Interaction of wild-type and mutant adeno-associated virus (AAV) Rep proteins on AAV hairpin DNA.

Both the Rep68 and Rep78 proteins of adeno-associated virus type 2 (AAV) bind to AAV terminal repeat hairpin DNA and can mediate site-specific nicking in vitro at the terminal resolution site (trs) within the terminal repeats. To define the regions of the Rep proteins required for these functions, a series of truncated Rep78 derivatives was created. Wild-type and mutant proteins were synthesized by in vitro translation and analyzed for AAV hairpin DNA binding, trs endonuclease activity, and interaction on hairpin DNA. Amino-terminal deletion mutants which lacked the first 29 or 79 amino acid residues of Rep78 did not bind hairpin DNA, which is consistent with our previous identification of a DNA-binding domain in this region. Progressive truncation of the carboxyl-terminal region of Rep78 did not eliminate hairpin DNA binding until the deletion reached amino acid 443. The electrophoretic mobility of the Rep-specific protein-DNA complexes was inversely related to the molecular weight of the Rep derivative. Analysis of the C-terminal deletion mutants by the trs endonuclease assay identified a region (amino acids 467 to 476) that is essential for nicking but is not necessary for DNA binding. When endonuclease-positive, truncated Rep proteins that bound hairpin DNA were mixed with full-length Rep78 or Rep68 protein in electrophoretic mobility shift assays, a smear of protein-DNA complexes was observed. This smear migrated at an intermediate position with respect to the bands generated by the proteins individually. An antibody recognizing only the full-length protein produced a novel supershift band when included in a mixed binding assay containing Rep68 and a truncated Rep mutant. These experiments suggest that the Rep proteins can form hetero-oligomers on the AAV hairpin DNA.     

The amino acid linker between the endonuclease and helicase domains of adeno-associated virus type 5 Rep plays a critical role in DNA-dependent oligomerization

The adeno-associated virus (AAV) genome encodes four Rep proteins, all of which contain an SF3 helicase domain. The larger Rep proteins, Rep78 and Rep68, are required for viral replication, whereas Rep40 and Rep52 are needed to package AAV genomes into preformed capsids; these smaller proteins are missing the site-specific DNA-binding and endonuclease domain found in Rep68/78. Other viral SF3 helicases, such as the simian virus 40 large T antigen and the papillomavirus E1 protein, are active as hexameric assemblies. However, Rep40 and Rep52 have not been observed to form stable oligomers on their own or with DNA, suggesting that important determinants of helicase multimerization lie outside the helicase domain. Here, we report that when the 23-residue linker that connects the endonuclease and helicase domains is appended to the adeno-associated virus type 5 (AAV5) helicase domain, the resulting protein forms discrete complexes on DNA consistent with single or double hexamers. The formation of these complexes does not require the Rep binding site sequence, nor is it nucleotide dependent. These complexes have stimulated ATPase and helicase activities relative to the helicase domain alone, indicating that they are catalytically relevant, a result supported by negative-stain electron microscopy images of hexameric rings. Similarly, the addition of the linker region to the AAV5 Rep endonuclease domain also confers on it the ability to bind and multimerize on nonspecific double-stranded DNA. We conclude that the linker is likely a key contributor to Rep68/78 DNA-dependent oligomerization and may play an important role in mediating Rep68/78's conversion from site-specific DNA binding to nonspecific DNA unwinding.     

In vitro resolution of adeno-associated virus DNA hairpin termini by wild-type Rep protein is inhibited by a dominant-negative mutant of rep.

