Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1998 Aug;72(8):6699-709.
doi: 10.1128/JVI.72.8.6699-6709.1998.

Viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells

S Waggoner  1 , P Sarnow
Affiliations

Viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells

S Waggoner et al. J Virol. 1998 Aug.

Abstract

The poliovirus 3' noncoding region (3'NCR) is involved in the efficient synthesis of viral negative-stranded RNA molecules. A strong interaction between a 105-kDa host protein and the wild-type 3'NCR, but not with a replication-defective mutant 3'NCR, was detected. This 105-kDa protein was identified as nucleolin which predominantly resides in the nucleolus and has been proposed to function in the folding of rRNA precursor molecules. A functional role for nucleolin in viral genome amplification was examined in a cell-free extract which has been shown to support the assembly of infectious virus from virion RNA. At early times of viral gene expression, extracts depleted of nucleolin produced less infectious virus than extracts depleted of fibrillarin, another resident of the nucleolus, indicating a functional role of nucleolin in the early stages of the viral life cycle in this in vitro system. Immunofluorescence analysis of uninfected and infected cells showed a nucleocytoplasmic relocalization of nucleolin, but not of fibrillarin, in poliovirus-infected cells. Relocalization of nucleolin was not simply a consequence of virally induced inhibition of translation or transcription, because inhibitors of translation or transcription did not induce nucleolar-cytoplasmic relocalization of nucleolin. These findings suggest a novel virus-induced mechanism by which certain nucleolar proteins are selectively redistributed in infected cells.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Viral RNA synthesis in cells infected with polioviruses containing various 3′NCR mutations. [3H]uridine incorporation in cells infected with the wild type (⊡), mutant 3NC202 (⧫), revertant R5 (▪), and revertant R9 (◊) is shown at the indicated times postinfection.
FIG. 2
FIG. 2
Nucleotide sequences of wild-type and mutant 3′NCRs. Shown are the sequences from nt 7373 to the 3′ terminal polyadenosine sequences of wild-type, mutant (3NC202), and revertant (R5 and R9) viral RNA genomes. The translation termination codons are underlined. The mutant 3NC202 has an 8-nt insertion, and the revertants R5 and R9 have nucleotide changes relative to the wild-type poliovirus indicated in bold and overlined.
FIG. 3
FIG. 3
Interactions of proteins with the poliovirus 3′NCR RNA. UV cross-linking of cellular proteins to the wild-type 3′NCR was performed as described in Materials and Methods. Lanes 1 and 2 contain samples of the radiolabeled RNA before and after treatment with RNase A, respectively. Lanes 3 and 4 contain nuclease-treated, cross-linked reaction mixtures without and with treatment with proteinase K, respectively. Numbers on the right indicate the migration of marker proteins of known molecular weights (in thousands). The autoradiograph was scanned using a Microtek ScanMaker E6, and the resulting image was labeled using Adobe Photoshop version 3.0.
FIG. 4
FIG. 4
(A) Formation of wild-type-105-kDa complexes in the presence of wild-type, mutant, or revertant 3′NCR-containing competitor RNAs. UV cross-linking was performed as described in Materials and Methods, except that unlabeled wild-type (WT), mutant (3NC202), or revertant (R5, R9) RNAs were preincubated with the HeLa extract prior to the addition of radiolabeled wild-type RNA (12 nM). The triangles at the top represent the molar excess (50-, 100-, and 875-fold) of each unlabeled RNA over the radiolabeled RNA. The arrow identifies the 105-kDa RNA-binding protein. (B) Quantitation of the formation of 105-kDa protein-RNA complexes in the presence of competitor RNAs. The amount of UV-cross-linked complexes formed was set to 100% in the absence of competitor RNA. The x axis indicates the concentrations of wild-type (▪), mutant (▴), or revertant (R5, ○; R9, ◊) RNAs relative to that of radiolabeled wild-type RNA (12 nM). (C) Formation of wild-type-, mutant-, and revertant-105-kDa protein complexes. UV cross-linking was performed as described above with labeled wild-type (WT), mutant (3NC202), or revertant (R5, R9) RNAs. The arrow denotes the migration of the 105-kDa RNA binding activity. The autoradiographs in panels A and C were scanned using a Microtek ScanMaker E6, and the resulting images were labeled using Adobe Photoshop version 3.0.
