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1.  OneBac: Platform for Scalable and High-Titer Production of Adeno-Associated Virus Serotype 1–12 Vectors for Gene Therapy 
Human Gene Therapy  2013;25(3):212-222.
Scalable and genetically stable recombinant adeno-associated virus (rAAV) production systems combined with facile adaptability for an extended repertoire of AAV serotypes are required to keep pace with the rapidly increasing clinical demand. For scalable high-titer production of the full range of rAAV serotypes 1–12, we developed OneBac, consisting of stable insect Sf9 cell lines harboring silent copies of AAV1–12 rep and cap genes induced upon infection with a single baculovirus that also carries the rAAV genome. rAAV burst sizes reach up to 5×105 benzonase-resistant, highly infectious genomic particles per cell, exceeding typical yields of current rAAV production systems. In contrast to recombinant rep/cap baculovirus strains currently employed for large-scale rAAV production, the Sf9rep/cap cell lines are genetically stable, leading to undiminished rAAV burst sizes over serial passages. Thus, OneBac combines full AAV serotype options with the capacity for stable scale-up production, the current bottleneck for the transition of AAV from gene therapy trials to routine clinical treatment.
PMCID: PMC3955967  PMID: 24299301
2.  Antibody-Mediated Enhancement of Parvovirus B19 Uptake into Endothelial Cells Mediated by a Receptor for Complement Factor C1q 
Journal of Virology  2014;88(14):8102-8115.
Despite its strong host tropism for erythroid progenitor cells, human parvovirus B19 (B19V) can also infect a variety of additional cell types. Acute and chronic inflammatory cardiomyopathies have been associated with a high prevalence of B19V DNA in endothelial cells of the myocardium. To elucidate the mechanisms of B19V uptake into endothelium, we first analyzed the surface expression of the well-characterized primary B19V receptor P antigen and the putative coreceptors α5β1 integrins and Ku80 antigen on primary and permanent endothelial cells. The receptor expression pattern and also the primary attachment levels were similar to those in the UT7/Epo-S1 cell line regarded as functional for B19V entry, but internalization of the virus was strongly reduced. As an alternative B19V uptake mechanism in endothelial cells, we demonstrated antibody-dependent enhancement (ADE), with up to a 4,000-fold increase in B19V uptake in the presence of B19V-specific human antibodies. ADE was mediated almost exclusively at the level of virus internalization, with efficient B19V translocation to the nucleus. In contrast to monocytes, where ADE of B19V has been described previously, enhancement does not rely on interaction of the virus-antibody complexes with Fc receptors (FcRs), but rather, involves an alternative mechanism mediated by the heat-sensitive complement factor C1q and its receptor, CD93. Our results suggest that ADE represents the predominant mechanism of endothelial B19V infection, and it is tempting to speculate that it may play a role in the pathogenicity of cardiac B19V infection.
IMPORTANCE Both efficient entry and productive infection of human parvovirus B19 (B19V) seem to be limited to erythroid progenitor cells. However, in vivo, the viral DNA can also be detected in additional cell types, such as endothelial cells of the myocardium, where its presence has been associated with acute and chronic inflammatory cardiomyopathies. In this study, we demonstrated that uptake of B19V into endothelial cells most probably does not rely on the classical receptor-mediated route via the primary B19V receptor P antigen and coreceptors, such as α5β1 integrins, but rather on antibody-dependent mechanisms. Since the strong antibody-dependent enhancement (ADE) of B19V entry requires the CD93 surface protein, it very likely involves bridging of the B19V-antibody complexes to this receptor by the complement factor C1q, leading to enhanced endocytosis of the virus.
PMCID: PMC4097764  PMID: 24807719
3.  Differential Adeno-Associated Virus Serotype-Specific Interaction Patterns with Synthetic Heparins and Other Glycans 
Journal of Virology  2014;88(5):2991-3003.
