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. 2003 Nov 1;31(21):6206-13.
doi: 10.1093/nar/gkg827.

ssDNA-dependent colocalization of adeno-associated virus Rep and herpes simplex virus ICP8 in nuclear replication domains

Regine Heilbronn  1 , Markus Engstler, Stefan Weger, Antje Krahn, Christian Schetter, Michael Boshart
Affiliations

ssDNA-dependent colocalization of adeno-associated virus Rep and herpes simplex virus ICP8 in nuclear replication domains

Regine Heilbronn et al. Nucleic Acids Res. .

Abstract

The subnuclear distribution of replication complex proteins is being recognized as an important factor for the control of DNA replication. Herpes simplex virus (HSV) single-strand (ss)DNA-binding protein, ICP8 (infected cell protein 8) accumulates in nuclear replication domains. ICP8 also serves as helper function for the replication of adeno-associated virus (AAV). Using quantitative 3D colocalization analysis we show that upon coinfection of AAV and HSV the AAV replication protein Rep and ICP8 co-reside in HSV replication domains. In contrast, Rep expressed by a recombinant HSV, in the absence of AAV DNA, displayed a nuclear distribution pattern distinct from that of ICP8. Colocal ization of Rep and ICP8 was restored by the reintroduction of single-stranded AAV vector genomes. In vitro, ICP8 displayed direct binding to Rep78. Single-stranded recombinant AAV DNA strongly stimulated this interaction, whereas double-stranded DNA was ineffective. Our findings suggest that ICP8 by its strong ssDNA-binding activity exploits the unique single-strandedness of the AAV genome to form a tripartite complex with Rep78 and AAV ssDNA. This novel mechanism for recruiting components of a functional replication complex directs AAV to subnuclear HSV replication compartments where the HSV replication complex can replicate the AAV genome.

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Figures

Figure 1
Figure 1
Construction of rHSVrep/cap. (A) The cloning strategy into the single XbaI site at nucleotide position 143.969 of HSV-1, strain 1802 flanked by non-essential HSV genes is depicted. The AAV-2 rep and cap gene expression cassette (nucleotides 191–4485) includes the AAV promoters and polyA+ signal, and is linked to an SV40 controlled lacZ gene. LacZ positive HSV clones were plaque purified and analyzed for AAV Rep and Cap expression. Lysates of rHSVrep/cap-infected cells were prepared at different times p.i. Samples equaling 1 × 104 cells were loaded per lane. Western blots were reacted with the indicated mAb, followed by peroxidase-coupled second antibody and enhanced chemiluminescence. (B) AAV capsid (VP) proteins analyzed with mAb B1. (C) AAV Rep detected with mAb 303.9. HSV + AAV infected cell extracts were prepared at 12 h p.i.
Figure 2
Figure 2
Subnuclear colocalization of AAV Rep and HSV ICP8. BHK cells were grown on coverslips, infected as indicated and fixed at 5 h p.i. Anti-Rep hybridoma 76.3 reactivity was visualized with Cy3-conjugated F(ab)-fragments of goat anti mouse IgG in the ‘red’ channel. The anti-ICP8 hybridoma 39S was applied and detected with Alexa 350-conjugated goat anti-mouse IgG in the ‘blue’ channel. Since blue color poorly reproduces on printed color panels, the Alexa 350 staining is represented as green false color (anti-ICP8 panel). For 3D-colocalization analysis the raw data set was restored using the ICTM algorithm of the Huygens 2 software (see Materials and Methods). The digitally deconvolved 2-channel 3D image was exported to the colocalization 1 software and to ImarisColoc for quantitative analysis, respectively. Following channel definition, a 2D histogram was calculated and a polygonal region was selected. This region was chosen by creating a selection proposal from the AAV + HSV data set. The threshold values and the histogram region were stored and applied to all data sets. After calculating the colocalization map, the colocalization voxels were displayed in blue color. The standard deviation of the intensities of all colocalization voxels in a map was found to be ≤8%. To facilitate navigation within the image the highlighted colocalization voxels were merged to a summed image (colocalization panel). Values given in the upper left edges of green and red panels are maximum fluorescence intensities measured within the raw 3D data set. The percent of green channel volume colocalized with red was calculated in ImarisColoc (Bitplane AG) for n = 6 nuclei and is given as mean ± SD in the upper left edges of the colocalization panel. The Pearson channel correlation in colocalized volume is given in the upper right edges of the colocalization panel (1 is perfect colocalization, 0 no correlation and –1 perfect inverse correlation). Recombinant AAV is transfected pTR-UF5 DNA.
Figure 3
Figure 3
AAV ssDNA-dependent in vitro interaction of AAV Rep and HSV ICP8. In vitro-translated, 35S-labeled Rep78 was incubated with either GST–ICP8 or GST alone. Protein–protein interaction was studied either alone, or in the presence of varying amounts of gel-purified AAV template DNAs. The DNA was either in the double-stranded (ds) conformation, or was heat-denatured to adopt a single-stranded conformation, as indicated. (A and C) Autoradiographies of SDS–PAGE after separation of GST-pull-down products; +RBS, ssDNA flanked by AAV-ITRs with Rep-binding sites. –RBS, ssDNA without ITRs and deleted internal RBS homology sequences. Lanes 1 represent an aliquot of in vitro translated Rep78 alone. The faint high molecular weight band appears to represent multimerized Rep78. (B and D) Phosphoimager quantification of three separate experiments as displayed in (A) and (C).
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