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. 2013 Apr;87(7):3640-54.
doi: 10.1128/JVI.02882-12. Epub 2013 Jan 2.

5-(Perylen-3-yl)ethynyl-arabino-uridine (aUY11), an arabino-based rigid amphipathic fusion inhibitor, targets virion envelope lipids to inhibit fusion of influenza virus, hepatitis C virus, and other enveloped viruses

Che C Colpitts  1 , Alexey V Ustinov, Raquel F Epand, Richard M Epand, Vladimir A Korshun, Luis M Schang
Affiliations

5-(Perylen-3-yl)ethynyl-arabino-uridine (aUY11), an arabino-based rigid amphipathic fusion inhibitor, targets virion envelope lipids to inhibit fusion of influenza virus, hepatitis C virus, and other enveloped viruses

Che C Colpitts et al. J Virol. 2013 Apr.

Abstract

Entry of enveloped viruses requires fusion of viral and cellular membranes. Fusion requires the formation of an intermediate stalk structure, in which only the outer leaflets are fused. The stalk structure, in turn, requires the lipid bilayer of the envelope to bend into negative curvature. This process is inhibited by enrichment in the outer leaflet of lipids with larger polar headgroups, which favor positive curvature. Accordingly, phospholipids with such shape inhibit viral fusion. We previously identified a compound, 5-(perylen-3-yl)ethynyl-2'-deoxy-uridine (dUY11), with overall shape and amphipathicity similar to those of these phospholipids. dUY11 inhibited the formation of the negative curvature necessary for stalk formation and the fusion of a model enveloped virus, vesicular stomatitis virus (VSV). We proposed that dUY11 acted by biophysical mechanisms as a result of its shape and amphipathicity. To test this model, we have now characterized the mechanisms against influenza virus and HCV of 5-(perylen-3-yl)ethynyl-arabino-uridine (aUY11), which has shape and amphipathicity similar to those of dUY11 but contains an arabino-nucleoside. aUY11 interacted with envelope lipids to inhibit the infectivity of influenza virus, hepatitis C virus (HCV), herpes simplex virus 1 and 2 (HSV-1/2), and other enveloped viruses. It specifically inhibited the fusion of influenza virus, HCV, VSV, and even protein-free liposomes to cells. Furthermore, aUY11 inhibited the formation of negative curvature in model lipid bilayers. In summary, the arabino-derived aUY11 and the deoxy-derived dUY11 act by the same antiviral mechanisms against several enveloped but otherwise unrelated viruses. Therefore, chemically unrelated compounds of appropriate shape and amphipathicity target virion envelope lipids to inhibit formation of the negative curvature required for fusion, inhibiting infectivity by biophysical, not biochemical, mechanisms.

