Abstract
BackgroundTargeting RNA is a promising yet underdeveloped modality for the selective killing of cells infected with HIV-1. The secretory ribonucleases (RNases) found in vertebrates have cytotoxic ribonucleolytic activity that is kept in check by a cytosolic ribonuclease inhibitor protein, RI.MethodsWe engineered amino acid substitutions that enable human RNase 1 to evade RI upon its cyclization into a zymogen that is activated by the HIV-1 protease. In effect, the zymogen has an HIV-1 protease cleavage site between the termini of the wild-type enzyme, thereby positioning a cleavable linker over the active site that blocks access to a substrate.ResultsThe amino acid substitutions in RNase 1 diminish its affinity for RI by 106-fold and confer high toxicity for T-cell leukemia cells. Pretreating these cells with the zymogen leads to a substantial drop in their viability upon HIV-1 infection, indicating specific toxicity toward infected cells.ConclusionsThese data demonstrate the utility of ribonuclease zymogens as biologic prodrugs.
Highlights
Numerous HIV antiviral compounds effectively target multiple aspects of the viral lifecycle [1]
The cytotoxic ribonucleolytic activity of RNases is kept in check by the cytosolic ribonuclease inhibitor protein, RI
We combine these strategies to create a ribonuclease zymogen that is Windsor et al AIDS Res Ther (2021) 18:77 activated within human cells upon HIV-1 infection, providing a targeted approach to disrupt the production of infectious HIV viral particles
Summary
We engineered amino acid substitutions that enable human RNase 1 to evade RI upon its cyclization into a zymogen that is activated by the HIV-1 protease. The zymogen has an HIV-1 protease cleavage site between the termini of the wild-type enzyme, thereby positioning a cleavable linker over the active site that blocks access to a substrate. Results: The amino acid substitutions in RNase 1 diminish its affinity for RI by 106-fold and confer high toxicity for T-cell leukemia cells. Pretreating these cells with the zymogen leads to a substantial drop in their viability upon HIV-1 infection, indicating specific toxicity toward infected cells
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