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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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Effect of siRNA on HSV-1 plaque formation and relative expression of capsid-related genes in HSV-1-infected Vero cells.(A–F) Effects of siRNA on relative mRNA expression levels of capsid genes (UL18, UL19, UL26, UL26.5, UL35 and UL38, respectively) compared with the virus group were analyzed by qPCR. (a–f) Effects of siRNA against different capsid genes (UL18, UL19, UL26, UL26.5, UL35 and UL38, respectively) on HSV-1 proliferation were evaluated by a plaque reduction assay. Vero cells were treated with different siRNAs. Cultures were infected with HSV-1, and plaques were counted 72 h later. The relative survival rate of each group was compared with the virus control set at 100%. Each sample was analyzed in triplicate, and data are expressed as the mean ± SEM. *P<0.05 vs. viral group.
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pone-0096623-g002: Effect of siRNA on HSV-1 plaque formation and relative expression of capsid-related genes in HSV-1-infected Vero cells.(A–F) Effects of siRNA on relative mRNA expression levels of capsid genes (UL18, UL19, UL26, UL26.5, UL35 and UL38, respectively) compared with the virus group were analyzed by qPCR. (a–f) Effects of siRNA against different capsid genes (UL18, UL19, UL26, UL26.5, UL35 and UL38, respectively) on HSV-1 proliferation were evaluated by a plaque reduction assay. Vero cells were treated with different siRNAs. Cultures were infected with HSV-1, and plaques were counted 72 h later. The relative survival rate of each group was compared with the virus control set at 100%. Each sample was analyzed in triplicate, and data are expressed as the mean ± SEM. *P<0.05 vs. viral group.

Mentions: In this experiment, 21 pairs of siRNAs targeting the HSV-1 capsid protein encoding gene were designed to evaluate the therapeutic efficiency of siRNA against HSV-1, including UL18 (capsid triplex protein VP23), UL19 (major capsid protein VP5), UL26 (capsid maturation protease VP24/21), UL26.5 (capsid scaffold protein VP22a), UL35 (small capsid protein VP26) and UL38 (capsid triplex protein VP19C). First, qPCR was performed to determine whether the siRNAs could efficiently silence the expression of these genes. As shown in Figure 2, UL18-specific siRNAs inhibited the expression of the UL18 gene significantly compared with the virus control. Expression of the UL18 gene dropped to 13.22% and 12.75% with the use of siUL18-1 and siUL18-3, respectively (Figure 2A). The equivalent amounts of UL18, siUL19-1, siUL19-3 and siUL19-4 reduced the expression of the UL19 gene to 20.29%, 18.48% and 21.14%, respectively (Figure 2B). Expression of the UL26 gene decreased to 18.79% with the use of siUL26-2 (Figure 2C), while that of the UL26.5 gene was reduced to 47.88% by siUL26.5 (Figure 2D). siUL35-2 reduced the expression of the UL35 gene to 44.05% (Figure 2E), and siUL38-3 reduced that of the UL38 gene to 17.52% (Figure 2F). The negative control group showed no statistically significant difference compared with the virus-infected groups (P>0.05).


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)

Effect of siRNA on HSV-1 plaque formation and relative expression of capsid-related genes in HSV-1-infected Vero cells.(A–F) Effects of siRNA on relative mRNA expression levels of capsid genes (UL18, UL19, UL26, UL26.5, UL35 and UL38, respectively) compared with the virus group were analyzed by qPCR. (a–f) Effects of siRNA against different capsid genes (UL18, UL19, UL26, UL26.5, UL35 and UL38, respectively) on HSV-1 proliferation were evaluated by a plaque reduction assay. Vero cells were treated with different siRNAs. Cultures were infected with HSV-1, and plaques were counted 72 h later. The relative survival rate of each group was compared with the virus control set at 100%. Each sample was analyzed in triplicate, and data are expressed as the mean ± SEM. *P<0.05 vs. viral group.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0096623-g002: Effect of siRNA on HSV-1 plaque formation and relative expression of capsid-related genes in HSV-1-infected Vero cells.(A–F) Effects of siRNA on relative mRNA expression levels of capsid genes (UL18, UL19, UL26, UL26.5, UL35 and UL38, respectively) compared with the virus group were analyzed by qPCR. (a–f) Effects of siRNA against different capsid genes (UL18, UL19, UL26, UL26.5, UL35 and UL38, respectively) on HSV-1 proliferation were evaluated by a plaque reduction assay. Vero cells were treated with different siRNAs. Cultures were infected with HSV-1, and plaques were counted 72 h later. The relative survival rate of each group was compared with the virus control set at 100%. Each sample was analyzed in triplicate, and data are expressed as the mean ± SEM. *P<0.05 vs. viral group.
Mentions: In this experiment, 21 pairs of siRNAs targeting the HSV-1 capsid protein encoding gene were designed to evaluate the therapeutic efficiency of siRNA against HSV-1, including UL18 (capsid triplex protein VP23), UL19 (major capsid protein VP5), UL26 (capsid maturation protease VP24/21), UL26.5 (capsid scaffold protein VP22a), UL35 (small capsid protein VP26) and UL38 (capsid triplex protein VP19C). First, qPCR was performed to determine whether the siRNAs could efficiently silence the expression of these genes. As shown in Figure 2, UL18-specific siRNAs inhibited the expression of the UL18 gene significantly compared with the virus control. Expression of the UL18 gene dropped to 13.22% and 12.75% with the use of siUL18-1 and siUL18-3, respectively (Figure 2A). The equivalent amounts of UL18, siUL19-1, siUL19-3 and siUL19-4 reduced the expression of the UL19 gene to 20.29%, 18.48% and 21.14%, respectively (Figure 2B). Expression of the UL26 gene decreased to 18.79% with the use of siUL26-2 (Figure 2C), while that of the UL26.5 gene was reduced to 47.88% by siUL26.5 (Figure 2D). siUL35-2 reduced the expression of the UL35 gene to 44.05% (Figure 2E), and siUL38-3 reduced that of the UL38 gene to 17.52% (Figure 2F). The negative control group showed no statistically significant difference compared with the virus-infected groups (P>0.05).

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