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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.


siRNA-knockdown of exogenous gene expression in MRC5 cells.(A) Fluorescence analysis shows inhibition of pAcGFP-N and pAcGFP plasmid expression. RVF N-specific si605N/si46N cotransfection (+) inhibited expression of GFP-N fusion protein in contrast to the untreated control (-); GFP-specific si475GFP cotransfection (+) inhibited expression of the green fluorescent protein. si689RL, the scrambled non-specific Renilla luciferase-siRNA, shows no inhibition of GFP-N protein expression. (B) Western blot analysis of siRNA knockdown of exogenous protein expression. Lane 1: expression of GFP (without siRNA treatment; arrow shows an estimated 27 kDa molecular weight protein); lane 2: knockdown of GFP expression with si475GFP; lane 3: knockdown of GFP-N fusion protein with si605N/si46N; lane 4: expression of GFP-N fusion protein (without siRNA treatment; arrow shows an estimated 54 kDa molecular weight protein); lane 5: cells only control. β-actin serves as loading control.
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Figure 1: siRNA-knockdown of exogenous gene expression in MRC5 cells.(A) Fluorescence analysis shows inhibition of pAcGFP-N and pAcGFP plasmid expression. RVF N-specific si605N/si46N cotransfection (+) inhibited expression of GFP-N fusion protein in contrast to the untreated control (-); GFP-specific si475GFP cotransfection (+) inhibited expression of the green fluorescent protein. si689RL, the scrambled non-specific Renilla luciferase-siRNA, shows no inhibition of GFP-N protein expression. (B) Western blot analysis of siRNA knockdown of exogenous protein expression. Lane 1: expression of GFP (without siRNA treatment; arrow shows an estimated 27 kDa molecular weight protein); lane 2: knockdown of GFP expression with si475GFP; lane 3: knockdown of GFP-N fusion protein with si605N/si46N; lane 4: expression of GFP-N fusion protein (without siRNA treatment; arrow shows an estimated 54 kDa molecular weight protein); lane 5: cells only control. β-actin serves as loading control.

Mentions: Knockdown of the expression of the RVFV N-GFP fusion protein encoded by the plasmid pAcGFP-N was performed in MRC-5 cells using the RVFV nucleoprotein-specific siRNAs si605N/si46N. Co-transfection of the pAcGFP-N plasmid with the siRNAs si605N/si46N resulted in inhibition of N-GFP fusion protein expression as demonstrated by the absence of specific GFP fluorescence (Figure 1A) and the fusion protein-specific band in Western blot analysis (Figure 1B). Treatment with the positive control siRNA, si475GFP, targeting the GFP gene, abrogated expression or fluorescence of the GFP-N fusion fusion protein (Figures 1A,B). In contrast, GFP fluorescence was observed in all mock-treated cells transfected with the recombinant plasmids pAcGFP-N or pAcGFP (Figure 1A). Treatment of the MRC5 cells with the scrambled negative control siRNA, si689RL, did not have any effect on fusion protein expression as demonstrated by clear expression of the N-GFP fusion protein (Figure 1A).


Short Interfering RNA Inhibits Rift Valley Fever Virus Replication and Degradation of Protein Kinase R in Human Cells
siRNA-knockdown of exogenous gene expression in MRC5 cells.(A) Fluorescence analysis shows inhibition of pAcGFP-N and pAcGFP plasmid expression. RVF N-specific si605N/si46N cotransfection (+) inhibited expression of GFP-N fusion protein in contrast to the untreated control (-); GFP-specific si475GFP cotransfection (+) inhibited expression of the green fluorescent protein. si689RL, the scrambled non-specific Renilla luciferase-siRNA, shows no inhibition of GFP-N protein expression. (B) Western blot analysis of siRNA knockdown of exogenous protein expression. Lane 1: expression of GFP (without siRNA treatment; arrow shows an estimated 27 kDa molecular weight protein); lane 2: knockdown of GFP expression with si475GFP; lane 3: knockdown of GFP-N fusion protein with si605N/si46N; lane 4: expression of GFP-N fusion protein (without siRNA treatment; arrow shows an estimated 54 kDa molecular weight protein); lane 5: cells only control. β-actin serves as loading control.
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Figure 1: siRNA-knockdown of exogenous gene expression in MRC5 cells.(A) Fluorescence analysis shows inhibition of pAcGFP-N and pAcGFP plasmid expression. RVF N-specific si605N/si46N cotransfection (+) inhibited expression of GFP-N fusion protein in contrast to the untreated control (-); GFP-specific si475GFP cotransfection (+) inhibited expression of the green fluorescent protein. si689RL, the scrambled non-specific Renilla luciferase-siRNA, shows no inhibition of GFP-N protein expression. (B) Western blot analysis of siRNA knockdown of exogenous protein expression. Lane 1: expression of GFP (without siRNA treatment; arrow shows an estimated 27 kDa molecular weight protein); lane 2: knockdown of GFP expression with si475GFP; lane 3: knockdown of GFP-N fusion protein with si605N/si46N; lane 4: expression of GFP-N fusion protein (without siRNA treatment; arrow shows an estimated 54 kDa molecular weight protein); lane 5: cells only control. β-actin serves as loading control.
Mentions: Knockdown of the expression of the RVFV N-GFP fusion protein encoded by the plasmid pAcGFP-N was performed in MRC-5 cells using the RVFV nucleoprotein-specific siRNAs si605N/si46N. Co-transfection of the pAcGFP-N plasmid with the siRNAs si605N/si46N resulted in inhibition of N-GFP fusion protein expression as demonstrated by the absence of specific GFP fluorescence (Figure 1A) and the fusion protein-specific band in Western blot analysis (Figure 1B). Treatment with the positive control siRNA, si475GFP, targeting the GFP gene, abrogated expression or fluorescence of the GFP-N fusion fusion protein (Figures 1A,B). In contrast, GFP fluorescence was observed in all mock-treated cells transfected with the recombinant plasmids pAcGFP-N or pAcGFP (Figure 1A). Treatment of the MRC5 cells with the scrambled negative control siRNA, si689RL, did not have any effect on fusion protein expression as demonstrated by clear expression of the N-GFP fusion protein (Figure 1A).

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.