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Silencing herpes simplex virus type 1 capsid protein encoding genes by siRNA: a promising antiviral therapeutic approach.

Jin F, Li S, Zheng K, Zhuo C, Ma K, Chen M, Wang Q, Zhang P, Fan J, Ren Z, Wang Y - PLoS ONE (2014)

Bottom Line: Plaque numbers and intracellular virions were significantly reduced by simultaneous knockdown of UL18 and UL19.The total intracellular viral genome loads were also significantly decreased in the UL18 and UL19 knockdown groups compared with the viral control.In conclusion, interfering with UL18 and UL19 gene expression could inhibit HSV-1 replication efficiently in vitro.

View Article: PubMed Central - PubMed

Affiliation: Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou, Guangdong, China; College of Pharmacy, Jinan University, Guangzhou, Guangdong, China.

ABSTRACT
Herpes simplex virus type 1 (HSV-1), a member of the herpesviridae, causes a variety of human viral diseases globally. Although a series of antiviral drugs are available for the treatment of infection and suppression of dissemination, HSV-1 remains highly prevalent worldwide. Therefore, the development of novel antiviral agents with different mechanisms of action is a matter of extreme urgency. During the proliferation of HSV-1, capsid assembly is essential for viral growth, and it is highly conserved in all HSV-1 strains. In this study, small interfering RNAs (siRNAs) against the HSV-1 capsid protein were screened to explore the influence of silencing capsid expression on the replication of HSV-1. We designed and chemically synthesized siRNAs for the capsid gene and assessed their inhibitory effects on the expression of target mRNA and the total intracellular viral genome loads by quantitative real-time PCR, as well as on the replication of HSV-1 via plaque reduction assays and electron microscopy. Our results showed that siRNA was an effective approach to inhibit the expression of capsid protein encoding genes including UL18, UL19, UL26, UL26.5, UL35 and UL38 in vitro. Interference of capsid proteins VP23 (UL18) and VP5 (UL19) individually or jointly greatly affected the replication of clinically isolated acyclovir-resistant HSV-1 as well as HSV-1/F and HSV-2/333. Plaque numbers and intracellular virions were significantly reduced by simultaneous knockdown of UL18 and UL19. The total intracellular viral genome loads were also significantly decreased in the UL18 and UL19 knockdown groups compared with the viral control. In conclusion, interfering with UL18 and UL19 gene expression could inhibit HSV-1 replication efficiently in vitro. Our research offers new targets for an RNA interference-based therapeutic strategy against HSV-1.

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Related in: MedlinePlus

Optimal transfection conditions.(A) Cell viability with siRNA and transfection reagent siRNA-Mate at different concentrations was assessed with the MTT assay. (B) qPCR was conducted to analyze the knockdown efficiency of transfecting different ratios of siRNA:siRNA-Mate/cm2. Each sample was analyzed in triplicate, and data are expressed as the mean ± SEM. *P<0.05 vs. virus control group. (C) FAM-tagged siRNA was transfected into Vero cells with a transfection ratio of 20 pmol siRNA:1 µl siRNA-Mate/cm2. The transfection efficiency and durability of the siRNA were detected by fluorescence microscopy on days 1, 2 and 3 after transfection.
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pone-0096623-g001: Optimal transfection conditions.(A) Cell viability with siRNA and transfection reagent siRNA-Mate at different concentrations was assessed with the MTT assay. (B) qPCR was conducted to analyze the knockdown efficiency of transfecting different ratios of siRNA:siRNA-Mate/cm2. Each sample was analyzed in triplicate, and data are expressed as the mean ± SEM. *P<0.05 vs. virus control group. (C) FAM-tagged siRNA was transfected into Vero cells with a transfection ratio of 20 pmol siRNA:1 µl siRNA-Mate/cm2. The transfection efficiency and durability of the siRNA were detected by fluorescence microscopy on days 1, 2 and 3 after transfection.

Mentions: Cytotoxicity levels of the siRNAs and transfection regents, as measured by the MTT assay, were very low even in high concentrations (Figure 1A). The cell viability of the 20 pmol siRNA: 1.5 µl siRNA-Mate/cm2 groups reached up to 81%. To screen for the most efficient knockdown ratios of siRNA:siRNA-Mate, an effective siRNA targeting the capsid protein VP23 encoding gene UL18, siUL18-1 (data from pre-experiment), was chosen as a representative of siRNAs for further experiments. The interference efficiency for each group approached 70%. The highest interference efficiency was obtained using 20 pmol of siRNA with 1 µl of siRNA-Mate/cm2 (Figure 1B), and this transfection ratio was used in further experiments. The transfection efficiency and durability of siRNAs in cells were determined by fluorescence microscopy. As shown in Figure 1C, the transfection efficiency reached up to 95% in this condition on the first day. Two days after transfection, the percentage of positive cells was maintained at 70%, but it decreased to 20% three days later.


