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Short Interfering RNA Inhibits Rift Valley Fever Virus Replication and Degradation of Protein Kinase R in Human Cells

View Article: PubMed Central - PubMed

ABSTRACT

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen causing severe outbreaks in humans and livestock in sub-Saharan Africa and the Arabian Peninsula. Human infections are characterized by fever, sometimes leading to encephalitis, retinitis, hemorrhagic fever, and occasionally death. There are currently no fully licensed vaccines or effective therapies for human use. Gene silencing mediated by double-stranded short interfering RNA (siRNA) is a sequence-specific, highly conserved mechanism in eukaryotes, which serves as an antiviral defense mechanism. Here, we demonstrate that siRNA duplexes directed against the RVFV nucleoprotein can effectively inhibit RVFV replication in human (MRC5 cells) and African green monkey cells (Vero E6 cells). Using these cells, we demonstrate that individual or complex siRNAs, targeting the RVFV nucleoprotein gene completely abrogate viral protein expression and prevent degradation of the host innate antiviral factor, protein kinase R (PKR). Importantly, pre-treatment of cells with the nucleoprotein-specific siRNAs markedly reduces the virus titer. The antiviral effect of the siRNAs was not attributable to interferon or the interferon response effector molecule, PKR. Thus, the antiviral activity of RVFV nucleoprotein-specific siRNAs may provide novel therapeutic strategy against RVFV infections in animals and humans.

No MeSH data available.


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qRT-PCR analysis of RVFV replication in response to RVFV-specific siRNA treatment.(A)Ct values of viral RNA amplification using supernatants from the various treatments; the Ct values of the siRNA treatments differed significantly from the untreated virus control (P < 0.05). (B) Illustrates log10 fold reduction in viral titers relative to the untreated virus control. si605N and si46N treatments show the highest fold-reductions. Asterisks (∗∗) above horizontal bars compare differences between treatments and denote significant differences. Fold reductions in viral titers of si605N or si46N were not different from siPooledN viral titers (P > 0.05).
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Figure 4: qRT-PCR analysis of RVFV replication in response to RVFV-specific siRNA treatment.(A)Ct values of viral RNA amplification using supernatants from the various treatments; the Ct values of the siRNA treatments differed significantly from the untreated virus control (P < 0.05). (B) Illustrates log10 fold reduction in viral titers relative to the untreated virus control. si605N and si46N treatments show the highest fold-reductions. Asterisks (∗∗) above horizontal bars compare differences between treatments and denote significant differences. Fold reductions in viral titers of si605N or si46N were not different from siPooledN viral titers (P > 0.05).

Mentions: As shown above, RVFV N-specific siRNAs inhibit viral nucleoprotein expression and prevent degradation host encoded PKR. Here, their potential to inhibit viral replication was assessed using qRT-PCR, flow cytometry and virus plaque assays. qRT-PCR cycle threshold (Ct) values obtained in cells infected with MP12 and treated with RVFV N-specific siRNAs were significantly higher when compared to the untreated control cells (P < 0.05) (Figure 4A). The amount of viral RNA produced after treatment with N-specific siRNAs, individually or as pooled complex, were at least 3 logs lower when compared to the untreated MP12-infected control (P < 0.05). Specifically, si605 and si46 displayed the highest fold reductions in virus replication of log10 3.74 and 3.63, respectively (Figure 4B). Flow cytometry analysis confirmed these results. Only background fluorescence was detectable in all N-specific siRNA treatments indicating effective inhibition of MP12 replication. In contrast, a strong positive fluorescence was detected in the untreated virus-infected control cells (Figure 5). Quantitation by plaque assay confirmed the above results. Production of viral progeny was significantly inhibited by the N-specific siRNA treatments when compared to the untreated infected control (P < 0.05) (Figure 6A). Meanwhile, si605N, si46N, and siPooledN exhibited stronger inhibition than si252N and si476N (Figure 6A). The magnitude of inhibition of virus replication was demonstrated by the absence of viral plaques in a 104 dilution of culture supernatants treated with N-specific siRNAs (Figure 6B). In contrast, viral plaques were easily detected in the untreated infected control (Figure 6B).