FIG. 4
FIG. 4
(A) Formation of wild-type-105-kDa complexes in the presence of wild-type, mutant, or revertant 3′NCR-containing competitor RNAs. UV cross-linking was performed as described in Materials and Methods, except that unlabeled wild-type (WT), mutant (3NC202), or revertant (R5, R9) RNAs were preincubated with the HeLa extract prior to the addition of radiolabeled wild-type RNA (12 nM). The triangles at the top represent the molar excess (50-, 100-, and 875-fold) of each unlabeled RNA over the radiolabeled RNA. The arrow identifies the 105-kDa RNA-binding protein. (B) Quantitation of the formation of 105-kDa protein-RNA complexes in the presence of competitor RNAs. The amount of UV-cross-linked complexes formed was set to 100% in the absence of competitor RNA. The x axis indicates the concentrations of wild-type (▪), mutant (▴), or revertant (R5, ○; R9, ◊) RNAs relative to that of radiolabeled wild-type RNA (12 nM). (C) Formation of wild-type-, mutant-, and revertant-105-kDa protein complexes. UV cross-linking was performed as described above with labeled wild-type (WT), mutant (3NC202), or revertant (R5, R9) RNAs. The arrow denotes the migration of the 105-kDa RNA binding activity. The autoradiographs in panels A and C were scanned using a Microtek ScanMaker E6, and the resulting images were labeled using Adobe Photoshop version 3.0.
FIG. 4
FIG. 4
(A) Formation of wild-type-105-kDa complexes in the presence of wild-type, mutant, or revertant 3′NCR-containing competitor RNAs. UV cross-linking was performed as described in Materials and Methods, except that unlabeled wild-type (WT), mutant (3NC202), or revertant (R5, R9) RNAs were preincubated with the HeLa extract prior to the addition of radiolabeled wild-type RNA (12 nM). The triangles at the top represent the molar excess (50-, 100-, and 875-fold) of each unlabeled RNA over the radiolabeled RNA. The arrow identifies the 105-kDa RNA-binding protein. (B) Quantitation of the formation of 105-kDa protein-RNA complexes in the presence of competitor RNAs. The amount of UV-cross-linked complexes formed was set to 100% in the absence of competitor RNA. The x axis indicates the concentrations of wild-type (▪), mutant (▴), or revertant (R5, ○; R9, ◊) RNAs relative to that of radiolabeled wild-type RNA (12 nM). (C) Formation of wild-type-, mutant-, and revertant-105-kDa protein complexes. UV cross-linking was performed as described above with labeled wild-type (WT), mutant (3NC202), or revertant (R5, R9) RNAs. The arrow denotes the migration of the 105-kDa RNA binding activity. The autoradiographs in panels A and C were scanned using a Microtek ScanMaker E6, and the resulting images were labeled using Adobe Photoshop version 3.0.
FIG. 5
FIG. 5
Purification of the 105-kDa protein using affinity chromatography with viral 3′NCR RNA. A silver-stained SDS-polyacrylamide gel which displays various fractions of 105-kDa protein-containing extracts after RNA affinity chromatography is shown. Lanes: M, molecular weight markers; I, input fraction; FT, flowthrough fraction; W, wash fraction. Fractions after elution with 0.3, 0.5, and 1.0 M KCl are shown. The arrow identifies the 105-kDa protein that was prepared for microsequencing. The migration of marker proteins of known molecular weights (in thousands) is indicated at the left. Note that the 1 M eluate was erroneously loaded prior to the 0.5 M eluate. The autoradiograph was scanned using a Microtek ScanMaker E6, and the resulting image was labeled using Adobe Photoshop version 3.0.
FIG. 6
FIG. 6
Immunoprecipitation of cross-linked RNA-protein complexes with antinucleolin and anti-2A antibodies. The 0.5 M KCl fraction obtained after 3′NCR-RNA affinity chromatography was cross-linked to the wild-type poliovirus 3′NCR, and immunoprecipitation experiments were performed. Lane I, cross-linked RNA-protein complex before immunoprecipitation. The supernatant (S) and pellet (P) fractions following immunoprecipitation with antinucleolin (N) or anti-2A (2A) antibodies are displayed. The arrow indicates the migration of the 105-kDa protein-RNA. The migration of a marker protein of known molecular weight (in thousands) is indicated at the right. The autoradiograph was scanned using a Microtek ScanMaker E6, and the resulting image was labeled using Adobe Photoshop version 3.0.
