Phosphorylation of the Hepatitis B Virus Large Envelope Protein.

Marie-Laure Fogeron,Laura Cole,Michael Nassal,Frédéric Delolme,Guillaume David,Roland Montserret,Lauriane Lecoq,Adeline Page,Anja BöCkmann

doi:10.3389/fmolb.2021.821755

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.

Highlights

The three hepatitis B virus (HBV) envelope proteins L, M, and S [large, middle and small hepatitis B surface antigen (HBsAg)] form the viral envelope
We recently reported a similar observation for the duck HBV L protein, where it resulted from alternative translation initiation in addition to phosphorylation (David et al, 2019)
We identified a total of nine phosphorylation sites, with several observed in different constructs by mass spectrometry and two confirmed by NMR as being major

Summary

Introduction

The three hepatitis B virus (HBV) envelope proteins L, M, and S [large, middle and small hepatitis B surface antigen (HBsAg)] form the viral envelope. PreS1 and PreS2 together, collectively termed PreS, are suspected to represent an intrinsically disordered protein domain. A third region of interest is localized at the PreS1/PreS2 border, comprising residues between approximately amino acids 90–120, which is believed to be involved in interactions with the viral capsid during particle formation (Gudima et al, 2007; Xi et al, 2021). Another site in PreS1 is involved in the interaction with the Hsc chaperone, reported to be a determinant in the i-PreS orientation observed in immature viral particles (Prange et al, 1999; Prange, 2012). Several specific sites in preS have been identified to be involved in the important functional interactions of L

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Conclusion