Role of the terminal repeat GAGC trimer, the major Rep78 binding site, in adeno‐associated virus DNA replication

Bishop, Brian M.; Santin, Alessandro D.; Quirk, J. Gerald; Hermonat, Paul L.

doi:10.1016/s0014-5793(96)01149-0

Cited by 12 publications

(10 citation statements)

References 33 publications

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“…Previously, we found that E1 aa353–649 was able to provide some helper function, in boosting basal autonomous wt AAV2 DNA replication in differentiating keratinocytes [36]. However, wt AAV2 biology studies and rAAV production are almost always carried out in HEK293 cells.…”

Section: Resultsmentioning

confidence: 99%

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The HPV16 E1 Carboxyl Domain Provides a Helper Function for Adeno-Associated Virus Replication

et al. 2018

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Background/Aims: Recombinant adeno-associated virus (rAAV) is now in the clinic, yet production of rAAV remains problematic. We previously determined that human papillomavirus type 16 (HPV16) E1 protein boosts rAAV yields and E1 enhances AAV Rep78’s replication-related biochemistries. Here, we deletion-mapped the helper domain within E1 to help glean its mechanism of action. Methods: Rep78-E1 interaction was analyzed by Gal4-based yeast two-hybrid (Y2H)-cDNA assay. rAAV DNA replication was studied by AAV/helper plasmid transfection into HEK293 cells and Southern blot. Gene expression analysis was made of AAV and E1 plasmid transfection, cDNA generation, and then quantitative polymerase chain reaction. NCBI protein BLAST was used for the homology analysis. Results: Gal4-Y2H- cDNA assay found in vivo Rep78-E1-binding activity across E1, but the carboxyl-third (amino acids [aa] 421–649) of E1 contained the predominant DNA replication helper domain. The amino-half of E1 (aa 1–337) inhibited transcription of rep (p5 promoter) and cap (p40, trending lower) from non-replicating helper plasmid by quantitative (q)RT-PCR. Conclusions: The aa 421–649 helper domain of HPV16 E1 includes the ATP-binding/helicase region of E1 which boosts rAAV production and has homology with the analogous region of parvovirus NS-1/Rep78 by NCBI protein BLAST, suggesting these biochemistries are responsible for the mechanism of action in E1 helper function.

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Section: Resultsmentioning

confidence: 99%

“…Five days after transfection, low-molecular-weight Hirt DNA was isolated [1, 6], agarose gel-electrophoresed, subjected to Southern blotting as described previously [36-38], and then probed with 32 P-eGFP DNA. No Dpn I digestion is needed at this late time of harvest as all transfected input plasmid DNA has been degraded by day 3.…”

Section: Methodsmentioning

confidence: 99%

The HPV16 E1 Carboxyl Domain Provides a Helper Function for Adeno-Associated Virus Replication

et al. 2018

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“…The AAV terminal repeat (TR) substrate was generated by the ligation of three separate synthetic oligonucleotides as described previously (43). The TR was 5Ј end-labeled with polynucleotide kinase using […”

Section: Methodsmentioning

confidence: 99%

Binding of the Human Papillomavirus Type 16 p97 Promoter by the Adeno-associated Virus Rep78 Major Regulatory Protein Correlates with Inhibition

