Construction Of Phage Library Of HBV PreS And Screening For PreS Specific Binding Ligand: From Oligo-peptide To Protein

The PreS domain of the surface protein of hepatitis B virus (HBV) plays a key role in virus maturation, assembly and infection. Therefore, specific PreS-interacting peptide will be both a valuable tool for the study of these processes and a potential therapeutics against viral infection.Presently, high-throughput screening for target-interacting peptides with phage display is one of the most efficient methods to obtain lead molecules. In the current study, M13 pâ…¢ based phage display random peptide libraries were constructed. The PreS1~65 domain was produced in E.coli as a fusion protein with maltose binding protein(MBP). Then, MBP fusion protein was purified by affinity chromatography and displayed an ideal solubility and stability. The fusion protein was used as target protein to screen phage display random peptide libraries for PreS-interacting peptides.Several specific PreS-interacting phages were selected. The sequences of the peptides encoded in these phages were determined. In vitro binding assays using some of these peptides indicates that they can interact with the PreS domain specifically. Importantly, these peptides interact with the aa21-47 region of PreS1, a site thought to be necessary for HBV particles to attach to host hepatocytes. The results also indicate that these peptides interact with an identical region of PreS, which makes it possible to align their sequences to deduce a common or consensus sequence that is required for the interaction with the PreS domain. A consensus sequence -W1T2X3W4W5- is deduced from the sequence alignment. Indeed, the deduced consensus peptide can also interact with the PreS domain.It is possible that the consensus sequence also resides in PreS-interacting cellular proteins. By blasting the consensus sequence in GenBank, a few cellular proteins bearing this motif were found. A close analysis of the structures of these proteins suggest that one protein, the lipoprotein lipase may interact with the PreS domain.In summary, in the present study, 1) several specific PreS-interacting peptides have been selected. These peptides may serve as inhibitors to block viral attachment to host hepatocytes;2)we find that the lipoprotein lipase(LPL) is demonstrated to interact with HBV particles. This result may support the result of Dr Deng Qiang who is oneof our group ,which show that the lipoprotein lipase was demonstrated to interact with HBV particles from constructed M13 pVIII based phage display random peptide libraries and used thio-PreS and CBD-PreS fusion proteins as target proteins to screen phage display random peptide libraries for PreS-interacting peptides, suggesting that it is could be a new candidate for HBV cellular receptor.