Adeno-associated virus and adenovirus coinfection induces a cellular DNA damage and repair response via redundant phosphatidylinositol 3-like kinase pathways.

During adeno-associated virus and adenovirus (AAV/Ad) coinfection, accumulation of viral genomes and proteins can alter cellular stress responses. To determine how AAV/Ad coinfection affects the host we screened over 60 cellular proteins for their responses. AAV/Ad coinfections induce a robust DNA damage response (DDR) that is distinct from that induced by Ad infection alone. Using chemical inhibitors, deficient cell lines and siRNA knockdowns of the DDR kinases, ATM, ATR and DNA-PK, we determined that DNA-PK and ATM kinases are the initial transducers of this response. AAV/Ad coinfection induces ATM- and DNA-PK mediated phosphorylation of RPA2, NBS1, H2AX and the checkpoint kinases CHK1/2. Inhibition of one or more of the DDR kinases reduces the level of phosphorylation of downstream targets but does not dramatically reduce Ad or AAV protein expression. However, AAV DNA levels are moderately affected by kinase inhibition. These experiments provide new insights into the cellular responses to AAV/Ad coinfections.

Adeno-associated virus infection of murine fibroblasts with help provided by mouse adenovirus.

Adeno-associated virus (AAV-2) replicates to high titers when host cells are coinfected with a helper virus. Here we analyzed the coinfection of AAV-2 and mouse adenovirus (MAV-1) in murine fibroblasts. We observed that AAV-2/MAV-1 coinfected NIH 3T3 cells produced approximately 10-40-fold less AAV-2 DNAse resistant particles than Hela cells. Levels of AAV-2 DNA replication were approximately 30-fold less in 3T3 cells as compared to Hela cells coinfected with human adenovirus (Ad-5). A study of these lower levels of infection in 3T3 cells compared to Hela cells revealed that receptor binding and internalization of AAV-2 in 3T3 and Hela cells was comparable. However, AAV-2 did not enter into the nucleus of mouse cells as efficiently as it does in human cells. Furthermore, viral DNA replication levels of AAV-2 DNA were found to be lower in mouse cells than human cells, indicating limitations in the murine nucleus for viral replication.

Activation of the ATPase activity of adeno-associated virus Rep68 and Rep78.

Rep68 and Rep78 DNA helicases, encoded by adeno-associated virus 2 (AAV2), are required for replication of AAV viral DNA in infected cells. They bind to imperfect palindromic elements in the inverted terminal repeat structures at the 3'- and 5'-ends of virion DNA. The ATPase activity of Rep68 and Rep78 is stimulated up to 10-fold by DNA containing the target sequence derived from the inverted terminal repeat; nontarget DNA stimulates ATPase activity at 50-fold higher concentrations. Activation of ATPase activity of Rep68 by DNA is cooperative with a Hill coefficient of 1.8 +/- 0.2. When examined by gel filtration at 0.5 M NaCl in the absence of DNA, Rep68 self-associates in a concentration-dependent manner. In the presence of DNA containing the binding element, Rep68 (and Rep78) forms protein-DNA complexes that exhibit concentration-dependent self-association in gel filtration analysis. The ATPase activity of the isolated Rep68-DNA and Rep78-DNA complexes is not activated by additional target DNA. Results of sedimentation velocity experiments in the presence of saturating target DNA are consistent with Rep68 forming a hexamer of the protein with two copies of the DNA element. Activation of the ATPase activity of Rep68 is associated with the formation of a protein-DNA oligomer.

Coupled ATP and DNA binding of adeno-associated virus Rep40 helicase.

