The adenovirus DNA binding protein enhances intermolecular DNA renaturation but inhibits intramolecular DNA renaturation

Zijderveld, D. C.; Stuiver, Maarten H.; Vliet, Peter C. van der

doi:10.1093/nar/21.11.2591

Cited by 21 publications

(27 citation statements)

References 49 publications

(56 reference statements)

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“…This would suggest that the addition of NC to an oligomer capable of forming a stable hairpin should actually promote hairpin formation. One possibility is that NC could unfold single-stranded DNA and hold it in a linear form, in a manner similar to the adenovirus DNA binding protein (40). This would also be consistent with reports that NC can unfold secondary structures in RNA and DNA, leading to increased RT polymerization rates through regions containing secondary structure (19,38).…”

Section: Resultssupporting

confidence: 83%

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Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Can Prevent Self-Priming of Minus-Strand Strong Stop DNA by Promoting the Annealing of Short Oligonucleotides to Hairpin Sequences

Driscoll

Hughes

2000

J Virol

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Understanding how viral components collaborate to convert the human immunodeficiency virus type 1 genome from single-stranded RNA into double-stranded DNA is critical to the understanding of viral replication. Not only must the correct reactions be carried out, but unwanted side reactions must be avoided. After minus-strand strong stop DNA (؊sssDNA) synthesis, degradation of the RNA template by the RNase H domain of reverse transcriptase (RT) produces single-stranded DNA that has the potential to self-prime at the imperfectly base-paired TAR hairpin, making continued DNA synthesis impossible. Although nucleocapsid protein (NC) interferes with ؊sssDNA self-priming in reverse transcription reactions in vitro, NC alone did not prevent self-priming of a synthetic ؊sssDNA oligomer. NC did not influence DNA bending and therefore cannot inhibit self-priming at hairpins by directly blocking hairpin formation. Using DNA oligomers as a model for genomic RNA fragments, we found that a 17-base DNA fragment annealed to the 3 end of the ؊sssDNA prevented self-priming in the presence of NC. This implies that to avoid self-priming, an RNA-DNA hybrid that is more thermodynamically stable than the hairpin must remain within the hairpin region. This suggests that NC prevents self-priming by generating or stabilizing a thermodynamically favored RNA-DNA heteroduplex instead of the kinetically favored TAR hairpin. In support of this idea, sequence changes that increased base pairing in the DNA TAR hairpin resulted in an increase in self-priming in vitro. We present a model describing the role of NC-dependent inhibition of self-priming in first-strand transfer.Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) synthesizes minus-strand strong stop DNA (ϪsssDNA) by extending tRNA 3 Lys bound at the primer binding site (PBS) in the RNA genome (8,15,37). To synthesize ϪsssDNA, RT copies the U5 and R regions; the R region contains two large hairpins known as the poly(A) hairpin and the TAR hairpin (9). When this RNA is copied into DNA, hairpins that correspond to the TAR and poly(A) hairpins of the RNA can form in the nascent ϪsssDNA; the formation of these hairpins depends on the digestion of the template RNA (17). The RNase H activity of RT cleaves the RNA portion of the RNA-DNA heteroduplex during polymerization, and there is additional RNase H cleavage after ϪsssDNA synthesis is complete (8,12,18,27,33). Although it is possible for either the nascent TAR and poly(A) DNA to form hairpins that could self-prime, such self-priming events are not detected when the HIV-1 genome is copied into DNA in infected cells. Because the TAR DNA hairpin forms at the end of R, the likelihood of self-priming is higher for TAR than for the poly(A) hairpin. Instead of self-priming, the 3Ј end of the nascent DNA is efficiently transferred to the R sequence on the 3Ј end of the template RNA, where synthesis continues. This event is known as the first-strand transfer. Nucleocapsid protein (NC) has been shown to prevent synthesis of selfpri...

