Adenovirus core protein VII contains distinct sequences that mediate targeting to the nucleus and nucleolus, and colocalization with human chromosomes

Lee, Tim W. R.; Blair, G. Eric; Matthews, David A.

doi:10.1099/vir.0.19546-0

Cited by 51 publications

(60 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The strong DNA binding of protein VII and the timing of its nuclear import after infection make it a good candidate for facilitating the nuclear import of the adenovirus genome (5,16,30,47). Recent studies by Lee et al (30) have shown that green fluorescent protein (GFP) fusions containing protein VII or fragments thereof accumulate in the nucleus in transfected cells.…”

mentioning

confidence: 99%

Adenovirus Core Protein pVII Is Translocated into the Nucleus by Multiple Import Receptor Pathways

Wodrich

Cassany

D’Angelo

et al. 2006

J Virol

102

View full text Add to dashboard Cite

Adenoviruses are nonenveloped viruses with an ϳ36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin ␣, importin ␤, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome.Adenoviruses (Ads) are nonenveloped, double-stranded DNA viruses with a diameter of ϳ90 nm. They contain an outer capsid shell with icosahedral symmetry composed of 12 vertices and 20 facets surrounding the viral core with the genomic DNA. Extending from each of the 12 vertices of the capsid is the spike-like fiber protein, which is anchored to the vertices by the penton protein. Ad infection of cells is initiated by attachment of the fiber to the Coxsackie adenovirus receptors of cells, followed by association of the penton with ␣V integrins. This secondary penton interaction is required for fiber release and viral uptake into clathrin-coated pits of the early endosomal pathway (10, 43, 51; reviewed in references 9 and 34). In the early endosome, a poorly understood mechanism involving a drop in pH induces conformational changes in the capsid, which appears to release proteins of the vertex region, including protein VI and penton (17). An amphipathic helix at the N terminus of protein VI is thought to mediate disruption of the endosomal membrane and aid in the release of the remaining partially uncoated capsid into the cytoplasm (52). The nucleocapsid then moves toward the nucleus in a microtubule-and dynein-dependent mechanism (26) and docks at the nuclear pore complex (NPC), the proteinaceous channel that mediates transport across the nuclear envelope. Finally, the viral genome is imported into the nucleus prior to initiation of viral replication (16,44).In contrast to the Ad capsid, the core does not display a well-ordered symmetry or the coaxial coiling of DNA previously observed with most bacteriopha...

show abstract

mentioning

confidence: 99%

Adenovirus Core Protein pVII Is Translocated into the Nucleus by Multiple Import Receptor Pathways

Wodrich

Cassany

D’Angelo

et al. 2006

J Virol

102

View full text Add to dashboard Cite

show abstract

“…Several other Ad proteins have been shown to localize to nucleoli, including IVa2 (29), the viral core proteins V (30, 31), Mu (26), and protein VII (25). The Ad E4orf4 protein is also targeted to nucleoli (33).…”

Section: Discussionmentioning

confidence: 99%

Identification of a New Human Adenovirus Protein Encoded by a Novel Late l -Strand Transcription Unit

Tollefson

Bai

Doronin

et al. 2007

J Virol

View full text Add to dashboard Cite

A short open reading frame named the "U exon," located on the adenovirus (Ad) l-strand (for leftward transcription) between the early E3 region and the fiber gene, is conserved in mastadenoviruses. We have observed that Ad5 mutants with large deletions in E3 that infringe on the U exon display a mild growth defect, as well as an aberrant Ad E2 DNA-binding protein ( An open reading frame (ORF) named the "U exon," located on the l strand (for leftward transcription) between the early E3 region and the fiber gene, was first identified after the sequencing of Ad40 (9). Davison et al. (9) reported that the U exon that is conserved in mastadenoviruses would encode just over 50 amino acids (aa) for most mastadenoviruses and might encode the N terminus of a larger protein. U exon sequences are conserved within each of the four Ad genera but not between genera (10). Davison et al. (10) suggested that the U exon is an ancient feature in adenoviruses (Ads; all Ad genera genomes encode capsid proteins and the E2 proteins within the central region of the Ad genome; the U exon is encoded within this region).In our work with a number of Ad E3 deletion mutants, we noted that mutants with deletions extending into the U exon have a mild growth defect and exhibit an altered Ad DNA-binding protein (DBP) (also known as E2 72K protein) intranuclear localization pattern when analyzed by indirect immunofluorescence. Reconstruction of the U exon DNA sequence reversed the phenotype of these mutants. Polyclonal and monoclonal antibodies were generated against peptides predicted from the putative U exon protein (UXP) ORF. We describe here the identification and characterization of UXP. MATERIALS AND METHODS Cell lines and adenoviruses. A549 cells (American Type Culture Collection[ATCC], Manassas, VA), HEK293 cells (Microbix, Toronto, Ontario, Canada), 21f1 cells (HeLa cells stably expressing DBP) (6), and A549E1 cells (A549 cells stably transfected with E1 sequences [bp 552 to 4090] under control of a mouse mammary tumor virus promoter; obtained from Genetic Therapy, Inc., Rockville, MD) were grown in Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS). HEL299 cells (human diploid embryonic lung cells; ATCC) were grown in DMEM with 10% FBS supplemented with nonessential amino acids and sodium pyruvate (1 mM). KB suspension culture cells were grown in Joklik's suspension culture medium containing 5% horse serum. Wildtype viruses used were Ad5 (ATCC VR-5), Ad1 (ATCC 1078-VR), Ad2, and Ad6 (ATCC 1083-VR). The Ad5-derived mutants used were dl327 (16), VRX-006, VRX-007 (13), VRX-021 (see below), dl7000 (42), and dl7001 (17, 42). The Ad E3 mutants dl722 (⌬12.5K) and dl762 (⌬14.7K) (5) were also used. Figure 1A is a schematic of the E3 genes present in these mutants, and Fig. 1B shows the U exon sequence present in these mutants.Construction of VRX-006 and VRX-021. The construction of mutants VRX-006 and VRX-007 has been described (13). In VRX-006 all E3 sequences between the L4 poly(A) site and a site (bp 30883) downstream ...

