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Binding of the 5'-untranslated region of coronavirus RNA to zinc finger CCHC-type and RNA-binding motif 1 enhances viral replication and transcription.

Tan YW, Hong W, Liu DX - Nucleic Acids Res. (2012)

Bottom Line: This interaction was subsequently confirmed in coronavirus infectious bronchitis virus (IBV).MADP1 was found to be translocated to the cytoplasm and partially co-localized with the viral replicase/transcriptase complexes (RTCs) in IBV-infected cells, deviating from its usual nuclear localization in a normal cell using indirect immunofluorescence.Using small interfering RNA (siRNA) against MADP1, defective viral RNA synthesis was observed in the knockdown cells, therefore indicating the importance of the protein in coronaviral RNA synthesis.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.

ABSTRACT
Coronaviruses RNA synthesis occurs in the cytoplasm and is regulated by host cell proteins. In a screen based on a yeast three-hybrid system using the 5'-untranslated region (5'-UTR) of SARS coronavirus (SARS-CoV) RNA as bait against a human cDNA library derived from HeLa cells, we found a positive candidate cellular protein, zinc finger CCHC-type and RNA-binding motif 1 (MADP1), to be able to interact with this region of the SARS-CoV genome. This interaction was subsequently confirmed in coronavirus infectious bronchitis virus (IBV). The specificity of the interaction between MADP1 and the 5'-UTR of IBV was investigated and confirmed by using an RNA pull-down assay. The RNA-binding domain was mapped to the N-terminal region of MADP1 and the protein binding sequence to stem-loop I of IBV 5'-UTR. MADP1 was found to be translocated to the cytoplasm and partially co-localized with the viral replicase/transcriptase complexes (RTCs) in IBV-infected cells, deviating from its usual nuclear localization in a normal cell using indirect immunofluorescence. Using small interfering RNA (siRNA) against MADP1, defective viral RNA synthesis was observed in the knockdown cells, therefore indicating the importance of the protein in coronaviral RNA synthesis.

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Related in: MedlinePlus

Two hundred and fifty pico moles (250 pmol) of either siRNA to EGFP or siRNA pools against Madp1 were transfected into H1299 cells twice and infected with recombinant luciferase-IBV at 72 h after the first transfection. (A) Volumes (in microliters) of each 50 -μM siRNA used in the siRNA pools. (B) Luciferase activity of the infected cells measured at 20 h post-infection showed a decrease in viral activity after silencing Madp1 with the different siRNA pools.
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gks165-F6: Two hundred and fifty pico moles (250 pmol) of either siRNA to EGFP or siRNA pools against Madp1 were transfected into H1299 cells twice and infected with recombinant luciferase-IBV at 72 h after the first transfection. (A) Volumes (in microliters) of each 50 -μM siRNA used in the siRNA pools. (B) Luciferase activity of the infected cells measured at 20 h post-infection showed a decrease in viral activity after silencing Madp1 with the different siRNA pools.

Mentions: To eliminate the possibility that the phenotype observed in MADP1-silenced cells during IBV infection was due to an off-target effect of the siRNA duplex used, four additional siRNA duplexes targeting different regions of MADP1 were used in various combinations with siMadp1 (Figure 6) to check their effect on IBV infection, as illustrated by the expression of the luciferase gene (Figure 6B). All six combinations of five different siRNA duplexes resulted in a reduction in the luciferase activity of the infected cells by either 70% (siCombi 3 and 4), without siMadp1 or more than 90% (siCombi 1, 2, 5 and 6) with siMadp1, compared to negative control, siEGFP-transfected cells (Figure 6B). This implies that, in general, knocking down MADP1 with any siRNA results in a reduction of virus infection.Figure 6.


Binding of the 5'-untranslated region of coronavirus RNA to zinc finger CCHC-type and RNA-binding motif 1 enhances viral replication and transcription.

Tan YW, Hong W, Liu DX - Nucleic Acids Res. (2012)

Two hundred and fifty pico moles (250 pmol) of either siRNA to EGFP or siRNA pools against Madp1 were transfected into H1299 cells twice and infected with recombinant luciferase-IBV at 72 h after the first transfection. (A) Volumes (in microliters) of each 50 -μM siRNA used in the siRNA pools. (B) Luciferase activity of the infected cells measured at 20 h post-infection showed a decrease in viral activity after silencing Madp1 with the different siRNA pools.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367200&req=5

gks165-F6: Two hundred and fifty pico moles (250 pmol) of either siRNA to EGFP or siRNA pools against Madp1 were transfected into H1299 cells twice and infected with recombinant luciferase-IBV at 72 h after the first transfection. (A) Volumes (in microliters) of each 50 -μM siRNA used in the siRNA pools. (B) Luciferase activity of the infected cells measured at 20 h post-infection showed a decrease in viral activity after silencing Madp1 with the different siRNA pools.
Mentions: To eliminate the possibility that the phenotype observed in MADP1-silenced cells during IBV infection was due to an off-target effect of the siRNA duplex used, four additional siRNA duplexes targeting different regions of MADP1 were used in various combinations with siMadp1 (Figure 6) to check their effect on IBV infection, as illustrated by the expression of the luciferase gene (Figure 6B). All six combinations of five different siRNA duplexes resulted in a reduction in the luciferase activity of the infected cells by either 70% (siCombi 3 and 4), without siMadp1 or more than 90% (siCombi 1, 2, 5 and 6) with siMadp1, compared to negative control, siEGFP-transfected cells (Figure 6B). This implies that, in general, knocking down MADP1 with any siRNA results in a reduction of virus infection.Figure 6.

Bottom Line: This interaction was subsequently confirmed in coronavirus infectious bronchitis virus (IBV).MADP1 was found to be translocated to the cytoplasm and partially co-localized with the viral replicase/transcriptase complexes (RTCs) in IBV-infected cells, deviating from its usual nuclear localization in a normal cell using indirect immunofluorescence.Using small interfering RNA (siRNA) against MADP1, defective viral RNA synthesis was observed in the knockdown cells, therefore indicating the importance of the protein in coronaviral RNA synthesis.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.

ABSTRACT
Coronaviruses RNA synthesis occurs in the cytoplasm and is regulated by host cell proteins. In a screen based on a yeast three-hybrid system using the 5'-untranslated region (5'-UTR) of SARS coronavirus (SARS-CoV) RNA as bait against a human cDNA library derived from HeLa cells, we found a positive candidate cellular protein, zinc finger CCHC-type and RNA-binding motif 1 (MADP1), to be able to interact with this region of the SARS-CoV genome. This interaction was subsequently confirmed in coronavirus infectious bronchitis virus (IBV). The specificity of the interaction between MADP1 and the 5'-UTR of IBV was investigated and confirmed by using an RNA pull-down assay. The RNA-binding domain was mapped to the N-terminal region of MADP1 and the protein binding sequence to stem-loop I of IBV 5'-UTR. MADP1 was found to be translocated to the cytoplasm and partially co-localized with the viral replicase/transcriptase complexes (RTCs) in IBV-infected cells, deviating from its usual nuclear localization in a normal cell using indirect immunofluorescence. Using small interfering RNA (siRNA) against MADP1, defective viral RNA synthesis was observed in the knockdown cells, therefore indicating the importance of the protein in coronaviral RNA synthesis.

Show MeSH
Related in: MedlinePlus