May 2006
Volume 47, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2006
MCMV–Specific siRNAs Enhance Virus Replication in vitro at Low Multiplicities of Infection
Author Affiliations & Notes
  • B. Marshall
    Cellular, Medical, Augusta, GA
  • S.S. Atherton
    Cellular, Medical, Augusta, GA
  • Footnotes
    Commercial Relationships  B. Marshall, None; S.S. Atherton, None.
  • Footnotes
    Support  NIH Grant EY009169
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 3066. doi:
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      B. Marshall, S.S. Atherton; MCMV–Specific siRNAs Enhance Virus Replication in vitro at Low Multiplicities of Infection . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3066.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : The aim of this work was to investigate the feasibility of inhibiting the in vitro replication of murine cytomegalovirus (MCMV) using siRNA targeted to MCMV immediate early (IE) gene expression. MCMV infection of the murine retina is an animal model for CMV retinitis which frequently occurs in immunosuppressed patients.

Methods: : Mouse M2–10B4 cells are a mouse stromal bone marrow–derived cell line which supports MCMV replication to high titer in vitro and is readily transfectable. M2–10B4 cells were infected with MCMV (Smith strain) at m.o.i.s of 2, 0.2, 0.02, or 0.002 and virus titer was measured at days 2 and 5 post–inoculation. siRNAs were prepared from both the MCMV IE–1 and IE–3 genes using a Silencer siRNA Cocktail Kit (Ambion Inc). This strategy produces a cocktail of siRNAs from the target gene of interest by amplification of a selected sub–region of the gene followed by dsRNA production and RNase III cleavage. For the IE–1 gene, a region of 362bp from the IE–1 specific exon 4 was amplified, while for the IE–3 gene, a region of 559bp from the IE–3 specific exon 5 was amplified. siRNA cocktails were transfected into M2–10B4 cells using Lipofectamine 2000 (Invitrogen) 4 hours before cells were infected with MCMV.

Results: : Initial studies indicated transfection efficiencies of approximately 80% for M2–10B4 cells, confirming their suitability for this study. Both IE–1 specific and IE–3 specific siRNAs demonstrated only modest knockdowns of virus titer at m.o.i.s of 2 and 0.2. Decreases in virus titers were typically between 2–fold and 10–fold. Surprisingly, at lower m.o.i.s, both IE–1 and IE–3 specific siRNAs appeared to enhance MCMV replication. Increases in virus titer of up to 50–fold were observed. In order to determine if this phenomenon was specifically related to the regulatory role of the immediate early genes, or was a more general feature of MCMV itself, siRNAs were made from the M86 major capsid protein, an MCMV structural gene with no known regulatory function. Once again, enhancement of virus replication was observed at lower m.o.i.s, suggesting that siRNA effects on MCMV replication may be m.o.i–dependent.

Conclusions: : The effects of siRNAs on MCMV replication may not be easily predictable and such unpredictability may be a confounding factor for in vivo use of siRNAs to prevent/reduce CMV replication.

Keywords: retina • antiviral drugs • pathology: experimental 
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