VIROLOGICA SINICA, February 2011, 26 (1):40-46 DOI 10.1007/s12250-011-3162-9

© Wuhan Institute of Virology, CAS and Springer-Verlag Berlin Heidelberg 2011

Effect of siRNAs on HSV-1 Plaque Formation and Relative

Expression Levels of RR mRNA* Zhe Ren 1,2#, Shen Li 1,3#, Qiao-li Wang1,3, Yang-fei Xiang 1,3, Yun-xia Cui 1,3, Yi-fei Wang 1,3**, Ren-bin Qi 2, Da-xiang Lu 2**, Shu-min Zhang 4** and Pei-zhuo Zhang 5 (1. Biomedicine research and development center of Jinan University, Guangdong, Guangzhou, 510632, China; 2. Medical College of Jinan University, Guangdong, Guangzhou, 510632, China; 3. National Engineering Research Center of Genetic Medicine, Guangdong, Guangzhou, 510632, China; 4. National Institute for the Control of Pharmaceutical and Biological Products, Beijing, 100050,China; 5. Shanghai GenePharma Co., Ltd, Shanghai, 201203, China)

Abstract: RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1 (HSV-1) RR is composed of two distinct homodimeric subunits encoded by UL39 and UL40, respectively. In this study, we applied siRNAs targeting the UL39 and UL40 genes of HSV-1. We showed that synthetic siRNA silenced effectively and specifically UL39 and UL40 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.

Key words: Small interfering RNA (siRNA); Herpes simplex virus type I (HSV-1); Ribonucleotide reductase (RR); Gene

The Herpesvirus family is divided into three sub- families: Alphaherpesvirinae, Betaherpesvirinae and

Received: 2010-08-03, Accepted: 2010-09-30 Gammaherpesvirinae. Herpes simplex virus type I # These authors contributed equally to this work. (HSV-1) is a neurotropic alpha-herpesvirus that only * Foundation items: The Nation “863” Program of China (2006AA02A226); The Joint Funds of National Science naturally occurs in humans with no known animal Foundation of China (U0632010); The State Key reservoirs. HSV-1 is a DNA virus with rapid pro- Laboratory of Phytochemistry and Plant Resources in West China; Chinese Academy of Sciences (O807 liferation and high infectivity. It is a common human B11211, O807E21211) and “211 grant of MOE”. pathogen that occurs worldwide with 60% to 95% of ** Corresponding author. Yi-fei Wang: Fax: +86-20-85223426, infection in adults, and is responsible for a broad E-mail: [email protected] range of human infectious diseases such as gingiva- Da-xiang Lu: Phone: +86-20-85220004, E-mail: [email protected] stomatitis, keratitis, cutaneous herpes, genital herpes Shu-min Zhang: Phone: +86-10-67095598, and encephalitis [25]. E-mail: [email protected]

