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.

Keywords: L HBsAg; NMR; cell-free (CF) protein synthesis; hepatitis B; mass spectrometry; phosphorylation.

FIGURE 1

Strep-Tactin© affinity purification of PreS1tag (A), PreS_tag (B), _tagPreS (C), and _tagL (D). Purification steps have been analyzed by SDS-PAGE, followed by Coomassie blue staining; CFS, total cell-free sample; P, pellet; SN, supernatant; E#, elution fractions.

FIGURE 2

Identification of acetylation and phosphorylation sites in PreS1, PreS, and HBV L using mass spectrometry and NMR. Mass spectrometry analysis has been performed on fractions from purified protein samples shown on Figure 1 for cell-free synthesized PreS1_tag (A), PreS_tag (B), _tagPreS (C), _tagL (D); and MAPK14- ^{E. coli} PreS_tag (E). Horizontal gray bars below the sequences represent the sequence coverage by LC-MS/MS. Red bars represent peptides where one or more phosphorylation sites have been confirmed; the residues in black correspond to the phosphorylation sites formally identified, and the ones in gray correspond to possible but not confirmed sites. The PreS1 part of the amino-acid sequence is typed in black, the PreS2 part in green, the beginning of the S part in yellow (the full _tagL protein was analyzed, but the sequence coverage of S was poor and no phosphorylation sites were detected), and the tag sequences in gray. Additional information extracted from NMR chemical shifts (see below) is shown as purple squares for acetylation and red squares for phosphorylation. Major phosphorylation sites identified by mass spectrometry and NMR are highlighted by vertical yellow bars; minor sites (only mass spectrometry) by vertical gray bars. (F) Functional regions of the HBV PreS: binding to NTCP (Yan et al., 2012); possible fusion peptides (Pérez-Vargas et al., 2021); the MD (Bruss and Thomssen, 1994); and a possible cell-permeable peptide (Oess and Hildt, 2000).

FIGURE 3

Partial phosphorylation is detected by NMR for PreS and PreS1 produced using cell-free synthesis: (A) Solution NMR ¹H-¹⁵N BEST-TROSY spectra of ²H-¹³C-¹⁵N PreS1_tag at pH 7.5 (in blue), PreS_tag at pH 6 (in green) and _tagPreS at pH 6 (in red). (B) Extract of 2D BEST-TROSY spectra showing a peak corresponding to unphosphorylated S98 for the 3 constructs on the top right. The peak is weak for both PreS_tag and _tagPreS, with another peak corresponding to phosphorylated S98 detected at the bottom left. This means that S98 is mainly, but not fully phosphorylated in PreS. The Cβ chemical shift was used to confirm the phosphorylation state of this serine (Supplementary Figure S10). (C) Sequence of the three constructs showing assigned residues for PreS1_tag (top, blue), PreS_tag (middle, green) and _tagPreS (bottom, red). Assigned residues are colored, while unassigned residues are in black type. Proline residues are colored when their Cα, Cβ and C′ are assigned. Residues which display two forms on the NMR spectra are shown in bold, and residues with chemical-shifts typical of phosphorylation and acetylation are indicated by a star with the corresponding color-code. Processed residue M1 is shown in brackets.