Short Interfering RNA Inhibits Rift Valley Fever Virus Replication and Degradation of Protein Kinase R in Human Cells
qRT-PCR analysis of RVFV replication in response to RVFV-specific siRNA treatment.(A)Ct values of viral RNA amplification using supernatants from the various treatments; the Ct values of the siRNA treatments differed significantly from the untreated virus control (P < 0.05). (B) Illustrates log10 fold reduction in viral titers relative to the untreated virus control. si605N and si46N treatments show the highest fold-reductions. Asterisks (∗∗) above horizontal bars compare differences between treatments and denote significant differences. Fold reductions in viral titers of si605N or si46N were not different from siPooledN viral titers (P > 0.05).
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Figure 4: qRT-PCR analysis of RVFV replication in response to RVFV-specific siRNA treatment.(A)Ct values of viral RNA amplification using supernatants from the various treatments; the Ct values of the siRNA treatments differed significantly from the untreated virus control (P < 0.05). (B) Illustrates log10 fold reduction in viral titers relative to the untreated virus control. si605N and si46N treatments show the highest fold-reductions. Asterisks (∗∗) above horizontal bars compare differences between treatments and denote significant differences. Fold reductions in viral titers of si605N or si46N were not different from siPooledN viral titers (P > 0.05).
Mentions: As shown above, RVFV N-specific siRNAs inhibit viral nucleoprotein expression and prevent degradation host encoded PKR. Here, their potential to inhibit viral replication was assessed using qRT-PCR, flow cytometry and virus plaque assays. qRT-PCR cycle threshold (Ct) values obtained in cells infected with MP12 and treated with RVFV N-specific siRNAs were significantly higher when compared to the untreated control cells (P < 0.05) (Figure 4A). The amount of viral RNA produced after treatment with N-specific siRNAs, individually or as pooled complex, were at least 3 logs lower when compared to the untreated MP12-infected control (P < 0.05). Specifically, si605 and si46 displayed the highest fold reductions in virus replication of log10 3.74 and 3.63, respectively (Figure 4B). Flow cytometry analysis confirmed these results. Only background fluorescence was detectable in all N-specific siRNA treatments indicating effective inhibition of MP12 replication. In contrast, a strong positive fluorescence was detected in the untreated virus-infected control cells (Figure 5). Quantitation by plaque assay confirmed the above results. Production of viral progeny was significantly inhibited by the N-specific siRNA treatments when compared to the untreated infected control (P < 0.05) (Figure 6A). Meanwhile, si605N, si46N, and siPooledN exhibited stronger inhibition than si252N and si476N (Figure 6A). The magnitude of inhibition of virus replication was demonstrated by the absence of viral plaques in a 104 dilution of culture supernatants treated with N-specific siRNAs (Figure 6B). In contrast, viral plaques were easily detected in the untreated infected control (Figure 6B).

View Article: PubMed Central - PubMed

ABSTRACT

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen causing severe outbreaks in humans and livestock in sub-Saharan Africa and the Arabian Peninsula. Human infections are characterized by fever, sometimes leading to encephalitis, retinitis, hemorrhagic fever, and occasionally death. There are currently no fully licensed vaccines or effective therapies for human use. Gene silencing mediated by double-stranded short interfering RNA (siRNA) is a sequence-specific, highly conserved mechanism in eukaryotes, which serves as an antiviral defense mechanism. Here, we demonstrate that siRNA duplexes directed against the RVFV nucleoprotein can effectively inhibit RVFV replication in human (MRC5 cells) and African green monkey cells (Vero E6 cells). Using these cells, we demonstrate that individual or complex siRNAs, targeting the RVFV nucleoprotein gene completely abrogate viral protein expression and prevent degradation of the host innate antiviral factor, protein kinase R (PKR). Importantly, pre-treatment of cells with the nucleoprotein-specific siRNAs markedly reduces the virus titer. The antiviral effect of the siRNAs was not attributable to interferon or the interferon response effector molecule, PKR. Thus, the antiviral activity of RVFV nucleoprotein-specific siRNAs may provide novel therapeutic strategy against RVFV infections in animals and humans.

No MeSH data available.


Related in: MedlinePlus