All currently identified primary receptors of adeno-associated virus (AAV) are glycans. Depending on the AAV serotype, these carbohydrates range from heparan sulfate proteoglycans (HSPG), through glycans with terminal α2-3 or α2-6 sialic acids, to terminal galactose moieties. Receptor identification has largely relied on binding to natural compounds, defined glycan-presenting cell lines, or enzyme-mediated glycan modifications. Here, we describe a comparative binding analysis of highly purified, fluorescent-dye-labeled AAV vectors of various serotypes on arrays displaying over 600 different glycans and on a specialized array with natural and synthetic heparins. Few glycans bind AAV specifically in a serotype-dependent manner. Differential glycan binding was detected for the described sialic acid-binding AAV serotypes 1, 6, 5, and 4. The natural heparin binding serotypes AAV2, -3, -6, and -13 displayed differential binding to selected synthetic heparins. AAV7, -8, -rh.10, and -12 did not bind to any of the glycans present on the arrays. For discrimination of AAV serotypes 1 to 6 and 13, minimal binding moieties are identified. This is the first study to differentiate the natural mixed heparin binding AAV serotypes 2, 3, 6, and 13 by differential binding to specific synthetic heparins. Also, sialic acid binding AAVs display differential glycan binding specificities. The findings are relevant for further dissection of AAV host cell interaction. Moreover, the definition of single AAV-discriminating glycan binders opens the possibility for glycan microarray-based discrimination of AAV serotypes in gene therapy.
PMCID: PMC3958061  PMID: 24371066
4.  The Nontoxic Cell Cycle Modulator Indirubin Augments Transduction of Adeno-Associated Viral Vectors and Zinc-Finger Nuclease-Mediated Gene Targeting 
Human Gene Therapy  2012;24(1):67-77.
Parameters that regulate or affect the cell cycle or the DNA repair choice between non-homologous end-joining and homology-directed repair (HDR) are excellent targets to enhance therapeutic gene targeting. Here, we have evaluated the impact of five cell-cycle modulating drugs on targeted genome engineering mediated by DNA double-strand break (DSB)-inducing nucleases, such as zinc-finger nucleases (ZFNs). For a side-by-side comparison, we have established four reporter cell lines by integrating a mutated EGFP gene into either three transformed human cell lines or primary umbilical cord–derived mesenchymal stromal cells (UC-MSCs). After treatment with different cytostatic drugs, cells were transduced with adeno-associated virus (AAV) vectors that encode a nuclease or a repair donor to rescue EGFP expression through DSB-induced HDR. We show that transient cell-cycle arrest increased AAV transduction and AAV-mediated HDR up to six-fold in human cell lines and ten-fold in UC-MSCs, respectively. Targeted gene correction was observed in up to 34% of transduced cells. Both the absolute and the relative gene-targeting frequencies were dependent on the cell type, the cytostatic drug, the vector dose, and the nuclease. Treatment of cells with the cyclin-dependent kinase inhibitor indirubin-3′-monoxime was especially promising as this compound combined high stimulatory effects with minimal cytotoxicity. In conclusion, indirubin-3′-monoxime significantly improved AAV transduction and the efficiency of AAV/ZFN-mediated gene targeting and may thus represent a promising compound to enhance DSB-mediated genome engineering in human stem cells, such as UC-MSCs, which hold great promise for future clinical applications.
Rahman and colleagues evaluate the impact of five cell cycle-modulating drugs on targeted genome engineering mediated by zinc-finger nucleases (ZFNs). They show that treatment of cells with the cyclin-dependent kinase inhibitor indirubin-3′-monoxime is especially promising as this compound significantly improves AAV transduction and the efficiency of AAV/ZFN-mediated gene targeting in vitro.
PMCID: PMC3555098  PMID: 23072634
5.  Versatile and Efficient Genome Editing in Human Cells by Combining Zinc-Finger Nucleases With Adeno-Associated Viral Vectors 
Human Gene Therapy  2011;23(3):321-329.
