ABSTRACTMany viral suppressors (VSRs) counteract antiviral RNA silencing, a central component of the plant’s immune response by sequestration of virus-derived antiviral small interfering RNAs (siRNAs). Here, we addressed how VSRs affect the activities of cellular microRNAs (miRNAs) during a viral infection by characterizing the interactions of two unrelated VSRs, the Tombusvirus p19 and the Cucumovirus 2b, with miRNA 162 (miR162), miR168, and miR403. These miRNAs regulate the expression of the important silencing factors Dicer-like protein 1 (DCL1) and Argonaute proteins 1 and 2 (AGO1 and AGO2), respectively. Interestingly, while the two VSRs showed similar binding profiles, the miRNAs were bound with significantly different affinities, for example, with the affinity of miR162 greatly exceeding that of miR168. In vitro silencing experiments revealed that p19 and 2b affect miRNA-mediated silencing of the DCL1, AGO1, and AGO2 mRNAs in strict accordance with the VSR’s miRNA-binding profiles. In Tombusvirus-infected plants, the miRNA-binding behavior of p19 closely corresponded to that in vitro. Most importantly, in contrast to controls with a Δp19 virus, infections with wild-type (wt) virus led to changes of the levels of the miRNA-targeted mRNAs, and these changes correlated with the miRNA-binding preferences of p19. This was observed exclusively in the early stage of infection when viral genomes are proposed to be susceptible to silencing and viral siRNA (vsiRNA) concentrations are low. Accordingly, our study suggests that differential binding of miRNAs by VSRs is a widespread viral mechanism to coordinately modulate cellular gene expression and the antiviral immune response during infection initiation.