The Jnk Pathway Is Stimulated By Cowpox Virus Which Is Critical For Viral Biology

Cowpoxvirus (CPV) is an orthopoxvirus, i.e. a double-stranded DNA virus that replicates in the cytoplasm of infected cells. As Vaccinia virus (VV) is the prototype of the orthopoxvirus genus and since we have been studying the participation of stress-activated protein kinases (SAPKs) JNK1/2 and found it as necessary for maximal VV multiplication (Pereira et al., 2005, manuscript in preparation), we asked whether CPV and VV share some common strategies to manipulate this signal transduction pathway. To that end, A31 cells were serum-deprived for 12 h and then infected at multiplicity of infection (MOI) 5.0 in the presence or absence of JNK, tyrosine kinase, viral DNA synthesis, microtubule and actin inhibitors (SP600125, genistein, AraC, nocodazole or citochalasin D), respectively. Cell lysates were then prepared to: western blot and immunoblotted with anti-phospho JNK1/2, anti-H3L or anti-ERK1/2 antibodies; northern blot assay and probed with viral thymidine kinase (TK) gene radiolabeled; or dot blot to analyzing viral DNA synthesis. We found that JNK was activated throughout the whole virus infection cycle, being that the phosphorylation increased significantly at late times of infection. In addition, phosphorylation of transcription factor c-Jun occurred in parallel to JNK kinetic. JNK1/2 stimulation was dependent on early steps of virus multiplication, since CPV inactivated upon UV irradiation was not able to phosphorylate JNK1/2 and the same was true by treating the cells with cycloheximide prior to virus infection. On the contrary, viral DNA replication was required only to increase JNK phosphorylation at late times, since pre-incubation with AraC resulted in decreased phosphorylation levels, similar to those verified at early times of the infection. Moreover, the maximal expression of viral TK early-gene and DNA synthesis were dependent of JNK phosphorylation. Our data also demonstrated that CPV stimulates JNK1/2 specifically through a tyrosine kinase-dependent path (Tyk), because it was blocked when the infection was carried out in the presence of genistein. Furthemore neither actin nor microtubule polymerization are required to JNK phosphorylation induced by CPV. The JNK inhibition still resulted in a deep decreasing on expression of late viral protein H3L. Collectively, the recruitment of JNK upon CPV infection is biologically relevant, since its disruption by using SP600125 or genistein leads to a drastic reduction in viral yield (2 log₁₀). Financial support: FAPEMIG, CNPq and CAPES