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Junín virus (JUNV), the etiological agent of the Argentine hemorrhagic fever, has a single-stranded RNA genome with ambisense expression which encodes for five proteins. In previous works we have demonstrated that the Z arenavirus matrix protein represents an attractive target for antiviral therapy. With the aim of studying a new alternative therapeutic mechanism, four Z-specific siRNAs (Z1- to Z4-siRNAs) were tested showing variable efficacy. The most effective inhibitor was Z2-siRNA targeted at the region encompassed by nt 179-197 of Z gene. The efficacy of this Z2-siRNA against JUNV was also demonstrated in virus-infected cells, by testing infectious virus plaque formation (92.8% JUNV yield reduction), viral RNA level or antigen expression, as well as in cells transfected with Z-specific reporter plasmids (91% reduction in expression of Z-EGFP fusion protein). Furthermore, the lack of effect of this Z-siRNA on the expression of other JUNV proteins, such as N and GPC, confirmed the specificity of action exerted by Z2-siRNA on Z transcript. Thus, the present study represents the first report of virus inhibition mediated by RNA interference for a New World arenavirus.

Our previous studies reported the inhibitory action against arenaviruses of antiretroviral zinc finger-reactive compounds provided by the National Cancer Institute (USA). These compounds were able to inactivate virions as well as to reduce virus yields from infected cells. Here, the inactivation of the arenavirus Junín (JUNV), agent of Argentine hemorrhagic fever, by the aromatic disulfide NSC20625 was analyzed. The treatment of purified JUNV with this compound eliminated infectivity apparently through irreversible modifications in the matrix Z protein detected by: (a) alterations in the electrophoretic migration profile of Z under non-reducing conditions; (b) an electrodense labeling in the internal layer beneath the envelope and around the matrix Z protein, in negatively stained preparations; (c) changes in the subcellular localization of Z in cells transfected with a recombinant fusion protein JUNVZ-eGFP. The infection of Vero cells with JUNV inactivated particles was blocked at the uncoating of viral nucleocapsid from endosomes, providing new evidence for a functional role of Z in this stage of arenavirus cycle. Furthermore, the inactivated JUNV particles retained the immunoreactivity of the surface glycoprotein GP1 suggesting that this disulfide may be useful in the pursuit of an inactivating agent to obtain a vaccine antigen or diagnostic tool.