scholarly journals Yeast Ty1 retrotransposon: the minus-strand primer binding site and acis-acting domain of the Ty1 RNA are both important for packaging of primer tRNA inside virus-like particles

1994 ◽  
Vol 22 (22) ◽  
pp. 4560-4565 ◽  
Author(s):  
M. Wilhelm ◽  
F.X. Wilhelm ◽  
G. Keith ◽  
B. Agoutin ◽  
T. Heyman
Keyword(s):  
Binding Site ◽  
Primer Binding Site ◽  
Minus Strand ◽  
Virus Like Particles ◽  
Ty1 Retrotransposon

scholarly journals Extended Minus-Strand DNA as Template for R-U5-Mediated Second-Strand Transfer in Recombinational Rescue of Primer Binding Site-Modified Retroviral Vectors

1998 ◽  
Vol 72 (3) ◽  
pp. 2519-2525 ◽  
Author(s):  
Jacob Giehm Mikkelsen ◽  
Anders H. Lund ◽  
Karen Dybkær ◽  
Mogens Duch ◽  
Finn Skou Pedersen
Keyword(s):  
Binding Site ◽  
Leukemia Virus ◽  
Retroviral Vectors ◽  
Primer Binding Site ◽  
Template Switching ◽  
Strand Transfer ◽  
Minus Strand ◽  
Endogenous Virus ◽  
Recombination System ◽  
Virus Rna

ABSTRACT We have previously demonstrated recombinational rescue of primer binding site (PBS)-impaired Akv murine leukemia virus-based vectors involving initial priming on endogenous viral sequences and template switching during cDNA synthesis to obtain PBS complementarity in second-strand transfer of reverse transcription (Mikkelsen et al., J. Virol. 70:1439–1447, 1996). By use of the same forced recombination system, we have now found recombinant proviruses of different structures, suggesting that PBS knockout vectors may be rescued through initial priming on endogenous virus RNA, read-through of the mutated PBS during minus-strand synthesis, and subsequent second-strand transfer mediated by the R-U5 complementarity of the plus strand and the extended minus-strand DNA acceptor template. Mechanisms for R-U5-mediated second-strand transfer and its possible role in retrovirus replication and evolution are discussed.

scholarly journals Interactions between Ty1 Retrotransposon RNA and the T and D Regions of the tRNAiMet Primer Are Required for Initiation of Reverse Transcription In Vivo

1998 ◽  
Vol 18 (2) ◽  
pp. 799-806 ◽  
Author(s):  
S. Friant ◽  
T. Heyman ◽  
A. S. Byström ◽  
M. Wilhelm ◽  
F. X. Wilhelm
Keyword(s):  
Reverse Transcription ◽  
Primer Binding Site ◽  
Minus Strand ◽  
Genomic Rna ◽  
Complementary Sequences ◽  
Compensatory Mutations ◽  
Ty1 Retrotransposon ◽  
Ty1 Retrotransposition ◽  
Strong Stop

ABSTRACT Reverse transcription of the Saccharomyces cerevisiaeTy1 retrotransposon is primed by tRNAi Met base paired to the primer binding site (PBS) near the 5′ end of Ty1 genomic RNA. The 10-nucleotide PBS is complementary to the last 10 nucleotides of the acceptor stem of tRNAi Met. A structural probing study of the interactions between the Ty1 RNA template and the tRNAi Met primer showed that besides interactions between the PBS and the 3′ end of tRNAi Met, three short regions of Ty1 RNA, named boxes 0, 1, and 2.1, interact with the T and D stems and loops of tRNAi Met. To determine if these sequences are important for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNAi Metwere tested for the ability to support transposition. We show that the Ty1 boxes and the complementary sequences in the T and D stems and loops of tRNAi Met contain bases that are critical for Ty1 retrotransposition. Disruption of 1 or 2 bp between tRNAi Met and box 0, 1, or 2.1 dramatically decreases the level of transposition. Compensatory mutations which restore base pairing between the primer and the template restore transposition. Analysis of the reverse transcription intermediates generated inside Ty1 virus-like particles indicates that initiation of minus-strand strong-stop DNA synthesis is affected by mutations disrupting complementarity between Ty1 RNA and primer tRNAi Met.

scholarly journals Stimulation of HIV-1 Minus Strand Strong Stop DNA Transfer by Genomic Sequences 3′ of the Primer Binding Site

2006 ◽  
Vol 281 (34) ◽  
pp. 24227-24235 ◽  
Author(s):  
Min Song ◽  
Mini Balakrishnan ◽  
Yan Chen ◽  
Bernard P. Roques ◽  
Robert A. Bambara
Keyword(s):  
Binding Site ◽  
Dna Transfer ◽  
Genomic Sequences ◽  
Primer Binding Site ◽  
Minus Strand ◽  
Hiv 1 ◽  
Strong Stop ◽  
Stimulation Of

scholarly journals HIV-1 Employs Multiple Mechanisms To Resist Cas9/Single Guide RNA Targeting the Viral Primer Binding Site

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Zhen Wang ◽  
Wenzhou Wang ◽  
Ya Cheng Cui ◽  
Qinghua Pan ◽  
Weijun Zhu ◽  
...  
Keyword(s):  
Binding Site ◽  
Reverse Transcription ◽  
Gene Editing ◽  
Viral Escape ◽  
Primer Binding Site ◽  
Minus Strand ◽  
Guide Rna ◽  
Target Dna ◽  
Almost All ◽  
Hiv 1

ABSTRACTThe clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (Cas9) gene-editing technology has been used to inactivate viral DNA as a new strategy to eliminate chronic viral infections, including HIV-1. This utility of CRISPR-Cas9 is challenged by the high heterogeneity of HIV-1 sequences, which requires the design of the single guide RNA (sgRNA; utilized by the CRISPR-Cas9 system to recognize the target DNA) to match a specific HIV-1 strain in an HIV patient. One solution to this challenge is to target the viral primer binding site (PBS), which HIV-1 copies from cellular tRNA3Lysin each round of reverse transcription and is thus conserved in almost all HIV-1 strains. In this study, we demonstrate that PBS-targeting sgRNA directs Cas9 to cleave the PBS DNA, which evokes deletions or insertions (indels) and strongly diminishes the production of infectious HIV-1. While HIV-1 escapes from PBS-targeting Cas9/sgRNA, unique resistance mechanisms are observed that are dependent on whether the plus or the minus strand of the PBS DNA is bound by sgRNA. Characterization of these viral escape mechanisms will inform future engineering of Cas9 variants that can more potently and persistently inhibit HIV-1 infection.IMPORTANCEThe results of this study demonstrate that the gene-editing complex Cas9/sgRNA can be programmed to target and cleave HIV-1 PBS DNA, and thus, inhibit HIV-1 infection. Given that almost all HIV-1 strains have the same PBS, which is copied from the cellular tRNA3Lysduring reverse transcription, PBS-targeting sgRNA can be used to inactivate HIV-1 DNA of different strains. We also discovered that HIV-1 uses different mechanisms to resist Cas9/sgRNAs, depending on whether they target the plus or the minus strand of PBS DNA. These findings allow us to predict that a Cas9 variant that uses the CCA sequence as the protospacer adjacent motif (PAM) should more strongly and persistently suppress HIV-1 replication. Together, these data have identified the PBS as the target DNA of Cas9/sgRNA and have predicted how to improve Cas9/sgRNA to achieve more efficient and sustainable suppression of HIV-1 infection, therefore improving the capacity of Cas9/sgRNA in curing HIV-1 infection.

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