Virol. Sin. (2011) 26: 40-46 41

HSV-1 encodes its own ribonucleotide reductase The HSV-1 strain F (ATCC VR-733) was obtained (RR), which reduces ribonucleoside diphosphates to from Hong Kong University. The Vero cell line the corresponding deoxyribonucleotides and is essential (ATCC CCL 81), obtained from Wuhan Institute of for the formation of substrates for DNA synthesis[30]. Virology, Chinese Academy of Sciences, was The HSV-1 RR is composed of two distinct maintained in Dulbecco’s modified Eagle medium homodimeric subunits. The large subunit (Mr140000), (DMEM) supplemented with 10% heat-inactivated designated ICP6 [9,10], is encoded by the UL39 gene; fetal bovine serum (FBS), 0.22% sodium bicarbonate the small subunit (Mr38000) is encoded by the UL40 (Sigma, St. Louis, USA), and 50 μg/mL gentamycin gene[20]. The RR of HSV-1 is composed of two (Gibco). For experiments, the Vero cells were cultured subunits arranged in a tight complex of α2β2 structure, in DMEM without serum and antibiotics. The HSV-1 and association of the subunits is necessary for strains were grown in the Vero cells at 37℃. enzyme activity [8,13,21,22]. HSV-1 can not utilize Synthesis of siRNAs cellular ribonucleotide reductase and therefore is The siRNAs used in this study consisted of dependent upon its own reductase for replication. It 21-nucleotide double-stranded RNAs, each strand of has been demonstrated that the viral enzyme may be which contained a 19-nucleotide target sequence and a required for virus growth in non-dividing cells and for two-uracil (U) overhang at the 3′ end. According to the viral pathogenesis and reactivation from latency in design rules for RNAi[7], four fragments of UL40 gene infected hosts in animal models studies [3, 4, 12, 14, 32]. from HSV-1strain F were predicted to have RNAi RNA interference (RNAi) is an innate cellular anti- capacity. These fragments were used as the target virus mechanism. RNAi technology, by use of siRNA (siRNA1, siRNA2, siRNA3, and siRNA4). synthesized small interfering RNA (siRNA) duplexes, The siN.C，not targeting any known gene，was used has been reported as an effective tool for treatment of as negative control (GenePharma) (Table 1). The infectious diseases by silencing viral genes to inhibit siRNA39 targeting the UL39 gene was designed as replication [1]. We have demonstrated previously that siRNA392 described previously[25]. All siRNAs were synthetic siRNA targeting UL39 gene silenced synthesized by Shanghai GenePharma Co.,Ltd. effectively and specifically UL39 mRNA expression Plaque assays and inhibited HSV-1 replication[25]. In this study, we Vero cells were grown in 24-well plates to 80% - examined the effects of siRNAs targeting the UL39 90% confluence and then transfected with specific or and UL40 genes on HSV-1 replication in Vero cells. control siRNA (80 nmol/L) using Lipofectamine 2000. The effect of siRNA on the expression of target genes After 4 h, the cells were infected with 25 PFU of was measured by real-time quantitative reverse tran- HSV-1 strain F. Cells were overlaid with 1 mL of a scription PCR. 1:1 mixture of NaCMC and 2× Dulbecco’s modified Eagle medium without serum to allow only cell-to-cell MATERIALS AND METHODS spread of virus. At 48-72 h post-infection, plates were Viruses, bacterial strains and cells fixed with 10% paraformaldehyde for 2 min and then

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Table 1. Design of siRNA targeting the UL40 gene siRNA Nucleotide sequence of siRNA Nucleotide location(bp)a siRNA1 Sense: 5’-GACGACCUGGUUACGGAAAdTdT-3’ 280-298 Antisense: 5’-UUUCCGUAACCAGGUCGUCGG-3’ siRNA2 Sense: 5’-AAUGCAUCGAAGUCGUACAdTdT-3’ 362-380 Antisense: 5’-UGUACGACUUCGAUGCAUUCC-3’ siRNA3 Sense: 5’-UCACCUGCCAGUCAAACGAdTdT-3’ 626-644 Antisense: 5’-UCGUUUGACUGGCAGGUGACC-3’ siRNA4 Sense: 5’-CCGACAAACACACCAAUUUdTdT-3’ 953-971 Antisense: 5’-AAAUUGGUGUGUUUGUCGGUG-3’ siN.C Sense: 5'-UUCUCCGAACGUGUCACGUdTdT-3’ Antisense: 5'-ACGUGACACGUUCGGAGAAdTdT-3’ stained with 1% crystal violet for 20 min to count the ACGAATGCCCC-3′. number of plaques per well and to photograph the Each real-time PCR reaction was performed for 40 monolayer. Overlapping plaques and plaques at the cycles at 95℃ for 1 min, 95℃ for 15 sec, 58℃ for edge of the well were counted as a single plaque. 15 sec and 72℃ for 40 sec. The relative expression of Real-time RT-PCR UL40 gene was normalized to the house-keeping gene Vero cells were grown in 24-well plates to 80% - 18s RNA and was calculated using the formula[2]: Rel 90% confluence and then transfected with specific or Exp =2(-ΔΔCT) Each real-time PCR reaction was control siRNA (80 nmol/L) using Lipofectamine 2000. performed in triplicate. After 4 h, the cells were infected with 25 PFU of Statistical analyses HSV-1 strain F. After 48 h, total RNA from the Results were given as means ± S.D. and statistical infected cells was extracted using Trizol (Invitrogen). significance was determined by the t test; P values of RNAs were reverse transcribed to yield single- less than 0.05 were considered as statistically significant. stranded cDNA. Real-time PCR reactions were performed using RESULTS SYBR Green Real-time PCR Master Mix (TAKARA, Effect of UL40-specific siRNA on HSV-1 infection Japan) according to the manufacturer’s instructions, HSV-1 produces visible plaques on serum-starved by using the ABI Prism 7000 Sequence Detection Vero cells at 48h post inoculation that develop central System (Applied Biosystems). The following primers clearing as the virus spreads outward (Table 2). Vero were used for amplification. UL40 sequence specific cells were transfected with UL40-specific siRNAs primers: Forward, 5′-CTTCCTCTTCGCTTTCCTGT (siRNA1, siRNA2, siRNA3 and siRNA4) or negative CG-3′; Reverse, 5′-CGCTTCCAGCCAGTCCACCT control siN.C and then infected with 25 PFU of T-3′. UL39 sequence specific primers: Forward, 5′-G HSV-1. Compared with virus control, transfection GCTGCAATCGGCCCTGAAGTA-3′; Reverse, 5′-G with siN.C or UL40-specific siRNA1 had no effect GTGGTCGTAGAGGCGGTGGAA-3′. 18s RNA upon viral plaque formation following infection (P > sequence specific primers: Forward, 5′-CCTGGATA 0.05). In contrast, HSV-1 infection of Vero cells that CCGCAGCTAGGA-3′; Reverse, 5′-GCGGCGCAAT were transfected with UL40-specific siRNAs (siRNA2,

