An antiviral mechanism of nitric oxide: inhibition of a viral protease

Abstract

Although nitric oxide (NO) kills or inhibits the replication of a variety of intracellular pathogens, the antimicrobial mechanisms of NO are unknown. Here, we identify a viral protease as a target of NO. The life cycle of many viruses depends upon viral proteases that cleave viral polyproteins into individual polypeptides. NO inactivates the Coxsackievirus protease 3C, an enzyme necessary for the replication of Coxsackievirus. NO S-nitrosylates the cysteine residue in the active site of protease 3C, inhibiting protease activity and interrupting the viral life cycle. Substituting a serine residue for the active site cysteine renders protease 3C resistant to NO inhibition. Since cysteine proteases are critical for virulence or replication of many viruses, bacteria, and parasites, S-nitrosylation of pathogen cysteine proteases may be a general mechanism of antimicrobial host defenses.

Figure 1

The NO Donor SNAP Inhibits CVB3 Replication HeLa cells were infected with CVB3 at a multiplicity of infection of 10. SNAP (200 μM) was added at various times after infection as indicated. The amount of CVB3 after 10 hr of infection was measured by the plaque assay. To one set of infected cells (C), 200 μM AP was added 1 hr after infection. To another set of cells (-1), SNAP was added 1 hr before infection. n = 3 ± SD.

Figure 2

Figure 2. NO Inhibits Proteolysis of CVB3 Polypeptide 2BC in Infected Cells (A) Exogenous NO donor SNAP inhibits proteolysis of CVB3 polypeptide. HeLa cells were infected with CVB3 at a multiplicity of infection of 10. After 1 hr, 200 μM of SNAP (+) or AP (-) was added, and after 3.5 hr guanidine was added to inhibit new viral RNA synthesis. HeLa cells were harvested at various times indicated after guanidine treatment. Cell lysates were fractionated by SDS-PAGE and immunoblotted with an antibody to CVB3 polypeptide 2C. These experiments were repeated twice with similar results. (B) Endogenous NO from activated macrophages inhibits proteolysis of CVB3 polypeptide. HeLa cells were infected with CVB3 at a multiplicity of infection of 10. After 1 hr, plastic inserts containing resting (R) or LPS/IFN-stimulated (S) macrophages were added to the wells containing infected HeLa cells. Some cultures were also treated with the NOS inhibitor NAME (0–10 mM). Cells were harvested 5 hr after infection and cell lysates analyzed for CVB3 polypeptide 2C as above. This experiment was repeated five times with similar results. (C) LPS or IFN do not directly affect CVB3 replication. HeLa cells were infected with CVB3 at a multiplicity of infection of 10. After 1 hr, LPS or IFN or media alone was added to the wells containing infected HeLa cells. Some cultures were also treated with the NOS inhibitor NAME (0–10 mM). Cells were harvested 5 hr after infection and cell lysates analyzed for CVB3 polypeptide 2C as above.

Figure 3

NO Inhibits Cleavage of a Viral Target by Purified 3C^pro (A) 3C^pro was expressed in bacteria and purified. Bacterial lysates were subjected to SDS-PAGE: lane 1, noninduced bacteria; lane 2, bacteria induced with IPTG; lane 3, high-speed supernatant of induced bacteria; lane 4, flow-through from a Ni⁺ column; lanes 5–9, sequential column washes with 10 mM imidazole; and lanes 10–13, fractions from a Ni⁺ column eluted with 500 mM imidazole. (B) The purified fusion protein GST-3B-3C_T, which contains an authentic 3C^pro cleavage site, was incubated with buffer, purified 3C^pro, or 3C^pro with the control compound AP, or the NO donor SNAP, or SNAP and DTT. The reaction mixture was analyzed by immunoblot with an antibody to GST and quantitated by densitometry. n = 3 ± SD, and * p < 0.05. (C) The cleavage experiment was then repeated using spermine NONOate (SP-NO) as an NO donor or its control compound spermine (SP). n = 3 ± SD, and * p < 0.05.

Figure 4

NO Inhibits 3C^pro Cleavage of Luciferase (A) Purified 3C^pro was added to luciferase, which contains a 3C^pro consensus cleavage motif, and the activity of luciferase remaining at various times was assayed using a luminometer. n = 3 ± SD. (B) Increasing amounts of 3C^pro were added to luciferase for 3 hr, and the amount of luciferase assayed as above. n = 3 ± SD. (C) Luciferase was incubated alone, or in the presence of 3C^pro with the following additions: the control compound AP, the NO donor SNAP, SNAP and DTT, the control compound spermine (SP), the NO donor spermine NONOate (SP-NO), or spermine NONOate and DTT. n = 3 ± SD.

Figure 5

NO Eliminates a Sulfhydryl Group from 3C^pro and Nitrosylates 3C^pro Purified 3C^pro was incubated with AP, SNAP, spermine, or spermine NONOate. (A) Analysis of the 3C^pro sulfhydryl group. Treated 3C^pro was incubated with an excess of 5,5 dithiobis(2-nitrobenzoate) (DNTB) and its absorption measured at 412 nm. n = 4 ± SD. (B) Nitrosylation of the 3C^pro active site Cys¹⁴⁷. 3C^pro was exposed to NO donors or their controls, then incubated with HgCl₂, and the amount of nitrite released was measured by the Griess reaction. n = 4 ± SD.

Figure 6

Mutation of the 3C^pro Active Site Cys¹⁴⁷ Renders 3C^pro Resistant to NO A mutant 3C^pro with serine substituted for cysteine at amino acid residue 147 was purified from bacteria. Wild-type 3C^pro (1 μg/ml) and mutated 3C^pro (5 μg/ml) were incubated for 3 hr with luciferase and AP, SNAP, or SNAP and DTT, and the activity of luciferase assayed in a luminometer. n = 3 ± SD, repeated twice with similar results.