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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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Over-expressed Flag-tagged MADP1 partially colocalized with viral RTCs. Vero cells over-expressing either Flag-tagged MADP1 or empty vector were infected with IBV, treated with actinomycin D at 3 h post-infection, and transfected with bromo-UTP at 7 h post-infection. Cells were fixed at 10 h post-infection and permeabilized with Triton-X 100. Immunofluorescence was performed with antibodies to Flag and BrdU followed by secondary antibodies conjugated with Alexa Fluor 488 and 594, respectively. Vector-transfected Vero cells infected with IBV were used as negative controls. H122 cells transfected with Flag-tagged MADP1 and infected with IBV, as described for Vero cells.
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gks165-F2: Over-expressed Flag-tagged MADP1 partially colocalized with viral RTCs. Vero cells over-expressing either Flag-tagged MADP1 or empty vector were infected with IBV, treated with actinomycin D at 3 h post-infection, and transfected with bromo-UTP at 7 h post-infection. Cells were fixed at 10 h post-infection and permeabilized with Triton-X 100. Immunofluorescence was performed with antibodies to Flag and BrdU followed by secondary antibodies conjugated with Alexa Fluor 488 and 594, respectively. Vector-transfected Vero cells infected with IBV were used as negative controls. H122 cells transfected with Flag-tagged MADP1 and infected with IBV, as described for Vero cells.

Mentions: MADP1 was identified as a component of the 18 S U11/12 snRNP (37) and its subcellular localization was determined to be in the nucleoplasm (35). IBV replication and transcription, on the other hand, take place in the cytoplasm of the infected cells. Therefore, to validate the likelihood of MADP1 interacting with the viral 5′-UTR, immunofluorescence was used to track the subcellular localization of both Flag-tagged MADP1 and de novo synthesized viral RNA in both mock-infected and IBV-infected cells. Flag-tagged MADP1 was over-expressed in cultured Vero cells, which were then infected with IBV and treated with actinomycin D to inhibit host transcription. The newly synthesized viral RNA, a marker for the RTCs, was labeled with BrUTP. The cells were fixed at 10 h post-infection to allow sufficient labeling of the newly synthesized viral RNA and to minimize the formation of large syncytial cells. In uninfected cells, Flag-tagged MADP1 was localized in the nucleus exclusively (Figure 2). Upon infection by IBV, Flag-tagged MADP1 appeared to be present in the cytoplasm as well (Figure 2). Interestingly, the cytoplasmic localization pattern of Flag-tagged MADP1 appears to be partially overlapped with that for the RTCs, although further studies would be required to ascertain if MADP1 would be a part of the RTCs (Figure 2). As a negative control for the over-expressed protein, vector transfected cells probed with Flag antibody showed negative staining for the over-expressed protein (Figure 2). Similar colocalization patterns were also observed in IBV-infected H1299 cells (Figure 2).Figure 2.


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)

Over-expressed Flag-tagged MADP1 partially colocalized with viral RTCs. Vero cells over-expressing either Flag-tagged MADP1 or empty vector were infected with IBV, treated with actinomycin D at 3 h post-infection, and transfected with bromo-UTP at 7 h post-infection. Cells were fixed at 10 h post-infection and permeabilized with Triton-X 100. Immunofluorescence was performed with antibodies to Flag and BrdU followed by secondary antibodies conjugated with Alexa Fluor 488 and 594, respectively. Vector-transfected Vero cells infected with IBV were used as negative controls. H122 cells transfected with Flag-tagged MADP1 and infected with IBV, as described for Vero cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3367200&req=5

gks165-F2: Over-expressed Flag-tagged MADP1 partially colocalized with viral RTCs. Vero cells over-expressing either Flag-tagged MADP1 or empty vector were infected with IBV, treated with actinomycin D at 3 h post-infection, and transfected with bromo-UTP at 7 h post-infection. Cells were fixed at 10 h post-infection and permeabilized with Triton-X 100. Immunofluorescence was performed with antibodies to Flag and BrdU followed by secondary antibodies conjugated with Alexa Fluor 488 and 594, respectively. Vector-transfected Vero cells infected with IBV were used as negative controls. H122 cells transfected with Flag-tagged MADP1 and infected with IBV, as described for Vero cells.
Mentions: MADP1 was identified as a component of the 18 S U11/12 snRNP (37) and its subcellular localization was determined to be in the nucleoplasm (35). IBV replication and transcription, on the other hand, take place in the cytoplasm of the infected cells. Therefore, to validate the likelihood of MADP1 interacting with the viral 5′-UTR, immunofluorescence was used to track the subcellular localization of both Flag-tagged MADP1 and de novo synthesized viral RNA in both mock-infected and IBV-infected cells. Flag-tagged MADP1 was over-expressed in cultured Vero cells, which were then infected with IBV and treated with actinomycin D to inhibit host transcription. The newly synthesized viral RNA, a marker for the RTCs, was labeled with BrUTP. The cells were fixed at 10 h post-infection to allow sufficient labeling of the newly synthesized viral RNA and to minimize the formation of large syncytial cells. In uninfected cells, Flag-tagged MADP1 was localized in the nucleus exclusively (Figure 2). Upon infection by IBV, Flag-tagged MADP1 appeared to be present in the cytoplasm as well (Figure 2). Interestingly, the cytoplasmic localization pattern of Flag-tagged MADP1 appears to be partially overlapped with that for the RTCs, although further studies would be required to ascertain if MADP1 would be a part of the RTCs (Figure 2). As a negative control for the over-expressed protein, vector transfected cells probed with Flag antibody showed negative staining for the over-expressed protein (Figure 2). Similar colocalization patterns were also observed in IBV-infected H1299 cells (Figure 2).Figure 2.

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