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Section: Resultssupporting

confidence: 83%

“…Therefore, the annealing of a strand transfer acceptor cannot be the only mechanism that prevents self-priming. One possibility is that NC interferes with the formation of hairpins by preventing bending of the hairpin DNA (24,40). However, we present evidence that this is not the mechanism that prevents self-priming.…”

mentioning

confidence: 54%

Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Can Prevent Self-Priming of Minus-Strand Strong Stop DNA by Promoting the Annealing of Short Oligonucleotides to Hairpin Sequences

Driscoll

Hughes

2000

J Virol

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“…However, no helicase is needed in the elongation phase of adenovirus replication. Recently we have shown that DBP by itself stimulates DNA unwinding when the duplex has a singlestranded extension [31], which suggests that DBP is the unwinding protein during chain elongation. Thus unwinding occurs independent of ATP and is optimal at low M$ concentrations, similar to the conditions required for in vitro adenovirus DNA replication [31].…”

Section: Discussionmentioning

confidence: 98%

“…Employing templates in which the origin is partially or completely single stranded no stimulation *Corresponding author. Fax: (31) (30) 888443. by ATP was observed suggesting that ATP might be required for origin opening [5,9,10]. Furthermore, none of the proteins involved in replication has ATPase activity, moreover, non-hydrolysable ATP analogues such as ATPyS, AMP-PNP and AMP-PCP also stimulate the reaction [lo].…”

Section: Introductionmentioning

confidence: 99%

Replication of adenovirus DNA in vitro is ATP‐independent

1994

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Using a reconstituted system for adenovirus DNA replication we tested the requirements for ATP and divalent cations. At the standard M$ concentration ATP stimulated initiation 5 to lo-fold. However, this effect was caused by complexing Mg2'. At the optimal Mg2' concentration ATP was not required for initiation or elongation. Besides Mg'+ also Mn", Ca2+ and Ba*' were shown to support initiation whereas for elongation only Mg2' was accepted. Since Mn2+ could efficiently be used for DNA chain elongation on synthetic templates we hypothesize that M$ is essential for the transition of initiation to elongation.

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“…Such intramolecular interactions are thought to occur during Ad replication when the ITRs in single-stranded intermediates form a so-called pan-handle structure (7,19). However, in vitro stud-ies have shown that the Ad DNA-binding protein, when present in sufficient amounts, inhibits intramolecular renaturation and enhances intermolecular renaturation (22). The inhibitory effect was attributed to the rigid structure of single-stranded DNA bound to DNA-binding protein and which might, therefore, be weaker on longer templates.…”

Section: Discussionmentioning

confidence: 99%

Common Structure of Rare Replication-Deficient E1-Positive Particles in Adenoviral Vector Batches

Murakami¹,

Havenga

Fawaz³

et al. 2004

J Virol

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The use of the PER.C6 adenovirus packaging cell line in combination with a designated vector plasmid system, whereby the cell line and vector with E1 deleted have no sequence overlap, eliminates the generation of replication-competent adenovirus during vector production. However, we have found cytopathic effect (CPE)-inducing particles in 2 out of more than 40 large-scale manufacturing lots produced in PER.C6 cells. The CPE inducer was detected at a frequency of 1 event in 7.5 ؋ 10 12 vector particles. Despite amplification, it was not readily purified, indicating that the agent itself is replication deficient and requires the parental recombinant adenovirus serotype 5 (rAd5) vector for replication and packaging. Therefore, we designated the agent as a helper-dependent E1-positive region containing viral particle (HDEP). Here, we report the molecular structure of the HDEP genome, revealing an Ad comprised of E1 sequences derived from PER.C6 cells flanked by inverted terminal repeat, packaging signal, and transgene sequences. These sequences form a palindromic structure devoid of E2, E3, E4, and late genes. Since only 5 bp were shared between E1 sequences in the PER.C6 genome and viral vector sequences, the data strongly suggested that insertion of genomic DNA into an adenoviral genome had occurred essentially via nonhomologous recombination. HDEPs have been found in unrelated virus batches and appear to share a common structure that may explain their mechanism of generation. This finding allowed development of an HDEP assay to screen batches of rAd5 produced on the PER.C6 cell line and resulted in detection of seven HDEP agents from four different transgene-virus vector constructs in separate batches of Ad.

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The adenovirus DNA binding protein enhances intermolecular DNA renaturation but inhibits intramolecular DNA renaturation

Cited by 21 publications

References 49 publications

Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Can Prevent Self-Priming of Minus-Strand Strong Stop DNA by Promoting the Annealing of Short Oligonucleotides to Hairpin Sequences

Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Can Prevent Self-Priming of Minus-Strand Strong Stop DNA by Promoting the Annealing of Short Oligonucleotides to Hairpin Sequences

Replication of adenovirus DNA in vitro is ATP‐independent

Common Structure of Rare Replication-Deficient E1-Positive Particles in Adenoviral Vector Batches

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