An adeno-associated virus (AAV) genome with a Lys-to-His (K340H) mutation in the consensus nucleotide triphosphate binding site of the rep gene has a dominant-negative DNA replication phenotype in vivo. We expressed both wild-type (Rep78) and mutant (Rep78NTP) proteins in two helper-free expression systems consisting of either recombinant baculoviruses in insect cells or the human immunodeficiency virus type 1 long terminal repeat promoter in human 293 cell transient transfections. We analyzed nuclear extracts from both expression systems for the ability to complement uninfected HeLa cell cytoplasmic extracts in an in vitro terminal resolution assay in which a covalently closed AAV terminal hairpin structure is converted to an extended linear duplex. Although both Rep78 and Rep78NTP bound to AAV terminal hairpin DNA in vitro, Rep78 but not Rep78NTP complemented the terminal resolution assay. Furthermore, Rep78NTP was trans dominant for AAV terminal resolution in vitro. We propose that the dominant-negative replication phenotype of AAV genomes carrying the K340H mutation is mediated by mutant Rep proteins binding to the terminal repeat hairpin.     

Productive replication of adeno-associated virus can occur in human papillomavirus type 16 (HPV-16) episome-containing keratinocytes and is augmented by the HPV-16 E2 protein

We used a sensitive assay to test whether an adeno-associated virus (AAV) productive replication cycle can occur in immortalized human keratinocytes carrying episomal human papillomavirus type 16 (HPV-16) DNA. Following transfection with cloned AAV DNA, infectious AAV was produced, and the infectivity was blocked by anti-AAV antiserum. The HPV-16 E2 protein substantially increased the yield of AAV. Other HPV early proteins did not, in our experiments, show this ability. E2 has been shown to be able to affect p53 levels and to block cell cycle progression at mitosis. We tested the effect of changes in p53 expression on AAV replication and found that large differences in the level of p53 did not alter AAV DNA replication. In extension of this, we found that cellular help for AAV in response to stress was also independent of p53. To test if a mitotic block could trigger AAV DNA replication, we treated the cells with the mitotic inhibitor nocodazole. AAV DNA replication was stimulated by the presence of nocodazole in these and a number of other cell types tested. Yields of infectious virus, however, were not increased by this treatment. We conclude that the HPV-16 E2 protein stimulates AAV multiplication in these cells and propose that this occurs independently of the effects of E2 on p53 and cell cycle progression. Since the effect of E2 was not seen in keratinocytes lacking the HPV-16 episome, we suggest that E2 can help AAV by working in concert with other HPV-16 proteins.     

Adeno-associated virus Rep78 binds to E2-responsive element 1 of bovine papillomavirus type 1

Adeno-associated virus type-2 (AAV-2) is a helper-dependent parvovirus that has been implicated in the inhibition of replication and oncogenic transformation of bovine papillomavirus type-1 (BPV-1) and other transforming DNA viruses. Previous studies have suggested that the Rep78 protein of AAV-2 is a key player mediating this effect. In this report we have analyzed the effect of AAV-2 Rep78 protein on the regulation of gene expression of a reporter gene under the control of the long control region (LCR) of BPV-1. Our results show that Rep78 is capable of down-regulating the promoter activity of the LCR in vivo in tissue culture cells. Inhibition of LCR activity in vivo suggested the need for Rep78 to bind to a region of the LCR promoter spanning the E2-responsive elements of BPV-1. This observation was further confirmed in vitro with gel shift assays showing specific binding of Rep78 to DNA oligonucleotides containing E2-responsive element 1 (E2RE1) sequences of BPV-1 LCR. Our results expand the understanding of the mechanism of trans-regulation mediated by Rep78 and involving this protein and DNA sequences with complex secondary structure.     

