beames

Notvall

et al. 2000

J Virol

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Previous mutagenesis studies with hepatitis B virus (HBV) suggest that continued interactions with core are required for several steps in genomic replication. To examine core-polymerase (Pol) interactions, insect cells were coinfected with baculovirus constructs that independently expressed core and Pol. The results demonstrated several features with implications that core plays an interactive role with HBV Pol: (i) core coprecipitated with constructs expressing full-length Pol as well as the terminal protein (TP), reverse transcriptase (RT) and RNase H domains of Pol, independently; (ii) coprecipitation of core was not dependent on the presence of an epsilon stem-loop sequence; and (iii) core-Pol complexes migrated as intact capsid particles, as detected by sucrose gradient analysis. To analyze the structural and sequence requirements of core in recognition of Pol, a series of core mutants with two-to four-amino-acid insertions or carboxy-terminal deletions were assessed for Pol interaction. The results indicated that capsid formation is required but not sufficient for interaction with Pol and that the TP and RT domains of Pol have different requirements for interaction with core. To map the core binding sites on Pol, a panel of amino-and carboxy-terminal deletion mutants of the TP and RT domains of Pol were analyzed for interaction with core. At least three separate core binding sites on Pol were detected. This analysis begins to define basic requirements for core-Pol interactions, but further study is necessary to delineate the effects of these interactions on encapsidation and genome replication.Hepatitis B virus (HBV) is a member of the Hepadnaviridae family. Mature HBV virions are enveloped and contain partially double stranded, non-covalently closed, circular DNA with the viral polymerase (Pol) covalently bound to the first deoxyribonucleotide of the 5Ј end of the minus-strand DNA. HBV replicates by reverse transcription of a pregenomic RNA molecule which occurs inside of a capsid particle (for reviews see references 11, 32, and 39). For encapsidation and subsequent genomic replication to occur, a ribonucleoprotein (RNP) complex forms between Pol and the epsilon stem-loop structure on a pregenomic RNA molecule (2). The epsilon stem-loop is present on both the 5Ј and 3Ј ends of pregenomic RNA, but only the 5Ј copy of epsilon functions in vivo as a packaging signal (14,16,18,34). A sequence in a bulge in the 5Ј copy of epsilon functions as a template for the first four nucleotides in DNA synthesis by a protein-primed reverse transcription reaction catalyzed by Pol (41, 42). Minus-strand DNA synthesis continues after a translocation step in which the primed-Pol complex translocates to a complementary sequence in the 3Ј copy of DR1 (8,26,29,(36)(37)(38)44). Whether the priming reaction occurs after packaging or prior to capsid assembly is not known, nor is the mechanism by which the RNA-Pol RNP complex interacts with the capsid protein to produce replication-competent HBV particles.The HBV capsid is composed ...

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“…A higher production and secretion level was reached because recombinant P36 protein was easily detected in Western blots in nonconcentrated supernatants. It has been shown that baculovirus-infected insect cells may secrete recombinant proteins (23). The fact that the protein was not observed in the supernatant culture at 4 dpi may indicate that strong proteolysis arises late in the infection, probably because cell lysis released proteases.…”

Section: Discussionmentioning

confidence: 99%

Expression of the Mycobacterium bovis P36 gene in Mycobacterium smegmatis and the baculovirus/insect cell system

Bigi

Taboga

Romanó

et al. 1999

Braz J Med Biol Res

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In the present study we evaluated different systems for the expression of mycobacterial antigen P36 secreted by Mycobacterium bovis. P36 was detected by Western blot using a specific antiserum. The P36 gene was initially expressed in E. coli, under the control of the T7 promoter, but severe proteolysis prevented its purification. We then tried to express P36 in M. smegmatis and insect cells. For M. smegmatis, we used three different plasmid vectors differing in copy number and in the presence of a promoter for expression of heterologous proteins. P36 was detected in the cell extract and culture supernatant in both expression systems and was recognized by sera from M. bovis-infected cattle. To compare the expression level and compartmentalization, the MPB70 antigen was also expressed. The highest production was reached in insect cell supernatants. In conclusion, M. smegmatis and especially the baculovirus expression system are good choices for the production of proteins from pathogenic mycobacteria for the development of mycobacterial vaccines and diagnostic reagents. Correspondence

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Expression of hepatitis B virus core and precore antigens in insect cells and characterization of a core-associated kinase activity

Cited by 57 publications

References 44 publications

Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids

Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids

Interaction between Hepatitis B Virus Core Protein and Reverse Transcriptase

Expression of the Mycobacterium bovis P36 gene in Mycobacterium smegmatis and the baculovirus/insect cell system

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