Gene Therapy Research Unit of the Childrens Medical Research Institute and The New Childrens Hospital1, and The University of Sydney Department of Paediatrics and Child Health2, PO Box 3515, Parramatta, NSW 2124, Australia
Author for correspondence: Ian Alexander (at Gene Therapy Research Unit, The New Childrens Hospital). Fax +61 2 9845 3082. e-mail iana{at}nch.edu.au
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Abstract |
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Introduction |
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While rAAV stocks can be generated that have extremely high virus titre, the proportion of single-stranded input genomes successfully converting to transcriptionally active double-stranded molecules varies dramatically with different target cell types (Halbert et al., 1995 ; Xiao et al., 1996
; Russell & Kay, 1999
) and as a function of the cell cycle (Russell et al., 1994
). This critical dependence of rAAV transduction efficiency on the state of the intracellular milieu remains poorly understood, and currently limits the potential utility of rAAV in many gene therapy applications. The demonstration, however, that rAAV transduction can be dramatically enhanced by exposing target cells to genotoxic stress (Alexander et al., 1994
; Russell et al., 1995
; Ferrari et al., 1996
) or adenovirus infection (Fisher et al., 1996
; Ferrari et al., 1996
) offers the prospect of overcoming this apparent biological limitation and optimizing the potential of this gene delivery system.
Adenovirus-mediated enhancement of rAAV transduction has previously been mapped to the E4 ORF6 gene product, the function of which has been described as necessary and sufficient to mediate this effect (Fisher et al., 1996 ; Ferrari et al., 1996
). An additional contribution from the E1 region has also been recognized, but considered to be cooperative and dependent on E4 ORF6 (Fisher et al., 1996
). Even less is known about the mechanism by which genotoxic stress enhances rAAV transduction, although induction of DNA repair activities has been proposed (Alexander et al., 1994
). We are interested in identifying the cellular proteins and activities mediating the enhancement of rAAV transduction efficiency, and initially hypothesized that p53 may subserve a crucial function in this process because p53 is upregulated in response to genotoxic stress (Levine, 1997
; Steele et al., 1998
), and conversely is targeted for degradation by interaction with the E4 ORF6 protein during an adenovirus infection (Querido et al., 1997
; Steegenga et al., 1998
). We therefore chose to establish whether or not these cellular insults that result in enhanced rAAV transduction are dependent on the p53 pathway.
In the current study, using HeLa cells and two p53-null cell lines, H1299 and Saos-2, we show that cellular p53 is not essential for the enhancement of rAAV transduction efficiency following exposure of target cells to genotoxic stress or adenovirus infection, confirming the recent report of Grifman et al. (1999) . We further demonstrate that, in the cell lines examined, the E1b 55 kDa protein subserves a function in adenovirus-mediated enhancement of rAAV transduction equally as important as that already attributed to the E4 ORF6 gene product. Indeed, the rAAV enhancement effect of these adenovirus proteins is best characterized as cooperative and interdependent and this paper reports the first empirically derived data supporting this hypothesis. Ongoing analysis of the mechanism by which adenovirus infection changes the intracellular milieu in favour of rAAV transduction should therefore be focused on the cooperative effects of the E4 ORF6 and E1b 55 kDa proteins that do not involve interaction with p53. This information should ultimately lead to an improved understanding of the cellular proteins and activities mediating rAAV transduction.
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Methods |
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Cell culture and DNA transfection.
HeLa cells, 293 cells and the p53-null cell lines H1299 and Saos-2 have been described elsewhere (Gey et al., 1952 ; Graham et al., 1977
; Mitsudomi et al., 1992
; Diller et al., 1990
). Cells were maintained in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% heat inactivated (30 min at 56 °C) calf serum (Starrate, Bethungra, Australia) and 2 mM glutamine at 37 °C in a 5% CO2 humidified atmosphere. The previously described p53 deletions in the H1299 and Saos-2 cell lines were confirmed by Southern blot analysis (data not shown).
