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
. 2012 Oct 25;432(2):283-95.
doi: 10.1016/j.virol.2012.05.028. Epub 2012 Jun 18.

Adenoviral protein V promotes a process of viral assembly through nucleophosmin 1

Hideyo Ugai  1 , George C Dobbins, Minghui Wang, Long P Le, David A Matthews, David T Curiel
Affiliations

Adenoviral protein V promotes a process of viral assembly through nucleophosmin 1

Hideyo Ugai et al. Virology. .

Abstract

Adenoviral infection induces nucleoplasmic redistribution of a nucleolar nucleophosmin 1/NPM1/B23.1. NPM1 is preferentially localized in the nucleoli of normal cells, whereas it is also present at the nuclear matrix in cancer cells. However, the biological roles of NPM1 during infection are unknown. Here, by analyzing a pV-deletion mutant, Ad5-dV/TSB, we demonstrate that pV promotes the NPM1 translocation from the nucleoli to the nucleoplasm in normal cells, and the NPM1 translocation is correlated with adenoviral replication. Lack of pV causes a dramatic reduction of adenoviral replication in normal cells, but not cancer cells, and Ad5-dV/TSB was defective in viral assembly in normal cells. NPM1 knockdown inhibits adenoviral replication, suggesting an involvement of NPM1 in adenoviral biology. Further, we show that NPM1 interacts with empty adenovirus particles which are an intermediate during virion maturation by immunoelectron microscopy. Collectively, these data implicate that pV participates in a process of viral assembly through NPM1.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. Adenoviral protein V regulates subcellular localization of NPM1 during infection
HPAEC cells were infected with adenovirus at an MOI of 10 PFU/cell for various times, followed by immunofluorescent staining. Subcellular localization of adenoviral pV and endogenous NPM1 was observed in infected cells at various times post-infection. Viral and endogenous cellular proteins in infected cells were visualized by staining with anti-pV and anti-NPM1 followed by secondary antibody conjugated with fluorescent probes. Uninfected and infected cells were stained for nuclear DNA using Hoechst 33342 and fluorescent signals were visualized by epifluorescence microscopy.
Fig. 2
Fig. 2. Protein V is required for adenoviral replication in normal cells
(A–C) Primary cells were infected with adenovirus at an MOI of 10 PFU/cell for various times. (A) Total DNA was extracted from infected cells, and 50 ng of total DNA was analyzed by qPCR. Plots represent as the copy number of the E4 gene per 50 ng of total DNA. (B) One-step growth curve kinetics of adenoviruses was analyzed in primary cell lines. (C) Proteins were extracted from infected primary cells at various times post-infection and 40 µg of total proteins were subjected to Western blot with the indicated antibodies. (D) Primary cells were infected with adenovirus at an MOI of 10, 50 or 100 PFU/cell for 1 and 48 hours. Crude proteins were extracted from infected cells, and 10 µg of proteins was subjected to Western blot. Ctrl: 1.0 × 109 viral particles (VPs) of purified Ad5 were applied as a control for the pV expression. (E) Production level of infectious adenovirus in primary cell lines at 48 h.p.i. was measured by plaque assay. Results were reported as mean ± SD. N.S.; no significant difference
Fig. 3
Fig. 3. Lack of pV affects the E1B-55K and pIX expression
HPAEC cells were infected with adenovirus at an MOI of 10 PFU/cell for various times. (A and B) Proteins were extracted from infected primary cells at various times post-infection and 40 µg of total proteins were subjected to Western blot with the indicated antibodies. (C) Gene expression of E1b-55k, pIX, and hexon mRNAs. Gene expression levels of E1b-55k, pIX, and hexon mRNAs at various times post-infection were determined by qRT-PCR. The data are expressed as the mean values ± SD (n = 4). N.S.; no significant difference.
Fig. 4
Fig. 4. Protein V determines property of adenoviral replication in primary cells
HPAEC cells were infected with adenovirus at an MOI of 10 PFU/cell for various times. (A) An equivalent of 40 µg of total proteins was analyzed by Western blot with the indicated antibodies. (B) One-step growth curve kinetics of adenoviruses was analyzed in HPAEC cells. Infectious titer was determined by plaque assay.
Fig. 5
Fig. 5. Knockdown of NPM1 inhibits adenoviral replication
(A and B) HPAEC cells were transfected with siRNA for NPM1 mRNA (siNPM1) or a control non-targeting RNA (siNeg) for 48 hours, followed by qRT-PCR to determine knockdown level of NPM1 mRNA (A); Data was shown as mean ± S.E.M., * P<0.01, ** P<0.05; two-tailed unpaired Student’s t-tests between groups, and Western blot analysis (B). (C) HPAEC cells were transfected with 10 nM siRNA for 48 hours, followed by immunofluorescent staining. (D) One-step growth curve analysis of adenoviruses in siNPM1-treated primary cells. Primary cells transfected with siRNA for 48 hours were infected with adenovirus at an MOI of 10 PFU/cell for various times. Infectious titer was determined by plaque assay.
