siRNA Targeting ICP4 Gene Reduces HSV–2 Infection of Cultured Rabbit Skin Cells

Abstract

Abstract: : Purpose: Recurrent corneal infections by Herpes simplex virus types 1 and 2 are a major cause of infectious blindness. Our goal is to design and test small interfering RNAs (siRNA) that target the mRNA of essential HSV genes for their ability to reduce viral replication. Methods: An essential immediate early gene, ICP4, was selected as a target for siRNA interference. Five potential siRNA target sites were identified on the ICP4 gene that consisted of 21– nucleotide sequences that start with AA sequence, contained 30–50% of GC content, and did not have more than four T bases, four A bases, or three G bases in a row. An 21–mer RNA oligonucletide corresponding to nucleotides 19–40 was chemically synthesized (Dharmacon, Lafeytte, CO), and annealed with its complementary sequence 21–mer to generate double stranded siRNA molecules. Triplicate cultures of rabbit skin (RS) cells were transfected with 150 nM siRNA and oligofectamine, and 24 hours later the cells were infected with HSV–2 virus (HG52) at a multiplicity of infection (MOI) of 1. Twelve hours after infection with HSV–2, virus was harvested by scraping the dishes and three cycles of freeze/thawing, and viral replication was measured by plaque assays. Controls included mock transfection with siRNA and transfection with scrambled sequence siRNA. Results: The average plaque forming units (pfu) in mock transfected control group was 3.8 ± 0.2 x 10⁵ pfu and in the scramble siRNA control group was 5.7 ± 0.6 x 10⁵ pfu. In contrast, virus production was reduced 3.25–fold in cells transfected with ICP4 siRNA group to an average of 1.2 ± 0.16 x 10⁵. The siRNA was not toxic to RS cells since virus production was not reduced in cells transfected with the scrambled control siRNA. Conclusions:Replication of HSV–2 virus in RS cells can be significantly reduced by siRNA that selectively targets ICP4 mRNA. RNA interference may provide an new therapeutic strategy against herpes simplex keratitis.

Keywords: herpes simplex virus • keratitis • microbial pathogenesis: experimental studies