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 A Succession of Mechanisms Stimulate Efficient Reconstituted HIV-1 Minus Strand Strong Stop DNA Transfer†

Biochemistry ◽  
2009 ◽  
Vol 48 (8) ◽  
pp. 1810-1819 ◽  
Author(s):  
Min Song ◽  
Mini Balakrishnan ◽  
Robert J. Gorelick ◽  
Robert A. Bambara
Keyword(s):  
Dna Transfer ◽  
Minus Strand ◽  
Hiv 1 ◽  
Strong Stop

scholarly journals Proximity and Branch Migration Mechanisms in HIV-1 Minus Strand Strong Stop DNA Transfer

2007 ◽  
Vol 283 (6) ◽  
pp. 3141-3150 ◽  
Author(s):  
Min Song ◽  
Vandana P. Basu ◽  
Mark N. Hanson ◽  
Bernard P. Roques ◽  
Robert A. Bambara
Keyword(s):  
Dna Transfer ◽  
Minus Strand ◽  
Branch Migration ◽  
Hiv 1 ◽  
Strong Stop

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.

scholarly journals Determination of the Ex Vivo Rates of Human Immunodeficiency Virus Type 1 Reverse Transcription by Using Novel Strand-Specific Amplification Analysis

2007 ◽  
Vol 81 (9) ◽  
pp. 4798-4807 ◽  
Author(s):  
David C. Thomas ◽  
Yegor A. Voronin ◽  
Galina N. Nikolenko ◽  
Jianbo Chen ◽  
Wei-Shau Hu ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Reverse Transcription ◽  
Ex Vivo ◽  
Dna Transfer ◽  
Minus Strand ◽  
Immunodeficiency Virus ◽  
Specific Amplification ◽  
Kinetics Of ◽  
Hiv 1

ABSTRACT Replication of human immunodeficiency virus type 1 (HIV-1), like all organisms, involves synthesis of a minus-strand and a plus-strand of nucleic acid. Currently available PCR methods cannot distinguish between the two strands of nucleic acids. To carry out detailed analysis of HIV-1 reverse transcription from infected cells, we have developed a novel strand-specific amplification (SSA) assay using single-stranded padlock probes that are specifically hybridized to a target strand, ligated, and quantified for sensitive analysis of the kinetics of HIV-1 reverse transcription in cells. Using SSA, we have determined for the first time the ex vivo rates of HIV-1 minus-strand DNA synthesis in 293T and human primary CD4+ T cells (∼68 to 70 nucleotides/min). We also determined the rates of minus-strand DNA transfer (∼4 min), plus-strand DNA transfer (∼26 min), and initiation of plus-strand DNA synthesis (∼9 min) in 293T cells. Additionally, our results indicate that plus-strand DNA synthesis is initiated at multiple sites and that several reverse transcriptase inhibitors influence the kinetics of minus-strand DNA synthesis differently, providing insights into their mechanism of inhibition. The SSA technology provides a novel approach to analyzing DNA replication processes and should facilitate the development of new antiretroviral drugs that target specific steps in HIV-1 reverse transcription.

scholarly journals Mutations in the U5 Region Adjacent to the Primer Binding Site Affect tRNA Cleavage by Human Immunodeficiency Virus Type 1 Reverse Transcriptase In Vivo

2007 ◽  
Vol 82 (2) ◽  
pp. 719-727 ◽  
Author(s):  
Jangsuk Oh ◽  
Mary Jane McWilliams ◽  
John G. Julias ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Rnase H ◽  
Primer Binding Site ◽  
Cleavage Specificity ◽  
Immunodeficiency Virus ◽  
Trna Primer ◽  
Hiv 1

ABSTRACT In retroviruses, the first nucleotide added to the tRNA primer defines the end of the U5 region in the right long terminal repeat, and the subsequent removal of this tRNA primer by RNase H exactly defines the U5 end of the linear double-stranded DNA. In most retroviruses, the entire tRNA is removed by RNase H cleavage at the RNA/DNA junction. However, the RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase cleaves the tRNA 1 nucleotide from the RNA/DNA junction at the U5/primer binding site (PBS) junction, which leaves an rA residue at the U5 terminus. We made sequence changes at the end of the U5 region adjacent to the PBS in HIV-1 to determine whether such changes affect the specificity of tRNA primer cleavage by RNase H. In some of the mutants, RNase H usually removed the entire tRNA, showing that the cleavage specificity was shifted by 1 nucleotide. This result suggests that the tRNA cleavage specificity of the HIV-1 RNase domain H depends on sequences in U5.

How the HIV-1 Nucleocapsid Protein Binds and Destabilises the (−)Primer Binding Site During Reverse Transcription

2008 ◽  
Vol 383 (5) ◽  
pp. 1112-1128 ◽  
Author(s):  
Sarah Bourbigot ◽  
Nick Ramalanjaona ◽  
Christian Boudier ◽  
Gilmar F.J. Salgado ◽  
Bernard P. Roques ◽  
...  
Keyword(s):  
Binding Site ◽  
Reverse Transcription ◽  
Nucleocapsid Protein ◽  
Primer Binding Site ◽  
Hiv 1

scholarly journals Mechanism of Minus Strand Strong Stop Transfer in HIV-1 Reverse Transcription

2002 ◽  
Vol 278 (10) ◽  
pp. 8006-8017 ◽  
Author(s):  
Yan Chen ◽  
Mini Balakrishnan ◽  
Bernard P. Roques ◽  
Philip J. Fay ◽  
Robert A. Bambara
Keyword(s):  
Reverse Transcription ◽  
Minus Strand ◽  
Hiv 1 ◽  
Strong Stop

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.

Sign in / Sign up

Export Citation Format

Share Document