The calcium phosphate coprecipitation technique was used for all transfections (Gorman, 1985 ). Cells were plated at a density of 5x105 to 1x106 cells per 25 cm2 tissue culture flask the day before transfection; 5 to 10 µg of plasmid DNA per culture flask was used in each transfection experiment. Six hours post-transfection the monolayers were washed and media replenished and, after a further 18 h, transfected cells were trypsinized and replated in 12-well tissue culture plates at densities of 1·5 to 3x105 cells per well. Experimental manipulations were commenced 6 h after replating. Stable E4 ORF6-expressing and control cell lines were derived from H1299 and Saos-2 cells by G418-selection after transfection with pCIE4ORF6 or pCI-neo, respectively. Two days after transfection cultures were subcultured at a 1:10 dilution into medium containing 800 µg/ml G418 (active). Complete G418-selection was achieved in H1299 and Saos-2 cells by 14 and 28 days respectively, as defined by the death of all cells in control untransfected cultures. Colonies surviving G418-selection were pooled to produce polyclonal populations for each cell line.
Recombinant AAV vector production and analysis of transduction.
All experiments were performed using CsCl gradient-purified rAAV vector stocks encoding bacterial -galactosidase under the transcriptional control of a CMV immediate-early promoter. Vector stocks were generated as previously described, initially by the conventional cotransfectionadenovirus infection strategy (Samulski et al., 1989
) and subsequently by the recently developed adenovirus-free strategy (Xiao et al., 1998
). The vector plasmid pAB11, the AAV helper plasmid pAAV/Ad and adenovirus dl309 were used for the conventional strategy, while the vector plasmid pCMVnLacZ, the AAV helper plasmid pXX2 and the adenovirus helper plasmid pXX6 were used for the adenovirus-free strategy. A single vector stock, rAAV-CMV-LacZ (Lot #120845), produced using the adenovirus-free strategy, was obtained directly from the Vector Core at the University of North Carolina, Chapel Hill, NC, USA. Transduction titres for individual vector stocks were measured on 293 cells in the absence of adenovirus by counting
-galactosidase-positive foci 24 h after vector exposure. Expression of
-galactosidase was detected by histochemical staining as previously described (Halbert et al., 1995
). All rAAV transduction enhancement experiments were carried out using low multiplicities of infection (m.o.i.), typically 10-3 to 10-4 transducing units per cell. These low m.o.i.s were employed so that the absolute number of rAAV transductants in control wells (10 to 100) and treated wells (up to 10000) could be readily counted. The basal rAAV transduction titres measured on 293 cells were 1·25- and 3·3-fold higher than those measured on H1299 and Saos-2 cells, respectively.
Propagation and analysis of adenovirus mutants.
Adenovirus stocks were propagated on 293 cells. The adenovirus E3 mutant dl309, and the E1b 55 kDa mutants dl338 and dl1520, have been described (Pilder et al., 1986 ; Barker & Berk, 1987
). The dl338 mutant has a 524 bp in-frame deletion between nucleotides 2805 and 3329 and does not produce E1b 55 kDa (Pilder et al., 1986
). The dl1520 mutant has an 827 bp deletion combined with a point mutation at nucleotide 2022 that introduces a premature stop codon (Barker & Berk, 1987
). Stocks of dl309 were purified by equilibrium density-gradient centrifugation in CsCl and stocks of the E1b 55 kDa mutants were made from clarified crude cell lysates as described (Snyder et al., 1996
). Virus titre was assigned for each preparation by plaque assay on 293 cells (Snyder et al., 1996
). To test the ability of adenovirus mutants to enhance rAAV transduction, cells were seeded into 12-well plates at a density of 1·5 to 3x105 cells per well in 0·5 ml of medium. Six hours after replating cells were simultaneously exposed to rAAV vector and the adenovirus mutant under investigation. Each adenovirus mutant was used at an m.o.i. of approximately 5 p.f.u. per cell.
UV irradiation.
A Stratalinker 1800 (Stratagene) was used to expose cell monolayers to 15 J/m2 UV irradiation (254 nm). Tissue culture medium was aspirated from cell monolayers which were then exposed to UV irradiation immediately prior to rAAV transduction.
