Analysis of the effects of charge cluster mutations in adeno-associated virus Rep68 protein in vitro.

Adeno-associated virus type 2 (AAV) is a human parvovirus with a single-stranded, linear DNA genome containing inverted terminal repeats which function as origins of replication (14, 40, 44, 53). AAV is nonpathogenic and usually requires an adenovirus or herpesvirus as a helper for efficient replication (7). The AAV rep gene encodes at least four overlapping multifunctional nonstructural proteins transcribed from two promoters. Rep68 and Rep78 (Rep68/78) are encoded by spliced and unspliced transcripts, respectively, from the promoter at map position 5; hence, the first 529 amino acids of Rep78 and Rep68 are identical (9, 35, 52, 56). Rep40 and Rep52 are encoded by spliced and unspliced transcripts, respectively, from the promoter at map position 19 (9, 35, 52, 56). The terminal palindromic repeat sequences of AAV DNA fold into hairpin structures and thereby serve as primers for the synthesis of the complementary strand (8, 52). The resulting closed-end intermediates are resolved by a process called terminal resolution, which involves cutting by a site- and strand-specific endonuclease at the terminal resolution site (trs) followed by unwinding and replication of the hairpin (17, 50, 51). Rep68/78 display activities which are required for AAV DNA replication, including the ability to bind specifically to sequences within the AAV terminal hairpin DNA (17–19, 34, 38, 63) and strand-specific nicking at the trs (17, 19, 50). The larger Rep proteins also have DNA helicase (17, 19, 26) and DNA-RNA helicase activities (65), as well as ATPase activity (65). Furthermore, the Rep68/78 proteins regulate AAV promoters (4, 27, 28, 30, 55) and have been shown to regulate numerous heterologous promoters (1, 15, 29, 66, 68). They are also thought to be involved in the preferential integration of AAV genomes into a region on the q arm of human chromosome 19 (13, 23–25, 31, 46, 57, 63). Rep68/78, like many transcriptional regulatory proteins of DNA viruses, such as the 32-kDa E1A protein of adenovirus; E1, E7, L1, and L2 of human papillomavirus; and VP2 and large T antigen of Simian virus 40, carry charge clusters in their primary sequences (for reviews, see the work of Karlin and Brendel [20, 21]). Ionic interactions of charge clusters within proteins are associated with protein-protein and protein-DNA interactions (58, 64, 72) and appear to be important in protein transport, nuclear localization, and transcriptional regulation (6). Mapping studies of the Rep68/78 proteins by site-specific mutation, truncation, and deletion reveal that the Rep proteins are composed of distinct, but interdependent, functional domains (see Fig. ​Fig.1)1) (33, 37, 38, 59, 60, 69). Rep68/78 DNA binding functions are believed to be bipartite, with binding specificity being associated with the first 241 amino acids (33, 38, 62, 70) and stabilizing interactions being associated with amino acids 242 to 476 (33, 38, 62, 70). The first 476 amino acids of Rep68/78 have been shown to be sufficient for nucleoside triphosphate-dependent endonuclease activity (62). The first 522 amino acids of Rep68/78 or amino acids 225 to 621 (Rep52) are sufficient for DNA helicase activity (26, 47). The observation that Rep68/78 proteins produce multiple shifted bands in electrophoretic mobility shift assays (EMSAs) with hairpin DNA (17–19, 33, 37, 70), along with the existence of dominant-negative mutant Rep68/78 proteins (26, 36, 59, 65) and the detection of multimeric Rep78 complexes in gel filtration, protein cross-linking, and coimmunoprecipitation experiments (48), strongly suggests that Rep68/78 proteins function as multimers. FIG. 1 Schematic representation of the Rep moiety of MBP-Rep68Δ showing the locations of charge cluster mutations. The locations of the charge-to-alanine mutations are labeled and shown as vertical black bars. The horizontal black bars indicate the regions ... We have previously demonstrated that a Rep68/78 truncation mutant protein containing amino acids 1 to 476 also produces a multiple-shifted-band pattern in EMSAs with AAV hairpin DNA and that these shifted bands have a faster migration than those formed with the wild-type Rep68. When this mutant protein with amino acids 1 to 476 was mixed with wild-type Rep68, there was a loss of the distinct multiple shifted bands and instead there was a smear of protein-DNA complexes with intermediate rates of migration (62). Truncations and site-specific mutagenesis studies suggest that there are at least two separate self-association domains between amino acids 164 and 484, including a 3,4 hydrophobic heptad repeat between amino acids 164 and 182 (48) and a region between amino acids 332 and 346, which has also been shown to be important for ATPase (48, 60) and helicase activities (60). In this study, we have mutated charged residues to alanines in areas of Rep68 with three or more charged residues within a continuous stretch of five. An overlap extension PCR method (16) was used to create these charge-to-alanine mutations in a maltose binding protein (MBP)-Rep68 fusion protein (MBP-Rep68Δ). MBP-Rep68Δ exhibits all of the functional activities reported for the wild-type Rep68 when it is assayed in vitro (10). We have assessed the effects of these mutations on AAV hairpin DNA binding, AAV trs endonuclease activity, and DNA helicase activity in vitro. In addition, these mutants were used in coimmunoprecipitation experiments with radiolabeled, in vitro-translated Rep68 in order to assess their ability to associate with wild-type Rep68.