Positive and negative effects of adenovirus type 5 helper functions on adeno-associated virus type 5 (AAV5) protein accumulation govern AAV5 virus production

Full replication of adeno-associated virus type 5 (AAV5) is sustained by adenovirus type 5 (Ad5) helper functions E1a, E1b, E2a, E4Orf6, and virus-associated (VA) RNA; however, their combined net enhancement of AAV5 replication was comprised of both positive and negative individual effects. Although Ad5 E4Orf6 was required for AAV5 genomic DNA replication, it also functioned together with E1b to degrade de novo-expressed, preassembled AAV5 capsid proteins and Rep52 in a proteosome-dependent manner. VA RNA enhanced accumulation of AAV5 protein, overcoming the degradative effects of E4Orf6, and was thus required to restore adequate amounts of AAV5 proteins necessary to achieve efficient virus production.     

The Mre11/Rad50/Nbs1 complex limits adeno-associated virus transduction and replication

Adeno-associated virus (AAV) is a parvovirus with a small single-stranded DNA genome that relies on cellular replication machinery together with functions supplied by coinfecting helper viruses. The impact of host factors on AAV infection is not well understood. We explored the connection between AAV helper functions supplied by adenovirus and cellular DNA repair proteins. The adenoviral E1b55K/E4orf6 proteins induce degradation of the cellular Mre11 repair complex (MRN) to promote productive adenovirus infection. These viral proteins also augment recombinant AAV transduction and provide crucial helper functions for wild-type AAV replication. Here, we show that MRN poses a barrier to AAV and that the helper function provided by E1b55K/E4orf6 involves MRN degradation. Using a fluorescent method to visualize the viral genome, we show an effect at the viral DNA level. MRN components accumulate at AAV replication centers and recognize the viral inverted terminal repeats. Together, our data suggest that AAV is targeted by MRN and has evolved to exploit adenoviral proteins that degrade these cellular factors.     

Nuclear export of the E1B 55-kDa and E4 34-kDa adenoviral oncoproteins mediated by a rev-like signal sequence.

The E1B 55-kDa and E4 34-kDa oncoproteins of adenovirus type 5 (abbreviated here as E1B-55kD and E4-34kD) promote the export of viral mRNA and inhibit the export of most cellular mRNA species. We show that the intracellular complex containing E1B-55kD and E4-34kD continuously shuttles between the nucleus and the cytoplasm, and may thus serve as a nucleocytoplasmic transporter for viral mRNA. We present evidence that within this complex, it is the E4-34kD protein that directs both nuclear import and nuclear export. E4-34kD contains a functional nuclear export signal similar to corresponding sequences found in the retroviral proteins rev and rex. This sequence element is required for nuclear export of the complex, and it can function autonomously when fused to a carrier protein and microinjected in HeLa cell nuclei. When E4-34kD is expressed alone, a portion of the protein that contains a predicted arginine-rich amphipathic alpha-helical structure mediates nuclear retention of the protein. This retention, however, can be abolished by the association with E1B-55kD or by a specific point mutation within the arginine-rich motif. The export of E4-34kD can be blocked by an HTLV-rex derived competitive inhibitor and overexpressed E4-34kD inhibits rev-mediated transport, suggesting that the export pathways accessed by the adenoviral and retroviral proteins share components. The interplay between two polypeptides as well as the involvement of a dominant nuclear retention domain are novel features that might contribute to the efficiency and regulation of the adenovirus export system.     

Adeno-associated virus replication. The effect of L-canavanine or a helper virus mutation on accumulation of viral capsids and progeny single-stranded DNA

We have studied the relationship between adeno-associated virus (AAV) DNA replication and virus particle assembly. Formation of empty or full particles and accumulation of AAV capsid proteins was prevented in the presence of the arginine analogue, L-canavanine, or when a temperature-sensitive helper adenovirus was used at the nonpermissive temperature. In each case there was a concomitant inhibition of AAV single-stranded (progeny) DNA accumulation but little or no effect upon synthesis of AAV duplex, replicating form DNA. These results indicate that AAV protein, perhaps in the form of assembled capsids, is required for AAV single-stranded progeny DNA accumulation.     

Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression.

A method is described for the production of recombinant adeno-associated virus (AAV) stocks that contain no detectable wild-type helper AAV. The recombinant viruses contained only the terminal 191 nucleotides of the AAV chromosome bracketing a nonviral marker gene. trans-Acting AAV functions were provided by a helper DNA in which the terminal 191 nucleotides of the AAV chromosome were substituted with adenovirus terminal sequences. Although the helper DNA did not appear to replicate, it expressed AAV functions at a substantially higher level than did DNA molecules that contained neither AAV nor adenovirus termini. Since the recombinant viruses with AAV termini contained no sequence homology to the helper DNA, no wild-type AAV was generated by homologous recombination within infected cells. Since the terminal region of the AAV chromosome is required for replication and encapsidation, only recombinant DNAs were amplified and packaged into AAV virions. When human cells were infected at a high multiplicity with a recombinant virus carrying a drug resistance marker gene, approximately 70% of the infected cells gave rise to colonies stably expressing the marker. The recombinant virus gene was then used to generate drug-resistant human cell lines subsequent to infection. These cells contained stably integrated copies of the recombinant viral DNA which could be excised, replicated, and encapsidated by infection with wild-type AAV plus adenovirus. Thus, AAV gene expression is not required for normal integration of an infecting DNA containing AAV termini.     

Regulation of adeno-associated virus DNA replication by the cellular TAF-I/set complex

The Rep proteins of the adeno-associated virus (AAV) are required for viral replication in the presence of adenovirus helper functions and as yet poorly characterized cellular factors. In an attempt to identify such factors, we purified Flag-Rep68-interacting proteins from human cell lysates. Several polypeptides were identified by mass spectrometry, among which was ANP32B, a member of the acidic nuclear protein 32 family which takes part in the formation of the template-activating factor I/Set oncoprotein (TAF-I/Set) complex. The N terminus of Rep was found to specifically bind the acidic domain of ANP32B; through this interaction, Rep was also able to recruit other members of the TAF-I/Set complex, including the ANP32A protein and the histone chaperone TAF-I/Set. Further experiments revealed that silencing of ANP32A and ANP32B inhibited AAV replication, while overexpression of all of the components of the TAF-I/Set complex increased de novo AAV DNA synthesis in permissive cells. Besides being the first indication that the TAF-I/Set complex participates in wild-type AAV replication, these findings have important implications for the generation of recombinant AAV vectors since overexpression of the TAF-I/Set components was found to markedly increase viral vector production.     

Analysis of adenovirus early region 4-encoded polypeptides synthesized in productively infected cells.

Peptide-specific antisera were developed to analyze the products encoded by adenovirus type 5 early region 4 (E4) open reading frames 6 and 7. Reading frame 6 previously was shown to encode a 34-kilodalton polypeptide (34K polypeptide) that forms a complex with the early region 1B (E1B)-55K antigen and is required for efficient viral growth in lytic infection. Antisera that were generated recognized the E4-34K protein as well as a family of related polypeptides generated by the fusion of open reading frames 6 and 7. These polypeptides shared amino-terminal sequences with the 34K protein. Short-pulse analysis suggested that the heterogeneity observed with the 6/7 fusion products resulted from differential splicing patterns of related E4 mRNAs. An antiserum directed against the amino terminus of reading frame 6 recognized only the free form of the 34K antigen that was not associated with the E1B-55K protein. This observation allowed the determination of the stability of the free and complexed form of this polypeptide. Pulse-chase analyses demonstrated that both forms of the 34K protein had half-lives greater than 24 h, suggesting that complex formation did not result in stabilization of this gene product. The half-lives of the 6/7 fusion products were approximately 4 h. The 34K protein also was shown to have a nuclear localization within infected cells. Finally, analysis of a mutant carrying deletions in both the E4-34K and E1B-55K polypeptides indicated that the complex formed between these two proteins was a functional unit in lytic infection.     