Charge-to-Alanine Mutagenesis of the Adeno-Associated Virus Type 2 Rep78/68 Proteins Yields Temperature-Sensitive and Magnesium-Dependent Variants

The adeno-associated virus type 2 (AAV) replication (Rep) proteins Rep78 and 68 (Rep78/68) exhibit a number of biochemical activities required for AAV replication, including specific binding to a 22-bp region of the terminal repeat, site-specific endonuclease activity, and helicase activity. Individual and clusters of charged amino acids were converted to alanines in an effort to generate a collection of conditionally defective Rep78/68 proteins. Rep78 variants were expressed in human 293 cells and analyzed for their ability to mediate replication of recombinant AAV vectors at various temperatures. The biochemical activities of Rep variants were further characterized in vitro by using Rep68 His-tagged proteins purified from bacteria. The results of these analyses identified a temperature-sensitive (ts) Rep protein (D40,42,44A-78) that exhibited a delayed replication phenotype at 32 degrees C, which exceeded wild-type activity by 48 h. Replication activity was reduced by more than threefold at 37 degrees C and was undetectable at 39 degrees C. Stability of the Rep78 protein paralleled replication levels at each temperature, further supporting a ts phenotype. Replication differences resulted in a 3-log-unit difference in virus yields between the permissive and nonpermissive temperatures (2.2 x 10(6) and 3 x 10(3), respectively), demonstrating that this is a relatively tight mutant. In addition to the ts Rep mutant, we identified a nonconditional mutant with a reduced ability to support viral replication in vivo. Additional characterization of this mutant demonstrated an Mg(2+)-dependent phenotype that was specific to Rep endonuclease activity and did not affect helicase activity. The two mutants described here are unique, in that Rep ts mutants have not previously been described and the D412A Rep mutant represents the first mutant in which the helicase and endonuclease functions can be distinguished biochemically. Further understanding of these mutants should facilitate our understanding of AAV replication and integration, as well as provide novel strategies for production of viral vectors.     

Asymmetric replication in vitro from a human sequence element is dependent on adeno-associated virus Rep protein.

The DNA of human parvovirus adeno-associated virus type 2 (AAV) integrates preferentially into a defined region of human chromosome 19. Southern blots of genomic DNA from latently infected cell lines revealed that the provirus was not simply inserted into the cellular DNA. Both the proviral and adjoining cellular DNA organization indicated that integration occurred by a complex, coordinated process involving limited DNA replication and rearrangements. However, the mechanism for targeted integration has remained obscure. The two larger nonstructural proteins (Rep68 and Rep78) of AAV bind to a sequence element that is present in both the integration locus (P1) and the AAV inverted terminal repeat. This binding may be important for targeted integration. To investigate the mechanism of targeted integration, we tested the cloned integration site subfragment in a cell-free replication assay in the presence or absence of recombinant Rep proteins. Extensive, asymmetric replication of linear or open-circular template DNA was dependent on the presence of P1 sequence and Rep protein. The activities of Rep on the cloned P1 element are analogous to activities on the AAV inverted terminal repeat. Replication apparently initiates from a 3'-OH generated by the sequence-specific nicking activity of Rep. This results in a covalent attachment between Rep and the 5'-thymidine of the nick. The complexity of proviral structures can be explained by the participation of limited DNA replication facilitated by Rep during integration.     

Adeno-associated virus site-specific integration is regulated by TRP-185

Adeno-associated virus (AAV) integrates site specifically into the AAVS1 locus on human chromosome 19. Although recruitment of the AAV nonstructural protein Rep78/68 to the Rep binding site (RBS) on AAVS1 is thought to be an essential step, the mechanism of the site-specific integration, particularly, how the site of integration is determined, remains largely unknown. Here we describe the identification and characterization of a new cellular regulator of AAV site-specific integration. TAR RNA loop binding protein 185 (TRP-185), previously reported to associate with human immunodeficiency virus type 1 TAR RNA, binds to AAVS1 DNA. Our data suggest that TRP-185 suppresses AAV integration at the AAVS1 RBS and enhances AAV integration into a region downstream of the RBS. TRP-185 bound to Rep68 directly, changing the Rep68 DNA binding property and stimulating Rep68 helicase activity. We present a model in which TRP-185 changes the specificity of the AAV integration site from the RBS to a downstream region by acting as a molecular chaperone that promotes Rep68 complex formation competent for 3'-->5' DNA helicase activity.