Poster abstracts

Poster number 101 submitted by Zetang Wu

HSV-1 encodes one or more silencing supressors

Zetang Wu (MCDB), Yali Zhu (MVIMG), Kenneth Buckley (MG), David M. Bisaro (MG), Deborah S. Parris (MVIMG)

Abstract:
Herpes Simplex Virus Encodes One or More RNA Silencing Suppressors
Zetang Wu3, Yali Zhu1, Paul Nolan1, Kenneth Buckley2, David M. Bisaro,1,2 and Deborah S. Parris1,2
1The Department of Molecular Virology, Immunology and Medical Genetics
2The Department of Molecular Genetics
3Program in Molecular, Cellular, and Developmental Biology

Abstract: It was hypothesized that HSV-1 encodes one or more silencing suppressors. Transient silencing in mammalian cells was induced by co-transfecting cells with a target plasmid encoding EGFP and a plasmid encoding a short imperfect hairpin directed to EGFP. Controls were co-transfected with the EGFP-expressing plasmid and one encoding a short imperfect hairpin directed to bacterial lacZ. 52 hr after transfection, the steady-state EGFP mRNA levels in silenced cells were 5-7 fold lower than those in control cells. The reduced EGFP mRNA level was associated with a decrease in the half-life of EGFP mRNA from 5.6 to 2.7 hr. In HSV-1-infected silenced cells, EGFP mRNA levels were enhanced up to 5-fold and this increase corresponded with an increased half-life of EGFP mRNA. Surprisingly, the half-life of EGFP mRNA in silenced and control cells was the same (4.3 hr), indicating the HSV-1 infection completely reversed established silencing. Enhanced EGFP mRNA levels were observed as early as 4 hr after HSV-1 infection of silenced cells, and increased through ~10 hr post-infection. Increased EGFP mRNA levels were also observed in the absence of viral DNA synthesis, ruling out a requirement for de novo synthesis of late proteins. To assess the biological relevance of silencing for HSV replication, we knocked-down a component of the RNA silencing machinery using siRNA directed to argonaut 2 (Ago2) mRNA. Preliminary results indicate that wild-type HSV replicates to higher levels in cells knocked-down for Ago2 expression compared to that in cells that received a control siRNA.

Keywords: RNA silencing, HSV-1, Silencing suppressor