The viral transactivator HBx protein exhibits a high potential for regulation via phosphorylation through an evolutionarily conserved mechanism

Abstract

Background: Hepatitis B virus (HBV) encodes an oncogenic factor, HBx, which is a multifunctional protein that can induce dysfunctional regulation of signaling pathways, transcription, and cell cycle progression, among other processes, through interactions with target host factors. The subcellular localization of HBx is both cytoplasmic and nuclear. This dynamic distribution of HBx could be essential to the multiple roles of the protein at different stages during HBV infection. Transactivational functions of HBx may be exerted both in the nucleus, via interaction with host DNA-binding proteins, and in the cytoplasm, via signaling pathways. Although there have been many studies describing different pathways altered by HBx, and its innumerable binding partners, the molecular mechanism that regulates its different roles has been difficult to elucidate.

Methods: In the current study, we took a bioinformatics approach to investigate whether the viral protein HBx might be regulated via phosphorylation by an evolutionarily conserved mechanism.

Results: We found that the phylogenetically conserved residues Ser25 and Ser41 (both within the negative regulatory domain), and Thr81 (in the transactivation domain) are predicted to be phosphorylated. By molecular 3D modeling of HBx, we further show these residues are all predicted to be exposed on the surface of the protein, making them easily accesible to these types of modifications. Furthermore, we have also identified Yin Yang sites that might have the potential to be phosphorylated and O-β-GlcNAc interplay at the same residues.

Conclusions: Thus, we propose that the different roles of HBx displayed in different subcellular locations might be regulated by an evolutionarily conserved mechanism of posttranslational modification, via phosphorylation.

Figure 1

Hepatitis B virus X protein (HBx). A) Schematic representation of HBx domain organization. As shown, HBx is functionally organized into an N-terminal, and a C-terminal domain. The N-terminal third of HBx corresponds to the negative regulatory domain, which includes a Ser/Pro-rich region. The remaining C-terminal two-thirds of the protein comprises the transactivation domain. B) Multiple sequence alignment of HBx proteins from all main HBV genotypes using ClustalW. Accession numbers are indicated in the left column, followed by the HBV genotype of each isolate. Conserved Ser, Thr, and Tyr residues are shown in bold across the sequences. At the bottom of the alignment, the consensus sequence is marked by an asterisk, conserved substitutions by a double dot, and a semiconserved substitution by a single dot.

Figure 2

Predicted O-glycosylation and Yin Yang sites on the HBx protein. The O-β-GlcNAc modification potential of each Ser, and Thr residues is shown by green vertical lines, and the light blue horizontal wavy line indicates the threshold for modification potential. The Yin Yang sites that were positively predicted are shown by red asterisks at the top. The profile was obtained with server YinOYang 1.2, using the HBx sequence of our HBV isolate 4.5 as a reference. The numbers at the top of lines of O-glycosylation potential indicate the positions of conserved HBx Ser, Thr or Tyr residues. As shown, Ser25 represents a FN Yin Yang site.

Figure 3

Homology 3D modeling of HBx. The HBx sequence of our isolate 4.5 used as a reference was submitted to server I-Tasser for protein structure prediction. Out of five models developed by the server, we selected the model with the highest C-value. To view and analyze HBx 3D structure, both RasMol v. 2.7.5.2., and MVP (Macromolecular Visualization and Processing, v. 1.0) software were used. The positions of Ser25, Ser41, and Thr81 are shown in the model.