E1B 55-Kilodalton-Associated Protein: a Cellular Protein with RNA-Binding Activity Implicated in Nucleocytoplasmic Transport of Adenovirus and Cellular mRNAs

The adenovirus type 5 (Ad5) early 1B 55-kDa protein (E1B-55kDa) is a multifunctional phosphoprotein that regulates viral DNA replication and nucleocytoplasmic RNA transport in lytically infected cells. In addition, E1B-55kDa provides functions required for complete oncogenic transformation of rodent cells in cooperation with the E1A proteins. Using the far-Western technique, we have isolated human genes encoding E1B-55kDa-associated proteins (E1B-APs). The E1B-AP5 gene encodes a novel nuclear RNA-binding protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that is highly related to hnRNP-U/SAF-A. Immunoprecipitation experiments indicate that two distinct segments in the 55-kDa polypeptide which partly overlap regions responsible for p53 binding are required for complex formation with E1B-AP5 in Ad-infected cells and that this protein interaction is modulated by the adenovirus E4orf6 protein. Expression of E1B-AP5 efficiently interferes with Ad5 E1A/E1B-mediated transformation of primary rat cells. Furthermore, stable expression of E1B-AP5 in Ad-infected cells overcomes the E1B-dependent inhibition of cytoplasmic host mRNA accumulation. These data suggest that E1B-AP5 might play a role in RNA transport and that this function is modulated by E1B-55kDa in Ad-infected cells.     

Identification of Rep-Associated Factors in Herpes Simplex Virus Type 1-Induced Adeno-Associated Virus Type 2 Replication Compartments

Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase δ was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.Adeno-associated virus (AAV) is a human parvovirus that is currently used as a gene transfer vector (14). AAV particles consist of a small icosahedral capsid protecting a single 4.7-kb single-stranded DNA (ssDNA) genome with two open reading frames, rep and cap, surrounded by inverted terminal repeats (ITRs). The ITRs are the only sequences required in cis for genome replication and packaging. The rep gene encodes four nonstructural Rep proteins: Rep78, -68, -52, and -40. The two larger isoforms, Rep78 and -68, have origin binding, helicase, and site-specific endonuclease activities and are involved in AAV gene expression and genome processing, including replication and site-specific integration (39). The two smaller Rep isoforms are not required for AAV DNA replication but are involved in the control of viral gene expression and packaging of viral DNA (30).When wild-type (wt) AAV infects a cell in the absence of a helper virus, it enters latency. Latent AAV genomes persist in cells either as episomes or as integrated genomes, preferentially at a specific locus (named AAVS1) on human chromosome 19. In most instances, no detectable viral gene expression or genome replication occurs unless the cell is co- or superinfected by a helper virus, such as adenovirus, herpes simplex virus type 1 (HSV-1), or HSV-2. Under these conditions, AAV replication and assembly take place in large intranuclear domains called replication compartments (RCs) that frequently colocalize with replication domains formed by the helper virus itself (81). The viral genome replicates by leading-strand synthesis and generates new ssDNA molecules by a strand displacement mechanism that occurs after strand- and site-specific cleavage of viral DNA by Rep78/68 within the ITRs (39).Studies conducted on the relationship between AAV and its helper viruses are important not only to identify helper activities that can be used to produce recombinant AAV vectors but also to understand how AAV adapts its replication strategy to the helper virus and to the nuclear environment in general. Adenovirus helper functions have historically been the first and most extensively studied functions. These studies have shown that adenovirus helps AAV by stimulating viral gene expression and by enhancing AAV genome replication, mostly indirectly (19). Indeed, early studies showed that the adenovirus polymerase (E2b) is dispensable for AAV replication (8) and that the viral DNA-binding protein (DBP), the product of the E2a gene, is able to modestly enhance the processivity of AAV genome replication in vitro (77). More recently, the adenovirus proteins E1b55k and E4orf6 were shown to stimulate AAV genome replication by degrading the cellular Mre11/Rad50/Nbs1 (MRN) complex that restricts AAV genome replication during adenovirus coinfection (32). The concept that AAV genome replication can rely mostly, if not uniquely, on direct help from cellular factors was further strengthened by the demonstration that purified proteins such as replication protein A (RPA), replication factor C (RFC), proliferating cell nuclear antigen (PCNA), minichromosome maintenance (MCM) proteins, and DNA polymerase δ (Pol δ) were sufficient to replicate the AAV genome in vitro in the presence of Rep (40-41, 43). The involvement of these cellular proteins during AAV genome replication was also confirmed by the proteomic analysis of factors associated with Rep proteins during adenovirus-induced AAV replication (42).Interestingly, studies conducted on HSV-1 helper activities suggest that the strategy of AAV replication may vary depending on the helper virus. Indeed, previous studies showed that the HSV-1 helicase-primase (HP) complex (UL5/8/52) and DBP (ICP8) could replicate transfected AAV-2 plasmids (80) and that the helicase activity, but not primase activity, of the HP complex was required for this effect (62, 66). More recently, a comprehensive study of HSV-1 helper activities demonstrated that the HSV-1 immediate-early proteins ICP0, ICP4, and ICP22 could stimulate rep gene expression, probably by diminishing intrinsic antiviral effects (1, 18). In addition, the HSV-1 DNA polymerase encoded by UL30, along with its associated processivity factor (UL42), although not strictly required, was demonstrated to significantly increase AAV replication levels induced in the presence of the HP complex and ICP8. Interestingly, the HSV-1 HP complex, DBP, and polymerase were also shown to be sufficient to replicate AAV DNA in vitro in the presence of Rep proteins without any cellular protein (78). Altogether, these observations indicate that in the context of an HSV-1 coinfection, AAV relies extensively on viral activities provided by the helper that directly participate in AAV genome replication.To further elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis to identify the cellular and HSV-1 factors associated with Rep proteins and, consequently, potentially recruited within AAV RCs. To analyze Rep-associated proteins in the presence and absence of HSV-1 DNA replication, this analysis was performed using wt HSV-1 and an HSV-1 mutant in which the DNA polymerase encoded by the UL30 gene is absent (HSVΔUL30). This study resulted in the identification of approximately 60 cellular proteins, among which the largest functional categories corresponded to factors involved in DNA and RNA metabolism. Immunofluorescence analyses confirmed that in the presence of HSV-1, a basal set of cellular DNA replication enzymes, including RPA, RFC, and PCNA, was recruited within AAV RCs, with the exception of the MCM helicases. The cellular DNA polymerases, in particular Pol δ, were not identified by this analysis but subsequently were shown to be recruited in AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, our results indicate that AAV replication induced by HSV-1 is associated with the recruitment of DNA repair factors, including components of the MRN complex, the Ku proteins, PARP-1, and factors of the mismatch repair (MMR) pathway. Finally, several HSV-1 proteins, most notably the UL12 protein, were also identified within AAV RCs. Our analyses confirmed the association between UL12 and Rep and demonstrated for the first time that this viral exonuclease plays a critical role during AAV replication by enhancing the formation of discrete AAV replicative forms and the production of AAV particles.Altogether, these results indicate that in the presence of HSV-1, AAV may replicate by using a basal set of cellular DNA replication enzymes but also relies extensively on HSV-1-derived proteins for its replication, including UL12, a newly discovered helper factor. These results suggest that AAV may be able to differentially adapt its replication strategy to the nuclear environment induced by the helper virus.     

