Peroxynitrite inhibition of Coxsackievirus infection by prevention of viral RNA entry

Abstract

Although peroxynitrite is harmful to the host, the beneficial effects of peroxynitrite are less well understood. We explored the role of peroxynitrite in the host immune response to Coxsackievirus infection. Peroxynitrite inhibits viral replication in vitro, in part by inhibiting viral RNA entry into the host cell. Nitrotyrosine, a marker for peroxynitrite production, is colocalized with viral antigens in the hearts of infected mice but not control mice. Nitrotyrosine coprecipitates with the viral polypeptide VP1 as well. Guanidinoethyl disulfide, a scavenger of peroxynitrite, blocks peroxynitrite inhibition of viral replication in vitro and permits an increase in viral replication in vivo. These data suggest that peroxynitrite is an endogenous effector of the immune response to viruses.

Fig. 1.

Peroxynitrite decreases CVB3 replication. (A) Dose–response. CVB3 was pretreated with increasing doses of peroxynitrite and then titered on HeLa cells (n = 3 ± SD) *, P < 0.01 versus 0 μM peroxynitrite. (B) HeLa cells were infected with CVB3 (MOI = 10) and treated with 100 μM peroxynitrite at various times after infection. Eight hours after infection, the amount of virus produced was measured by titering (n = 3 ± SD) *, P < 0.01 versus control.

Fig. 2.

Peroxynitrite inhibits RNA entry into cells but does not affect viral binding or viral RNA. (A) Peroxynitrite has no significant effect on CVB3 binding. HeLa cells were incubated with [³⁵S]CVB3 at 4°C for 20 min and washed. The amount of bound virus was then measured in a scintillation counter. As a control, 50-fold excess nonlabeled virus was added to labeled virus (white bar) (n = 3 ± SD; P > 0.1 for all peroxynitrite samples versus 0 μM). (B) Peroxynitrite has a minor effect on viral RNA. CVB3 RNA was pretreated with increasing doses of peroxynitrite and transfected into HeLa cells. The amounts of virus in cell lysates prepared 8 h after transfection were quantified by the plaque assay (n = 3 ± SD). (C) Peroxynitrite blocks viral RNA entry into host cells. CVB3 was pretreated or not with 1 μM peroxynitrite and incubated with HeLa cells (MOI = 10) for 1 h at 37°C. HeLa cells were then harvested, cytoplasm was collected from cell lysates by ultracentrifugation, and total RNA was isolated and analyzed by RT-PCR for CVB3 RNA. Some virions were pretreated with the peroxynitrite scavenger GED before exposure to peroxynitrite. ONOO^–, peroxynitrite. This experiment was repeated twice with similar results.

Fig. 3.

Peroxynitrite nitrates and cross-links CVB3 capsid proteins. (A) Immunoblot for VP1. CVB3 was treated with peroxynitrite, fractionated by SDS/PAGE, and immunoblotted with antibody to VP1. (B) Immunoblot for nitrotyrosine. CVB3 was treated with peroxynitrite, fractionated by SDS/PAGE, and immunoblotted with antibody to nitrotyrosine. Nitrated SOD was used as a positive control. (C) Immunoprecipitation for nitrotyrosine followed by immunoblot for VP1. CVB3 was treated with peroxynitrite, immunoprecipitated with antibody to nitrotyrosine, and immunoblotted with antibody to VP1.

Fig. 4.

Nitrotyrosine and viral proteins colocalize and coprecipitate in infected mice. Mice were infected with 10³ pfu of CVB3; hearts were harvested 5 d after infection; and serial sections of the heart were stained for VP1, nitrotyrosine, or both. (A) VP1 expression. VP1 is localized to individual inflammatory cells and to foci of inflammation. (B) Nitrotyrosine localization. Nitrotyrosine localizes to foci of inflammation. (C) VP1 and nitrotyrosine colocalization. Serial sections of the heart were stained with hematoxylin/eosin or with antibodies to both VP1 (red) and nitrotyrosine (brown). Nitrotyrosine and VP1 colocalize to foci of inflammation. Double-staining was performed on 3–4 mice per group with similar results. (D) VP1 and nitrotyrosine coimmunoprecipitate. Hearts from mice infected or not as above were lysed, immunoprecipitated with antibody to nitrotyrosine, and immunoblotted with antibody to VP1 (Top). Lysates from the same mice were also immunoblotted with antibody to VP1 (Middle) or GAPDH (Bottom). This experiment was repeated twice with similar results.

Fig. 5.

The peroxynitrite scavenger GED permits increased viral replication. (A) GED blocks peroxynitrite inhibition of viral replication in cells. CVB3 was pretreated with or without GED and then treated or not with 100 μM peroxynitrite. HeLa cells were then infected with the pretreated CVB3 for 8 h, and the amount of virus replication was measured by the plaque assay. (B) GED blocks peroxynitrite nitration of viral peptide VP1 in vitro. Shown is an immunoprecipitation for nitrated VP1. CVB3 was pretreated with or without GED and then treated or not with 500 μM peroxynitrite, immunoprecipitated with antibody to nitrotyrosine, and immunoblotted with antibody to VP1. (C) The peroxynitrite scavenger GED permits an increase in viral replication in mice. Mice were treated with the peroxynitrite scavenger GED from days 0–3. On day 0, mice were infected with 10³ pfu of CVB3, and the amount of virus was measured in the heart 5 d after infection. (n = 3 ± SD) *, P < 0.05 for 30 versus 0 mg·kg^–1·d^–1. (D) The NOS inhibitor L-NAME permits an increase in viral replication in mice. Mice were pretreated for 1 d with increasing amounts of L-NAME orally and then infected with 10³ pfu of CVB3 i.p. Treatment with control or L-NAME continued for 5 d, and then the hearts were harvested and analyzed for viral titers as above (n = 3 ± SD).