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

Abstract

During adenovirus infection, following capsid dissociation, core protein VII enters the host cell nucleus complexed with adenovirus DNA. In order to determine whether protein VII may have an active role in this nuclear import, regions of the preVII gene were amplified by PCR, and further oligonucleotide mutants were designed with site-directed mutation of codons for the basic amino acids arginine and lysine. Fragments were cloned into a mammalian expression plasmid to express the peptides as N-terminal fusions to enhanced green fluorescent protein. Results demonstrate that preVII protein contains both nuclear and nucleolar targeting sequences. Such signals may be important in the delivery of adenovirus DNA to the host cell nucleus during adenovirus infection. Furthermore, the data suggest that protein VII may bind to human chromosomes by means of two distinct domains, one sharing homology with the N-terminal regulatory tail of histone H3.Enclosed by approximately 12 capsid proteins the human adenovirus genome, comprising approximately 36 kb of linear double-stranded DNA, is non-covalently bound to the viral core proteins Mu, V, and VII (Vayda et al., 1983). Proteins VII and Mu are tightly associated with the viral DNA (Vayda et al., 1983;Chatterjee et al., 1985), whilst protein V may form a link between the viral DNA-core protein complex and the viral capsid (Matthews & Russell, 1998).In human adenoviruses, both protein VII and Mu are encoded by the late transcription unit L2 (Alestrom et al., 1984). Mature protein VII of human adenovirus 2 and 5 contains 174 amino acids and is formed from its precursor by adenovirus-encoded protease-mediated cleavage of a 24 amino acid N-terminal segment (Sung et al., 1983;Alestrom et al., 1984;Webster et al., 1989). The mature protein has four highly basic domains containing arginineand lysine-rich sequences, separated by predicted alphahelices. The interaction of protein VII with DNA appears to be charge-based between these basic regions and the phosphate backbone of 90-150 DNA bp (Vayda & Flint, 1987), resulting in the DNA being considerably condensed as a result of superfolding (Black & Center, 1979;Sato & Hosokawa, 1984). Protein VII associates more efficiently with double-stranded DNA than single-stranded (Sato & Hosokawa, 1984). Binding of both proteins VII and Mu to DNA is not DNA sequence-specific (Russell & Precious, 1982).During adenovirus infection, following capsid dissociation, protein VII remains complexed to adenovirus DNA, and this complex enters the nucleus via the nuclear pore (Greber et al., 1997). Protein VII inhibits adenovirus DNA synthesis (Korn & Horwitz, 1986) and transcription in vitro (Nakanishi et al., 1986) and thus is then likely to dissociate from DNA to allow these processes to occur. Antibodies raised against protein Mu for use in Western blots and ELISA cross-react with protein VII, presumably due to the homologous unusual arginine-rich sequences present in both proteins (Lunt et al., 1988). Protein VII has homologues in every adenovirus studi...

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

“…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.…”

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

“…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).…”

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

“…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:…”

“…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).…”

“…In the chimeric expression system, both the native, unmodified core protein and the fusion counterpart were expressed from the same vector. We initially applied the AdEasy system to construct E1 expression vectors under the control of the constitutive cytomegalovirus (CMV) promoter ( (Lee et al, 2003(Lee et al, , 2004Matthews, 2001). …”