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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

Election microscopy of infected cells.Electron microscopic images of (A) cell control group, (B) viral group, (C) siN.C-treated group, (D) siUL18-3 treated group and (E) siUL19-1 treated group. Black arrows indicate viral particles. Bar, 500 nm. (F) The numbers of particles in each group were counted from six fields and the mean particles per field were calculated. Data are expressed as the mean ± SEM. *P<0.05 vs. viral group.
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pone-0096623-g006: Election microscopy of infected cells.Electron microscopic images of (A) cell control group, (B) viral group, (C) siN.C-treated group, (D) siUL18-3 treated group and (E) siUL19-1 treated group. Black arrows indicate viral particles. Bar, 500 nm. (F) The numbers of particles in each group were counted from six fields and the mean particles per field were calculated. Data are expressed as the mean ± SEM. *P<0.05 vs. viral group.

Mentions: The capsid is an indispensable component of the HSV virus. From the above results, we found that defects in the expression of VP23 and VP5 greatly inhibited the proliferation of HSV-1, as well as ACV-resistant HSV-1 strains and HSV-2. However, whether the effects were induced by defects in capsid formation or virus egressing from the infected cells was unknown. Thus, to investigate this issue, election microscopy was performed, and virus particles were observed in the nucleus, cytoplasm and extracellular compartments. As shown in figure 6A–E, the viral particles were greatly decreased in siRNA interference groups compared with the virus group and the negative group. The numbers of particles in each group were counted and statistics performed as shown in figure 6F. The inhibition rates on the cellular viral particles by siUL18-3 and siUL19-1 were 91.11% and 86.67%, respectively. Thin sections of HSV-infected cells showed nucleocapsids within the nucleus of both siRNA-treat and untreated Vero cells. The virus group (Figure S1) and negative group (Figure S2) had numerous extracellular virions attached to the cell membrane, as well as a number of virions within the cytoplasm and nucleus. In contrast, after treatment with siUL18 (Figure S3) and siUL19 (Figure S4), numbers of virions at the cell surface and in the nucleus and cytoplasm were significantly decreased.


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)

Election microscopy of infected cells.Electron microscopic images of (A) cell control group, (B) viral group, (C) siN.C-treated group, (D) siUL18-3 treated group and (E) siUL19-1 treated group. Black arrows indicate viral particles. Bar, 500 nm. (F) The numbers of particles in each group were counted from six fields and the mean particles per field were calculated. Data are expressed as the mean ± SEM. *P<0.05 vs. viral group.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0096623-g006: Election microscopy of infected cells.Electron microscopic images of (A) cell control group, (B) viral group, (C) siN.C-treated group, (D) siUL18-3 treated group and (E) siUL19-1 treated group. Black arrows indicate viral particles. Bar, 500 nm. (F) The numbers of particles in each group were counted from six fields and the mean particles per field were calculated. Data are expressed as the mean ± SEM. *P<0.05 vs. viral group.
Mentions: The capsid is an indispensable component of the HSV virus. From the above results, we found that defects in the expression of VP23 and VP5 greatly inhibited the proliferation of HSV-1, as well as ACV-resistant HSV-1 strains and HSV-2. However, whether the effects were induced by defects in capsid formation or virus egressing from the infected cells was unknown. Thus, to investigate this issue, election microscopy was performed, and virus particles were observed in the nucleus, cytoplasm and extracellular compartments. As shown in figure 6A–E, the viral particles were greatly decreased in siRNA interference groups compared with the virus group and the negative group. The numbers of particles in each group were counted and statistics performed as shown in figure 6F. The inhibition rates on the cellular viral particles by siUL18-3 and siUL19-1 were 91.11% and 86.67%, respectively. Thin sections of HSV-infected cells showed nucleocapsids within the nucleus of both siRNA-treat and untreated Vero cells. The virus group (Figure S1) and negative group (Figure S2) had numerous extracellular virions attached to the cell membrane, as well as a number of virions within the cytoplasm and nucleus. In contrast, after treatment with siUL18 (Figure S3) and siUL19 (Figure S4), numbers of virions at the cell surface and in the nucleus and cytoplasm were significantly decreased.

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