A helper virus-free packaging system for recombinant adeno-associated virus vectors

Adeno-associated virus (AAV) is a human parvovirus that is currently receiving widespread attention for its potential use as a gene therapy vector. Construction of the recombinant AAV vector (rAAV) involves replacing most of the viral genome with a transgene of interest and then packaging this recombinant genome into an infectious virion. Most current protocols for generating rAAV entail the co-transfection of a vector plasmid and a packaging plasmid that expresses the viral replication and structural genes onto adenovirus (Ad) infected cells growing in culture. Limitations of this procedure include (1) contamination of rAAV with the Ad helper virus, (2) low yields of rAAV and (3) production of replication-competent AAV. In this report we describe new helper plasmids (pSH3 and pSH5) that eliminate the Ad co-infection requirement. The helper plasmids express the AAV rep and cap genes and the Ad E2A, VAI and E4 genes. When the helper plasmids are co-transfected onto human 293 cells with a vector plasmid in the absence of Ad infection, the rAAV vector yield is up to 80-fold greater than those obtained with the pAAV/Ad packaging plasmid. Moreover, replication competent AAV in the rAAV preparations is less than 0.00125%. The major advantages of this system are (1) the absence of infectious adenovirus and (2) the use of only two plasmids, which enhances transfection efficiencies and hence vector production. We believe that this two-plasmid transfection system will allow for more widespread use of the AAV vector system because of its simplicity and high yields. This system will be especially useful for preclinical analyses of multiple rAAV vectors.     

Complete in vitro reconstitution of adeno-associated virus DNA replication requires the minichromosome maintenance complex proteins

Adeno-associated virus (AAV) replicates its DNA exclusively by a leading-strand DNA replication mechanism and requires coinfection with a helper virus, such as adenovirus, to achieve a productive infection. In previous work, we described an in vitro AAV replication assay that required the AAV terminal repeats (the origins for DNA replication), the AAV Rep protein (the origin binding protein), and an adenovirus-infected crude extract. Fractionation of these crude extracts identified replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and polymerase δ as cellular enzymes that were essential for AAV DNA replication in vitro. Here we identify the remaining factor that is necessary as the minichromosome maintenance (MCM) complex, a cellular helicase complex that is believed to be the replicative helicase for eukaryotic chromosomes. Thus, polymerase δ, RFC, PCNA, and the MCM complex, along with the virally encoded Rep protein, constitute the minimal protein complexes required to reconstitute efficient AAV DNA replication in vitro. Interfering RNAs targeted to MCM and polymerase δ inhibited AAV DNA replication in vivo, suggesting that one or more components of the MCM complex and polymerase δ play an essential role in AAV DNA replication in vivo as well as in vitro. Our reconstituted in vitro DNA replication system is consistent with the current genetic information about AAV DNA replication. The use of highly conserved cellular replication enzymes may explain why AAV is capable of productive infection in a wide variety of species with several different families of helper viruses.     

Effect of deletions in adenovirus early region 1 genes upon replication of adeno-associated virus.

The growth of adeno-associated virus (AAV) is dependent upon helper functions provided by adenovirus. We investigated the role of adenovirus early gene region 1 in the AAV helper function by using six adenovirus type 5 (Ad5) host range mutants having deletions in early region 1. These mutants do not grow in human KB cells but are complemented by and grow in a line of adenovirus-transformed human embryonic kidney cells (293 cells); 293 cells contain and express the Ad5 early region 1 genes. Mutants having extensive deletions of adenovirus early region 1a (dl312) or regions 1a and 1b (dl313) helped AAV as efficiently as wild-type adenovirus in 293 cells, but neither mutant helped in KB cells. No AAV DNA, RNA, or protein synthesis was detected in KB cells in the presence of the mutant adenoviruses. Quantitative blotting experiments showed that at 20 h after infection with AAV and either dl312 or dl313 there was less than one AAV genome per cell. In KB cells infected with AAV alone, the unreplicated AAV genomes were detected readily. Apparently, infection with adenovirus mutant dl312 or dl313 results in degradation of most of the infecting AAV genomes. We suggest that at least an adenovirus region 1b product (and perhaps a region 1a product also) is required for AAV DNA replication. This putative region 1b function appears to protect AAV DNA from degradation by an adenovirus-induced DNase. We also tested additional Ad5 mutants (dl311, dl314, sub315, and sub316). All of these mutants were inefficient helpers, and they showed varying degrees of multiplicity leakiness. dl312 and dl313 complemented each other for the AAV helper function, and each was complemented by Ad5ts125 at the nonpermissive temperature. The defect in region 1 mutants for AAV helper function acts at a different stage of the AAV growth cycle than the defect in the region 2 mutant ts125.     