Zinc-finger nucleases (ZFNs) have become a valuable tool for targeted genome engineering. Based on the enzyme's ability to create a site-specific DNA double-strand break, ZFNs promote genome editing by activating the cellular DNA damage response, including homology-directed repair (HDR) and nonhomologous end-joining. The goal of this study was (i) to demonstrate the versatility of combining the ZFN technology with a vector platform based on adeno-associated virus (AAV), and (ii) to assess the toxicity evoked by this platform. To this end, human cell lines that harbor enhanced green fluorescence protein (EGFP) reporters were generated to easily quantify the frequencies of gene deletion, gene disruption, and gene correction. We demonstrated that ZFN-encoding AAV expression vectors can be employed to induce large chromosomal deletions or to disrupt genes in up to 32% of transduced cells. In combination with AAV vectors that served as HDR donors, the AAV-ZFN platform was utilized to correct a mutation in EGFP in up to 6% of cells. Genome editing on the DNA level was confirmed by genotyping. Although cell cycle profiling revealed a modest G2/M arrest at high AAV-ZFN vector doses, platform-induced apoptosis could not be detected. In conclusion, the combined AAV-ZFN vector technology is a useful tool to edit the human genome with high efficiency. Because AAV vectors can transduce many cell types relevant for gene therapy, the ex vivo and in vivo delivery of ZFNs via AAV vectors will be of great interest for the treatment of inherited disorders.
Händel and colleagues characterize the genome-editing ability and toxicity of adeno-associated viral (AAV) vectors encoding zinc finger nucleases (ZFNs). ZFN-encoding AAV vectors induce large chromosomal deletions and disrupt genes in up to 32% of transduced cells harboring an enhanced green fluorescent protein (EGFP) reporter gene, and can correct an EGFP mutation in up to 6% of cells when combined with AAV vectors serving as homology-directed repair donors. No cellular apoptosis was detected.
PMCID: PMC3300077  PMID: 21980922
6.  Roles of E4orf6 and VA I RNA in Adenovirus-Mediated Stimulation of Human Parvovirus B19 DNA Replication and Structural Gene Expression 
Journal of Virology  2012;86(9):5099-5109.
Despite its very narrow tropism for erythroid progenitor cells, human parvovirus B19 (B19V) has recently been shown to replicate and form infectious progeny virus in 293 cells in the presence of early adenoviral functions provided either by infection with adenovirus type 5 or by addition of the pHelper plasmid encoding the E2a, E4orf6, and VA RNA functions. In the present study we dissected the individual influence of these functions on B19V genome replication and expression of structural proteins VP1 and VP2. We show that, in the presence of the constitutively expressed E1A and E1B, E4orf6 alone is able to promote B19V DNA replication, resulting in a concomitant increase in VP expression levels. The stimulatory effects of E4orf6 require the integrity of the BC box motifs, which target cellular proteins such as p53 and the Mre11 DNA repair complex for proteosomal degradation through formation of an E3 ubiquitin ligase complex with E1B. VA RNA also strongly induces VP expression but, in contrast to E4orf6, in a replication-independent manner. This stimulation could be attributed exclusively to the VA I RNA transcript and does not involve major activating effects at the level of the B19V p6 promoter, but the nucleotide residues required for the well-defined pathway of VA I RNA mediated stimulation of translation through functional inactivation of protein kinase R. These data show that the cellular pathways regulating B19V replication may be very similar to those governing the productive cycle of the helper-dependent parvoviruses, the adeno-associated viruses.
PMCID: PMC3347340  PMID: 22357277
8.  DNA-Binding Activity of Adeno-Associated Virus Rep Is Required for Inverted Terminal Repeat-Dependent Complex Formation with Herpes Simplex Virus ICP8 
Journal of Virology  2012;86(5):2859-2863.
