Characterization of Adeno-Associated Virus Rep Protein Inhibition of Adenovirus E2a Gene Expression

AbstractAdeno-associated virus (AAV) replication (Rep) proteins are pleiotropic effectors of viral DNA replication, RNA transcription, and site-specific integration into chromosome 19. In addition to regulating AAV gene expression, the Rep proteins modulate expression of a variety of cellular and viral genes. In this report we investigate Rep-mediated effects on expression of the adenovirus (Ad) E2a gene and the Ad major late promoter. We have found that all four Rep proteins repress E2a expression at the protein level, with Rep40 showing the weakest repression. Mutations in the purine nucleotide binding (PNB) site weakened each of the protein's abilities to repress expression. Analysis of steady-state E2a mRNA showed that Rep proteins decreased mRNA levels, but to a lesser extent than E2a protein levels. Analysis of mRNA stability demonstrated that neither Rep78 nor Rep52 affected E2a mRNA stability, suggesting that the decrease in mRNA is due to Rep-mediated inhibition of Ad E2a transcription. To determine if Rep68 proteins could directly inhibit RNA transcription, we performed in vitro transcription assays using HeLa nuclear extracts supplemented with Rep68 and Rep68PNB. We demonstrate that Rep68, but not mutant Rep68PNB, blocked in vitro transcription of a template containing the Ad major late promoter. These results provide insight into how AAV and its encoded Rep proteins interact with Ad and provide a model system for the study of AAV and host-cell interactions

Enhancement of Recombinant Adeno-Associated Virus Type 2-Mediated Transgene Expression in a Lung Epithelial Cell Line by Inhibition of the Epidermal Growth Factor Receptor

Recombinant adeno-associated viruses (rAAVs) have attracted considerable interest as gene delivery systems because they show long-term expression in vivo and transduce numerous cell types. Limitations to successful gene transduction from rAAVs have prompted investigations of a variety of treatments to enhance transgene expression from rAAV vectors. Tyrphostin-1, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, dramatically enhances rAAV transgene expression. Elegant studies have demonstrated that a single-strand D-sequence-binding protein (ssDBP) is phosphorylated by EGFR and binds to the D sequence element in the AAV terminal repeat (TR). Binding of the Tyr-phosphorylated ssDBP prevents conversion of single-stranded vector DNA to a double-strand conformation. We observed dramatic increases in transgene expression in lung epithelial cells (IB3) with tyrphostin treatment. Gel shift analysis of ssDBP revealed that its DNA binding characteristics were unchanged after tyrphostin treatment or adenovirus infection. Tyrphostin stimulated rAAV transgene expression to a greater extent than adenovirus coinfection. Southern hybridizations revealed that the vector DNA remained in the single-strand conformation in tyrphostin-treated cells but double-stranded replicative form monomer DNA was most abundant in adenovirus-infected cells. Northern analyses revealed that tyrphostin treatment enhanced mRNA accumulation more than in adenovirus-infected cultures even though replicative form DNA was undetectable. Analysis of the JNK, ERK, and p38K mitogen-activated protein kinase pathways revealed that tyrphostin treatment stimulated the activity of JNK and p38K. Our data suggest that tyrphostin-induced alteration of stress response pathways results in dramatic enhancement of transcription on linear vector DNA templates in the IB3 cell line. These results expand the downstream targets of the EGFR in regulating rAAV transduction

Effects of Adeno-Associated Virus on Adenovirus Replication and Gene Expression during Coinfection

Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10- to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the E3 gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis

Identification of an Adeno-Associated Virus Rep Protein Binding Site in the Adenovirus E2a Promoter

Adeno-associated virus (AAV) and other parvoviruses inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we investigated AAV's interaction with adenovirus (Ad), AAV's most efficient helper virus. Coinfection with Ad and AAV results in an AAV-mediated inhibition of Ad5 gene expression and replication. The AAV replication proteins (Rep) activate and repress gene expression from AAV and heterologous transcription promoters. To investigate the role of Rep proteins in the suppression of Ad propagation, we performed chromatin immunoprecipitation analyses that demonstrated in vivo AAV Rep protein interaction with the Ad E2a gene promoter. In vitro binding of purified AAV Rep68 protein to the Ad E2a promoter was characterized by electrophoretic mobility shift assays (K(d) = 200 ± 25 nM). A 38 bp, Rep68-protected region (5′-TAAGAGTCAGCGCGCAGTATTTACTGAAGAGAGCCT-3′) was identified by DNase I footprint analysis. The 38-bp protected region contains the weak E2a TATA box, sequence elements that resemble the Rep binding sites identified by random sequence oligonucleotide selection, and the transcription start site. These results suggest that Rep binding to the E2a promoter contributes to the inhibition of E2a gene expression from the Ad E2a promoter and may affect Ad replication