Virol. Sin. (2011) 26: 40-46 43 siRNA3 and siRNA4) produced much less plaques (P with negative control siN.C. In UL40-specific siRNAs < 0.01). The inhibition rates of siRNA1, siRNA2, and UL39-specific siRNA39 co-transfected, UL40- siRNA3 and siRNA4 on HSV-1 plaque formation was specific siRNAs and UL39-specific siRNA39 remar- 1.6%, 50%,41.9% and 62.9%, respectively. kably inhibited the expression of the RR genes (Fig. Effect of UL40-specific siRNA and UL39-specific 2). Compared with virus control, siRNA1 and siRNA co-transfected on HSV-1 infection siRNA39 reduced the expression of UL40 gene and HSV-1 produces visible plaques on serum-starved UL39 gene to levels that were 98%(P>0.05) and Vero cells at 48h post inoculation that develop central 68.5% (P < 0.01) respectively; siRNA2 and siRNA39 clearing as the virus spread outward (Table 3). Vero reduced the expression of UL40 gene and UL39 gene cells were co-transfected with UL40-specific siRNAs to levels that were 36.52%(P <0.01) and 32.38% (P < (siRNA1, siRNA2, siRNA3 or siRNA4) and 0.01) respectively; siRNA3 and siRNA39 reduced the UL39-specific siRNA39 and then infected with 25 expression of UL40 gene and UL39 gene to levels that PFU of HSV-1. Compared with virus control, were 33.89%(P<0.01) and 31.23% (P<0.01) respectively; transfection with siN.C had no effect upon viral siRNA4 and siRNA39 reduced the expression of plaque formation following infection (P > 0.05). In UL40 gene and UL39 gene to levels that were contrast, HSV-1 infection of Vero cells that were 19.43%(P<0.01) and 28.69% (P<0.01) respectively. co-transfected with UL40-specific siRNAs (siRNA1, siRNA2, siRNA3 or siRNA4) and siRNA39 produced DISCUSSION much less plaques (P<0.05 or P<0.01).The inhibition Despite the impact of RNAi in probing gene function rates of siRNA1+siRNA39, siRNA2+siRNA39, siRNA3+siRNA39 and siRNA4+siRNA39 on HSV-1 Table 2. Effect of siRNA on HSV-1 plaque formation Plaques Inhibition plaque formation was 22.3%, 49.2%, 62.3% and siRNA (mean±S.D,n=3) Rate (%) a 73.8%, respectively. siRNA1 20.33±0.58 1.6 c siRNA2 10.33±0.58 50.0 Effect of siRNA on the expression of RR genes c siRNA3 12.00±1.00 41.9 c To determine whether the decrease in the number of siRNA4 7.67±0.58 62.9 a siN.C 19.67±1.15 HSV-1 plaques was due to siRNA silencing Virus Control 20.67±0.58 a c expression of the UL40 gene or UL39 gene, a real- P>0.05, P<0.01. time PCR analyses was performed as above. The results indicated UL40-specific siRNAs but siRNA1 Table 3. Effect of siRNAs on HSV-1 plaque formation Plaques Inhibition remarkably inhibited the expression of the UL40 gene siRNAs (mean±S.D,n=3) Rate(%) (Fig. 1). Compared with virus control, siRNA1、 siRNA1+siRNA39 15.67±1.15b 22.3 siRNA2+siRNA39 10.33±0.58 c 49.2 siRNA2、siRNA3 and siRNA4 reduced the expression siRNA3+siRNA39 7.67±0.58 c 62.3 of UL40 gene to levels that were 97.27%(P >0.05)、 siRNA4+siRNA39 5.33±0.58 c 73.8 siN.C 20.00±1.00 a 30.17%(P < 0.01)、32.06% (P < 0.01) and 18.69% (P Virus Control 20.33±0.58 a b c <0.01) respectively less than those seen in cells treated P>0.05, P<0.05, P<0.01.