Zhan

Santin

Liu

et al. 1999

Journal of Biological Chemistry

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Human papillomavirus type 16 (HPV-16) infection is positively associated with cervical cancer, whereas adeno-associated virus (AAV) infection is negatively associated with this same cancer. In earlier studies these two virus types have been shown to directly interact, with AAV inhibiting or enhancing papillomavirus functions depending upon the specific circumstances. One defined interaction between these two viruses is the ability of the AAV Rep78 major regulatory protein to inhibit gene expression of the E6 promoter of BPV-1 (bovine papillomavirus type 1) and HPV types 16 and 18. As Rep78 is a DNA binding transcription factor, we considered whether Rep78 might bind HPV-16 DNA. Here, Rep78 is demonstrated to bind a 44-base pair region (nucleotides 14 -56) within the HPV-16 p97 promoter using the electrophoretic mobility shift assay. This region is important for HPV-16 because it includes functional Sp1 and E2 protein binding motifs as well as part of the origin of replication. Furthermore, two Rep78 amino acid substitution mutants, at positions 77 or 64 -65, were identified that did not recognize p97 DNA. Both of these Rep78 mutants were found to be defective for inhibition of p97 promoter activity in HeLa and T-47D nuclear extracts in vitro, in a transient chloramphenicol acetyltransferase assay, as well as defective for full inhibition of HPV-16-directed focus formation. These data, taken together, strongly suggest that the Rep78-p97 promoter interaction is at least partially responsible for Rep78-mediated inhibition of HPV-16. Finally, the finding that Rep78 specifically recognizes p97 DNA is surprising because the p97 promoter region contains no GAGC motifs, the core motif for Rep78 recognition. These data suggest that the p97 promoter may represent a new prototypical DNA target type for Rep78.Infection and DNA integration by certain human papillomavirus types (HPV) 1 are central events in the generation of cervical cancer (1, 2). The most common HPV type associated with cervical cancer is HPV-16; roughly two-thirds of cervical cancers contain the DNA of this virus. Adeno-associated virus (AAV) is another virus commonly found in the anogenital region (3-6). Infection by AAV, in sharp contrast to the HPVs, is negatively associated with cervical cancer as demonstrated by the prevalence or titer of anti-AAV antibodies (7, 8). Bidirectional interaction has been observed between AAV and papillomaviruses. One such interaction is that papillomaviruses might serve as helpers for AAV (AAV is a helper-dependent parvovirus) (9), allowing for AAV DNA replication and virion production. Our laboratory is exploring the hypothesis that AAV may regulate the role of HPVs in cervical carcinogenesis. In earlier studies (10 -12) we demonstrated that AAV inhibits bovine papillomavirus type 1 (BPV-1) and HPV-16-induced oncogenic transformation. Others (13-15) have also observed AAV inhibition of BPV-1 and HPV-18. The effect has been mapped to the AAV encoded Rep78 protein, and this protein has been shown to inhibit expression of...

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“…Mutational analysis has identified a core 22-bp sequence required for stable Rep binding to linear TR substrates (27). This RBE includes the tetrameric GAGC repeat identified by several groups as necessary for stable Rep binding to both linear and hairpinned TR substrates (4,8,19,20,32,36). Moreover, chemical interference assays indicate that all major Rep contacts within the linear portion of the hairpinned TR fall within the RBE (1,25,27).…”

mentioning

confidence: 99%

“…Although the contribution of the RBE to Rep-catalyzed trs nicking has not been determined, mutant AAV genomes containing multiple transversions in the RBE replicate at much lower levels than do wild-type (wt) genomes (4). This observation has led to the conclusion that stable Rep binding to the AAV TR is necessary for efficient origin nicking and subse-quent viral replication.…”

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confidence: 99%

Mechanism of Rep-Mediated Adeno-Associated Virus Origin Nicking

Brister

Muzyczka

2000

J Virol

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The single-stranded adeno-associated virus type 2 (AAV) genome is flanked by terminal repeats (TRs) that fold back on themselves to form hairpinned structures. During AAV DNA replication, the TRs are nicked by the virus-encoded Rep proteins at the terminal resolution site (trs). This origin function apparently requires three sequence elements, the Rep binding element (RBE), a small palindrome that comprises a single tip of an internal hairpin within the TR (RBE), and the trs. Previously, we determined the sequences at the trs required for Rep-mediated cleavage and demonstrated that the trs endonuclease reaction occurs in two discrete steps. In the first step, the Rep DNA helicase activity unwinds the TR, thereby extruding a stem-loop structure at the trs. In the second step, Rep transesterification activity cleaves the trs. Here we investigate the contribution of the RBE and RBE during this process. Our data indicate that Rep is tethered to the RBE in a specific orientation during trs nicking. This orientation appears to align Rep on the AAV TR, allowing specific nucleotide contacts with the RBE and directing nicking to the trs. Accordingly, alterations in the polarity or position of the RBE relative to the trs greatly inhibit Rep nicking. Substitutions within the RBE also reduce Rep specific activity, but to a lesser extent. Interestingly, Rep interactions with the RBE and RBE during nicking seem to be functionally distinct. Rep contacts with the RBE appear necessary for both the DNA helicase and trs cleavage steps of the endonuclease reaction. On the other hand, RBE contacts seem to be required primarily for TR unwinding and formation of the trs stem-loop structure, not cleavage. Together, these results suggest a model of Rep interaction with the AAV TR during origin nicking through a tripartite cleavage signal comprised of the RBE, the RBE, and the trs.

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Role of the terminal repeat GAGC trimer, the major Rep78 binding site, in adeno‐associated virus DNA replication

Cited by 12 publications

References 33 publications

The HPV16 E1 Carboxyl Domain Provides a Helper Function for Adeno-Associated Virus Replication

The HPV16 E1 Carboxyl Domain Provides a Helper Function for Adeno-Associated Virus Replication

Binding of the Human Papillomavirus Type 16 p97 Promoter by the Adeno-associated Virus Rep78 Major Regulatory Protein Correlates with Inhibition

Mechanism of Rep-Mediated Adeno-Associated Virus Origin Nicking

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