Volume 15, Issue 7, Pages 1323-1330 (July 2007) A Dual Role of EGFR Protein Tyrosine Kinase Signaling in Ubiquitination of AAV2 Capsids and Viral Second-strand DNA Synthesis  Li Zhong, Weihong Zhao, Jianqing Wu, Baozheng Li, Sergei Zolotukhin, Lakshmanan Govindasamy, Mavis Agbandje-McKenna, Arun Srivastava  Molecular Therapy  Volume 15, Issue 7, Pages 1323-1330 (July 2007) DOI: 10.1038/sj.mt.6300170 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Adeno-associated virus 2 (AAV2)-mediated transgene expression in HeLa cells, pre-treated with or without Tyrphostin 23 (Tyr23), following transduction with either single-stranded AAV2-enhanced green fluorescence protein (ssAAV2-EGFP) or self-complementary AAV2 EGFP (scAAV2-EGFP) vectors. (a) Transgene expression was detected by fluorescence microscopy at 48 hours post-infection. Original magnification ×100. (b) Quantitative analyses of AAV2 transduction efficiency. Images from five visual fields were analyzed quantitatively using ImageJ analysis software. Transgene expression was assessed as total area of green fluorescence (pixel2) per visual field (mean ± SD). Analysis of variance was used to compare test results with the control and they were determined to be statistically significant. *P < 0.05 versus control + ssAAV2-EGFP; #P < 0.05 versus control + scAAV2-EGFP. Molecular Therapy 2007 15, 1323-1330DOI: (10.1038/sj.mt.6300170) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Adeno-associated virus-mediated transgene expression in HeLa cells mock-transfected, or stably transfected with wild-type (wt)- or C-S mutant (m) T-cell protein tyrosine phosphatase (TC-PTP) expression plasmids, following transduction with either self-stranded AAV2 enhanced green fluorescence protein (ssAAV2-EGFP) or self-complementary AAV2-EGFP (scAAV2-EGFP) vectors. (a) Transgene expression was detected by fluorescence microscopy at 48 hours post-infection. Original magnification ×100. (b) Quantitative analyses of AAV2 transduction efficiency was assessed as described in the legend to Figure 1, and were determined to be statistically significant. *P < 0.05 versus control + ssAAV2-EGFP; #P < 0.05 versus control + scAAV2-EGFP. Molecular Therapy 2007 15, 1323-1330DOI: (10.1038/sj.mt.6300170) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Intracellular trafficking of AAV2 vectors following perturbation of EGFR-PTK signaling, or proteasome inhibition. (a) Southern blot analyses of cytoplasmic and nuclear distribution of AAV2 genomes in HeLa cells following pre-treatment with Tyrphostin 23 (Tyr23), over-expression of wild-type T-cell protein tyrosine phosphatase (wtTC-PTP), or treatment with MG132, and (b) densitometric scanning of autoradiographs for the quantitation of relative amounts of viral genomes. These results are from two independent experiments. ssDNA, single stranded DNA, mTC-PTP, C-S mutant TC-PTP. Molecular Therapy 2007 15, 1323-1330DOI: (10.1038/sj.mt.6300170) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Comparative analyses of adeno-associated virus 2 (AAV2) transduction efficiency in HeLa cells with various treatments. (a) HeLa cells were mock-treated or treated with Tyrphostin 23 (Tyr23), MG132, or both, and cells stably transfected with the wild-type T-cell protein tyrosine phosphatase (wtTC-PTP) expression plasmid were either mock-treated or treated with MG132, followed by infection with AAV-lacZ vectors. Cells were fixed and stained with X-Gal. Transgene expression was detected by microscopy at 48 hours post-infection. Original magnification ×100. (b) AAV transduction efficiency was assessed by quantitative analyses as described in the legend to Figure 1, and the results were determined to be statistically significant. *P < 0.05 versus control + single-stranded AAV2-lacZ. Molecular Therapy 2007 15, 1323-1330DOI: (10.1038/sj.mt.6300170) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Comparative analyses of adeno-associated virus 2 (AAV2)-mediated transduction efficiency in HeLa cells with various treatments, following transduction with self-complementary AAV2-enhanced green fluorescence protein (scAAV2-EGFP) vectors. (a) HeLa cells were mock-treated or treated with Tyrphostin 23 (Tyr23), MG132, or both, and cells either mock-transfected or stably transfected with the wild-type T-cell protein tyrosine phosphatase (wtTC-PTP) or C-S mutant TC-PTP (mTC-PTP) expression plasmids were either mock-treated or treated with MG132. Transgene expression was detected by fluorescence microscopy at 48 hours post-infection (original magnification ×100). (b) AAV transduction efficiencies were assessed by quantitative analyses as described in the legend to Figure 1, and the results were determined to be statistically significant. *P< 0.05 versus control + scAAV2-EGFP. Molecular Therapy 2007 15, 1323-1330DOI: (10.1038/sj.mt.6300170) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 Western blot analyses of ubiquitinated proteins in HeLa cells following treatment with MG132 in the presence or absence of Tyrphostin 23 (Tyr23) or T-cell protein tyrosine phosphatase (TC-PTP). Whole cell lysates prepared from untreated cells (lanes 1 and 6), and following treatment with MG132 (lanes 2 and 7), Tyr23 (lane 3), or both (lane 8), and cells either stably transfected with the wild-type T-cell protein tyrosine phosphatase (wtTC-PTP) or C-S mutant TC-PTP (mTC-PTP) expression plasmids following either mock-treatment (lanes 4 and 5) or treatment (lanes 9 and 10) with MG132 were probed with anti-ubiquitin monoclonal antibody. Molecular Therapy 2007 15, 1323-1330DOI: (10.1038/sj.mt.6300170) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 7 Western blot analyses of ubiquitinated adeno-associated virus (AAV2) capsid proteins in HeLa cells treated with MG132 in the presence or absence of Tyr23 or wild-type T-cell protein tyrosine phosphatase (wtTC-PTP), following transduction with single- stranded AAV2 (ssAAV2)-RFP vectors. Whole cell lysates prepared from HeLa cells, untreated or treated with MG132 following mock-infection (lanes 1 and 2), and HeLa cells untreated (lane 3), treated with Tyrphostin 23 (Tyr23) (lane 4), MG132 (lane 5), or both (lane 6), or cells stably transfected with the wtTC-PTP expression plasmid following either mock-treatment (lane 7) or treatment with MG132 (lane 8), following infection with ssAAV2-RFP vectors, were immunoprecipitated with anti-AAV2 capsid antibody A20 followed by Western blot analyses with anti-ubiquitin (anti-Ub) monoclonal antibody. IgG, immunoglobulin G. Molecular Therapy 2007 15, 1323-1330DOI: (10.1038/sj.mt.6300170) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 8 A model for interaction between epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling and ubiquitin (Ub)/proteasome pathway in the regulation of intracellular trafficking as well as second-strand DNA synthesis of adeno-associated virus (AAV2) vectors. See text for details. *Indicates that proteasome inhibitors only affect the degradation step of AAV2 vectors, and **denotes that both the ubiquitination of AAV2 capsids and the viral second-stand DNA synthesis steps are affected by EGFR-PTK inhibitors. EE, early endosome; CP, clathrin-coated pits; LE, late endosome; F, FKBP52; P, phospho-tyrosine residues; HSPG, heparan sulfate proteoglycan. Molecular Therapy 2007 15, 1323-1330DOI: (10.1038/sj.mt.6300170) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions