| Summary: | A safe, potent and broad-spectrum antiviral is urgently needed to combat emerging viral respiratory diseases such as avian influenza H5N1 and H7N9, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). Previous studies carried out by PhD students in our lab found that mouse β-defenisn 4 (mBD4) shows highly antiviral activity in vitro. However, the recombinant mBD4 (rmBD4) expressed by E.coli is limited to very small scale of production and is very expensive.
Thus, in this study, we firstly screened 16 short peptides derived from mBD4 and other mouse and human β-defensins for identifying their antiviral effects. One short peptide P9 (30 amino acids), derived from mBD4, exhibited potent and broad-spectrum antiviral effects against multiple respiratory viruses, including influenza A viruses H1N1, H3N2, H5N1, H7N7 and H7N9, SARS coronavirus (SARS-CoV)and MERS coronavirus (MERS-CoV). This P9 showed very high selectivity index (970), which was higher than that of the full-length peptide of synthetic mBD4 (smBD4) and rmBD4 in vitro. Secondly, the prophylactic and therapeutic effects of P9 against the infection of H1N1 virus were further detected in animal model. The survival rate of P9-pretreated mice challenged by lethal dose of H1N1 virus was 100%. The therapeutic effects of P9 protecting mice from lethal challenge of H1N1 virus were also statistically significant. The survival rate of mice could reach up to 67% by intranasal inoculation and 56% by intraperitoneal injection, respectively.
To investigate the antiviral mechanism, we firstly elucidated that P9 could inhibit viral infection but not viral replication or release. Secondly, we detected whether P9 inhibited viral infection by binding to the surface of target cells or viral particles. The results showed that P9 only bound to viral particles but not to the cell surface. It was further identified that P9 bound to viral surface glycoprotein HA but not NA. Thirdly, we demonstrated that P9 did not inhibit virus binding to its receptor and block the virus entry into cells by endocytosis. Instead, P9 inhibited the acidification in late endosomes and thusP9 blocked virus-membrane fusion and subsequent viral disassembly and viral RNA release. Finally, we elucidated that the antiviral activity of P9 was attributed to its high binding affinity to viral HA and the abundance of basic amino acids in its composition.
In this study, we have demonstrated that a short peptide P9, which is derived from mBD4, showed potent antiviral activity against multiple respiratory viruses. This peptide can be developed to a new promising prophylactic and therapeutic agent with broad-spectrum antiviral activity and low possibility to cause drug resistance. Moreover, this study has also revealed a novel antiviral mechanism for P9 and paved a path for the development of new antiviral agents with broad-spectrum antiviral activity against emerging respiratory viruses, such as avian influenza H5N1 and H7N9, as well as SARS-CoV and MERS-CoV. === published_or_final_version === Microbiology === Doctoral === Doctor of Philosophy
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