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Figures

Fig 1
Fig 1
Chemical and three-dimensional (3D) structures of the RAFIs aUY11, dUY11, aUY12, dUY5, and dUY1. The 3D structures (11) are displayed in three orthogonal perspectives (gray, carbon; blue, hydrogen; red, oxygen; dark blue, nitrogen).
Fig 2
Fig 2
aUY11 is active against enveloped but otherwise unrelated viruses. (A) Infectivity of HSV-2, HCV, influenza virus, or Sindbis virus exposed to aUY11 (open squares) or dUY11 (filled circles), tested by plaquing efficiency or focus-forming efficiency (average ± standard deviation [SD]; n ≥ 3). (B) Infectivity of model DNA and RNA enveloped viruses exposed to aUY11 or dUY11, tested by plaquing efficiency or focus-forming efficiency (average ± SD; n ≥ 3). (C) Infectivity of nonenveloped poliovirus (open triangles), adenovirus (filled inverted triangles), or reovirus (filled diamonds) exposed to aUY11 (average ± range; n = 2).
Fig 3
Fig 3
aUY11 localizes to lipid membranes. (A) The emission spectra of aUY11 and dUY11 in virions or protein-free liposomes are most similar, and closely resemble their spectra in hydrophobic environments. aUY11 or dUY11 was added to aqueous buffer (gray) or to 1-octanol (black) to a final concentration of 48 nM or 0.48 nM, respectively. aUY11 or dUY11 (48 nM final concentration) was also added to 106 PFU of influenza virus A (blue), 106 FFU of HCV (orange), 107 PFU of HSV-1 (green), 107 PFU of HSV-2 (teal), or 107 PFU of VSV (pink) or to 2 nmol liposomes (red) in 2.5 ml of aqueous buffer. Fluorescence was excited at 455 nm. Emission spectra for dUY11 in VSV virions, liposomes, aqueous buffer, and octanol were determined previously (11). (B) The RAFI aUY11 localizes to cellular lipid membranes. Vero cell monolayers exposed to PKH26 general membrane dye for 10 min at 37°C were washed and then exposed to aUY11 or dUY11 for 1, 5, 15, 40, or 120 min at 37°C. Shown are confocal microscopy images; scale bars, 25 μm.
Fig 4
Fig 4
aUY11 and dUY11 protect cells from infection with influenza virus, HCV, or HSV-1. MDCK, Huh7.5, or Vero cells were treated with aUY11 (A) or dUY11 (B) for 1 h prior to infection with influenza virus, HCV, or HSV-1, respectively (pretreatment). Alternately, virions were preexposed to aUY11 (A) or dUY11 (B) prior to infection of cells (virion treatment). In both cases, infectivity was evaluated by plaquing or focus-forming efficiency and is expressed as a percentage relative to a DMSO vehicle-treated control. The error bars represent ranges from two experiments, with the exception of HSV-1 (B), which shows one experiment representative of two.
Fig 5
Fig 5
aUY11 inhibits the infectivity of influenza virus, HCV, and HSV-1 virions produced by infected cells. (A) Cells infected with 5 or 0.5 PFU or FFU per cell of influenza virus, HCV, or HSV-1 were incubated in the presence of aUY11 for 23 h (influenza virus and HSV-1) or 44 h (HCV). Supernatants and cell lysates were harvested and titrated for the presence of infectious virus. (B) Virions harvested in panel A were tested for the presence of aUY11 by examining its fluorescence spectra. aUY11 fluorescence was excited at 455 nm.
Fig 6
Fig 6
The RAFI aUY11 does not perturb membrane fluidity but prevents the formation of negative curvature in lipid structures. (A) Membrane fluidity was assessed by fluorescence polarization. aUY11 did not affect the fluidity of liposomes, whereas cholesterol decreased their fluidity, as expected and as shown by an increase in polarization. (B) Schematic representation of the lipid phase transitions measured by DSC. The transition from lamellar gel to lamellar liquid crystalline occurs at melting temperature (Tm); the transition from lamellar liquid crystalline to hexagonal phase occurs at temperature TH. (C) DSC of aUY11 in DEPE. The heating scans are shown with positive values and the corresponding cooling scans with negative values. The gel-to-liquid-crystalline phase transition temperature remains largely unaltered, at ∼37°C, with the addition of increasing amounts of aUY11 to DEPE, while the transition temperature to the hexagonal phase increases. This progression is shown in the enlarged graphs.
Fig 7
Fig 7
The RAFIs aUY11 and dUY11 inhibit fusion of VSV and influenza virus to cellular membranes. Shown are fluorescence dequenching of R18-labeled VSV virions preexposed to 60 nM aUY11 or DMSO vehicle during pH-dependent fusion to Vero cells (A) and influenza virus virions preexposed to 0.6 μM dUY11, aUY11, or DMSO vehicle during fusion to MDCK cells (average ± range; n = 2) (background, dequenching at neutral pH) (B).
Fig 8
Fig 8
aUY11 inhibits fusion of HCV to Huh7.5 cells in a new HCV fluorescence dequenching fusion assay. (A) Fusion of HCV to Huh7.5 cells was tested at several pHs. The pH requirements in our fusion assay match those for HCV infectivity. The error bars indicate SD. (B) HCV fused to Huh7.5 cells but, as expected, not to Vero cells, which is consistent with the ability of HCV to infect the former but not the latter. (C) Fluorescence dequenching of R18-labeled HCV JFH-1 virions preexposed to 0.6 μM dUY11, aUY11, or DMSO vehicle during pH-dependent fusion to Huh7.5 cells (average ± SD; n = 3); background is the level of dequenching observed at neutral pH.
Fig 9
Fig 9
aUY11 inhibits acid-induced liposome fusion to cells. Shown is fluorescence dequenching of R18-labeled DOPC-cholesterol liposomes preexposed to 2 μM aUY11 or DMSO vehicle during acid-induced fusion to Vero cells.
Fig 10
Fig 10
RAFIs inhibit VSV plaquing efficiency and fusion to Vero cells at similar concentrations. Shown are comparisons of inhibition of fusion (open squares) or infectivity (filled circles) by RAFIs. Lipid-mixing assays were conducted as described in the legend to Fig. 7. For plaquing assays, approximately 200 VSV virions were exposed to increasing concentrations of aUY11, dUY11, aUY12, dUY5, or dUY1 for 10 min at 37°C. The virions thus exposed were then used to infect 5 × 106 Vero cells seeded in 6-well plates. Plaquing efficiency was calculated as a percentage of plaques formed by virions exposed to DMSO vehicle.
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