Immunohistochemistry.
The mouse monoclonal antibody MAb3, which recognizes an epitope within the N terminus of E4 ORF6, was obtained from Tom Shenk, Princeton University (Marton et al., 1990 ). The mouse monoclonal antibody 2A6, specific for the E1b 55 kDa protein, was obtained from Arnold Levine, Princeton University (Sarnow et al., 1982
). Both antibodies were provided as hybridoma tissue culture supernatant and were diluted in TBS pH 7·42% BSA. The 2A6 antibody was detected with an indirect immunoperoxidase system according to the manufacturers instructions (Dako). Peroxidase activity was visualized with DAB (Sigma). MAb3 binding was detected with an FITC-labelled goat
-mouse secondary antibody (Becton Dickson) and visualized under epifluorescent illumination on a Leica DMLB upright microscope.
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Results |
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Transient transfection of a plasmid encoding the dl309 genome enhances rAAV transduction in H1299 and HeLa cells
The effect of transient transfection of plasmids containing the entire adenovirus dl309 genome (pFG140) or the E4 ORF6 gene alone (pCIE4ORF6) on the efficiency of rAAV transduction was compared in H1299 cells (p53-null) and HeLa cells (p53-positive). Transfection with the E4 ORF6-encoding plasmid failed to significantly enhance rAAV transduction in either cell line, whereas transfection with the plasmid encoding the entire adenoviral genome significantly enhanced rAAV transduction in both cell lines (Fig. 4).
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Discussion |
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Two separate groups, using mutant adenoviruses, have previously mapped the enhancement effect of adenovirus infection on rAAV transduction to E4 ORF6, and concluded that the E4 ORF6 gene product is necessary and sufficient to enhance rAAV transduction (Fisher et al., 1996 ; Ferrari et al., 1996
). Interestingly, one of these groups also noted, but did not further dissect, an additional contribution by the E1 region to enhanced rAAV transduction that was described as cooperative and dependent on expression of E4 ORF6 (Fisher et al., 1996
). We therefore anticipated that expression of E4 ORF6 alone would, at least partially, recapitulate the enhancement effect of adenovirus dl309 infection on rAAV transduction observed in H1299 and Saos-2 cells. Intriguingly, while transient expression of E4 ORF6 in 293 cells enhanced rAAV transduction as previously described (Fisher et al., 1996
; Ferrari et al., 1996
), neither transient nor stable expression of E4 ORF6 enhanced rAAV transduction in H1299 and Saos-2 cells. Important differences between 293 cells and H1299 cells or Saos-2 cells that may account for this observation include the presence of p53 and adenovirus E1 gene products in 293 cells. The ability of adenovirus infection to enhance rAAV transduction in a p53-null environment and the observation that p53 overexpression does not inhibit rAAV transduction (Grifman et al., 1999
) support the hypothesis that the relevant difference is the expression of adenoviral E1 proteins in 293 cells. This possibility is further supported by the observation that transient transfection of H1299 cells or HeLa cells with a plasmid encoding only E4 ORF6 failed to enhance rAAV transduction in either cell line. In contrast, transfection with a plasmid encoding the entire adenovirus dl309 genome, including the E1 and E4 regions, enhanced rAAV transduction in both cell lines. Collectively, these results demonstrate that in the cell lines examined E4 ORF6 expression alone is not sufficient to enhance rAAV transduction, and that a contribution from the E1 region is necessary.