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well-recognized target for antiviral agents given the direct role it plays in regulating DNA replication[5,26,29] and its indirect role in regulating other enzymes in the DNA synthesis pathway via control of the nucleotide pool. In the present report, we showed that HSV-1 was susceptible to the RNAi pathway, both the

siRNAs tested resulted in silencing of the HSV-1 RR Fig. 1. Relative expression levels of UL40 mRNA in HSV-1 gene and a reduction in viral plaque formation. infected Vero cells. The relative expression of UL40 mRNA in Although four siRNAs had sequences homologous cells treated with different siRNAs compared with virus control to the HSV-1 UL40 mRNA, they differed in their (100%) was determined by real-time PCR. The result was efficacy to establish RR silencing. In several normalized to the house-keeping gene 18s RNA and was calculated using the formula: Rel Exp = 2(-ΔΔCT). Data represent independent experiments, siRNA4 consistently the means ± S.D. of three wells on same plate. inhibited UL40 expression more efficiently than the

other siRNAs, indicating that the difference in efficacy can not be ascribed solely to differences in transfection efficiency. Variation in the silencing activity of several siRNAs targeting the same mRNA has also been reported previously [11,16,31]. The reasons for the differences in RNAi efficacy are not well understood, but has been postulated to be due to differences in the thermodynamic properties of the siRNAs [17], as well as secondary and tertiary RNA

structure of the target site that disables recognition by Fig. 2. Relative expression levels of RR mRNA in HSV-1 the siRNA[16,19,27]. infected Vero cells. The relative expression of RR mRNA [6] (UL39 mRNA and UL40 mRNA)in cells treated with different David et al demonstrated that HSV-1 ribonucleotide siRNAs compared with virus control (100%) was determined reductase activity is not essential for growth in by real-time PCR. The result was normalized to the cultured cells and that the cellular enzyme, when house-keeping gene 18s RNA and was calculated using the active, can support virus growth. They found hrR , formula: Rel Exp = 2(-ΔΔCT). Data represent the means ± S.D. of 3 three wells on same plate. which is incapable of inducing viral ribonucleotide reductase activity in untransformed cells, was capable and in the development of novel therapeutics and of synthesizing substantial amounts of viral DNA in antivirals, there have been only a few studies exponentially growing Vero cells (74% as compared exploring the potential for RNAi approaches to with wild-type levels). However, viral DNA synthesis HSV-1. These reports have focused primarily on the α of the mutant was more compromised when it was and γ genes of HSV-1[18,23,32]. RR, as a β gene, is a grown in serum-starved Vero cells (29% as compared

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