Fig 4.

Mode of anti-HRV-2 activity of AZ5385. (A) Structure of the VP1 hydrophobic pocket of HRV-2 showing localization of the S181 and T210 residues (in yellow), the T207 residue (in blue), and the C12 fatty acid “pocket factor” (in pink). Neighboring residues to S181 at the pocket entry gate and to T210 at the pocket wall are specified. Image was created based on crystal structure of HRV-2 (12; PDB ID: 1FPN) using UCSF ChimeraX. (B and C) AZ5385 exhibits modest HRV2-inactivating (virucidal) activity. (D) The drug-resistant passage 11 virus is not inactivated by AZ5385. The virus at 10⁵ TCID₅₀ was incubated for 15 min (B) or 60 min (C and D) at 37°C with 8.7 µM of AZ5385 or a corresponding volume of DMSO, and the mixture was immediately subjected to determination of virus titer. Three separate experiments were performed. (E) AZ5385 exhibited no effect on the virus attachment to cells. The compound-treated virus was attached to HeLa cells for 1 h at 4°C. The amount of bound virus was quantified by qPCR and expressed as a number of RNA copies. (F) AZ5385 did not block HRV-2 entry into cells. The HeLa cell-bound HRV-2 was incubated for 2 h at 34°C in the presence of AZ5385 or DMSO control, and the cells were quantified for infectivity and RNA copies. (G–J) The virus propagated in the presence of AZ5385 exhibits high RNAcopy/infectivity ratio. The progeny HRV-2 particles prepared from culture supernatant and infected cells after inoculation at MOI 1 (G) and 0.01 (H) or prepared from infected cells only (I) were concentrated by ultracentrifugation, and their infectivity and RNA copy number were determined. (J) AZ5385-resistant passage 11 virus showed no increased RNA copy/infectivity ratio. The virus was prepared and analyzed as in Fig. 4H. Statistically significant difference **P < 0.01; ns, non-significant.