Silencing herpes simplex virus type 1 capsid protein encoding genes by siRNA: a promising antiviral therapeutic approach.

Jin F, Li S, Zheng K, Zhuo C, Ma K, Chen M, Wang Q, Zhang P, Fan J, Ren Z, Wang Y - PLoS ONE (2014)

Optimal transfection conditions.(A) Cell viability with siRNA and transfection reagent siRNA-Mate at different concentrations was assessed with the MTT assay. (B) qPCR was conducted to analyze the knockdown efficiency of transfecting different ratios of siRNA:siRNA-Mate/cm2. Each sample was analyzed in triplicate, and data are expressed as the mean ± SEM. *P<0.05 vs. virus control group. (C) FAM-tagged siRNA was transfected into Vero cells with a transfection ratio of 20 pmol siRNA:1 µl siRNA-Mate/cm2. The transfection efficiency and durability of the siRNA were detected by fluorescence microscopy on days 1, 2 and 3 after transfection.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4008601&req=5

pone-0096623-g001: Optimal transfection conditions.(A) Cell viability with siRNA and transfection reagent siRNA-Mate at different concentrations was assessed with the MTT assay. (B) qPCR was conducted to analyze the knockdown efficiency of transfecting different ratios of siRNA:siRNA-Mate/cm2. Each sample was analyzed in triplicate, and data are expressed as the mean ± SEM. *P<0.05 vs. virus control group. (C) FAM-tagged siRNA was transfected into Vero cells with a transfection ratio of 20 pmol siRNA:1 µl siRNA-Mate/cm2. The transfection efficiency and durability of the siRNA were detected by fluorescence microscopy on days 1, 2 and 3 after transfection.
Mentions: Cytotoxicity levels of the siRNAs and transfection regents, as measured by the MTT assay, were very low even in high concentrations (Figure 1A). The cell viability of the 20 pmol siRNA: 1.5 µl siRNA-Mate/cm2 groups reached up to 81%. To screen for the most efficient knockdown ratios of siRNA:siRNA-Mate, an effective siRNA targeting the capsid protein VP23 encoding gene UL18, siUL18-1 (data from pre-experiment), was chosen as a representative of siRNAs for further experiments. The interference efficiency for each group approached 70%. The highest interference efficiency was obtained using 20 pmol of siRNA with 1 µl of siRNA-Mate/cm2 (Figure 1B), and this transfection ratio was used in further experiments. The transfection efficiency and durability of siRNAs in cells were determined by fluorescence microscopy. As shown in Figure 1C, the transfection efficiency reached up to 95% in this condition on the first day. Two days after transfection, the percentage of positive cells was maintained at 70%, but it decreased to 20% three days later.

Bottom Line: Plaque numbers and intracellular virions were significantly reduced by simultaneous knockdown of UL18 and UL19.The total intracellular viral genome loads were also significantly decreased in the UL18 and UL19 knockdown groups compared with the viral control.In conclusion, interfering with UL18 and UL19 gene expression could inhibit HSV-1 replication efficiently in vitro.

View Article: PubMed Central - PubMed

Affiliation: Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou, Guangdong, China; College of Pharmacy, Jinan University, Guangzhou, Guangdong, China.

ABSTRACT
Herpes simplex virus type 1 (HSV-1), a member of the herpesviridae, causes a variety of human viral diseases globally. Although a series of antiviral drugs are available for the treatment of infection and suppression of dissemination, HSV-1 remains highly prevalent worldwide. Therefore, the development of novel antiviral agents with different mechanisms of action is a matter of extreme urgency. During the proliferation of HSV-1, capsid assembly is essential for viral growth, and it is highly conserved in all HSV-1 strains. In this study, small interfering RNAs (siRNAs) against the HSV-1 capsid protein were screened to explore the influence of silencing capsid expression on the replication of HSV-1. We designed and chemically synthesized siRNAs for the capsid gene and assessed their inhibitory effects on the expression of target mRNA and the total intracellular viral genome loads by quantitative real-time PCR, as well as on the replication of HSV-1 via plaque reduction assays and electron microscopy. Our results showed that siRNA was an effective approach to inhibit the expression of capsid protein encoding genes including UL18, UL19, UL26, UL26.5, UL35 and UL38 in vitro. Interference of capsid proteins VP23 (UL18) and VP5 (UL19) individually or jointly greatly affected the replication of clinically isolated acyclovir-resistant HSV-1 as well as HSV-1/F and HSV-2/333. Plaque numbers and intracellular virions were significantly reduced by simultaneous knockdown of UL18 and UL19. The total intracellular viral genome loads were also significantly decreased in the UL18 and UL19 knockdown groups compared with the viral control. In conclusion, interfering with UL18 and UL19 gene expression could inhibit HSV-1 replication efficiently in vitro. Our research offers new targets for an RNA interference-based therapeutic strategy against HSV-1.

Show MeSH
Related in: MedlinePlus