Herpes simplex virus (HSV) helper functions for (AAV) replication comprise HSV ICP8 and helicase-primase UL5/UL52/UL8. Here we show that N-terminal amino acids of AAV Rep78 that contact the Rep-binding site within the AAV inverted terminal repeat (ITR) are required for ternary-complex formation with infected-cell protein 8 (ICP8) on AAV single-strand DNA (ssDNA) in vitro and for colocalization in nuclear replication domains in vivo. Our data suggest that HSV-dependent AAV replication is initiated by Rep contacting the AAV ITR and by cooperative binding of ICP8 on AAV ssDNA.
PMCID: PMC3302247  PMID: 22205745
9.  Selection-Independent Generation of Gene Knockout Mouse Embryonic Stem Cells Using Zinc-Finger Nucleases 
PLoS ONE  2011;6(12):e28911.
Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10−6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.
PMCID: PMC3237556  PMID: 22194948
12.  The Central and Basolateral Amygdala Are Critical Sites of Neuropeptide Y/Y2 Receptor-Mediated Regulation of Anxiety and Depression 
Anxiety is integrated in the amygdaloid nuclei and involves the interplay of the amygdala and various other areas of the brain. Neuropeptides play a critical role in regulating this process. Neuropeptide Y (NPY), a 36 aa peptide, is highly expressed in the amygdala. It exerts potent anxiolytic effects through cognate postsynaptic Y1 receptors, but augments anxiety through presynaptic Y2 receptors. To identify the precise anatomical site(s) of Y2-mediated anxiogenic action, we investigated the effect of site-specific deletion of the Y2 gene in amygdaloid nuclei on anxiety and depression-related behaviors in mice. Ablating the Y2 gene in the basolateral and central amygdala resulted in an anxiolytic phenotype, whereas deletion in the medial amygdala or in the bed nucleus of the stria terminalis had no obvious effect on emotion-related behavior. Deleting the Y2 receptor gene in the central amygdala, but not in any other amygdaloid nucleus, resulted in an added antidepressant-like effect. It was associated with a reduction of presumably presynaptic Y2 receptors in the stria terminalis/bed nucleus of the stria terminalis, the nucleus accumbens, and the locus ceruleus. Our results are evidence of the highly site-specific nature of the Y2-mediated function of NPY in the modulation of anxiety- and depression-related behavior. The activity of NPY is likely mediated by the presynaptic inhibition of GABA and/or NPY release from interneurons and/or efferent projection neurons of the basolateral and central amygdala.
PMCID: PMC3073168  PMID: 20445054
13.  Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion 
Nucleic Acids Research  2010;38(22):8269-8276.
Zinc-finger nucleases (ZFNs) have been successfully used for rational genome engineering in a variety of cell types and organisms. ZFNs consist of a non-specific FokI endonuclease domain and a specific zinc-finger DNA-binding domain. Because the catalytic domain must dimerize to become active, two ZFN subunits are typically assembled at the cleavage site. The generation of obligate heterodimeric ZFNs was shown to significantly reduce ZFN-associated cytotoxicity in single-site genome editing strategies. To further expand the application range of ZFNs, we employed a combination of in silico protein modeling, in vitro cleavage assays, and in vivo recombination assays to identify autonomous ZFN pairs that lack cross-reactivity between each other. In the context of ZFNs designed to recognize two adjacent sites in the human HOXB13 locus, we demonstrate that two autonomous ZFN pairs can be directed simultaneously to two different sites to induce a chromosomal deletion in ∼10% of alleles. Notably, the autonomous ZFN pair induced a targeted chromosomal deletion with the same efficacy as previously published obligate heterodimeric ZFNs but with significantly less toxicity. These results demonstrate that autonomous ZFNs will prove useful in targeted genome engineering approaches wherever an application requires the expression of two distinct ZFN pairs.
PMCID: PMC3001086  PMID: 20716517
14.  Integration Preferences of Wildtype AAV-2 for Consensus Rep-Binding Sites at Numerous Loci in the Human Genome 
PLoS Pathogens  2010;6(7):e1000985.
Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of rep-deficient AAV vectors revealed a highly significant overrepresentation of integration events near to consensus RBS. Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome.