FIG. 7
FIG. 7
Binding of purified nucleolin to the viral 3′NCR. Cross-linking was performed as described in Materials and Methods, except that various amounts of unlabeled wild-type 3′NCR was preincubated with purified nucleolin prior to the addition of radiolabeled wild-type RNA. Lanes 1, 2, and 3 contain 0, 10-, and 100-fold molar excess, respectively, of the unlabeled RNA over the radiolabeled RNA (12 nM). Lane 4 displays the pattern when the 0.5 M KCl fraction, obtained after 3′NCR RNA affinity chromatography, was cross-linked to the wild-type 3′NCR and displayed in the same gel. The arrow indicates the 105-kDa RNA binding activity. The autoradiograph was scanned using a Microtek ScanMaker E6, and the resulting image was labeled using Adobe Photoshop version 3.0.
FIG. 8
FIG. 8
Immunodepletion of HeLa S10 cell extracts. Extracts (lane 1) were subjected to four successive rounds of immunodepletion using monoclonal antibodies directed against fibrillarin (lane 2) or monoclonal antibodies directed against nucleolin (lane 3). The amount of nucleolin was visualized by Western blotting using an antibody directed against nucleolin, as described in Materials and Methods. The autoradiograph was scanned using a Microtek ScanMaker E6, and the resulting image was labeled using Adobe Photoshop version 3.0.
FIG. 9
FIG. 9
(A) Immunolocalization of nucleolin (Nuc), Sam68 (Sam), and TATA-binding protein (TBP) during poliovirus infection. Cells were stained with anti-nucleolin (Nuc), anti-Sam68 (Sam), or anti-TBP (TBP) at 0, 1.5, 3, and 4.5 h after infection with wild-type poliovirus. (B) Immunolocalization of fibrillarin during wild-type poliovirus infection. Cells were stained with anti-fibrillarin antibodies at 0 (Fib0) and 4.5 (Fib4.5) h after infection with wild-type poliovirus. Color slides were scanned using a Nikon 35-mm film scanner LS-1000, and the resulting image was labeled using Adobe Photoshop version 3.0.
FIG. 9
FIG. 9
(A) Immunolocalization of nucleolin (Nuc), Sam68 (Sam), and TATA-binding protein (TBP) during poliovirus infection. Cells were stained with anti-nucleolin (Nuc), anti-Sam68 (Sam), or anti-TBP (TBP) at 0, 1.5, 3, and 4.5 h after infection with wild-type poliovirus. (B) Immunolocalization of fibrillarin during wild-type poliovirus infection. Cells were stained with anti-fibrillarin antibodies at 0 (Fib0) and 4.5 (Fib4.5) h after infection with wild-type poliovirus. Color slides were scanned using a Nikon 35-mm film scanner LS-1000, and the resulting image was labeled using Adobe Photoshop version 3.0.
FIG. 10
FIG. 10
Immunolocalization of nucleolin in cycloheximide-treated cells. Cells were stained with monoclonal antibodies directed against nucleolin after 0 (Chx0) or 4.5 (Chx4.5) h of incubation with cycloheximide (20 μg/ml). Color slides were scanned using a Nikon 35-mm film scanner LS-1000, and the resulting image was labeled using Adobe Photoshop version 3.0.
FIG. 11
FIG. 11
Immunolocalization of nucleolin in uninfected (ActD; top panels) and poliovirus-infected (ActD-PV; bottom panels) cells which were treated with actinomycin D (5 μg/ml). Cells were stained with monoclonal antibodies directed against nucleolin after 0, 1.5, 3, or 4.5 h, as indicated. Color slides were scanned using a Nikon 35-mm film scanner LS-1000, and the resulting image was labeled using Adobe Photoshop version 3.0.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Andino R, Rieckhof G E, Achacosco P L, Baltimore D. Poliovirus RNA synthesis utilizes an RNP complex formed around the 5-end of viral RNA. EMBO J. 1993;12:3587–3598. - PMC - PubMed
    1. Andino R, Rieckhof G E, Baltimore D. A functional ribonucleoprotein complex forms around the 5′ end of poliovirus RNA. Cell. 1990;63:369–380. - PubMed
    1. Aris J P, Blobel G. cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera. Proc Natl Acad Sci USA. 1991;88:931–935. - PMC - PubMed
    1. Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K. Current protocols in molecular biology. New York, N.Y: Greene Publishing Associates, Inc., John Wiley and Sons, Inc.; 1989.
    1. Banerjee R, Echeverri A, Dasgupta A. Poliovirus-encoded 2C polypeptide specifically binds to the 3′-terminal sequences of viral negative-strand RNA. J Virol. 1997;71:9570–9578. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

-