show abstract

“…Recent intracellular localization and functional studies of Mu, pV, and pVII were performed with carboxy terminal fusion of EGFP to the native proteins (Lee et al, 2003(Lee et al, , 2004Matthews, 2001). Our attention was drawn to three established observations from these reports:…”

Section: Discussionmentioning

confidence: 99%

“…Protein VII (pVII) is the major core protein contributing over 800 copies per virion (Lehmberg et al, 1999). Expressed as a 174 amino acid precursor from which a 24 amino acid N-terminus is cleaved (Sung et al, 1983), its functional and sequence homology to histone H3 (Cai and Weber, 1993;Lee et al, 2003) indicates that pVII acts as a histone-like center around which viral DNA is wrapped to form nucleosome structures (Chatterjee et al, 1986;Mirza and Weber, 1982). Protein V (pV) is speculated to serve as a bridge between the genome core and the capsid through association with proteins Mu, VI, and VII as well as the viral DNA (Chatterjee et al, 1985;Matthews and Russell, 1998;Vayda and Flint, 1987).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

An Imaging System to Monitor Efficacy of Adenovirus-Based Virotherapy Agents

Curiel¹

2006

View full text Add to dashboard Cite

Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. (From -To) 1 Feb 04 -31 Jan 06 REPORT DATE (DD-MM-YYYY) February 2006 REPORT TYPE Final DATES COVERED TITLE AND SUBTITLEAn imaging system to monitor efficacy of adenovirus-based virotherapy agents PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBERThe University of Alabama at Birmingham Birmingham, AL 35294-0109 SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR'S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 SPONSOR/MONITOR'S REPORT NUMBER(S) DISTRIBUTION / AVAILABILITY STATEMENTApproved for Public Release; Distribution Unlimited SUPPLEMENTARY NOTES ABSTRACTOur preliminary data establish a number of important key points. Foremost, these results show that adenovirus can be genetically labeled with a fluorescent structural fusion protein through a complete replacement with IX-EGFP in a chimeric context. At least for our pIX-EGFP strategy, the label was incorporated into virions conferring a fluorescent property that allowed detection of individual particles. Ad-IX-EGFP binding and infection could both be detected via the fluorescent label. This capsid-labeling system is applicable to CRAds because it slightly decreased progeny yield but did not affect the cytopathic effect and spread of the virus. Notably, the level of pIX-EGFP fluorescence directly correlated with the amount of progeny production due to its dependence on E1 activity for expression. The data with pIX-EGFP fulfills all the requirements of the ideal monitoring system for CRAds except noninvasive detection which we propose to accomplish. Both our proposed capsid-labeling approaches demonstrate great promise for detection of viral replication and spread and hence monitoring of CRAds. To this end, we have conceptualized an approach to achieve direct biological labeling of adenoviruses for CRAd imaging. Specifically, we have identified a capsid protein of adenovirus, pIX, which allows genetic incorporation of heterologous peptides which are thereby presented on the surface of the virion. We have recently demonstrated that we can incorporate int...

show abstract

Adenovirus core protein VII contains distinct sequences that mediate targeting to the nucleus and nucleolus, and colocalization with human chromosomes

Cited by 51 publications

References 29 publications

Adenovirus Core Protein pVII Is Translocated into the Nucleus by Multiple Import Receptor Pathways

Adenovirus Core Protein pVII Is Translocated into the Nucleus by Multiple Import Receptor Pathways

Identification of a New Human Adenovirus Protein Encoded by a Novel Late l -Strand Transcription Unit

An Imaging System to Monitor Efficacy of Adenovirus-Based Virotherapy Agents

Contact Info

Product

Resources

About