Spatial and temporal organization of adeno-associated virus DNA replication in live cells

Upon cell entry, the genomes of herpes simplex virus type 1 (HSV-1) and adenovirus (Ad) associate with distinct nuclear structures termed ND10 or promyelocytic leukemia (PML) nuclear bodies (NBs). PML NB morphology is altered or disrupted by specific viral proteins as replication proceeds. We examined whether adeno-associated virus (AAV) replication compartments also associate with PML NBs, and whether modification or disruption of these by HSV-1 or Ad, both of which are helper viruses for AAV, is necessary at all. Furthermore, to add a fourth dimension to our present view of AAV replication, we established an assay that allows visualization of AAV replication in live cells. A recombinant AAV containing 40 lac repressor binding sites between the AAV inverted terminal repeats was constructed. AAV Rep protein and helper virus-mediated replication of this recombinant AAV genome was visualized by binding of enhanced yellow fluorescent protein-lac repressor fusion protein to double-stranded AAV replication intermediates. We demonstrate in live cells that AAV DNA replication occurs in compartments which colocalize with AAV Rep. Early after infection, the replication compartments were small and varied in numbers from 2 to more than 40 per cell nucleus. Within 4 to 8 h, individual small replication compartments expanded and fused to larger structures which filled out much of the cell nucleus. We also show that AAV replication compartments can associate with modified PML NBs in Ad-infected cells. In wild-type HSV-1-infected cells, AAV replication compartments and PML NBs did not coexist, presumably because PML was completely disrupted by the HSV-1 ICP0 protein. However, alteration or disruption of PML appears not to be a prerequisite for AAV replication, as the formation of replication compartments was normal when the ICP0 mutants HSV-1 dl1403 and HSV-1 FXE, which do not affect PML NBs, were used as the helper viruses; under these conditions, AAV replication compartments did not associate with PML NBs.     

Regulation of p53 levels by the E1B 55-kilodalton protein and E4orf6 in adenovirus-infected cells.

The adenovirus type 5 243R E1A protein induces p53-dependent apoptosis in the absence of the 19- and 55-kDa E1B polypeptides. This effect appears to result from an accumulation of p53 protein and is unrelated to expression of E1B products. We now report that in the presence of the E1B 55-kDa polypeptide, the 289R E1A protein does not induce such p53 accumulation and, in fact, is able to block that induced by E1A 243R. This inhibition also requires the 289R-dependent transactivation of E4orf6 expression. E4orf6 is known to form complexes with the E1B 55-kDa protein and to function both in the transport and stabilization of viral mRNA and in shutoff of host cell protein synthesis. We demonstrated that the block in p53 accumulation is not due to the generalized shutoff of host cell metabolism. Rather, it appears to result from a mechanism targeted specifically to p53, most likely involving a decrease in the stability of p53 protein. The E1B 55-kDa protein is known to interact with both E4orf6 and p53, and as demonstrated recently by others, we showed that E4orf6 also binds directly to p53. Thus, multiple interactions between all three proteins may regulate p53 stability, resulting in the maintenance of low levels of p53 following virus infection.