| Summary: | Previously, a designed ankyrin repeat protein, Ank<sup>GAG</sup>1D4, was generated for intracellular targeting of the HIV-1 capsid domain. The efficiency was satisfactory in interfering with the HIV assembly process. Consequently, improved Ank<sup>GAG</sup>1D4 binding affinity was introduced by substituting tyrosine (Y) for serine (S) at position 45. However, the intracellular anti-HIV-1 activity of Ank<sup>GAG</sup>1D4-S45Y has not yet been validated. In this study, the performance of Ank<sup>GAG</sup>1D4 and Ank<sup>GAG</sup>1D4-S45Y in inhibiting wild-type HIV-1 and HIV-1 maturation inhibitor-resistant replication in SupT1 cells was evaluated. HIV-1 p24 and viral load assays were used to verify the biological activity of Ank<sup>GAG</sup>1D4 and Ank<sup>GAG</sup>1D4-S45Y as assembly inhibitors. In addition, retardation of syncytium formation in infected SupT1 cells was observed. Of note, the defense mechanism of both ankyrins did not induce the mutation of target amino acids in the capsid domain. The present data show that the potency of Ank<sup>GAG</sup>1D4-S45Y was superior to Ank<sup>GAG</sup>1D4 in interrupting either HIV-1 wild-type or the HIV maturation inhibitor-resistant strain.
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