Author Summary
This is the first unbiased genome-wide analysis of wildtype AAV integration combined with a thorough bioinformatic analysis of preferred genomic motifs and patterns in the neighbourhood of the integration sites identified. The preference of Rep-dependent AAV integration near multiple consensus Rep-binding sites was lost in the case of AAV vector integration in the absence of Rep expression. Our findings challenge the commonly accepted notion of site-specific AAV targeting to AAVS1 on chromosome 19q13.42. Although AAVS1 contains a canonical Rep-binding site, numerous additional sites including the newly identified hotspots AAVS2 on chromosome 5p13.3 and AAVS3 on chromosome 3p24.3 harbour functional Rep-binding sites suitable for AAV integration. AAV vectors are quickly moving forward in the clinic and Rep-dependent vector targeting strategies are being actively pursued. Detailed information of AAV wildtype versus recombinant AAV vector integration sites and preferences are needed to evaluate the safety profile of AAV vectors in gene therapy.
PMCID: PMC2900306  PMID: 20628575
16.  Role of the Herpes Simplex Virus Helicase-Primase Complex during Adeno-Associated Virus DNA Replication 
Journal of Virology  2006;80(11):5241-5250.
A subset of DNA replication proteins of herpes simplex virus (HSV) comprising the single-strand DNA-binding protein, ICP8 (UL29), and the helicase-primase complex (UL5, UL8, and UL52 proteins) has previously been shown to be sufficient for the replication of adeno-associated virus (AAV). We recently demonstrated complex formation between ICP8, AAV Rep78, and the single-stranded DNA AAV genome, both in vitro and in the nuclear HSV replication domains of coinfected cells. In this study the functional role(s) of HSV helicase and primase during AAV DNA replication were analyzed. To differentiate between their necessity as structural components of the HSV replication complex or as active enzymes, point mutations within the helicase and primase catalytic domains were analyzed. In two complementary approaches the remaining HSV helper functions were either provided by infection with HSV mutants or by plasmid transfection. We show here that upon cotransfection of the minimal four HSV proteins (i.e., the four proteins constituting the minimal requirements for basal AAV replication), UL52 primase catalytic activity was not required for AAV DNA replication. In contrast, UL5 helicase activity was necessary for fully efficient replication. Confocal microscopy confirmed that all mutants retained the ability to support formation of ICP8-positive nuclear replication foci, to which AAV Rep78 colocalized in a manner strictly dependent on the presence of AAV single-stranded DNA (ssDNA). The data indicate that recruitment of AAV Rep78 and ssDNA to nuclear replication sites by the four HSV helper proteins is maintained in the absence of catalytic primase or helicase activities and suggest an involvement of the HSV UL5 helicase activity during AAV DNA replication.
PMCID: PMC1472166  PMID: 16699004
17.  ssDNA-dependent colocalization of adeno-associated virus Rep and herpes simplex virus ICP8 in nuclear replication domains 
Nucleic Acids Research  2003;31(21):6206-6213.
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.
PMCID: PMC275469  PMID: 14576307
18.  Packaging of Human Chromosome 19-Specific Adeno-Associated Virus (AAV) Integration Sites in AAV Virions during AAV Wild-Type and Recombinant AAV Vector Production 
Journal of Virology  2003;77(8):4881-4887.
Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus on human chromosome 19, called AAVS1. During the development of a sensitive real-time PCR assay for site-specific integration, AAV-AAVS1 junctions were reproducibly detected in highly purified AAV wild-type and recombinant AAV vector stocks. A series of controls documented that the junctions were packaged in AAV capsids and were newly generated during a single round of AAV production. Cloned junctions displayed variable AAV sequences fused to AAVS1. These data suggest that packaged junctions represent footprints of AAV integration during productive infection. Apparently, AAV latency established by site-specific integration and the helper virus-dependent, productive AAV cycle are more closely related than previously thought.