Among the proteins encoded by the E1 region we chose to investigate the contribution of the E1b 55 kDa gene product to adenovirus-mediated enhancement of rAAV transduction. This protein is known to form a functional complex with the E4 ORF6 gene product (Sarnow et al., 1984 ; Cutt et al., 1987
) and both proteins have been identified as providing helper function in the rescue and replication of wild-type AAV (Richardson & Westphal, 1984
; Samulski & Shenk, 1988
). Infection of H1299 cells with the E1b 55 kDa mutant adenoviruses dl338 and dl1520 resulted in a markedly diminished enhancement of rAAV transduction compared to infection with dl309. In contrast, all three mutant viruses enhanced rAAV transduction to a similar extent in 293 cells. In transient transfection experiments, free of the complicating influence of adenovirus infection, expression of the E1b 55 kDa gene product was required for the E4 ORF6-mediated effect on rAAV enhancement in HeLa, H1299 and Saos-2 cell lines. Unexpectedly, expression of the E1b 55 kDa gene product alone enhanced rAAV transduction in HeLa cells, although optimal levels of rAAV enhancement required coexpression of E4 ORF6. The explanation for this observation in HeLa cells remains unclear, and is confounded by the expression of papillomavirus type 18 E6 and E7 oncoproteins (Schneider-Gadicke & Schwarz, 1986
).
To our knowledge, the only published data directly supporting the conclusion that E4 ORF6 expression is both necessary and sufficient to enhance rAAV transduction were also generated in HeLa cells (Fisher et al., 1996 ). In these experiments a HeLa cell line stably expressing E4 ORF6 under the transcriptional control of a zinc-inducible sheep metallothionein promoter was used. Exposure of these cells to zinc sulphate at concentrations up to 250 µM correlated with enhanced rAAV transduction. Why these results differ from our own HeLa data remains a matter of conjecture. Possible explanations include (i) differences in the biological properties of the HeLa cell lines employed, (ii) differences in the experimental conditions used (such as the input m.o.i. of rAAV) and (iii) possibly the ability of zinc to act directly as a cellular stress. In support of this last possibility we have found that zinc treatment of unmodified HeLa cells enhances rAAV transduction (5-fold enhancement at 200 µM) (unpublished observation). This might also explain why there was little correlation between enhanced transduction following zinc treatment and the detection of duplex replicative form monomers in HeLa cells, which was observed in 293 cells (Fisher et al., 1996
). Recently published data provide evidence that, in contrast to adenovirus-mediated enhancement of rAAV transduction, genotoxic stress-mediated enhancement does not appreciably increase the appearance of replicative form rAAV genome intermediates (Sanlioglu et al., 1999
). In summary, while we concede that E4 ORF6 expression alone may enhance rAAV transduction in some cellular contexts, our data support the conclusion that E1b 55 kDa subserves an equally crucial, and largely overlooked function in this process.
The demonstrated interdependence of the E4 ORF6 and E1b 55 kDa proteins for enhancement of rAAV transduction in H1299 and Saos-2 cells directs analysis of the underlying mechanism to the cooperative functions performed by these proteins which are independent of cellular p53. The E4 ORF6 and E1b 55 kDa proteins have been shown to form a functional complex (Cutt et al., 1987 ), one effect of which is to promote the cytoplasmic accumulation of viral mRNAs (Samulski & Shenk, 1988
). Indeed, this effect of the E4 ORF6E1b 55 kDa protein complex has already been postulated to account for the previously observed contribution of the E1 region to adenovirus-mediated enhancement of rAAV transduction (Fisher et al., 1996
). While our data provide further support for this mechanism, it alone is insufficient to fully account for our results. Post-transcriptional effects mediated by the E4 ORF6E1b 55 kDa protein complex might well increase the amount of rAAV-encoded reporter protein expressed per transductant, but are unlikely to be sufficient to account for the E1b 55 kDa protein-dependent increase in the absolute number of individual transduction events observed. We therefore conclude that it is likely that the E1b 55 kDa protein also functions to enhance critical events earlier in the rAAV transduction pathway. Clearly, a key possibility is the conversion of input single-stranded rAAV genomes to transcriptionally active double-stranded molecules, a process that as already stated has been linked to the E4 ORF6 protein (Fisher et al., 1996
; Ferrari et al., 1996
). Candidate activities of the E4 ORF6 and E1b 55 kDa proteins, identified in the context of adenovirus infection, that might facilitate this genome conversion process include the redistribution and recruitment of cellular proteins to specific intranuclear sites for viral DNA replication (Doucas et al., 1996
). Adenovirus infection has also been shown to recruit input single-stranded rAAV genomes to intranuclear virus replication centres (Weitzman et al., 1996
). This rAAV mobilization phenomenon, however, can be achieved with either E4 or E1b mutant adenoviruses, thereby precluding a requirement for E4 ORF6 or E1b 55 kDa in this process.
