SP600125 inhibits Orthopoxviruses replication in a JNK1/2 -independent manner: Implication as a potential antipoxviral

Abstract

The pharmacological inhibitor SP600125 [anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone] has been largely employed as a c-JUN N-terminal kinase (JNK1/2) inhibitor. In this study, we evaluated whether pretreatment with SP600125 was able to prevent Orthopoxviruses Vaccinia virus (VACV), Cowpox virus (CPXV) and modified Vaccinia virus Ankara (MVA) replication. We found that incubation with SP600125 not only blocked virus-stimulated JNK phosphorylation, but also, significantly reduced virus production. We observed 1-3 log decline in viral yield depending on the cell line infected (A31, BSC-40 or BHK-21). The reduction in viral yield correlated with a dramatic impact on virus morphogenesis progress, intracellular mature viruses (IMV) were barely detected. Despite the fact that SP600125 can act as an efficient anti-orthopoxviral compound, we also provide evidence that this antiviral effect is not specifically exerted through JNK1/2 inhibition. This conclusion is supported by the fact that viral titers measured after infections of JNK1/2 knockout cells were not altered as compared to those of wild-type cells. In contrast, a decline in viral titers was verified when the infection of KO cells was carried out in the presence of the pharmacological inhibitor. SP600125 has been the focus of recent studies that have evaluated its action on diverse viral infections including DNA viruses. Our data support the notion that SP600125 can be regarded as a potential antipoxviral compound.

Fig. 1

VACV and CPXV infection stimulate JNK1/2 phosphorylation. BSC-40 cells were left uninfected (MOCK) or were infected with CPXV (A) or VACV (B) for the times shown. (C) Cells were left uninfected (MOCK) or infected (VACV) and treated with SP600125 (10, 20, 40 or 50 μM) prior to and during viral infection (lanes 4–7). Cell lysates (40 μg) were prepared, subjected to western blot and probed with anti-phospho JNK1/2 (Thr183/Tyr185) – upper panels, or probed with anti-ERK1/2 or anti-β actin antibody as a loading control – lower panels. Data are representative of three distinct experiments with similar results.

Fig. 2

SP600125 inhibits VACV, CPXV and MVA growth. Multi-step growth curves - A31 cells (A, B), BSC-40 (C, D) and BHK-21(E–G) were CPXV- (A, C, G), VACV- (B, D, F) and MVA- (E) infected at an MOI of 10, for the indicated times, either in the absence or presence of SP600125 (40 μM). Cell lysates were collected and viral yields were quantitated. Data are representative of three distinct experiments with similar results.

Fig. 3

SP600125 blocks virion maturation during orthopoxvirus morphogenesis. Electron microscopy analysis – A31 cells were either left untreated (A–C) or treated with SP600125 prior to VACV (D–G) infection at MOI of 2 for 18 h. Cells were then fixed and prepared for transmission electron microscopy. Electron micrographs are shown with their scale indicated by the bars. Abbreviations:^∗ – Crescents, IV – immature virus, IVN – immature virions with nucleoids, IMV – intracellular mature virus, N – nucleus, M – mitochondria, V - virosome. Panel G is a close up image of panel F. Data is representative of two independent experiments with similar results.

Fig. 4

Inhibition of VACV and CPXV growth by SP600125 is independent of JNK1/2. WT (A) and JNK1/2 KO (B) cells were either left untreated or treated with SP600125 (40 μM), or with JNK Inhibitor VIII (4 μM) prior to VACV or CPXV infection at an MOI of 10. At 24 h.p.i, total cell lysates were assayed for viral production. (C) Cells were left uninfected (MOCK) or were treated with SP600125 or JNKi at (4 μM) (lanes 5, 6 and 8, 9), as indicated. Total cell lysates (40 μg) were prepared, subjected to western blot and probed with anti-phospho c-JUN (Ser 73) – upper panels, or probed with anti-β tubulin antibody (1:3000) as a loading control – lower panels. Data are representative of three distinct experiments with similar results.