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RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells.

Zhao Z, Wu G, Zhu X, Yan X, Dou Y, Li J, Zhu H, Zhang Q, Cai X - Virol. J. (2012)

Bottom Line: The results showed that the ORF095-specific siRNA-70 effectively down-regulated the expression of ORF095.When Vero cells were transfected with shRNA expression vectors (p61/GFP, p70/GFP, p165/GFP and p296/GFP) and then infected with GTPV, GTPV-ORF095-70 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by GTPV.The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of GTPV (approximately 463. 5-fold reduction of viral titers) on Vero cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Animal virology of the Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences, Gansu, PR China.

ABSTRACT

Background: Goatpox is an economically important disease in goat and sheep-producing areas of the world. Many vaccine strategies developed to control the disease are not yet completely successful. Hairpin expression vectors have been used to induce gene silencing in a large number of studies on viruses. However, none of these studies has been attempted to study GTPV. In the interest of exploiting improved methods to control goat pox, it is participated that RNAi may provide effective protection against GTPV. In this study we show the suppression of Goatpox virus (GTPV) replication via knockdown of virion core protein using RNA interference.

Results: Four short interfering RNA (siRNA) sequences (siRNA-61, siRNA-70, siRNA-165 and siRNA-296) against a region of GTPV ORF095 were selected. Sense and antisense siRNA-encoding sequences separated by a hairpin loop sequence were designed as short hairpin RNA (shRNA) expression cassettes under the control of a human U6 promoter. ORF095 amplicon was generated using PCR, and then cloned into pEGFP-N1 vector, named as p095/EGFP. p095/EGFP and each of the siRNA expression cassettes (p61, p70, p165 and p296) were co-transfected into BHK-21 cells. Fluorescence detection, flow cytometric analysis, retro transcription PCR (RT-PCR) and real time PCR were used to check the efficiency of RNAi. The results showed that the ORF095-specific siRNA-70 effectively down-regulated the expression of ORF095. When Vero cells were transfected with shRNA expression vectors (p61/GFP, p70/GFP, p165/GFP and p296/GFP) and then infected with GTPV, GTPV-ORF095-70 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by GTPV. The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of GTPV (approximately 463. 5-fold reduction of viral titers) on Vero cells.

Conclusions: This study demonstrates that vector-based shRNA methodology can effectively inhibit GTPV replication on Vero cells. Simultaneously, this work represents a strategy for controlling goatpox, potentially facilitating new experimental approaches in the analysis of both viral and cellular gene functions during of GTPV infection.

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Transient expression of siRNAs conferred the sequence-specific inhibition of expression of GTPV ORF95 in BKH-21 cells. (A) Fluorescence detection of cotransfection of p095/EGFP with their corresponding siRNA expression plasmids 24 h posttransfection. (B) Flow cytometric analysis of cotransfection of p095/EGFP with their corresponding siRNA expression plasmids 48 h posttransfection. EGFP expression level in cells cotransfected with (a) pEGFP-N1 vector; (b) p095/EGFP; (c) p095/EGFP and p61; (d) p095/EGFP and p70; (e) p095/EGFP and p165; (f) p095/EGFP and p296; (g) p095/EGFP and pControl; (h) BHK-21 cell control. LR-Value means the rate of EGFP positive cells.
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Figure 3: Transient expression of siRNAs conferred the sequence-specific inhibition of expression of GTPV ORF95 in BKH-21 cells. (A) Fluorescence detection of cotransfection of p095/EGFP with their corresponding siRNA expression plasmids 24 h posttransfection. (B) Flow cytometric analysis of cotransfection of p095/EGFP with their corresponding siRNA expression plasmids 48 h posttransfection. EGFP expression level in cells cotransfected with (a) pEGFP-N1 vector; (b) p095/EGFP; (c) p095/EGFP and p61; (d) p095/EGFP and p70; (e) p095/EGFP and p165; (f) p095/EGFP and p296; (g) p095/EGFP and pControl; (h) BHK-21 cell control. LR-Value means the rate of EGFP positive cells.

Mentions: Different siRNAs suppressed the expression of fusion green fluorescent protein in BHK-21 cells is different. The siRNAs targeting to the conserved region of GTPV genome were generated in vitro by human recombinant Dicer enzyme, as described in Figure 1B. To identify an effective inhibitory effect of siRNAs, the cDNA cassettes of these regions were inserted into the 5' end of enhanced green fluorescent protein (EGFP) gene to construct reporter plasmids (Figure 2). The reporter plasmids were used to cotransfect BHK-21 cells with either the homologous siRNAs or the heterologous siRNAs. The results showed that the number of EGFP-expressing cell was markedly reduced in the sample transfected with homologous siRNAs than sample transfected with heterologous siRNAs or non-transfected (Figure 3A). FACS demonstrated that the levels of inhibition mediated by the siRNAs were similar among the different experiment groups and significantly higher than the control group (cotransfection with heterologous siRNAs or without siRNAs).


RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells.