In conclusion, our results provide further insight into the mechanisms subserving adenovirus-mediated enhancement of rAAV transduction efficiency. Importantly, we have shown that cellular p53 is not essential for this effect and that the E1b 55 kDa protein plays a more critical role than previously appreciated. Further dissection of the interactions of E4 ORF6E1b 55 kDa with the host cell will undoubtedly contribute to the elucidation of the cellular proteins and activities mediating rAAV transduction. Such knowledge will be of great value in fully exploiting the biology of rAAV for research and gene therapy applications.
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Acknowledgments |
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References |
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Atchison, R. W., Casto, B. C. & Hammon, W. M.(1965). Adenovirus-associated defective virus particles.Science149, 754-756.
Barker, D. D. & Berk, A. J.(1987). Adenovirus proteins from both E1B reading frames are required for transformation of rodent cells by viral infection and DNA transfection.Virology156, 107-121.[Medline]
Berns, K. I. & Rose, J. A.(1970). Evidence for a single-stranded adenovirus-associated virus genome: isolation and separation of complementary single strands.Journal of Virology5, 693-699.[Medline]
Buller, R. M., Janik, J. E., Sebring, E. D. & Rose, J. A.(1981). Herpes simplex virus types 1 and 2 completely help adenovirus-associated virus replication. Journal of Virology40, 241-247.[Medline]
Cutt, J. R., Shenk, T. & Hearing, P.(1987). Analysis of adenovirus early region 4-encoded polypeptides synthesized in productively infected cells.Journal of Virology61, 543-552.[Medline]
Diller, L., Kassel, J., Nelson, C. E., Gryka, M. A., Litwak, G., Gebhardt, M., Bressac, B., Ozturk, M., Baker, S. J. & Vogelstein, B.(1990). p53 functions as a cell cycle control protein in osteosarcomas.Molecular and Cellular Biology10, 5772-5781.[Medline]
Dobner, T., Horikoshi, N., Rubenwolf, S. & Shenk, T.(1996). Blockage by adenovirus E4orf6 of transcriptional activation by the p53 tumor suppressor. Science272, 1470-1473.[Abstract]
Doucas, V., Ishov, A. M., Romo, A., Juguilon, H., Weitzman, M. D., Evans, R. M. & Maul, G. G.(1996). Adenovirus replication is coupled with the dynamic properties of the PML nuclear structure. Genes & Development10, 196-207.[Abstract]
Ferrari, F. K., Samulski, T., Shenk, T. & Samulski, R. J.(1996). Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors.Journal of Virology70, 3227-3234.[Abstract]
Fisher, K. J., Gao, G. P., Weitzman, M. D., DeMatteo, R., Burda, J. F. & Wilson, J. M.(1996). Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis.Journal of Virology70, 520-532.[Abstract]
Flotte, T. R. & Carter, B. J.(1995). Adeno-associated virus vectors for gene therapy.Gene Therapy2, 357-362.[Medline]
Gey, G. O., Coffman, W. D. & Kubicek, M. T.(1952). Tissue culture studies of the proliferative capacity of cervical carcinoma and normal epithelium.Scientific Proceedings of the American Association for Cancer Research12, 264.
Goodman, S., Xiao, X., Donahue, R. E., Moulton, A., Miller, J., Walsh, C., Young, N. S., Samulski, R. J. & Nienhuis, A. W.(1994). Recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells.Blood84, 1492-1500.