Adeno-associated virus 2 Rep40 helicase is involved in packaging single-stranded genomic DNA into virions. ATPase activity was stimulated 5-10-fold by DNA, depending upon assay conditions. The concentration dependence of Rep40 ATPase activity in the absence and presence of DNA indicates that the monomer is inactive and that the active enzyme is at least a dimer. Binding to oligonucleotides, examined by fluorescence anisotropy, was positively cooperative and required ATP or ATPgammaS; ADP and AMPPCP did not promote binding. The cooperativity and the nucleotide requirement were also demonstrated by surface plasmon resonance. Although the Rep40 behaves as a monomer in solution, it binds to DNA as an oligomer. The requirement of a nucleotide for DNA binding and the stimulation of ATPase activity by DNA indicate that the two processes are linked. Glutaraldehyde cross-linking generated a species that migrates as a trimer on sodium dodecyl sulfate (SDS) gel electrophoresis; ATPS promoted the formation of this species and higher order oligomers. The predominant cross-linked species was a trimer in the absence of ATPgammaS, regardless of whether duplex or single-stranded DNA was present. In the presence of duplex or single-stranded DNA and ATPgammaS, glutaraldehyde cross-linking generated a species that behaved as a dimer on SDS gel elctrophoresis. Sucrose-gradient velocity sedimentation of Rep40 gave an S20,w of 3 in the absence of ligands or in the presence of a 26 bp duplex DNA. The S20,w was 3.5 in the presence of ATPgammaS and 7 and 7.6 in the presence of DNA and ATPgammaS.

Adeno-associated virus interactions with B23/Nucleophosmin: identification of sub-nucleolar virion regions.

Adeno-associated virus (AAV) is a human parvovirus that normally requires a helper virus such as adenovirus (Ad) for replication. The four replication proteins (Rep78, 68, 52 and 40) encoded by AAV are pleiotropic effectors of virus integration, replication, transcription and virion assembly. Using Rep68 column chromatography and mass spectrometry, we have identified the nucleolar, B23/Nucleophosmin (NPM) protein as an Rep-interacting partner. Rep-NPM interactions were verified by co-immunofluorescence and chemical cross-linking studies. We have found that there is demonstrable, but limited co-localization between Rep and NPM in co-infected cells. In contrast, there was significant co-localization between NPM and AAV Cap proteins. In vitro experiments using purified MBPRep78 and NPM show that NPM stimulates MBPRep78 interactions with the AAV ITR as well as endonuclease activity. These studies suggest that NPM plays a role in AAV amplification affecting Rep function and virion assembly.

Adeno-associated virus induces apoptosis during coinfection with adenovirus.

Adeno-associated virus (AAV) is a nonpathogenic parvovirus that efficiently replicates in the presence of adenovirus (Ad). Exogenous expression of the AAV replication proteins induces caspase-dependent apoptosis, but determining if AAV infection causes apoptosis during viral infection is complicated by Ad-mediated programmed cell death. To eliminate Ad-induced cytolysis, we used an E3 adenoviral death protein (ADP) mutant, pm534. AAV and pm534-coinfected cells exhibited increased cell killing compared to pm534 alone. Relative to cells infected with Ad alone, AAV and wild-type Ad-infected cells displayed decreased ADP expression, increased cytolysis until the third day of the infection, and decreased cytolysis thereafter. Biochemical and morphological characteristics of apoptosis were observed during coinfections with AAV and pm534 or Ad, including a moderate degree of caspase activation that was not present during infections with pm534 or Ad alone. AAV coinfection also increased extracellular pH. These studies suggest that AAV induces caspase-dependent and caspase-independent apoptosis.

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.

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.

Mechanisms of adeno-associated virus genome encapsidation.

The defective parvovirus, adeno-associated virus (AAV), is under close scrutiny as a human gene therapy vector. AAV's non-pathogenic character, reliance on helper virus co-infection for replication and wide tissue tropism, make it an appealing vector system. The virus' simplicity and ability to generate high titer vector preparations have contributed to its wide spread use in the gene therapy community. The single stranded AAV DNA genome is encased in a 20-25 nm diameter, icosahedral protein capsid. Assembly of AAV occurs in two distinct phases. First, the three capsid proteins, VP1-3, are rapidly synthesized and assembled into an empty virion in the nucleus. In the second, rate-limiting phase, single-strand genomic DNA is inserted into pre-formed capsids. Our rudimentary knowledge of these two phases comes from radioactive labeling pulse-chase experiments, cellular fractionation and immunocytological analysis of infected cells. Although the overall pattern of virus assembly and encapsidation is known, the biochemical mechanisms involved in these processes are not understood. Elucidation of the processes of capsid assembly and encapsidation may lead to improved vector production. While all of the parvoviruses share the characteristic icosahedral particle, differences in their surface topologies dictate different receptor binding and tissue tropism. Based on the analysis of the molecular structures of the parvoviruses and capsid mutagenesis studies, investigators have manipulated the capsid to change tissue tropism and to target different cell types, thus expanding the targeting potential of AAV vectors.

