Inhibition of Junín virus replication by small interfering RNAs - PubMed (original) (raw)
Inhibition of Junín virus replication by small interfering RNAs
María C Artuso et al. Antiviral Res. 2009 Oct.
Abstract
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.
Figures
Fig. 1
Effect of Z-siRNAs on virus infection. (A) Vero cells were transfected with different Z-siRNA (Z1–Z4) and infected at a MOI of 0.1 with the IV4454 (light grey bars) and the XJCl3 (dark grey bars) strains of JUNV, or TCRV (black bars). Supernatants collected at 24 h p.i. were titrated by plaque assay. Inhibition of virus yield was calculated comparatively to the titer obtained in cells transfected with X-siRNA. Each value represents the mean of two independent experiments performed in duplicate ± standard deviation (S.D.). (B) Vero cells were transfected with Z2-siRNA and infected with JUNV, MOI = 0.1. Supernatants were collected at 24 or 48 h p.i. and titrated by plaque assay. Inhibition of virus yield was calculated as above. (C) Vero cells were transfected with Z2-siRNA and infected with JUNV at different MOIs. Supernatants were collected at 24 h p.i., and titrated by plaque assay. Inhibition of virus yield was calculated as above. (D) Amount of viral RNA in Vero cells transfected with Z1-siRNA or Z2-siRNA and infected with JUNV was quantified by real time RT-PCR. Values, standardized to those of actin mRNAs, were expressed as relative RNA levels comparatively to the amount obtained in cells transfected with X-siRNA and represented as mean of two independent experiments performed in triplicate ± S.D. (E) Expression of viral antigens in Vero cells transfected with Z2-siRNA or X-siRNA and infected with JUNV was detected by immunofluorescence assay using a rabbit anti-JUNV polyclonal serum. Magnification = 400×.
Fig. 2
Gene regions targeted for Z-siRNA design. The sequence data for different strains of JUNV (A) and the other arenaviruses TCRV and LCMV (B) for the multiple alignments of the matrix protein Z were obtained from GenBank: IV4454 (DQ538136), XJCl3 (unpublished), Rumero (AY619640), XJ13 (NC_005080), Candid #1 (AY819707), TCRV (NC_004292) and LCMV (AF004519).
Fig. 3
Effect of Z-siRNAs on the expression of Z-recombinant fusion protein. (A) Vero cells grown on glass coverslips were co-transfected with pcDNA3.1-EGFP and X-siRNA (panel A), pcDNA3.1-EGFP and Z2-siRNA (panel B), pcDNA3.1-Z-EGFP and X-siRNA (panel C) or pcDNA3.1-Z-EGFP and the corresponding Z-siRNA (Z1–Z4) (panels D–G, respectively). At 48 h post-transfection, cells were fixed, mounted and observed in a fluorescence microscope (magnification = 400×). (C) Vero cells grown on glass coverslips were untransfected (panel A), co-transfected with pcDNA3.1-Z-Flag and X-siRNA (panel B) or pcDNA3.1-Z-Flag and Z2-siRNA (panel C). After 48 h, cells were fixed and Z-Flag expression was detected using a rabbit anti-Flag serum, followed by rhodamine-conjugated goat anti-rabbit IgG (magnification = 400×). (D) The same samples as (B) were analyzed by Western blot using a rabbit anti-Flag serum or a mouse monoclonal anti-actin antibody.
Fig. 4
Effect of Z-siRNA on JUNV nucleocapsid and glycoprotein expression. Vero cells grown on glass coverslips were untransfected (panels A and D), co-transfected with pcDNAHisMAX C-GPC or pcDNAHisMAX C-N and X-siRNA (panels B and E) or Z2-siRNA (panels C and F). At 48 h post-transfection, cells were fixed, and protein expression was detected using specific monoclonal antibodies against GPC/G1 and N, followed by FITC-conjugated goat anti-mouse IgG (magnification = 400×).
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