Goodrum, F. D., Shenk, T. & Ornelles, D. A.(1996). Adenovirus early region 4 34-kilodalton protein directs the nuclear localization of the early region 1B 55-kilodalton protein in primate cells. Journal of Virology70, 6323-6335.[Abstract]
Gorman, C.(1985). High efficiency gene transfer into mammalian cells. In DNA Cloning, 1st edn, pp. 143-165. Edited by D. M. Glover. Oxford & Washington, DC:IRL Press.
Graham, F. L.(1984). Covalently closed circles of human adenovirus DNA are infectious.EMBO Journal3, 2917-2922.[Abstract]
Graham, F. L., Smiley, J., Russell, W. C. & Nairn, R.(1977). Characteristics of a human cell line transformed by DNA from human adenovirus type 5.Journal of General Virology36, 59-74.[Abstract]
Grifman, M., Chen, N. N., Gao, G. P., Cathomen, T., Wilson, J. M. & Weitzman, M. D.(1999). Overexpression of cyclin A inhibits augmentation of recombinant adeno-associated virus transduction by the adenovirus E4orf6 protein.Journal of Virology73, 10010-10019.
Halbert, C. L., Alexander, I. E., Wolgamot, G. M. & Miller, A. D.(1995). Adeno-associated virus vectors transduce primary cells much less efficiently than immortalized cells.Journal of Virology69, 1473-1479.[Abstract]
Jones, N. & Shenk, T.(1979). Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells.Cell17, 683-689.[Medline]
Laughlin, C. A., Cardellichio, C. B. & Coon, H. C.(1986). Latent infection of KB cells with adeno-associated virus type 2.Journal of Virology60, 515-524.[Medline]
Levine, A. J.(1997). p53, the cellular gatekeeper for growth and division.Cell88, 323-331.[Medline]
Maass, G., Bogedain, C., Scheer, U., Michl, D., Horer, M., Braun-Falco, M., Volkenandt, M., Schadendorf, D., Wendtner, C. M., Winnacker, E. L., Kotin, R. M. & Hallek, M.(1998). Recombinant adeno-associated virus for the generation of autologous, gene-modified tumor vaccines: evidence for a high transduction efficiency into primary epithelial cancer cells.Human Gene Therapy9, 1049-1059.[Medline]
McLaughlin, S. K., Collis, P., Hermonat, P. L. & Muzyczka, N.(1988). Adeno-associated virus general transduction vectors: analysis of proviral structures.Journal of Virology62, 1963-1973.[Medline]
Marton, M. J., Baim, S. B., Ornelles, D. A. & Shenk, T.(1990). The adenovirus E4 17-kilodalton protein complexes with the cellular transcription factor E2F, altering its DNA-binding properties and stimulating E1A-independent accumulation of E2 mRNA.Journal of Virology64, 2345-2359.[Medline]
Mitsudomi, T., Steinberg, S. M., Nau, M. M., Carbone, D., DAmico, D., Bodner, S., Oie, H. K., Linnoila, R. I., Mulshine, J. L. & Minna, J. D.(1992). p53 gene mutations in non-small-cell lung cancer cell lines and their correlation with the presence of ras mutations and clinical features.Oncogene7, 171-180.[Medline]
Moore, M., Horikoshi, N. & Shenk, T.(1996). Oncogenic potential of the adenovirus E4orf6 protein.Proceedings of the National Academy of Sciences, USA93, 11295-11301.
Muzyczka, N.(1992). Use of adeno-associated virus as a general transduction vector for mammalian cells.Current Topics in Microbiology and Immunology158, 97-129.[Medline]
Nevels, M., Rubenwolf, S., Spruss, T., Wolf, H. & Dobner, T.(1997). The adenovirus E4orf6 protein can promote E1A/E1B-induced focus formation by interfering with p53 tumor suppressor function.Proceedings of the National Academy of Sciences, USA94, 1206-1211.
Pilder, S., Moore, M., Logan, J. & Shenk, T.(1986). The adenovirus E1B-55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs. Molecular and Cellular Biology6, 470-476.
