| It was hypothesized that herpes simplex virus type-1 (HSV-1) encodes one or more proteins capable of functioning as a silencing suppressor during productive infection. A transient silencing system was developed that relies on co-transfection of mammalian cells with a plasmid that expresses a silencing target (EGFP) and one that expresses either a perfect EGFP-specific hairpin (dsEGFP) or an imperfect hairpin (midsEGFP). Co-transfection of target plasmid with a plasmid that expresses perfect or imperfect hairpins specific for lacZ (dsLacZ or midsLacZ, respectively), was used as a control. Results demonstrated that dsEGFP and midsEGFP reduced the EGFP mRNA level by 2.5- and 7-fold, respectively, compared with control hairpins, and a 1:1 molar ratio of midsEGFP to target plasmid yielded maximum silencing of EGFP. Silencing by the midsEGFP hairpin decreased the half-life of EGFP mRNA ∼2-fold compared with the control imperfect hairpin.;Infection of transfected cells with HSV-1 wild-type strain KOS increased the steady-state amount of EGFP mRNA, regardless of whether or not the EGFP transcript contained an intron. This increase was due, at least in part, to an increase in the half-life of EGFP mRNA in silenced cells, consistent with silencing suppression. This increased EGFP mRNA half-life occurred despite the fact that host shut-off functions of HSV-1 are known to globally reduce the stability of mRNAs. Indeed viruses with loss-of-function mutations in these host shut-off genes increased the stability of EGFP mRNA in silenced cells to an even greater extent than the wild-type virus. Increased accumulation of target EGFP mRNA occurred as early as 4 hr after HSV-1 infection and did not require viral DNA synthesis, suggesting that the silencing suppressor activity was an immediate-early gene product or a virion component.;We tested several immediate-early genes and a virion component gene for ability to suppress silencing in an established plant assay and found that only the virion component, US11, could suppress silencing in plants in the absence of other HSV-1 genes. Further experiments suggest that US11 could play an important role in silencing suppression in mammalian cells.;Taken together these results demonstrate that infection by HSV-1 can suppress established silencing and suggest that this silencing suppression may have evolved as a means to counter a very early host silencing response following virus infection. A prediction of this hypothesis is that replication of HSV-1 would be enhanced if the host silencing response were abrogated. To test this prediction, siRNAs were transfected into cells to knock-down the RNA slicer activity, argonaut 2 (Ago-2), prior to virus infection. The yields of virus following HSV-1 infection of cells treated with siRNA to Ago-2, compared to those treated with RISC-free control RNA duplex, were significantly increased. |