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 (Kd= 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.

AAV vector delivery to cells in culture.

Adeno-associated virus (AAV) gene delivery vectors are being investigated as vehicles for gene therapy for a wide variety of hereditary and acquired human diseases. AAV's inability to self-propagate, ability to be maintained as an episome in the transduced cell, and relatively innocuous effects on the immune system make it the vector of choice for prolonged in vivo gene expression. AAV type 2 is the most commonly used serotype for gene delivery. AAV2 vectors will deliver DNA to a wide variety of cell types. The development of vectors derived from the other five serotypes has expanded the tissue tropism of the AAV vector system. Tropism depends on the presence of cell-surface receptor elements on the target cell. For AAV2, heparin sulfate proteoglycan (7), alpha2betaV integrin (8) and the fibroblast growth factor receptor-1 (FGFR-1) (9) are believed to mediate the initial internalization steps in infection. The ubiquity of these cell-surface components confers a wide tropism on AAV2 vectors.

A biochemical characterization of the adeno-associated virus Rep40 helicase.

The human adeno-associated virus (AAV) has generated much enthusiasm as a transfer vector for human gene therapy. Although clinical gene therapy trials have been initiated using AAV vectors, much remains to be learned regarding the basic mechanisms of virus replication, gene expression, and virion assembly. AAV encodes four nonstructural, or replication (Rep), proteins. The Rep78 and Rep68 proteins regulate viral DNA replication, chromosomal integration, and gene expression. The Rep52 and Rep40 proteins mediate virus assembly. To better understand Rep protein function, we have expressed the Rep40 protein in Escherichia coli and purified it to near homogeneity. Like the other Rep proteins, Rep40 possesses helicase and ATPase activity. ATP is the best substrate, and Mg2+ is the most efficient divalent metal ion for helicase activity. A Lys to His mutation in the purine nucleotide-binding site results in a protein that inhibits helicase activity in a dominant negative manner. Rep40 unwinds double-stranded DNA containing a 3' single-stranded end, or blunt end, unlike the Rep68 and Rep52 enzymes, which have a strict requirement for DNA duplexes containing a 3' single-stranded end. Values for KATP in the ATPase assay are 1.1 +/- 0.2 mM and 1.2 +/- 0.2 mM in the absence and presence, respectively, of single-stranded DNA. Values for Vmax are 220 +/- 10 and 1,500 +/- 90 nmol/min/mg in the absence and presence, respectively, of single-stranded DNA. These studies provide the first enzymatic characterization of the AAV Rep40 protein and elucidate important functional differences between the AAV helicases.

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.

Hyper-phosphorylation of the adeno-associated virus Rep78 protein inhibits terminal repeat binding and helicase activity.

The replication (Rep) proteins of adeno-associated virus (AAV) play prominent roles in regulation of viral DNA replication, RNA transcription, assembly of an infectious virion and establishment of the provirus. We have previously demonstrated that all four Rep proteins are phosphorylated on serine residues [Virology 23 (1997) 332-336]. Reversible phosphorylation may provide a mechanism for regulating Rep protein function. To test this hypothesis, we used the phosphatase inhibitor okadaic acid (OA) to obtain hyper-phosphorylated Rep proteins. OA treatment of AAV- and adenovirus (Ad)-infected cells and baculovirus-infected insect cells at a concentration of 100 nM resulted in a significant increase in Rep protein phosphorylation. This concentration suggests that protein phosphatase 2A (PP2A) is one of the enzymes involved in regulation of Rep phosphorylation. The increased phosphorylation occurred primarily on serine residues with a detectable amount of phosphate on threonine. Hyper-phosphorylation of Rep78 resulted in reduced binding to the AAV origin of DNA replication. Hyper-phosphorylated Rep78 also had diminished helicase activity. These results suggest that regulated phosphorylation of Rep78 plays a role in controlling Rep functions in the virus replication cycle.

Characterization of adeno-associated virus rep protein inhibition of adenovirus E2a gene expression.

Adeno-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.