PMCID: PMC152110  PMID: 12663794
19.  Kinetics and Frequency of Adeno-Associated Virus Site-Specific Integration into Human Chromosome 19 Monitored by Quantitative Real-Time PCR 
Journal of Virology  2002;76(15):7554-7559.
Adeno-associated virus type 2 (AAV-2) integrates specifically into a site on human chromosome 19 (chr-19) called AAVS1. To study the kinetics and frequency of chr-19-specific integration after AAV infection, we developed a rapid, sensitive, and quantitative real-time PCR assay for AAV inverted terminal repeat-chr-19-specific junctions. Despite the known variability of junction sites, conditions were established that ensured reliable quantification of integration rates within hours after AAV infection. The overall integration frequency was calculated to peak at between 10 and 20% of AAV-infected, unselected HeLa cells. At least 1 in 1,000 infectious AAV-2 particles was found to integrate site specifically up to day 4 postinfection in the absence of selection. Chromosomal breakpoints within AAVS1 agreed with those found in latently infected clonal cell lines and transgenic animals. Use of this quantitative real-time PCR will greatly facilitate the study of the early steps of wild-type and recombinant AAV vector integration.
PMCID: PMC136374  PMID: 12097568
20.  High Variability between Results of Different In-House Tests for Cytomegalovirus (CMV) Monitoring and a Standardized Quantitative Plasma CMV PCR Assay 
Journal of Clinical Microbiology  2002;40(6):2285-2287.
A total of 2,718 blood samples were analyzed in five virological laboratories for the presence of cytomegalovirus (CMV) by in-house tests and one standardized plasma PCR assay. Results from in-house tests showed remarkable variability. Detection of CMV pp65 antigen or DNA from cells was more sensitive than that by plasma CMV PCR assay.
PMCID: PMC130743  PMID: 12037112
21.  The Adeno-Associated Virus Type 2 Regulatory Proteins Rep78 and Rep68 Interact with the Transcriptional Coactivator PC4 
Journal of Virology  1999;73(1):260-269.
The adeno-associated virus type 2 (AAV-2) Rep78/Rep68 regulatory proteins are pleiotropic effectors of viral and cellular DNA replication, of cellular transformation by viral and cellular oncogenes, and of homologous and heterologous gene expression. To search for cellular proteins involved in mediating these functions, we used Rep68 as bait in the yeast two-hybrid system and identified the transcriptional coactivator PC4 as a Rep interaction partner. PC4 has been shown to mediate transcriptional activation by a variety of sequence-specific transcription factors in vitro. Rep amino acids 172 to 530 were sufficient and amino acids 172 to 224 were absolutely necessary for the interaction with PC4. The PC4 domains required for interaction were mapped to the C-terminal single-stranded DNA-binding domain of PC4. In glutathione S-transferase (GST) pull-down assays, in vitro-transcribed and -translated Rep78 or Rep68 proteins were bound specifically by GST-PC4 fusion proteins. Similarly, PC4 expressed in Escherichia coli was bound by GST-Rep fusion proteins, confirming the direct interaction between Rep and PC4 in vitro. Rep was found to have a higher affinity for the nonphosphorylated, transcriptionally active form of PC4 than for the phosphorylated, transcriptionally inactive form. The latter is predominant in nuclear extracts of HeLa or 293 cells. In the yeast system, but not in vitro, Rep-PC4 interaction was disrupted by a point mutation in the putative nucleotide-binding site of Rep68, suggesting that a stable interaction between Rep and PC4 in vivo is ATP dependent. This mutation has also been shown to impair Rep function in AAV-2 DNA replication and in inhibition of gene expression and inducible DNA amplification. Cytomegalovirus promoter-driven overexpression of PC4 led to transient accumulation of nonphosphorylated PC4 with concomitant downregulation of all three AAV-2 promoters in the absence of helper virus. In the presence of adenovirus, this effect was relieved. These results imply an involvement of the transcriptional coactivator PC4 in the regulation of AAV-2 gene expression in the absence of helper virus.
PMCID: PMC103830  PMID: 9847329

Results 1-21 (21)