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Phosphorylation of the Hepatitis B Virus Large Envelope Protein.

ABSTRACT: We here establish the phosphorylation sites in the human hepatitis B virus (HBV) large envelope protein (L). L is involved in several functionally important interactions in the viral life cycle, including with the HBV cellular receptor, HBV capsid, Hsc70 chaperone, and cellular membranes during fusion. We have recently shown that cell-free synthesis of the homologous L protein of duck HBV in wheat germ extract results in very similar phosphorylation events to those previously observed in animal cells. Here, we used mass spectrometry and NMR to establish the phosphorylation patterns of human HBV L protein produced by both in vitro cell-free synthesis and in E. coli with the co-expression of the human MAPK14 kinase. While in the avian virus the phosphorylation of L has been shown to be dispensable for infectivity, the identified locations in the human virus protein, both in the PreS1 and PreS2 domains, raise the intriguing possibility that they might play a functional role, since they are found at strategic sites predicted to be involved in L interactions. This would warrant the further investigation of a possible function in virion formation or cell entry.

SUBMITTER: Fogeron ML

PROVIDER: S-EPMC8904964 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Phosphorylation of the Hepatitis B Virus Large Envelope Protein.

Fogeron Marie-Laure ML Lecoq Lauriane L Cole Laura L Montserret Roland R David Guillaume G Page Adeline A Delolme Frédéric F Nassal Michael M Böckmann Anja A

Frontiers in molecular biosciences 20220223

We here establish the phosphorylation sites in the human hepatitis B virus (HBV) large envelope protein (L). L is involved in several functionally important interactions in the viral life cycle, including with the HBV cellular receptor, HBV capsid, Hsc70 chaperone, and cellular membranes during fusion. We have recently shown that cell-free synthesis of the homologous L protein of duck HBV in wheat germ extract results in very similar phosphorylation events to those previously observed in animal ...[more]

PMID: 35282608

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Project description:Hepatitis B virus (HBV) large (L) envelope protein is translated from 2.4-kb RNA. It contains preS1, preS2, and S domains and is detected in Western blotting as p39 and gp42. The 3.5-kb pregenomic RNA produces core and polymerase (P) proteins. We generated L-minus mutants of a genotype A clone and a genotype D clone from 1.1-mer or 1.3-mer construct, with the former overproducing pregenomic RNA. Surprisingly, mutating a preS1 ATG codon(s) or introducing a nonsense mutation soon afterwards switched secreted p39/gp42 to a p41/p44 doublet, with its amount further increased by a nonsense mutation in the core gene. A further-downstream preS1 nonsense mutation prevented p41/p44 production. Tunicamycin treatment confirmed p44 as the glycosylated form of p41. In this regard, splicing of 3.5-kb RNA to generate a junction at nucleotides (nt) 2447 to 2902 for genotype D enables translation of p43, with the N-terminal 47 residues of P protein fused to the C-terminal 371 residues of L protein. Indeed p41/p44 were detectable by an antibody against the N terminus of P protein and eliminated by a nonsense mutation at the 5' P gene or a point mutation to prevent that splicing. Therefore, lost L (and core) protein expression from the 1.1-mer or 1.3-mer construct markedly increased p41/p44 (p43), the P-L fusion protein. Cotransfection with an expression construct for L/M proteins reversed high extracellular p41/p44 associated with L-minus mutants, suggesting that L protein retains p43 in wild-type HBV to promote its intracellular degradation. Considering that p43 lacks N-terminal preS1 sequence critical for receptor binding, its physiological significance during natural infection and therapeutic potential warrant further investigation. IMPORTANCE The large (L) envelope protein of hepatitis B virus (HBV) is translated from 2.4-kb RNA and detected in Western blotting as p39 and gp42. Polymerase (P) protein is expressed at a low level from 3.5-kb RNA. The major spliced form of 3.5-kb RNA will produce a fusion protein between the first 47 residues of P protein and a short irrelevant sequence, although also at a low level. Another spliced form has the same P protein sequence fused to L protein missing its first 18 residues. We found that some point mutations to eliminate L and core protein expression from overlength HBV DNA constructs converted p39/gp42 to p41/gp44, which turned out to be the P-L fusion protein. Thus, the P-L fusion protein can be expressed at extremely high level when L protein expression is prevented. The underlying mechanism and functional significance of this variant form of L protein warrant further investigation.

Dataset Information

Phosphorylation of the Hepatitis B Virus Large Envelope Protein.

Publications

Phosphorylation of the Hepatitis B Virus Large Envelope Protein.

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