Adeno-Associated Virus Rep Protein-Mediated Inhibition of Transcription of the Adenovirus Major Late Promoter In Vitro

Adeno-associated virus (AAV) is a human parvovirus that normally requires a helper virus such as adenovirus (Ad) for replication. The four AAV replication proteins (Rep78, Rep68, Rep52, and Rep40) are pleiotropic effectors of virus integration, replication, transcription, and virion assembly. These proteins exert effects on Ad gene expression and replication. In transient plasmid transfection assays, Rep proteins inhibit gene expression from a variety of transcription promoters. We have examined Rep protein-mediated inhibition of transcription of the Ad major late transcription promoter (AdMLP) in vitro. Rep78/68 are the strongest transcription suppressors and the purine nucleotide binding site in the Rep proteins, and by implication, the ATPase activity or conformational change induced by nucleotide binding is required for full repression. Rep52 has modest effects, and Rep40 exerts no significant effect on transcription. Rep78/68 and their N-terminal 225-residue domain bind to a 55-bp AdMLP DNA fragment in gel shift assays, suggesting that protein-DNA interactions are required for inhibition. This interaction was confirmed in DNase I protection assays and maps to a region extending from the TATA box to the transcription initiation site. Gel shift, DNase I, and chemical cross-linking assays with TATA box-binding protein (TBP) and Rep68 indicate that both proteins interact with each other and with the promoter at adjacent sites. The demonstration of Rep interaction with TBP and the AdMLP suggests that Rep78/68 alter the preinitiation complex of RNA polymerase II transcription. These observations provide new insight into the mechanism of Rep-mediated inhibition of gene expression

The impact of mitotane therapy on serum free proteins in patients with adrenocortical carcinoma

Adrenocortical Carcinoma (ACC) is a rare malignancy of the adrenal cortex. Whilst surgery is the preferred treatment, adjunctive therapy with mitotane may be offered post-surgically to minimise the risk of recurrence or in the absence of surgery to attenuate progression.Aims: To evaluate the effects of mitotane treatment on serum protein concentrations in patients treated for ACC with mitotane therapy and compare this to patients with other adrenal neoplasms and a normal pregnant cohort.Methods: Serum cortisol, thyroid function tests, adrenocorticotrophic hormone (ACTH), cortisol binding globulin (CBG), thyroxine-binding globulin (TBG), gonadotrophins and androgens were measured on plasma and serum samples. Thirty-five patients with ACC were included, and mitotane levels noted to be sub-/supra-/therapeutic. Data were tested for normality, reported as Means ± SD, and compared to other two cohorts using paired-sample t-test with 5% p-value for significance and 95% confidence interval (CI).Results: Patients on mitotane therapy had higher mean serum CBG concentration compared to the adrenal neoplasm group (sub-therapeutic: 79.5 (95% CI:33.6, 125.4nmol/L), therapeutic: 85.3 (95% CI:37.1-133.6nmol/L), supra-therapeutic: 75.7 (95% CI: -19.3,170.6nmol/L): adrenal neoplasm 25.5 (95% CI:17.5,33.5nmol/L). Negative correlations between serum cortisol and CBG concentration were demonstrated within the supra-/therapeutic plasma mitotane and adrenal neoplasm groups.Conclusion: Patients with ACC and therapeutic plasma mitotane concentrations had higher serum CBG concentrations compared to those with adrenal neoplasms or pregnant women, and higher serum cortisol. While there was no direct correlation with cortisol and mitotane level, the negative correlation of cortisol with CBG may suggest that the direct effect of mitotane in increasing cortisol may also reflect that mitotane has a direct adrenolytic effect