Querido, E., Marcellus, R. C., Lai, A., Charbonneau, R., Teodoro, J. G., Ketner, G. & Branton, P. E.(1997). Regulation of p53 levels by the E1B 55-kilodalton protein and E4orf6 in adenovirus-infected cells.Journal of Virology71, 3788-3798.[Abstract]
Richardson, W. D. & Westphal, H.(1984). Requirement for either early region 1a or early region 1b adenovirus gene products in the helper effect for adeno-associated virus.Journal of Virology51, 404-410.[Medline]
Russell, D. W. & Kay, M. A.(1999). Adeno-associated virus vectors and hematology.Blood94, 864-874.
Russell, D. W., Miller, A. D. & Alexander, I. E.(1994). Adeno-associated virus vectors preferentially transduce cells in S phase. Proceedings of the National Academy of Sciences, USA91, 8915-8919.[Abstract]
Russell, D. W., Alexander, I. E. & Miller, A. D.(1995). DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors.Proceedings of the National Academy of Sciences, USA92, 5719-5723.[Abstract]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Samulski, R. J. & Shenk, T.(1988). Adenovirus E1B 55-Mr polypeptide facilitates timely cytoplasmic accumulation of adeno-associated virus mRNAs.Journal of Virology62, 206-210.[Medline]
Samulski, R. J., Chang, L. S. & Shenk, T.(1987). A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication.Journal of Virology61, 3096-3101.[Medline]
Samulski, R. J., Chang, L. S. & Shenk, T.(1989). Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression.Journal of Virology63, 3822-3828.[Medline]
Sanlioglu, S., Duan, D. & Engelhardt, J. F.(1999). Two independent molecular pathways for recombinant adeno-associated virus genome conversion occur after UV-C and E4orf6 augmentation of transduction. Human Gene Therapy10, 591-602.[Medline]
Sarnow, P., Sullivan, C. A. & Levine, A. J.(1982). A monoclonal antibody detecting the adenovirus type 5-E1b-58Kd tumor antigen: characterization of the E1b-58Kd tumor antigen in adenovirus-infected and -transformed cells.Virology120, 510-517.[Medline]
Sarnow, P., Hearing, P., Anderson, C. W., Halbert, D. N., Shenk, T. & Levine, A. J.(1984). Adenovirus early region 1B 58000-dalton tumor antigen is physically associated with an early region 4 25000-dalton protein in productively infected cells.Journal of Virology49, 692-700.[Medline]
Schneider-Gadicke, A. & Schwarz, E.(1986). Different human cervical carcinoma cell lines show similar transcription patterns of human papillomavirus type 18 early genes.EMBO Journal5, 2285-2292.[Abstract]
Snyder, R. O., Xiao, X. & Samulski, R. J. (1996). Production of recombinant adeno-associated viral vectors. In Current Protocols in Human Genetics, 12.0.112.1.24. Edited by N. Dracopoli, J. Haines, B. Krof, C. Moir, C. Morton, C. Seidman, J. Seidman & D. Smith. USA: New York: John Wiley & Sons.
Steegenga, W. T., Riteco, N., Jochemsen, A. G., Fallaux, F. J. & Bos, J. L.(1998). The large E1B protein together with the E4orf6 protein target p53 for active degradation in adenovirus infected cells.Oncogene16, 349-357.[Medline]
Steele, R. J., Thompson, A. M., Hall, P. A. & Lane, D. P.(1998). The p53 tumour suppressor gene.British Journal of Surgery85, 1460-1467.[Medline]
Weitzman, M. D., Fisher, K. J. & Wilson, J. M.(1996). Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers.Journal of Virology70, 1845-1854.[Abstract]
Xiao, X., Li, J. & Samulski, R. J.(1996). Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector.Journal of Virology70, 8098-8108.[Abstract]
Xiao, X., Li, J., McCown, T. J. & Samulski, R. J.(1997). Gene transfer by adeno-associated virus vectors into the central nervous system.Experimental Neurology144, 113-124.[Medline]
Xiao, X., Li, J. & Samulski, R. J.(1998). Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.Journal of Virology72, 2224-2232.
Received 31 May 2000;
accepted 17 August 2000.
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