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. 2017 Oct:510:46-54.
doi: 10.1016/j.virol.2017.07.007. Epub 2017 Jul 10.

The function of DNA binding protein nucleophosmin in AAV replication

Stifani Satkunanathan  1 , Robin Thorpe  1 , Yuan Zhao  2
Affiliations

The function of DNA binding protein nucleophosmin in AAV replication

Stifani Satkunanathan et al. Virology. 2017 Oct.

Abstract

Adeno-associated viruses (AAV) contain minimal viral proteins necessary for their replication. During virus assembly, AAV acquire, inherently and submissively, various cellular proteins. Our previous studies identified the association of AAV vectors with the DNA binding protein nucleophosmin (NPM1). Nucleophosmin has been reported to enhance AAV infection by mobilizing AAV capsids into and out of the nucleolus, indicating the importance of NPM1 in the AAV life cycle; however the role of NPM1 in AAV production remains unknown. In this study, we systematically investigated NPM1 function on AAV production using NPM1 knockdown cells and revealing for the first time the presence of G-quadruplex DNA sequences (GQRS) in the AAV genome, the synergistic NPM1-GQRS function in AAV production and the significant enhancement of NPM1 gene knockdown on AAV vector production. Understanding the role of cellular proteins in the AAV life cycle will greatly facilitate high titre production of AAV vectors for clinical use.

Keywords: AAV biology; G-quadruplex DNA sequences; NPM1; shRNA.

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Figures

Fig. 1
Fig. 1
Immunoblotting analysis of NPM1 and AAV protein expression in parental 293T cells after transient transfection of AAV packaging and helper plasmids, showing (A) overtime decrease in NPM1 expression in AAV 2 and AAV8 producer cells; (B) detection of AAV capsid (VP1, VP2 and VP3) and Rep proteins from 24 h after transfection; (C) NPM1 knockdown in LV-NPM1-shRNA transduced 293T cells carrying shRNA M1 to M5 sequences targeting NPM1; (D) sustained down-regulation of NPM1 in single cell clones and in culture for up to 90 days. Controls: 293T cell lysate without the transfection of any plasmids (293T), 293T cells with the transfection of pcDNA3.1 plasmids (Mock); 293T cells with a shRNA sequence targeting non-mammalian genes (Scramble) and without any shRNA sequences (293T) were used as gene knockdown controls. Housekeeping gene GAPDH was further used as a loading control.
Fig. 2
Fig. 2
Relative genome titres of AAV vectors produced from NPM1 gene knockdown cells compared to that from control scramble cells, showing up to 35 fold and 30 fold increases in (A) AAV2 and (B) AAV8 vector genome titres (VG/mL), respectively, compared to scramble cells and throughout 90 days in culture; (C) NPM1 expression in individual AAV producer cell lines at the last production time points. Different sympols, e.g. square or circle etc, represent independently established NPM1 knockdown cell lines. (D) in vitro transduction on HelaS3 cells (left panel, labelled in green, GFP) and in vivo transduction in Balb C mice (right panel, in blue, luciferase) showing comparable transduction efficiency when using AAV2 vectors produced from control scramble cells (Scr/AAV2luc) or from NPM1 knockdown cells (NPM/AAV2luc). Scale bars are 30 µm.
Fig. 3
Fig. 3
Protein-DNA binding assays showing (A) NPM1 interaction with GQRS structure within c-myc (myc, 1st lane) and AAV ITR sequences (ITRA, 2nd lane) in 293T cells after transfection with AAV packaging plasmids and a trace NPM1 interaction with c-myc (myc, 4th lane) and ITR (ITRA, 5th lane) GQRS in NPM1 knockdown cells after AAV plasmid transfection; a non-GQRS DNA oligonucleotide (NGQRS) was included as nonspecific DNA binding control (NGQRS, 6th lane); (B) Competition for AAV_ITR binding to NPM1 proteins using an increased amount of c-myc GQRS (top and middle panels): a progressively diminishing of ITR_A (top panel) or ITR_B GQRS (middle panel) binding to NPM1 proteins; a non-GQRS DNA oligonucleotide (NGQRS, bottom panel) was also used as assay control, showing that non-GQRS DNA oligonucleotides (NGQRS) cannot compete out the AAV ITR_A GQRS binding to NPM1 proteins (bottom panel) and that AAV ITR binding to NPM1 proteins is GQRS specific; (C) NPM1 expression in 293T and NPM1 knockdown cells before DNA binding assays. A non-GQRS DNA oligonucleotide (NGQRS) was included as nonspecific DNA binding control and Housekeeping gene GAPDH was used as a loading control.
Fig. 4
Fig. 4
Immunoblotting analysis of AAV protein showing a comparable (A)rep and (B)cap expression between control scramble cells and NPM1 knockdown cells and comparable distribution of Rep and Cap proteins in cytoplasm, nuclear and insoluble membrane fractions of control scramble (S) and NPM1 knockdown (N) producer cells.
Fig. 5
Fig. 5
Fluorescence microscopy of NPM1 and AAV Rep and Cap proteins; showing nuclear localisation of AAV Rep (labelled in red, left panels) and NPM1 proteins (in green, bottom panels) and cytoplasm localisation of AAV Cap protein (in red, right panels) in NPM1 knockdown cells (top panels) and parental 293T cells (middle and bottom panels) after transient transfection of AAV packaging and helper plasmids. Nuclei were labelled with Dapi in blue. Scale bars are 50 µm.
Fig. 6
Fig. 6
Confocal microscopy of assembled AAV2 particles (labelled in red): showing cytoplasm localisation of AAV particles in NPM1 knockdown cells (labelled in red, left panels) compared to nuclear localisation of AAV particles in control 293T cells (labelled in red, right panels). Nuclei were labelled with Dapi in blue. Scale bars are 200 µm for top panel and 15 µm for bottom panel.
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