Fig. 6
Fig. 6. NPM1 is involved in empty adenovirus particles
(A) HPAEC and A549 cells were infected with adenovirus at an MOI of 10 PFU/cell, followed by cell harvesting at 72 h.p.i. Uninfected and infected cells were fixed in 30 mM HEPES-NaOH buffer (pH 7.4) containing 1.25% glutaraldehyde and 2% paraformaldehyde, embedded, and sectioned for transmission electron microscopy. (i – iv) White framed areas display enlarged areas. (B) Analysis of protein composition of viral particles by GELCODE blue staining. Infectious and empty virus particles (VPs) of Ad5 were CsCl-purified from the nuclei fraction of infected A549 cells. A total of 1.0 × 1010 VPs of purified infectious and empty VPs was separated by electrophoresis in 10% SDS-PAGE, and the gel was stained with GELCODE Blue Stain Reagent. (C) Validation of purified infectious and empty VPs by PCR analysis. The fiber gene was amplified using a template which is equivalent to 1.0 × 1010 VPs of purified VPs. (D) Detection of NPM1 in the fraction of the Ad5 empty VPs. Infectious and empty VPs were denatured and separated by electrophoresis in 10% SDS-PAGE, followed by Western blot analysis using indicated antibodies. An equivalent of 10 µg total proteins which were extracted from A549 cells was applied for a control to detect cellular proteins. (E) Immunoelectron microscopic analysis of NPM1 accessibility on empty VPs. To test the accessibility of NPM1 on empty VPs, infectious and empty VPs were bound on copper grids with a carbon-coated Formvar film. The NPM1 epitope was detected with indicated antibodies, followed by donkey anti-mouse immunoglobulin conjugated with 10-nm gold and donkey anti-rabbit immunoglobulin conjugated with 25-nm gold. Electron micrographs for the adenoviral particles were taken with JEOL 1200 EX II Transmission Electron Microscope.
Fig. 6
Fig. 6. NPM1 is involved in empty adenovirus particles
(A) HPAEC and A549 cells were infected with adenovirus at an MOI of 10 PFU/cell, followed by cell harvesting at 72 h.p.i. Uninfected and infected cells were fixed in 30 mM HEPES-NaOH buffer (pH 7.4) containing 1.25% glutaraldehyde and 2% paraformaldehyde, embedded, and sectioned for transmission electron microscopy. (i – iv) White framed areas display enlarged areas. (B) Analysis of protein composition of viral particles by GELCODE blue staining. Infectious and empty virus particles (VPs) of Ad5 were CsCl-purified from the nuclei fraction of infected A549 cells. A total of 1.0 × 1010 VPs of purified infectious and empty VPs was separated by electrophoresis in 10% SDS-PAGE, and the gel was stained with GELCODE Blue Stain Reagent. (C) Validation of purified infectious and empty VPs by PCR analysis. The fiber gene was amplified using a template which is equivalent to 1.0 × 1010 VPs of purified VPs. (D) Detection of NPM1 in the fraction of the Ad5 empty VPs. Infectious and empty VPs were denatured and separated by electrophoresis in 10% SDS-PAGE, followed by Western blot analysis using indicated antibodies. An equivalent of 10 µg total proteins which were extracted from A549 cells was applied for a control to detect cellular proteins. (E) Immunoelectron microscopic analysis of NPM1 accessibility on empty VPs. To test the accessibility of NPM1 on empty VPs, infectious and empty VPs were bound on copper grids with a carbon-coated Formvar film. The NPM1 epitope was detected with indicated antibodies, followed by donkey anti-mouse immunoglobulin conjugated with 10-nm gold and donkey anti-rabbit immunoglobulin conjugated with 25-nm gold. Electron micrographs for the adenoviral particles were taken with JEOL 1200 EX II Transmission Electron Microscope.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Anderson CW, Young ME, Flint SJ. Characterization of the adenovirus 2 virion protein, mu. Virology. 1989;172(2):506–512. - PubMed
    1. Andersson MG, Haasnoot PC, Xu N, Berenjian S, Berkhout B, Akusjarvi G. Suppression of RNA interference by adenovirus virus-associated RNA. J Virol. 2005;79(15):9556–9565. - PMC - PubMed
    1. Barker DD, Berk AJ. Adenovirus proteins from both E1B reading frames are required for transformation of rodent cells by viral infection and DNA transfection. Virology. 1987;156(1):107–121. - PubMed
    1. Bischoff JR, Kirn DH, Williams A, Heise C, Horn S, Muna M, Ng L, Nye JA, Sampson-Johannes A, Fattaey A, McCormick F. An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science. 1996;274(5286):373–376. - PubMed
    1. Bodnar JW, Hanson PI, Polvino-Bodnar M, Zempsky W, Ward DC. The terminal regions of adenovirus and minute virus of mice DNAs are preferentially associated with the nuclear matrix in infected cells. J Virol. 1989;63(10):4344–4353. - PMC - PubMed

Publication types