Zhao Z, Wu G, Zhu X, Yan X, Dou Y, Li J, Zhu H, Zhang Q, Cai X - Virol. J. (2012)

Transient expression of siRNAs conferred the sequence-specific inhibition of expression of GTPV ORF95 in BKH-21 cells. (A) Fluorescence detection of cotransfection of p095/EGFP with their corresponding siRNA expression plasmids 24 h posttransfection. (B) Flow cytometric analysis of cotransfection of p095/EGFP with their corresponding siRNA expression plasmids 48 h posttransfection. EGFP expression level in cells cotransfected with (a) pEGFP-N1 vector; (b) p095/EGFP; (c) p095/EGFP and p61; (d) p095/EGFP and p70; (e) p095/EGFP and p165; (f) p095/EGFP and p296; (g) p095/EGFP and pControl; (h) BHK-21 cell control. LR-Value means the rate of EGFP positive cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298800&req=5

Figure 3: Transient expression of siRNAs conferred the sequence-specific inhibition of expression of GTPV ORF95 in BKH-21 cells. (A) Fluorescence detection of cotransfection of p095/EGFP with their corresponding siRNA expression plasmids 24 h posttransfection. (B) Flow cytometric analysis of cotransfection of p095/EGFP with their corresponding siRNA expression plasmids 48 h posttransfection. EGFP expression level in cells cotransfected with (a) pEGFP-N1 vector; (b) p095/EGFP; (c) p095/EGFP and p61; (d) p095/EGFP and p70; (e) p095/EGFP and p165; (f) p095/EGFP and p296; (g) p095/EGFP and pControl; (h) BHK-21 cell control. LR-Value means the rate of EGFP positive cells.
Mentions: Different siRNAs suppressed the expression of fusion green fluorescent protein in BHK-21 cells is different. The siRNAs targeting to the conserved region of GTPV genome were generated in vitro by human recombinant Dicer enzyme, as described in Figure 1B. To identify an effective inhibitory effect of siRNAs, the cDNA cassettes of these regions were inserted into the 5' end of enhanced green fluorescent protein (EGFP) gene to construct reporter plasmids (Figure 2). The reporter plasmids were used to cotransfect BHK-21 cells with either the homologous siRNAs or the heterologous siRNAs. The results showed that the number of EGFP-expressing cell was markedly reduced in the sample transfected with homologous siRNAs than sample transfected with heterologous siRNAs or non-transfected (Figure 3A). FACS demonstrated that the levels of inhibition mediated by the siRNAs were similar among the different experiment groups and significantly higher than the control group (cotransfection with heterologous siRNAs or without siRNAs).

Bottom Line: The results showed that the ORF095-specific siRNA-70 effectively down-regulated the expression of ORF095.When Vero cells were transfected with shRNA expression vectors (p61/GFP, p70/GFP, p165/GFP and p296/GFP) and then infected with GTPV, GTPV-ORF095-70 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by GTPV.The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of GTPV (approximately 463. 5-fold reduction of viral titers) on Vero cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Animal virology of the Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences, Gansu, PR China.

ABSTRACT

Background: Goatpox is an economically important disease in goat and sheep-producing areas of the world. Many vaccine strategies developed to control the disease are not yet completely successful. Hairpin expression vectors have been used to induce gene silencing in a large number of studies on viruses. However, none of these studies has been attempted to study GTPV. In the interest of exploiting improved methods to control goat pox, it is participated that RNAi may provide effective protection against GTPV. In this study we show the suppression of Goatpox virus (GTPV) replication via knockdown of virion core protein using RNA interference.

Results: Four short interfering RNA (siRNA) sequences (siRNA-61, siRNA-70, siRNA-165 and siRNA-296) against a region of GTPV ORF095 were selected. Sense and antisense siRNA-encoding sequences separated by a hairpin loop sequence were designed as short hairpin RNA (shRNA) expression cassettes under the control of a human U6 promoter. ORF095 amplicon was generated using PCR, and then cloned into pEGFP-N1 vector, named as p095/EGFP. p095/EGFP and each of the siRNA expression cassettes (p61, p70, p165 and p296) were co-transfected into BHK-21 cells. Fluorescence detection, flow cytometric analysis, retro transcription PCR (RT-PCR) and real time PCR were used to check the efficiency of RNAi. The results showed that the ORF095-specific siRNA-70 effectively down-regulated the expression of ORF095. When Vero cells were transfected with shRNA expression vectors (p61/GFP, p70/GFP, p165/GFP and p296/GFP) and then infected with GTPV, GTPV-ORF095-70 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by GTPV. The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of GTPV (approximately 463. 5-fold reduction of viral titers) on Vero cells.

Conclusions: This study demonstrates that vector-based shRNA methodology can effectively inhibit GTPV replication on Vero cells. Simultaneously, this work represents a strategy for controlling goatpox, potentially facilitating new experimental approaches in the analysis of both viral and cellular gene functions during of GTPV infection.

Show MeSH
Related in: MedlinePlus