RNA interference inhibits herpes simplex virus type 1 isolated from saliva samples and mucocutaneous lesions

Abstract

The aim of this study was to evaluate the use of RNA interference to inhibit herpes simplex virus type-1 replication in vitro. For herpes simplex virus type-1 gene silencing, three different small interfering RNAs (siRNAs) targeting the herpes simplex virus type-1 UL39 gene (sequence si-UL 39-1, si-UL 39-2, and si-UL 39-3) were used, which encode the large subunit of ribonucleotide reductase, an essential enzyme for DNA synthesis. Herpes simplex virus type-1 was isolated from saliva samples and mucocutaneous lesions from infected patients. All mucocutaneous lesions' samples were positive for herpes simplex virus type-1 by real-time PCR and by virus isolation; all herpes simplex virus type-1 from saliva samples were positive by real-time PCR and 50% were positive by virus isolation. The levels of herpes simplex virus type-1 DNA remaining after siRNA treatment were assessed by real-time PCR, whose results demonstrated that the effect of siRNAs on gene expression depends on siRNA concentration. The three siRNA sequences used were able to inhibit viral replication, assessed by real-time PCR and plaque assays and among them, the sequence si-UL 39-1 was the most effective. This sequence inhibited 99% of herpes simplex virus type-1 replication. The results demonstrate that silencing herpes simplex virus type-1 UL39 expression by siRNAs effectively inhibits herpes simplex virus type-1 replication, suggesting that siRNA based antiviral strategy may be a potential therapeutic alternative.

Keywords: Herpes simplex virus type 1; Real-time PCR; siRNA.

Fig. 1

Effect of different siRNAs on the expression levels of HSV-1 DNA. The expression level of HSV-1 DNA in cells treated with different concentrations (3–21 nM) of siRNAs (si-UL-39 1, si-UL-39 2, si-UL39 3) was determined by real-time quantitative PCR. The result was normalized to the housekeeping gene 18S RNA. NC – cells transfected with non-specific siRNA (negative control). CIN cells infected with HSV-1 but not transfected.

Fig. 2

Inhibition of HSV-1 replication in an infected patient. The inhibition of UL-39 gene was performed using siRNAs (si-UL-39 1, si-UL-39 2, si-UL39 3). NC, negative control; CIN, cells infected with HSV-1 but not transfected.