Compounds reactive against the arenavirus RING finger Z protein induce Z oligomerization and block its interaction with the PRH cellular protein

CC GARCÍA; M DJAVANI; M SALVATO; EB DAMONTE

Barcelona, España

Conferencia; Eighteenth International Conference on Antiviral Research; 2005

The Lymphocytic choriomeningitis virus (LCMV) is the Arenaviridae family prototype member. The virions contain bisegmented single-stranded RNA genomes with an ambisense coding strategy, expressing five proteins. One of them, the Z protein, presents a conserved RING finger motif that makes this protein an attractive target for antiviral therapy. Several antiretroviral Zn finger compounds were reported to establish disulfides linkages between Zn finger motifs in HIV nucleocapsid protein blocking virus replication. In the present study, we investigate the LCMV-Z protein multimer formation after the treatment with one of these compounds, the disulfide NSC20625, provided by the National Cancer Institute (USA). LCMV-Z protein was incubated for 1.5 h at 37°C with 10mM of the compound, analysed by SDS-PAGE in reducing and non reducing conditions, blotted into membranes and revealed with an antiserum anti-LCMV-Z. Interestingly, the treated purified protein exhibited significant altered pattern showing high-molecular-weight multimers. These results suggest that cysteine residues of Z RING finger may undergo conformational changes caused by the compound, establishing inter and intramolecular disulfide bonds, resulting in the formation of Z multimers. Furthermore, it has been reported that LCMV replication affect the subcellular localization of the Proline Rich Homeodomain (PRH) cellular protein in human hepatocytes and that PRH can be physically associated with the Z protein. To determine whether the treatment with the NSC20625 affects the distribution pattern of PRH, LCMV-infected cells were treated with the agent for 72 h. Then, cells were fixed, stained with a purified antibody to monitor PRH bodies and analysed by microscopy. No major differences between the PRH pattern in uninfected cells or infected-treated cells were observed, suggesting that the association of PRH bodies and Z proteins is being blocked by the compound, so it might be due through a PRH-Z RING finger interaction. These results will open the possibility of targeting Z protein as a new antiviral strategy to combat hemorrhagic fever arenaviruses.